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Research Collection
Doctoral Thesis
Understanding the toxicity of nanosilver for synthesis ofbiocompatible plasmonic-superparamagnetic nanocomposites
Author(s): Sotiriou, Georgios A.
Publication Date: 2011
Permanent Link: https://doi.org/10.3929/ethz-a-006712477
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
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Diss. ETH No. 19860
Understanding the Toxicity of Nanosilver for
Synthesis of Biocompatible Plasmonic-
Superparamagnetic Nanocomposites
A dissertation submitted to
ETH ZURICH
for the degree of
Doctor of Sciences
presented by
GEORGIOS A. SOTIRIOU
M.Sc. ETH Zurich, Switzerland
born July 25th, 1983
citizen of Greece
accepted on the recommendation of
Prof. Dr. Sotiris E. Pratsinis, examiner
Prof. Dr. Sven Panke, co-examiner
Zurich, 2011
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Cover page: “Nanosilver stars”
Silica-coated nanosilver particles dispersed in water and
visualized under dark-field illumination. The bright dots
correspond to the plasmonic nanosilver particles because of
their strong light scattering.
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“οὐδὲν μάθημα μετὰ δουλείας τὸν ἐλεύθερον χρὴ μανθάνειν,
οἱ μὲν γὰρ τοῦ σώματος πόνοι βίᾳ πονούμενοι
χεῖρον οὐδὲν τὸ σῶμα ἀπεργάζονται,
ψυχῇ δὲ βίαιον οὐδὲν ἔμμονον μάθημα”
άτης
από την “Πολιτεία” Πλάτωνος
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Acknowledgements
I am deeply grateful to Prof. Dr. Sotiris E. Pratsinis for his continuous
scientific and moral support throughout my thesis. Through his supervision and
numerous stimulating discussions he opened for me the door of professionalism and
high quality research. I would also like to express my gratitude to Prof. Dr. Sven
Panke for his interest in my work, providing scientific support and access to his
laboratory, and for co-advising this thesis.
This work was carried out at the Particle Technology Laboratory at ETH
Zurich and I would like to thank all of its members for creating such a nice
atmosphere to work in, as well as for many fruitful discussions. Many thanks go to
my officemates, Dr. Adrian Camenzind and Jesper Knijnenburg, for creating an
enjoyable environment to work in. Special thanks to Dr. Frank Krumeich whose
expertise in electron microscopy made it possible to visualize numerous small
particles and structures. I would also like to thank the workshop of the Institute of
Process Engineering and especially Rene Pluss, for his creative solutions to many
constructional or technical challenges. Many thanks to the IT support and especially
to Justina Palmer who was patient enough to deal with any computer issues that
arose. Special thanks to Agnes Rupacher who with her support made the
bureaucratic matters seem suddenly very simple and easy.
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This work would not have been accomplished without the support of Prof.
Janos Vörös and Dr. Takumi Sannomiya from the Laboratory of Biosensors and
Bioelectronics, Prof. Dr. Ann M. Hirt from the Institute of Geophysics, Dr. Pierre-
Yves Lozach from the Institute of Biochemistry, all at ETH Zurich. Special thanks
to Dr. Andreas Meyer from the Department of Biosystems Science and Engineering
who introduced me to many small fluorescent micro-organisms. Many thanks to Dr.
Justine Kusch and Dr. Sung-Sik Lee for their help with dark-field imaging, and to
Prof. Laura Sigg and Dr. Niksa Odzak for the DGT measurements. I would also like
to thank Prof. Dr. Philip Demokritou from Harvard University for his hospitality in
his lab and help to perform research in the nanoscale from a different point of view.
Several bachelor and master students performed their projects with me or
worked as assistants that made contributions to this thesis: Melanie Schneider,
Giovanni Matucci, Jara Schnyder, Christoph Blattmann, Samuel Gass, David
Wochner, Siebert Frieling, Sugeet Chopra, Sylvie Anthonioz, Silvan Staufert,
Sagrario Lira Ramos, Ivan Pahrebniakou, Gion-Andri Büsser, Denis Butscher,
Georg Balmer.
I would like to especially thank Dr. Alexandra Teleki not only for her
scientific contributions to this thesis, but also for providing endless joy and moral
support in my life within and beyond science.
Finally, I would like to express my deepest gratitude to my parents and my
sister for their unconditional love and support for every step that I make.
Financial support from the Swiss National Science Foundation (#200020-
126694) and the European Research Council is kindly acknowledged.
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Contents
Acknowledgements .............................................................................. i
Contents ......................................................................................... iii
Summary ......................................................................................... xi
Zusammenfassung ............................................................................ xvii
1. Engineering Nanosilver as Antibacterial, Biosensor and
Bioimaging material ..................................................................... 1
1.1 Introduction ............................................................................................... 2
1.1.1 Historical perspective of silver metal ............................................................... 2
1.1.2 Silver in the nanoscale .................................................................................. 3
1.1.3 Implications regarding use of nanosilver .......................................................... 6
1.2 Nanosilver synthetic routes and morphologies .............................................. 8
1.2.1 Pure and surface modified ............................................................................. 8
1.2.2 Supported on ceramic nanoparticles .............................................................. 10
1.2.3 Coated nanosilver ...................................................................................... 11
1.2.4 Heterodimer or “Janus-like” nanosilver particles ............................................ 11
1.3 Interactions of nanosilver with biological systems ....................................... 12
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1.3.1 Antimicrobial activity ................................................................................ 12
1.3.2 Cytotoxicity of nanosilver particles against mammalian cells ........................... 14
1.3.3 Nanosilver particles, Ag+ ions or both? .......................................................... 19
1.3.4 Ag+ ion release in aqueous suspensions.......................................................... 21
1.4 Biomedical Applications of Nanosilver ...................................................... 23
1.4.1 Antimicrobial agent ................................................................................... 23
1.4.2 Biosensors with plasmonic nanosilver ........................................................... 24
1.4.3 Bioimaging agent ....................................................................................... 27
1.5 Summary and concluding remarks ............................................................. 29
1.6 References ............................................................................................... 30
2. Antibacterial Activity of Nanosilver Ions and Particles ........................ 49
2.1 Introduction ............................................................................................. 50
2.2 Materials and methods .............................................................................. 51
2.2.1 Particle synthesis ....................................................................................... 51
2.2.2 Particle characterization ............................................................................. 52
2.2.3 Antibacterial Activity ................................................................................. 53
2.3 Results and discussion .............................................................................. 55
2.3.1 Nanosilver morphology and size ................................................................... 55
2.3.2 Ag+ ion release........................................................................................... 58
2.3.3 Antibacterial activity of nanosilver: Ag+ ions and Ag nanoparticles .................. 61
2.4 Conclusions ............................................................................................. 65
2.5 References ............................................................................................... 65
3. Nanosilver on Nanostructured Silica: Antibacterial Activity and
Ag Surface Area .......................................................................... 71
3.1 Introduction ............................................................................................. 72
3.2 Materials and methods .............................................................................. 74
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3.2.1 Particle synthesis and characterization .......................................................... 74
3.2.2 Antibacterial activity .................................................................................. 75
3.3 Results and discussion .............................................................................. 76
3.3.1 Effect of precursor composition ..................................................................... 76
3.3.2 Antibacterial activity .................................................................................. 84
3.4 Conclusions ............................................................................................. 90
3.5 References ............................................................................................... 91
4. Quantifying the Origin of Nanosilver Ions and their
Antibacterial Activity .................................................................. 97
4.1 Introduction ............................................................................................. 98
4.2 Materials and methods .............................................................................. 99
4.3 Results and discussion ............................................................................ 101
4.3.1 Morphology and composition of flame- and wet-made Ag/SiO2 nanoparticles .. 101
4.3.2 Ag+ ion release: Dissolution of the surface oxide layer by washing ................... 104
4.3.3 Ag+ ion release: Reduction under H2 of the surface oxide layer ....................... 108
4.3.4 Ag oxide layer thickness and Ag+ ion concentration ...................................... 110
4.3.5 Antibacterial activity ................................................................................ 112
4.4 Conclusions ........................................................................................... 116
4.5 References ............................................................................................. 117
5. Non-toxic Dry-coated Nanosilver for Plasmonic Biosensors ................ 121
5.1 Introduction ........................................................................................... 122
5.2 Materials and methods ............................................................................ 125
5.2.1 Nanosilver particle production and characterization ..................................... 125
5.2.2 Toxicity evaluation .................................................................................. 127
5.2.3 Biosensor performance .............................................................................. 128
5.3 Results and discussion ............................................................................ 129
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5.3.1 Hermetic SiO2 coating: ‘Curing’ nanosilver’s toxicity .................................... 129
5.3.2 Optical properties of SiO2-coated nanosilver: Agglomeration .......................... 134
5.3.3 Label-free biosensor performance ................................................................ 136
5.4 Conclusions ........................................................................................... 139
5.5 References ............................................................................................. 140
6. Hybrid, Silica-coated, Janus-like Plasmonic-Magnetic Nanoparticles ..... 145
6.1 Introduction ........................................................................................... 146
6.2 Materials and methods ............................................................................ 148
6.2.1 Hybrid SiO2-coated Ag/Fe2O3 nanoparticle synthesis .................................... 148
6.2.2 Particle characterization ........................................................................... 149
6.2.3 Biocompatibility of the hybrid biomarkers ................................................... 150
6.2.4 Bioimaging ............................................................................................. 151
6.3 Results and discussion ............................................................................ 151
6.3.1 Morphology ............................................................................................ 151
6.3.2 Magnetic and plasmonic performance ......................................................... 153
6.3.3 Stability in aqueous suspensions and buffer solutions .................................... 156
6.3.4 Biocompatibility of SiO2-coated hybrid plasmonic-magnetic biomarkers .......... 157
6.3.5 Bioimaging ............................................................................................. 161
6.4 Conclusions ........................................................................................... 163
6.5 References ............................................................................................. 164
7. Outlook and Research Recommendations ........................................ 169
7.1 References ............................................................................................. 172
A. Supplementary Information: Antibacterial Activity of Nanosilver
Ions and Particles.................................................................... 175
A.1 Morphology of flame-made nanosilver particles ........................................ 175
A.2 Ag+ ion release and stability in suspensions .............................................. 177
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A.3 Reference ............................................................................................... 179
B. Comparison of the Antibacterial Activity of as-prepared and washed
Nanosilver Particles................................................................. 181
B.1 Introduction ........................................................................................... 181
B.2 Materials and methods ............................................................................ 182
B.3 Results and discussion ............................................................................ 182
B.3.1 Comparison of antibacterial activities ......................................................... 182
B.3.2 Effective Ag+ ion concentration on E. coli viability ....................................... 185
B.3.3 Calculation of Ag mass concentration ......................................................... 186
B.3.4 Calculation of Ag surface area concentration ............................................... 187
C. Hermetically Silica-coated Nanosilver: Optimizing the
Coating Reactor...................................................................... 189
C.1 Introduction ........................................................................................... 190
C.2 Materials and methods ............................................................................ 191
C.3 Results and discussion ............................................................................ 192
C.3.1 Control of nanosilver size .......................................................................... 192
C.3.2 Ag+ ion release of composite Ag/SiO2 ......................................................... 193
C.3.3 Single coating ring ................................................................................... 194
C.3.4 Double coating ring .................................................................................. 197
C.4 References ............................................................................................. 198
D. Color-tunable Nanophosphors by Co-doping Flame-made Y2O3
with Tb and Eu....................................................................... 201
D.1 Introduction ........................................................................................... 202
D.2 Materials and methods ............................................................................ 204
D.3 Results and discussion ............................................................................ 205
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D.3.1 Phosphor morphology ............................................................................... 205
D.3.2 Photoluminescence of Y2O3:Tb3+ nanophosphors .......................................... 209
D.3.3 Co-doped Y2O3:Tb/Eu .............................................................................. 212
D.4 Conclusions ........................................................................................... 214
D.5 References ............................................................................................. 215
E. Flame Synthesis and Characterization of Silica-coated
Y2O3:Tb3+ Nanophosphors.......................................................... 219
E.1 Introduction ........................................................................................... 220
E.2 Materials and methods ............................................................................ 222
E.3 Results and discussion ............................................................................ 223
E.3.1 Morphology and crystallinity of Y2O3:Tb3+ nanoparticles .............................. 223
E.3.2 Phosphorescence of cubic and monoclinic Y2O3:Tb3+ nanoparticles .................. 225
E.3.3 Annealing of uncoated and SiO2-coated monoclinic Y2O3:Tb3+ nanoparticles ... 229
E.4 Conclusions ........................................................................................... 236
E.5 References ............................................................................................. 237
F. A Novel Platform for Pulmonary and Cardiovascular Toxicological
Characterization of Inhaled Engineered Nanomaterials...................... 243
F.1 Introduction ........................................................................................... 244
F.2 Materials and methods ............................................................................ 246
F.2.1 Versatile engineered nanomaterials generation system (VENGES) ................. 246
F.2.2 Performance characterization experiments ................................................... 249
F.2.3 Acute Pulmonary and cardiovascular effects of inhaled nanostructured Fe2O3 using
the VENGES platform and IVCL assay ...................................................... 251
F.3 Results and discussion ............................................................................ 252
F.3.1 Performance characterization experiments ................................................... 252
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F.3.2 Pulmonary and cardiovascular effects of inhaled nanostructured Fe2O3 using the
VENGES platform .................................................................................. 260
F.4 Conclusions ........................................................................................... 264
F.5 References ............................................................................................. 265
G. Multi-layer Polymer Nanocomposite Films..................................... 273
G.1 Introduction ........................................................................................... 274
G.2 Materials and methods ............................................................................ 274
G.3 Results and discussion ............................................................................ 275
G.4 Conclusions ........................................................................................... 287
G.5 References ............................................................................................. 287
Curriculum Vitae ............................................................................. 289
Publications and Presentations ............................................................ 291
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Summary
Nowadays, everyday life would be inconceivable without nanotechnology.
The biomedical field has already obtained a great benefit from nanotechnology as it
helps the diagnosis and even therapy of diseases. The unique physicochemical
properties of silver nanoparticles (nanosilver) have brought them to the foreground
of such nanotechnology-based products and applications. Nanosilver can kill micro-
organisms very efficiently which facilitates its employment as antimicrobial agent.
Furthermore, because of their small size, nanosilver particles interact in a special
way with light that gives rise to their plasmonic properties. Because of these
properties, nanosilver can be used in a variety of biomedical applications, such as
biosensing and bioimaging.
The potential benefits, however, that nanosilver can offer have to be
balanced with the potential adverse effects that its broad use may cause. Therefore,
a fundamental understanding of the mechanism that nanosilver interacts with
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biological systems needs to be established in order to employ its full capacity. The
last few years, this has ignited a number of studies that investigate the toxicity of
nanosilver particles against biological systems. However, a systematic
understanding of the physical properties that influence this toxicity has yet to be
established.
In chapter 1, an overview of the synthesis methods of nanosilver particles
and their biological interactions with bacteria and mammalian cells is presented.
The main nanosilver toxicity mechanisms are discussed focusing on the Ag+ ion
release and the physicochemical properties that influence it. The toxicity of
nanosilver with different sizes and surface coatings is compared, emphasizing the
limitations of the currently used systems. The biomedical applications of nanosilver
particles are also reviewed in respect to their antimicrobial and plasmonic
properties, summarizing the state-of-the-art processes and products but also
highlighting the further challenges.
In the second chapter, the effect of the released Ag+ ions on the antibacterial
activity of nanosilver particles is investigated. This was performed by synthesizing
nanosilver particles on nanostructured silica with a precise control over their size,
and investigating the Ag+ ion release of these particles in aqueous solutions. Smaller
nanosilver particles release higher fractions of Ag+ ions, indicating a size-dependent
phenomenon. The antibacterial activity against E. coli in the presence of nanosilver
particles and ions or only in the presence of ions is further investigated. When small
(<10 nm) nanosilver particles are employed that release high fractions of Ag+ ions,
the antibacterial activity is dominated by these ions. In contrast, when relatively
larger (>10 nm) nanosilver particles are used that do not release many Ag+ ions,
then the antibacterial activity of the particles and the ions is comparable.
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In chapter 3, the dose relations for the antibacterial activity of nanosilver on
nanostructured silica particles are investigated focusing on the small (<10 nm) size
range where Ag+ ion release is significant. The effect of the silicon and silver
precursors on the morphology of the synthesized nanoparticles is also investigated.
The resulting nanosilver particles have similar Ag+ ion release independently of the
precursors used. The antibacterial activity of these nanosilver particles against E. coli
is investigated at various concentrations and sizes. The nanosilver surface area
concentration in suspension correlates best with the antibacterial activity that is
observed rather than nanosilver mass or number concentration. This indicates that
the nanosilver dose expressions in toxicological studies might be most accurate
when assessed in terms of surface area concentration.
The origin of the released Ag+ ions from the nanosilver surface is
investigated in chapter 4. The Ag+ ion release of the composite Ag/SiO2
nanoparticles made by flame- or wet-chemistry is investigated and directly
correlated to their size, independently of their synthesis route. Furthermore, when
nanosilver particles dispersed in water are collected and re-suspended in fresh water,
their Ag+ ion release is minimal. Additionally, when nanosilver is reduced under H2
and converted to metallic, the Ag+ ion release is also at minimal levels. This
indicates that the Ag+ ions originate from the dissolution of the oxide layer on the
nanosilver surface. In fact, the Ag+ ion release can be quantitatively traced back to
the dissolution of one or two oxide surface layers depending on nanosilver size,
closing thus the mass balance. The antibacterial activity of washed nanosilver
particles is, therefore, lower than the one of as-prepared nanosilver.
The understanding obtained by the above results was employed in chapter 5
in order to design nanosilver particles that do not exhibit toxicity but retain their
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desired optical properties. So rather large (>30 nm) nanosilver particles were made
and coated in situ with a nanothin SiO2 layer. In that way, the fully-coated
nanosilver particles were not toxic against E. coli cells in contrast to the partially-
coated ones. The inert SiO2 coating enabled the easy dispersion of these nanosilver
particles in aqueous solutions and significantly prevented their flocculation. This
facilitated their employment as plasmonic biosensors. Their biosensing performance
was investigated by detecting bovine serum albumin in a flow cell and monitoring
the shift of the plasmon absorption band. The fully-coated nanosilver particles
outperformed the partially-coated ones, enabling them to be used as non-toxic
plasmonic biosensors.
Finally, in chapter 6, the further employment of nanosilver particles in
bioimaging was investigated. This time, nanosilver particles were synthesized along
with iron oxide particles, forming the so-called Janus-like structures. The composite
particles were also coated in situ by a nanothin SiO2 layer. These specially designed
multifunctional nanoparticles exhibited the desired plasmonic and magnetic
properties of the nanosilver and iron oxide particles, respectively. In addition, the
SiO2 coating prevented their flocculation in aqueous and biological buffer solutions
and most importantly, inhibited significantly the toxic Ag+ ion release from the
nanosilver particles. As a result, these multicomponent plasmonic-magnetic
nanoparticles exhibited no cytotoxicity against HeLa cells for 24 hours incubation.
Furthermore, their surface functionalization with an antibody was verified by
monitoring the shift in their plasmon absorption band. These biofunctionalized
multifunctional nanoparticles were selectively bound to target cells (HeLa and Raji
cells) and their detection was possible under dark field illumination.
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The potential of nanosilver to be used as a multifunctional biomaterial was
mainly limited because of its inherent toxicity. In this thesis, it was shown that
when a fundamental understanding on the parameters that influence this toxicity is
established, then nanosilver particles can be synthesized that maintain the desired
properties without exhibiting the adverse ones. This understanding could assist the
development of nanosilver products with superior performance that could be
employed in a broad range of applications.
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Zusammenfassung
Das Alltagsleben wäre heutzutage nahezu unvorstellbar ohne
Nanotechnologie. In der Biomedizin ist Nanotechnologie für das Erkennen und
Behandeln von Krankheiten schon von grossem Nutzen. Die besonderen
physikochemischen Eigenschaften von Silber Nanopartikel (Nanosilber) hat sie in
den Vordergrund von solchen, auf Nanotechnologie basierenden, Produkten und
Anwendungen gerückt. Nanosilber kann sehr effizient Mikroorganismen töten, was
die Anwendung als antimikrobieller Wirkstoff ermöglicht. Auf Grund ihrer kleinen
Grösse wechselwirken Silber Nanoteilchen in einer speziellen Art mit Licht und
plasmonischen Eigenschaften der Partikel zur Folge hat. Auf Grund dieser
Eigenschaften, kann Nanosilber in einer Vielfalt von biomedizinischen
Anwendungen, z.B. als Biosensoren und für Bioimaging, eingesetzt werden.
Der mögliche Nutzen von Nanosilber muss dennoch mit den eventuellen
nachteiligen Auswirkungen, die eine weit verbreitete Anwendung verursachen
könnte, abgeglichen werden. Um die volle Leistung von Nanosilber ausschöpfen zu
können, muss ein grundlegendes Verständnis der Wechselwirkungen von Nanosilber
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mit biologischen Systemen entwickelt werden. Dies hat in den letzten Jahren eine
grosse Anzahl von Studien angeregt, die die Toxizität von Nanosilber gegenüber
biologischen Systemen untersucht. Ein systematisches Verständnis der
physikalischen Eigenschaften die die Toxizität beeinflussen, muss dennoch erstellt
werden.
Im Kapitel 1, wird ein Überblick über die Herstellungsverfahren von Silber
Nanoteilchen und deren biologischen Wechselwirkungen mit Bakterien und
Säugetierzellen präsentiert. Es werden die wichtigsten Mechanismen der Nanosilber
Toxizität mit einem Schwerpunkt auf die Freilassung von Ag+ Ionen diskutiert
sowie die physikochemischen Eigenschaften, die diese beeinflussen. Die Toxizität
von Nanosilber von unterschiedlicher Grösse und mit verschiedener
Oberflächenbeschichtungen werden verglichen, um die Einschänkungen der heute
verwendeten Systeme zu unterstreichen. Die biomedizinischen Anwendungen von
Nanosilber werden auch in Bezug auf deren antimikrobiellen und plasmonischen
Eigenschaften besprochen, moderne Prozesse und Produkte werden
zusammengefasst, aber auch weitere Herausforderungen werden aufgezeigt.
Im zweiten Kapitel wird der Einfluss von freigesetzten Ag+ Ionen auf die
antibakterielle Aktivität von Silber Nanoteilchen untersucht. Dafür wurden Silber
Nanoteilchen mit genauer Kontrolle ihrer Grösse auf nanostrukturiertem
Siliziumdioxid hergestellt und die Freisetzung von Ag+ Ionen von diesen Teilchen in
wässrigen Lösungen erforscht. Kleinere Silber Nanoteilchen setzten höhere Anteile
von Ag+ Ionen frei, was auf ein grössenabhängiges Phänomen hindeutet. Die
antibakterielle Aktivität gegenüber E. coli in der Gegenwart von Nanosilberteilchen
und Ionen oder nur von Ionen wurde weiter untersucht. Wenn kleine (< 10 nm)
Nanosilberteilchen die einen hohen Anteil von Ag+ Ionen freisetzen, eingesetzt
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werden, wird die antibakterielle Aktivität von diesen Ionen beherrscht. Wenn aber
im Vergleich grössere (> 10 nm) Nanosilberteilchen, die nicht viele Ag+ Ionen
freisetzen, verwendet werden, ist die antibakterielle Aktivität der Partikel und der
Ionen vergleichbar.
Im dritten Kapitel, wird die Dosis-Beziehung der antibakteriellen Aktivität
von Nanosilber auf nanostrukturiertem Siliziumdioxidteilchen mit Schwerpunkt auf
dem kleinen Grössenbereich (< 10 nm) untersucht wo die Freisetzung von Ag+
Ionen von grosser Bedeutung ist. Der Einfluss der Silikon- und
Silberausgangsstoffen auf die Morphologie der hergestellten Nanoteilchen wird auch
erforscht. Diese Nanosilberteilchen haben ähnliche Ag+ Ionenfreisetzung
unabhängig von den verwendeten Ausgangsstoffen. Die antibakterielle Aktivität von
diesen Nanoteilchen gegenüber E. coli mit verschiedenen Konzentrationen und
Grössen wurde untersucht. Die Nanosilber Oberflächenkonzentration in der
Suspension, eher als Nanosilber Masse- oder Anzahlkonzentration, entspricht am
besten der gemessenen antibakteriellen Aktivität. Dies deutet darauf hin, dass
Nanosilber Dosis-Beziehungen in toxikologischen Studien am genausten sein
könnten, wenn sie hinsichtlich der Silber Oberfläche ausgewertet werden.
Der Ursprung der freigesetzten Ag+ Ionen von der Nanosilber Oberfläche
wird im Kapitel 4 untersucht. Die Ag+ Ionenfreisetzung von der Ag/SiO2
Verbundstruktur in den Nanoteilchen, die mittels Flammen- oder
Nassphasensynthese hergestellt worden sind, wird untersucht und in direkter
Verbindung zu ihrer Grösse gesetzt, unabhängig von dem Herstellungsverfahren.
Ausserdem ist die Ag+ Ionenfreisetzung minimal, wenn Nanosilber mittels H2
reduziert und in metallisch umgewandelt wird. Dies deutet darauf hin, dass die Ag+
Ionen von der Auflösung der Oxidschicht der Silberteilchen stammen. Tatsächlich
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kann die Ag+ Ionenfreisetzung quantitativ auf die Auflösung von eine oder zwei
Oxidoberflächensichten, abhängig von der Nanosilber Grösse, zurückgeführt
werden und somit wird die Massenbilanz geschlossen. Die antibakterielle Aktivität
von gewaschenen Nanosilberteilchen ist tiefer als die von den hergestellten,
unbehandelten Nanosilber, da Ag+ Ionen nicht freigesetzt werden. Stattdessen kann
die antibakterielle Aktivität auf den Kontakt der Bakterien mit der
Nanosilberoberfläche zurückgeführt werden.
Das von den oben erwähnten Ergebnissen erzielte Verständnis wurde
eingesetzt um Nanosilberteilchen, die keine Toxizität aber ihre erwünschten
optischen Eigenschaften aufweisen, im Kapitel 5 zu entwickeln. Eher grosse (> 30
nm) Nanosilberteilchen wurden hergestellt und in situ mit einer nanodünnen SiO2
Schicht umhüllt. Die vollständig beschichteten Nanosilberteilchen waren gegenüber
E.coli nicht toxisch im Gegensatz zu den nur teilweise beschichteten Partikeln. Die
inerte SiO2 Beschichtung ermöglichte die einfache Dispersion von diesen
Nanosilberteilchen in wässrigen Lösungen und verhinderte erheblich die
Flockenbildung. Dies vereinfachte deren Verwendung als plasmonische
Biosensoren. Die biosensorischen Eigenschaften wurden durch die Erkennung von
Rindenserumalbumin in einer Durchflusszelle untersucht und die Verschiebung von
dem plasmonischen Absorptionsband wurde verfolgt. Die vollständig umhüllten
Nanosilberteilchen übertrafen die teilweise Beschichteten, und sie könnten deshalb
als nicht-toxische plasmonische Biosensoren verwendet werden.
Schliesslich im Kapitel 6, wurde die weitere Anwendung von
Nanosilberteilchen in Bioabbildung untersucht. Hier wurden Nanosilberteilchen
zusammen mit Eisenoxidteilchen hergestellt, und sogenannte Janus-ähnliche
Strukturen sind entstanden. Die Verbundteilchen wurden auch in situ mit einer
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nanodünnen SiO2 Schicht umhüllt. Diese speziell entworfenen multifunktionellen
Nanoteilchen zeigten die jeweiligen erwünschten plasmonischen und magnetischen
Eigenschaften von Nanosilber und Eisenoxidteilchen. Ausserdem verhinderte die
SiO2 Schicht die Flokkulation der Teilchen in wässrigen und biologischen
Pufferlösungen und verhinderte erheblich die toxische Ag+ Ionenfreisetzung von den
Nanosilberteilchen. Daher wiesen diese mehrkomponenten plasmonisch-magnetisch
Nanoteilchen keine Zytotoxizität gegenüber HeLa Zellen während 24 Stunden
Inkubation auf. Des Weiteren, wurde ihre Oberflächenfunktionalisierung mit einem
Antikörper bestätigt indem die Verschiebung ihrer plasmonischen Absorptionsbande
verfolgt wurde. Diese biofunktionalisierten multifunktionellen Nanoteilchen wurden
selektiv an Zielzellen (HeLa und Raji Zellen) gebunden und konnten mittels in
Dunkelfeldbeleuchtung nachgewiesen werden.
Das Potential von Nanosilber, als multifunktionelles Biomaterial verwendet
zu werden, war hauptsächlich durch ihre inhärente Toxizität eingeschränkt. In
dieser Dissertation wurde gezeigt, dass, wenn ein grundlegendes Verständnis der
Kenngrössen die die Toxizität beeinflussen ermittelt wird, Nanosilberteilchen
hergestellt werden können, die die gewünschten Eigenschaften ohne den
nachteiligen aufweisen. Dieses Verständnis könnte bei der Entwicklung von
Nanosilberprodukten mit ausgezeichneten Leistungen helfen, die in einer Vielfalt
von Anwendungen eingesetzt werden könnten.
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CHAPTER 1
1. Engineering Nanosilver as Antibacterial,
Biosensor and Bioimaging material1
Abstract
Silver nanoparticles (nanosilver) exhibit unique physicochemical properties
that facilitate their use in a variety of applications, especially in the biomedical field.
The ability of nanosilver to destroy infectious micro-organisms has enabled it to be
used as an antimicrobial agent with strong efficiency. Furthermore, its special
interaction with light gives rise to its plasmonic properties that facilitate its
employment as a biosensor or bioimaging agent. Here, the interactions of nanosilver
particles with biological systems including bacteria and mammalian cells are
reviewed. The main nanosilver toxicity mechanisms are discussed focusing on the
Ag+ ion release when nanosilver is dispersed in aqueous solutions. The biomedical
applications of nanosilver in respect to its antimicrobial and plasmonic properties
are also presented, summarizing the main advantages but also its limitations and
challenges. The need of a fundamental understanding on the physical properties that
influence the toxicity of nanosilver is highlighted in order to employ the beneficial
properties of nanosilver with minimal impact on the environment and human
health.
1 Part of this chapter is published in Curr. Opin. Chem. Eng. 1, 3-10 (2011).
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1.1 Introduction
1.1.1 Historical perspective of silver metal
Throughout history, silver is a material known to mankind since the dawn of
civilization. It appears usually in nature combined with copper, lead or gold. Since
ancient times it attracted the attention of humans because of its lustrous, brilliant
metallic luster appearance and this facilitated its employment as jewelry or as a
medium of exchange (Figure 1.1). The most well-known ancient silver mine dates
back to 500 B.C. at Laurion, approximately 75 km south of Athens. The
consequences of the silver mined from this location during ancient times are
incalculable, as it was used to build the Athenian naval fleet [1] that played a
catalytic role on the victory against the invading Persian empire [2].
Figure 1.1: A silver tetradrachm of Athens (ca 480 B.C.) showing on one side the goddess
Athena and on the other side an owl, the sacred bird of Athena. The British Museum©.
Apart from the uses of silver as a precious metal such as ornaments and high-
value silverware, its unique physicochemical properties brought it to a variety of
modern technological applications. Silver has the highest electrical and thermal
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conductivity than any other metal and therefore it was introduced in electrical
contacts and conductors. Silver still occupies such a position in a variety of high-end
products where the energy loss by the tarnished copper is undesirable [3]. Other
silver compounds, namely silver halides, because of their high light sensitivity are
employed in photographic films and papers [4], while other forms of silver find
applications in explosives [5] and batteries [6].
Another physical property of silver is the ability to kill micro-organisms,
something known since Hippocrates, who had suggested that fine silver particles
have beneficial healing properties when treating ulcers [7]. Later on, water and wine
were preserved in silver vessels and silver compounds are employed in wound
treatments till modern times [8]. This ability of silver to protect mankind from
harmful bacteria and diseases has given it even mystical powers according to
folklore, being the only metal that can be used effectively against supernatural
creatures such as werewolves or vampires.
1.1.2 Silver in the nanoscale
When silver exists in its nanometer size scale (nanosilver), its antimicrobial
properties are amplified because of the much larger surface-to-volume ratio. In fact,
fine silver particles were found to be antibacterial already in 1912, when colloidal
solutions of silver and mercury particles exhibited strong toxicity against B. coli
communis [9]. After that, colloidal silver was introduced to the market as a strong
disinfectant elixir and ignited more studies of a similar nature. These suggested that
silver colloids have a strong germicidal action when administered orally or
hypodermically because of their acute toxicity to parasites [10]. Such a chronic
indigestion, however, of silver can cause argyria, a not life-threatening disease but
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with cosmetically undesirable outcomes as it is related to a blue or gray
discoloration of the skin [11].
Figure 1.2: Schematic diagram illustrating a localized surface plasmon. Adopted from [19].
Nanosilver is used as a catalyst widely in industry [12], and the most well-
known reaction that nanosilver catalyzes is the epoxidation of ethylene to form
ethylene oxide [13]. However, especially because of its unique optical properties,
nanosilver is also found in a variety of other applications including optoelectronics
and photonics [14], biological detection [15,16], surface enhanced Raman scattering
[17], and coloristic [18]. In fact, these properties originate from the interaction of
small metallic (gold, silver) particles with electromagnetic irradiation that gives rise
to the localized surface plasmons, that are collective oscillations of their surface
conduction electrons (Figure 1.2) [19]. When the particle size is comparable to the
wavelength of the incident light, this interaction influences its light absorption and
scattering [20] that affects also their color. This was already known empirically
among ancient Roman glass manufacturers, as they were employing gold and silver
along with the glass production that resulted in differently colored glasses depending
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on the direction of the light, with the most well-known example being the Lycurgus
cup [21] (Figure 1.3).
The first scientific approach for the color of small metallic particles,
however, was made experimentally by Faraday at 1857, when he presented that gold
colloids have a ruby-red color [22] and this was theoretically explained by Mie later
on [23]. Since then, many studies investigate the optical properties of fine metallic
particles, and a number of potential applications that employ them have emerged.
Among plasmonic materials, however, silver has the lowest optical losses in the
ultraviolet-visible spectrum [20], and thus is typically preferred over the more
expensive gold.
Figure 1.3: The Lycurgus cup (ca 300 A.D.) in reflected (left side) and transmitted (right side)
light. The color of the glass changes depending on the direction of the light. This difference in
the color is attributed to gold and silver nanoparticles in the glass matrix. The British
Museum©.
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The latest years, several studies have focused on the biomedical applications
of nanosilver particles that could be separated in two categories: the ones that
exploit the antimicrobial properties of nanosilver particles and how they can be used
in order to prevent infections, and the ones that facilitate the plasmonic properties of
nanosilver and explore its employment either as a diagnostic (e.g. biosensors, in-
vivo biomarkers) or therapeutic (e.g. photothermal treatment) tool. The unique
properties of nanosilver, however, have enabled it to be used in many, not only
biomedical, applications. As a matter of fact, nanosilver is the most commonly used
engineered nanomaterial in commercial products [24]. Such products include
antibacterial textiles [25], polymer films for food packaging [26], paints and
pigments [27], filters for water [28] or air [29] treatment, to name just a few.
1.1.3 Implications regarding use of nanosilver
The broad use of nanosilver, however, raises concerns regarding its fate and
potential adverse effect on the environment and human health. Actually, nanosilver
is the first nanomaterial to attract the attention of the U.S. Environmental
Protection Agency as not long ago petitions had been filed for nanosilver to be
regarded as pesticide [30]. Such concerns were formed when it was shown that some
nanosilver may “escape” to waste water treatment plants after washing products that
employ it [31]. This release of silver into the aquatic environments may be toxic for
beneficial for the environment micro-organisms and thus ignited a number of studies
that investigate the fate of nanosilver in the environment and its potential adverse
effects.
One of the main ways that silver can be released into the aquatic
environments is in the form of ions [32]. Even though silver metal is not soluble in
water, when in the nanometer size range Ag+ ions are released (leached) from its
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surface [32-35]. This release is related to the oxidation of metallic nanosilver by its
interaction with dissolved oxygen and protons [36]. Nanosilver particle formation
from such released Ag+ ions can occur under relevant environmental conditions
simulating the soil sediments [37], further emphasizing the environmental impact of
the released Ag+ ions and their potential toxic effects. Such nanosilver particles
transform to the less toxic silver sulfide nanoparticles in sewage sludge [38] that can
influence, however, the silver uptake and bioaccumulation into food chains [39] and
could originate partly from released or leached nanosilver particles from commercial
products [40]. The origin of the released Ag+ ions is not clear at the moment, while
a debate exists in the literature whether the released Ag+ ions or the nanosilver
particles themselves play the dominating role for their antibacterial activity
[34,41,42]. Therefore, the role of released Ag+ ions in the antimicrobial activity of
nanosilver, as well as the parameters that influence its Ag+ ion release in aquatic
environments need to be unraveled.
In this chapter, an overview of the synthesis methods of nanosilver with
selected morphologies is presented. The interactions of nanosilver with bacteria and
mammalian cells and the main toxicity mechanisms are discussed focusing on the
Ag+ ion release and the properties that influence it. The toxicity of nanosilver with
different sizes and surface morphologies is compared, pointing out the lack of a
unified perspective when examining such studies. Finally, the biomedical
applications of nanosilver as antimicrobial agent, biosensor and bioimaging agent
are discussed, summarizing the state-of-the-art processes and highlighting the further
limitations and challenges.
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1.2 Nanosilver synthetic routes and morphologies
Nanosilver can be made by various methods that can be categorized in wet-
and gas-phase synthetic routes. In general, wet chemical processes allow for a good
control on the polydispersity of the particles. Gas-phase processes, on the other
hand, combine several advantages such as formation of high purity products without
any byproducts, few process steps, they are easily scalable [43]. Furthermore,
nanosilver can be made in different morphologies such as pure, coated with a
ceramic or polymer layer, surface functionalized, supported on ceramics and as
heterodimers or “Janus-like” with other particles (Figure 1.4). In the following
paragraphs these different nanosilver morphologies will be discussed as obtained by
either wet- or gas-phase synthetic routes.
1.2.1 Pure and surface modified
Perhaps the most common method for nanosilver synthesis is the reduction
of silver nitrate (AgNO3) with sodium borohydride (NaBH4) [44]. With this method,
an aqueous solution of AgNO3 is mixed with an aqueous solution of NaBH4 under
continuous stirring. The resulting nanoparticles can range in primary particle size,
but typical values are 1-50 nm. Variations of this method involve the reduction of
different silver compounds such as silver perchlorate [45] or silver ethylhexanoate
[46] as well as the employment of different reducing agents like ascorbic acid [47] or
sodium hydroxide [48]. A detailed list of different reducing and stabilizing agents
can be found in [49]. The obtained nanosilver particles have a rather spherical shape
(Figure 1.4a) and their size depends on process parameters such as the Ag
concentration and temperature [45].
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Figure 1.4: Electron microscopy images of nanosilver particles with different morphology: (a)
pure [47], (b,c) supported on SiO2 [64,65], (d) SiO2-coated [66], (e) heterodimer or “Janus-
like” [67].
A main limitation of the wet-phase method of the reduction of silver salts is
the stability of the resulting colloidal solutions, since nanosilver particles tend to
form agglomerates (flocs) in suspension and thus losing their nanosize effect [47].
One way to overcome this agglomeration is to use capping agents on the nanosilver
particles which are typically organic molecules [50]. Such surface modified particles
are then made by adding the corresponding organic molecule (e.g. citrate [51],
arabic gum [52], poly(vinylpyrrolidone-PVP) [53]) during their synthesis. The
capping agent can influence the particle size and shape [54] and additionally, stable
nanosilver particle dispersions are formed in solvents.
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The first method to synthesize nanosilver particles in the gas-phase with
controlled size and polydispersity was introduced in 1983, by using the evaporation-
condensation technique. With this method the silver metal is evaporated at high
temperature in a tube flow reactor and subsequent particle nucleation occurs by
cooling [55]. The average nanosilver primary particle size obtained by such methods
ranges from 2 to 100 nm [56-60]. With the above method, the silver vapor can be
supplied also by other techniques such as arc evaporation [61]. Other methods for
the synthesis of pure nanosilver particles include the electro-exploding wire [62] and
laser ablation in aqueous solution [63].
1.2.2 Supported on ceramic nanoparticles
Noble metallic particles are also often anchored on the surface of other
ceramic particles, the so-called “support”. This morphology is especially desired in
catalysis, where specific reactions that small metallic particles catalyze occur in
elevated temperatures that could lead to grain growth of the metallic particles
through sintering and surface diffusion. The higher thermal stability of the ceramic
particles in addition to the typically corrugated structure inhibits this growth
significantly [68]. The support material may be spherical or nanostructured with the
nanosilver particles decorating its surface (Figure 1.4b,c). Nanosilver supported on a
variety of ceramic materials (e.g. SiO2 [64,65], TiO2 [69,70], Al2O3 [70], ZnO [71])
has been made by wet-phase methods. Typically, the nanosilver particles are formed
in later step by impregnation, after the initial synthesis of the support material.
Gas-phase processes have also been shown to be effective for the synthesis of
such particle morphologies. Nanosilver supported on SiO2 [26,72,73], TiO2 [33,72],
Fe2O3 [72], Al2O3 [72], ZnO [74], Ca3(PO4)2 [26] has been made by flame spray
pyrolysis, with which the product nanoparticles are formed after the liquid precursor
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combustion by nucleation from the gas-phase and subsequently grow by surface
reaction and coagulation [75]. The nanosilver primary particle size ranges from 1-10
nm and can be controlled by varying the Ag precursor concentration and it is
noteworthy that there is a finer crystal size control than wet-phase methods [74].
1.2.3 Coated nanosilver
For several applications it is desired that nanosilver particles are coated by a
SiO2 layer. Such coatings are typically made by wet-phase synthesis, most often by
using modifications of the Stöber technique [76]. The SiO2 coating thickness can be
controlled well but in order to obtain homogeneous coatings (Figure 1.4d), a
thickness of about 20 nm is common [66,76]. Such a SiO2 shell made by wet-phase
processes is typically porous [66,76,77] and therefore allows the Ag+ ion transport
[77]. This facilitates the controlled release of Ag+ ions when such particles are
dispersed in aqueous solutions. A nanothin SiO2 coating on nanosilver can be
applied also by photoinduced chemical vapor deposition, where the SiO2 coating on
nanosilver is made by the reaction of the Si-precursor on the freshly formed core
nanosilver particles in the gas-phase in one-step [78].
1.2.4 Heterodimer or “Janus-like” nanosilver particles
Another morphology of interest is the formation of heterodimer or dumbbell-
like, the so-called “Janus-like”, particles that involves two adjacent particles of
different materials (e.g. iron oxide [67], gold [79,80]). This morphology is desired in
applications that require an extra functionality, such as magnetic [67,81] by the
addition of an iron oxide nanoparticle next to the nanosilver (Figure 1.4e). Such
multifunctional nanoparticles can be used in multiple imaging techniques (MRI,
optical microscopy) for detection of cells and facilitates their magnetic guiding [67].
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1.3 Interactions of nanosilver with biological systems
1.3.1 Antimicrobial activity
There is an increasing interest in the antimicrobial activity of nanosilver
particles the last few years. The number of publications that deal with the
antibacterial activity of nanosilver has exponentially grown since 2004 [82]. The
potential toxic activity of nanosilver particles has been examined against a large
variety of prokaryotic organisms including bacteria and fungi or viruses [83].
Table 1.1 shows a list of some studies that investigate the antibacterial activity of
nanosilver with different morphologies and primary particle sizes, along with their
concentration range where an antibacterial activity was observed, the bacteria cell
line and the initial bacteria concentration expressed in colony forming units per mL
(CFU/mL). A more detailed list of studies dealing with the antibacterial activity of
nanosilver can be found in [84]. Escherichia coli (E. coli) is the most common bacteria
culture used but the nanosilver concentration range for an antibacterial activity
varies for the different studies. It is noteworthy that there is a wide range in the
nanosilver morphologies and sizes used as well as in the initial CFU/mL. This
makes it difficult to directly compare the results of such studies, even when the same
bacteria have been employed.
The biological mechanism of the nanosilver toxicity is not completely clear.
It has been suggested that nanosilver particles, as well as the released Ag+ ions from
their surface destroy sulfur and phosphorus containing compounds such as DNA
and proteins [93,97]. This has vast ramifications on the membrane stability of the
cell as well as on the functions of proteins leading to cell death (Figure 1.5).
Furthermore, it seems that higher nanosilver concentrations are required to inhibit
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Table 1.1: List of studies that investigate the antibacterial activity of nanosilver with different
morphologies and sizes against various prokaryotic biological systems.
Composition Primary particle
size (nm) Biol. System CFU/mL
Concentration range (mg/L)
Ref.
Ag+ Only Ag+ ions - E. coli - 0-10 [85]
S. aureus
Pure Ag 15, 75 E. coli 2·103 0-114 [86]
6 E. coli - 10-100 [87]
B. subtilis - “
10 P. chlororaphis 108 0-10 [88]
Surface modified A
12 E. coli 105 0-50 [89]
20 E. coli 5·106 0-40 [90]
2-5 E. coli 108 0-85 [91]
7 E. coli 105 0-6.25 [92]
S. aureus “ 0-7.5
29 E. coli “ 0-13
S. aureus “ 0-17
89 E. coli “ 0-12
S. aureus “ 0-34
Carbon-coated Ag 21 E. coli 5·107 0-75 [93]
S. typhus “ “
P. aeruginosa “ “
V. cholera “ “
PVP-coated Ag S. enterica - 6.25-100 [94]
B. cinerea - “
Citrate-coated Ag 9, 62 E. coli 108 0-108 [36]
Supported on SiO2 <10 E. coli - 0-5000a [95]
P. aeruginosa - “
S. aureus - “
E. cloacae - “
C. albicans - “
P. citrinum - “
A. niger - “
Supported on TiO2 2 E. coli - 0-35 [33] Supported on
Fe2O3 10-20 E. faecalis 106 0-150a [96]
S. aureus “ 0-78
E. coli “ 0-78
P. aeruginosa “ 0-150
S. epidermidis “ 0-150 aConcentration values correspond to the composite Ag/SiO2 or Ag/Fe2O3
nanoparticles.
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the growth of Gram-positive bacteria than Gram-negative ones [92]. This could be
attributed to the structural differences of the cell membrane between two families of
bacteria, as the Gram-positive ones have wider cellular wall than the Gram-negative
ones [98], and thus may inhibit the nanosilver particle or ion transport through it.
Bacteria are relatively easy to culture and perhaps could be considered a screening
tool for the toxicity of nanosilver particles before other more complex biological
systems are examined.
Figure 1.5: A TEM image of P. aeruginosa bacteria incubated with nanosilver particles. The
nanosilver particles have damaged the cell membrane significantly [93].
1.3.2 Cytotoxicity of nanosilver particles against mammalian cells
The broad use of nanosilver particles makes their exposure to humans
inevitable. Such exposure may occur either accidentally by the use of nanosilver
products, or deliberately when their theranostic properties are sought [99].
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Therefore, in order to correctly assess their potential hazards to human health and
environment, detailed toxicological characterization is needed. In particular, the
interactions of nanosilver particles with human or animal cells are necessary to be
studied. Such biological systems, however, are more complex than bacteria, and
therefore, high-throughput cytotoxicity assays and validated standard protocols are
essential [100,101]. Only in such a way the toxic effects of nanosilver particles can
be systematically reproduced and their potential hazards evaluated and compared.
There are various indications of the well being of a cell. The oxidative stress
that is correlated with the reactive oxygen species (ROS) is perhaps the most well-
known [102]. Such ROS can cause toxic effects through the production of free
radicals that influence the redox potential of the cell, thus damaging proteins, lipids
and DNA. Therefore, monitoring the ROS and the oxidative stress can be
considered a way to determine proinflammatory responses and cytotoxicity [102].
Other ways to measure the cell viability or cytotoxicity exploit specific functions
within the cell. These functions occur only when the cell is healthy and, therefore,
any absence of them can be considered an indication of toxicity. One example of
those is the function of mitochondrial enzymes, which can be detected by the use of
specific dyes. When these enzymes are functioning properly, they convert the color
of the dye, indicating the cell viability [103]. Another method to assess the toxicity
of nanosilver is to monitor the induced DNA damage, the so-called genotoxicity.
With this method, the DNA is stained with a specific dye and its structure is
monitored, since damaged DNA has a more relaxed structure than the undamaged
one [104]. Other methods include the monitoring of the cell membrane damage,
since specific dyes only penetrate the cell when their membrane is damaged [105].
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Table 1.2: List of studies that investigate the cytotoxicity of nanosilver with different
morphologies and sizes against various mammalian cell lines. The cytotoxicity assays used are
also indicated.
Composition Primary particle
size (nm) Biological System
Cell Conc.
Conc. range (mg/L)
Cytotoxicity Assay
Ref.
Pure Ag 15, 100 Rat liver cells - 0-50 Mitochondrial
function [106]
Membrane
damage
Oxidative stress
Hydrocarbon-coated Ag
15, 30, 55 Rat adrenal
pheochromocytome (PC12)
- 0-50 Mitochondrial
function [107]
Membrane
damage
Oxidative stress
Glutathione monitoring
Hydrocarbon-coated Ag
27.5 Murine
neuroblastoma 2.5·104 0-100
Mitochondrial function
[108]
Polysaccharide-coated Ag
26
Oxidative stress
Nerve growth
factor
PEI-coated Ag 7-10 Human
hepatocellular 5.5·104 0-3
Neutral red uptake
[109]
Gene altering
Starch-coated 6-20 Human lung
ATP assay [110]
Human glioblastoma
Oxidative stress
Necrotic and
Apoptotic cells
DNA damage
Mitochondrial
function
Glycolipid-coated Ag
15-20 Human
hepatocellular 1.5·104 0-50
Mitochondrial function
[99]
DNA damage
Citrate-coated
Ag 10
Rat adrenal pheochromocytome
(PC12)
- 0-3.24 Oxidative stress [111]
PVP-coated 10, 50
Trypan blue exclusion
DNA content
Membrane-total
protein
Supported on SiO2
5 Human alveolar
macrophages 105 0-50
Membrane damage
[105]
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Several studies have investigated the effects of nanosilver particles on
mammalian cells. A short list is given in Table 1.2. Like in the toxicity evaluation of
nanosilver against bacteria, a large variety of nanosilver with different morphologies
and primary particle sizes has been employed. Typical cell lines include
macrophages, liver cells, lung cells, as the target is to identify the toxicity that
nanosilver induces to cells that it may interact with. Macrophages, being the first
line of defense, are the first cells that nanosilver particles will encounter upon their
entrance in a body, as they will try to destroy them through phagocytosis [112].
Lung cells are of interest, as one of the major pathways that nanosilver, and other
engineered nanomaterials, may enter the human body is inhalation [105]. Finally,
liver cells are examined because in a human body nanosilver will be cleared through
the liver [106].
The majority of such studies emphasize that nanosilver particles can indeed
induce cytotoxic effects to mammalian cells. This is somehow expected, considering
the interactions of Ag+ ions and particles with biological compounds such as DNA
and proteins. In fact, nanosilver particles were found to be more cytotoxic to rat
liver cells when compared to other nanomaterials (including iron oxide, titania,
aluminum, molybdenum oxide), and it was concluded that this higher toxicity is
attributed to the generation of the ROS and oxidative stress [106]. When those cells
were incubated with nanosilver particles they became abnormal in size, displaying
cellular shrinkage, and obtained irregular shape [106]. In addition, even when
nanosilver particles of about 15-20 nm are coated with a biocompatible layer of
glycolipds, they still exhibit higher cytotoxicity than similarly sized and coated gold
nanoparticles, as measured by the mitochondrial function and DNA damage [99].
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Additionally, nanosilver particles tend to form agglomerates when internalized by
cells in vacuoles, as seen in Figure 1.6 [108].
Figure 1.6: Transmission electron microscopy images of nanosilver particles incubated for 24
hours with cells internalized in vacuoles, most probable endosomes. [108].
In order to identify the potential hazards of nanosilver, however, its toxicity
needs to be evaluated not only in comparison to other nanomaterials, but also
among nanosilver particles with different properties, such as size and surface
coatings. In fact, size dependencies have been observed for other nanomaterials,
such as TiO2 [113], that stimulate further studies involving the toxic effects of
nanosilver with various sizes [114]. Actually, nanosilver particles of about 15 nm in
diameter coated with a 2 nm hydrocarbon layer induced larger oxidative stress than
similar ones of 30 and 55 nm in diameter [107]. Extreme caution, however, is
required when evaluating such results, since surface coatings such as hydrocarbons
may interfere with the toxicity mechanism of pure nanosilver, as these coatings can
also induce toxicity and oxidative stress [115]. As a matter of fact, hydrocarbon-
coated nanosilver was found to be more toxic than similarly sized polysaccharide-
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coated nanosilver, further indicating that the hydrocarbon-coating may influence the
nanosilver toxicity [108].
1.3.3 Nanosilver particles, Ag+ ions or both?
As mentioned before, the Ag+ ion release of the nanosilver particle surface
plays a major role on their toxicity [116]. Actually, there is a debate in the literature
on identifying the toxicity induced by nanosilver or the released Ag+ ions from its
surface. Some of them claim that the effect of the nanosilver particles themselves is
negligible, and thus the toxicity stems mostly from the released Ag+ ions. Others
find that the ions do not really participate in the toxic effect, while some claim that
both particles and ions induce toxicity.
More specifically, Navarro et al. investigated the toxicity of nanosilver
particles against algae, and found that when the released Ag+ ions were inactivated
by a strong silver ligand (cysteine), the otherwise observed strong toxicity
diminished [34]. Thus, the observed nanosilver toxicity was mainly attributed to the
free Ag+ ions and not the particles. It should be noted, however, that H2O2 produced
by the algae may participate actively in the dissolution of nanosilver, thus causing
the release of more Ag+ ions [34]. In contrast, Fabrega et al. showed that the
released Ag+ ions did not participate in the toxicity that was observed against
aquatic bacteria and attributed that the particles themselves played the major role
[41].
The cytotoxic effect of both nanosilver particles and released ions against
human hepatic cells was also investigated by Kawata et al. [109]. They also bound
the released Ag+ ions from PEI-coated nanosilver (~7-10 nm) with cysteine and they
observed that the remaining nanosilver particles induced toxicity, indicating that
both Ag+ ions and particles contribute to this toxic effect. Similarly, Powers et al.
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concluded that the toxicity observed by nanosilver particles of about 10 nm (citrate
and PVP-coated) cannot only be attributed to released Ag+ ions, but also the
particles induce oxidative stress [111]. These different results make it difficult to
establish a good understanding of the toxicity mechanism of nanosilver particles and
their released Ag+ ions. Recently, By systematically varying the nanosilver size, it
has been shown that Ag+ ions dominate the toxicity of nanosilver less than about 10
nm in diameter [117] (Figure 1.7).
Figure 1.7: The toxicity of nanosilver as a function of particle size. For small (< 10 nm)
nanosilver, a large fraction of Ag+ ions is released from their oxidized and highly convex
surface (Kelvin effect) dominating the Ag toxicity. Silver oxide is readily dissolved in liquids
in contrast to metallic Ag. For larger (> 10 nm) particles, a small fraction of Ag+ ions is
released so the Ag toxicity is affected by both ions and direct contact with the nanosilver
particle surface.
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1.3.4 Ag+ ion release in aqueous suspensions
All above results indicate that the toxicity of nanosilver particles is a very
complex system and cannot be explained by simple models. Therefore, the Ag+ ion
release mechanism in aqueous solutions needs to be investigated in detail, as well as
the parameters that influence it, in order to connect the observed toxicity with
specific physico-chemical properties. In fact, it has been observed that the oxidation
state of nanosilver strongly influences its Ag+ ion release and therefore, its toxicity,
since oxidized nanosilver exhibited much stronger antibacterial activity against
E. coli [36]. This was also observed when silver oxide nanoparticles (~2 nm)
supported on photoactive TiO2 particles were reduced to metallic ones by UV
irradiation and did not exhibit antibacterial activity [33]. This is associated with the
Ag+ ion release from oxidized nanosilver, since silver oxide has higher solubility in
water than metallic silver [118]. When nanosilver particles of about 5 nm were
oxidized by O3 treatment, they released much higher Ag+ ion concentrations,
indicating also that the Ag+ ion release can be controlled by the oxidation state of
nanosilver [119].
Furthermore, it was shown that the Ag+ ion release of about 5 and 60 nm
nanosilver, as well as of a macro-sized silver foil (~120 m thickness) per unit mass
follows a mass-specific release rate. However, when the data are renormalized per
surface area they collapse from 5 orders of magnitude variation to less than 1
(Figure 1.8a,b) [119]. So the antibacterial activity of nanosilver against E. coli is
better expressed by surface area, rather than mass or number concentrations [120]
(Figure 1.8c,d).This clearly shows that surface area concentration plays a major role
on the Ag+ ion release, and further implies that perhaps dose relations concerning
nanosilver should be expressed in such terms.
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Ag mass concentration C, mg/L
0.0 0.5 1.0 1.5 2.0 2.5
E. coli growth, %
0
20
40
60
80
100
Ag surface area concentration C∙AgSSA, m2/L
0.00 0.05 0.10 0.15 0.20
E. coli growth, %
0
20
40
60
80
100
Figure 1.8: (a) The Ag+ ion release (Agdis) over the total Ag amount kinetics for different
nanosilver sizes and a macro-sized silver foil. (b) The same data renormalized on the basis of
surface area [119]. The extent of E. coli growth of all data at 210, 270 and 330 minutes as a
function of the Ag (c) mass concentration C in suspension and (d) surface area concentration
C·AgSSA [120].
There are several other factors that can influence the Ag+ ion release: pH,
temperature, organic matter and surface coatings [35,41]. This is one reason why
interpretations of the exhibited toxicity of nanosilver particles and their released
ions need to be made carefully. For example, different surface coatings can
influence the Ag+ ion release kinetics and the final degree of dissolution, as it was
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23
shown that the Ag+ ion release of citrate-coated nanosilver of about 50 nm was
lower than the release of PVP-coated similarly sized nanosilver [121].
The origin of these released ions still remains to be identified. It has been
speculated that the dissolved oxygen in the aqueous solution from the air may
induce the surface oxidation of nanosilver, and thus its dissolution [36]. The rate of
diffusion of oxygen from the environment, however, in the aqueous solution cannot
explain the observed Ag+ ion release behavior, since then one would expect the
nanosilver to fully dissolve when in suspension. Additionally, if the Ag+ ion release
would depend only on the initial dissolved oxygen concentration then there should
always be the same Ag+ ion concentration in suspension [121]. Other effects could
also influence the Ag+ ion release such as the curvature of the nanosized particles
(Kelvin effect). Therefore, a fundamental understanding on the mechanism and the
origin of the released Ag+ ions from nanosilver is essential in order to establish
correct dose and risk assessments of nanosilver.
1.4 Biomedical Applications of Nanosilver
1.4.1 Antimicrobial agent
In several health treatments such as in intravenous catheters, endotracheal
tubes, wound dressings, bone cements, oral cavities fillings, or implant surgery the
spread of an infection may result in poor outcomes for the living quality or even the
life of the patient. Therefore, it is common to treat patients with an antibiotic in
order to minimize this risk. However, several bacteria start exhibiting resistance to
antibiotics [122], thus making it difficult to prevent their spread with antibiotics.
This is the reason why other materials that exhibit antibacterial properties could be
employed in such applications, such as nanosilver.
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Nanosilver (5-50 nm) embedded in PMMA bone cement exhibited high
antibacterial activity when tested against antibiotic-resistant bacteria, with no
cytotoxic signs against human cells for identical nanosilver concentrations [123].
Similarly, plastic catheters were impregnated with nanosilver particles of about 10
nm and exhibited strong antibacterial activity. When these catheters were placed
within mice, a controlled release of Ag+ ions at the implantation site was achieved
[124]. The antibacterial properties of nanosilver particles dispersed in dental
adhesives were also demonstrated against streptococci [125]. In that case, the
nanosilver particles not only inhibited the bacteria growth, but also did not affect the
desired mechanical properties of the adhesive, enabling it to be used in orthodontic
treatments.
The employment of such nanosilver particles is not only limited to polymer
materials, but expanded also as antibacterial coatings on metallic surfaces. For
example, nanosilver particles can be deposited on the surface of titanium that could
be used as an implantable biomaterial. In such a case, it was shown that this
nanosilver coating exhibited strong antibacterial activity and additionally prevented
the attachment of bacteria on the metallic surface [126]. All above examples
highlight the potential that nanosilver particles have in such biomedical applications
as an alternative to common antibiotics antibacterial agent.
1.4.2 Biosensors with plasmonic nanosilver
There is a lot of interest in biomedical applications of nanosilver employing
its plasmonic properties. These properties strongly depend on the nanoparticle size,
shape and dielectric medium that surrounds it [20]. For example, by increasing the
nanosilver size, its color changes (Figure 1.9a) and its plasmon absorption band
shifts to higher wavelengths [50,127]. However, this shift to higher wavelengths
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occurs also when the refractive index of the medium that surrounds the nanoparticle
increases (Figure 1.9b) [128]. In fact, the latter dependency can be exploited in order
to detect biomolecules, such as proteins, that have typically a larger refractive index
than the buffer solutions [15,129]. Therefore, the detection of specific analytes at
low concentrations can be achieved by monitoring the shift of the peak position on
the extinction (absorption and scattering) spectrum of plasmonic nanostructures
during the adsorption of these analytes on the plasmonic surface [19,130].
Figure 1.9: (a) Suspensions of nanosilver particles with an increasing average diameter from
64 to 158 nm (from left to right) [127]. (b) The peak position of the planar nanosilver array
(100 nm) plasmon absorption band as a function of the reactive index of the surrounding
medium [128].
The dependence of the optical properties of nanosilver particles on the
dielectric properties of the surrounding medium (Figure 1.9b) can be exploited for
biosensing applications. Most biomolecules have a higher refractive index than
buffer solutions so when they attach on the nanosilver particles, the local refractive
index increases causing a shift on the extinction (absorption and scattering)
spectrum. Biosensors utilizing plasmonic nanostructures (local surface plasmon
resonance-LSPR) are advantageous over the already commercialized thin plasmonic
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continuous films (surface plasmon resonance-SPR) that are used for similar
bioapplications [131] because they exhibit less interference from the refractive index
of the buffer solution and possess greater spatial resolution [132].
Triangular nanosilver particles made by nanosphere lithography were
deposited on substrates in order to monitor interactions between biomolecules, the
well-studied biotin-streptavidin system, reaching picomolar limit-of-detection [15].
Yonzon et al. used the same triangular nanosilver particles for the detection of
concanavalin A [133], Haes et al. to monitor the interaction of two biomolecules
that are related to the Alzheimer’s disease [134] and detect them at low
concentrations [135]. Other nanosilver structures such as nanocubes or rhombes can
also be employed in such biosensing applications of protein interactions [136,137].
Lately, nanosilver plasmonic biosensors start being employed in cancer detection
with promising results [138].
Even though silver is more efficient than gold in terms of plasmonic
performance, the latter is typically used in such biosensing applications because
silver oxidizes and easily forms plasmonically unattractive compounds like halides
in biological solutions. Furthermore, surface sulfidation may deteriorate the
plasmonic performance of nanosilver that can occur even when nanosilver is
exposed to ambient laboratory conditions [139]. This is the reason why often a
protective coating is applied on the surface of nanosilver that further facilitates its
easy dispersion in solutions [50]. However, till today, most plasmonic
nanostructures employing nanosilver are made by multiple step lithographic
techniques and no plasmonic biosensing array has been made by using spherical
nanosilver particles, perhaps partially attributed to the limitations mentioned above.
Recently, the employment of silica-coated nanosilver as biosensors for the detection
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of bovine serum albumin (BSA) was demonstrated with excellent solution dispersion
and limited antibacterial activity [140].
1.4.3 Bioimaging agent
Plasmonic particles, such as nanosilver, can be detected by a number of
optical microscopy techniques that include light scattering [141], dark-field
illumination [142], two-photon fluorescence imaging [67] and photon illumination
confocal microscopy [143]. In fact, the detection of nanosilver particles under these
techniques is advantageous over the commonly used fluorescent organic dyes, as the
latter decompose during imaging, exhibiting the so-called photobleaching. In
contrast, nanosilver particles are photostable allowing thus their employment as
biological probes to monitor continuously dynamic events for an extended period of
time [144]. This versatile detection of plasmonic particles has brought them to a
number of studies where they are selectively bound to cells for in-vitro bioimaging
[141,142,145], allowing therefore their easy detection under a microscope. The
plasmonic properties of such small metallic nanoparticles enable them also to be
employed further as an in-vivo therapeutic tool. Plasmonic particles are conjugated
to biological targets [146] such as cancer cells or tissues and are used to absorb light
and convert the light energy into thermal energy [146,147]. This destroys the targets
by thermal ablation, enabling such particles to be used in a non-invasive cancer
treatment [131,148]. Typically, gold nanoparticles are used for such applications,
because they are relatively less toxic to the cells that are monitored than nanosilver
particles [99]. In fact, this potential toxicity of the plasmonically superior nanosilver
is their main limitation, since they may destroy the cell [108]. Therefore, only a few
studies have investigated the potential of nanosilver as bioimaging agent.
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Figure 1.10: Interaction of biofunctionalized nanosilver particles with fibroblast cells that
express a specific protein on their surface (a) and that do not (b). The nanosilver particles have
been selectively bound on the protein sites on the surface of the cells that express the
corresponding ligand protein (a) [149].
Typically, there are two ways to employ the nanosilver particles as
bioimaging agents: either incubated with cells and monitor their physical
interactions and uptake, or to functionalize the nanosilver surface with a
biomolecule that binds specifically to sites on the cell membrane. The former is
relatively easier to perform, since for the latter a specific biofunctionalization
molecule is needed. Nanosilver particles incubated with neuroblastoma cells were
detected under dark-field illumination exhibiting their interaction, however, there
was a toxic effect observed [108]. Similarly, nanosilver particles attached on iron
oxide nanoparticles forming heterodimers were incubated with macrophages cell
and allowed for their easy detection by two-photon imaging after their uptake by the
cells [67]. The potential of nanosilver as bioimaging agent has also been
demonstrated with zebrafish embryos, where their detection under dark-field
illumination facilitated the dynamic imaging of their transport in and out of the
embryos and their toxicity assessment and concentrations [144]. The specific
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attachment of nanosilver particles has also been demonstrated by binding them to
specific receptors on the membrane of fibroblast cells (Figure 1.10) [149], or by
attaching them to intracellular proteins and monitoring their internalization through
the cell membrane of neuroblastoma cells [150]. The selective binding on HeLa and
Raji cells of silica-coated, Janus-like silver-iron oxide nanoparticles (Figure 1.4d)
that could be magnetically manipulated was achieved by hDC-SIGN antibody [151].
The presence of a thin (about 2 nm) silica coating blocked quite effectively the
toxicity of nanosilver.
1.5 Summary and concluding remarks
Nanotechnology has opened up a number of possibilities ranging from high-
tech products to everyday-life applications, with nanosilver being one of the striking
examples. The unique physicochemical properties that arise when silver is in the
nanoscale, such as to fight infectious germs with superb efficiency and to interact
with light in such a special way, have brought it on the first firing line of
nanotechnology products. The biomedical field is perhaps the first field of many to
come that has explored the potential of nanosilver particles. The employment of
nanosilver in a variety of biomedical applications, such as antimicrobial agent to
prevent the spread of micro-organisms or as biosensor and bioimaging agent, has
demonstrated its great capacity to improve the living quality of humans.
The potential positive outcomes of nanosilver, however, have to be balanced
with any potential adverse effects that it may induce to the environment and human
health. In order to relish the increasing beneficial applications of nanosilver, an
extremely thorough investigation needs to be performed on its interactions with
biological systems. Extreme caution, however, is required when investigating the
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toxicity of nanosilver against biological systems. Particles without any surface
modification would be preferred, in order to attribute the observed results to
nanosilver, and not to their surface material. However, more often than not, several
nanosilver synthetic routes involve such surface modifications that are essential for
its stability in solutions. Therefore, perhaps synthetic routes that do not influence
the surface properties of nanosilver particles, but still facilitate their easy dispersion
should be preferred, such as nanosilver supported on an inert material.
Therefore, the systematic investigation of the nanosilver toxicity is facilitated
by a careful selection of the nanosilver particle morphology and biological system. A
fundamental understanding has to be established on the physical properties that
influence these interactions. This understanding may help to design and synthesize
safer nanosilver particles that possess the desired properties for the target application
and at the same time overcome any adverse toxic effect. This would result in
sustainable engineered nanomaterials in a rather “eco-friendly” way that offers their
sought-out performance and minimize risks to human health and environment.
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CHAPTER 2
2. Antibacterial Activity of Nanosilver
Ions and Particles1
Abstract
The antibacterial activity of nanosilver against Gram negative Escherichia coli
bacteria is investigated by immobilizing nanosilver on nanostructured silica particles
and closely controlling Ag content and size. These Ag/SiO2 nanoparticles were
characterized by S/TEM, EDX spectroscopy, X-ray diffraction and the exposed Ag
surface area was measured qualitatively by O2 chemisorption. Furthermore, the
fraction of dissolved nanosilver was determined by measuring the released (leached)
Ag+ ion concentration in aqueous suspensions of such Ag/SiO2 particles. The
antibacterial effect of Ag+ ions was distinguished from that of nanosilver particles by
monitoring the growth of E. coli populations in the presence and absence of Ag/SiO2
particles. The antibacterial activity of nanosilver was dominated by Ag+ ions when
fine Ag nanoparticles (less than about 10 nm in average diameter) were employed
that release high concentrations of Ag+ ions. In contrast, when relatively larger Ag
nanoparticles were used, the concentration of the released Ag+ ions was lower. Then
the antibacterial activity of the released Ag+ ions and nanosilver particles was
comparable.
1 Part of this chapter is published in Environ. Sci. Technol. 44, 5649-5654 (2010).
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2.1 Introduction
The unique physicochemical properties of silver nanoparticles (nanosilver)
have made them one of the most commercialized nanomaterials in health care [1].
Apart from nanosilver’s “traditional” applications in heterogeneous catalysis [2], its
antibacterial properties make it attractive in new applications as an additive in
textiles [3] and food packaging [4]. This antibacterial activity, however, is
undesirable when nanosilver is disposed and ends up dissolving and leaching ions
[3,5-7] acting against aquatic micro-organisms [8]. So regulatory agencies worldwide
monitor nanosilver [9] prompting research for a detailed understanding of its
toxicity [10,11]. That way, correct risk assessments can be made and its safe use can
be established for minimal, if any, environmental impact.
There is an ongoing debate regarding the role of released Ag+ ions from
nanosilver and its toxicity against micro-organisms [12]. More specifically, Navarro
et al. [13] concluded that nanosilver alone has minimal toxicity and it serves mostly
as a source of Ag+ ions. Miao et al. [14] showed also that dissolved Ag+ ions dictate
nanosilver’s toxicity. In contrast, Fabrega et al. [5] concluded that the effect of
released Ag+ ions is not significant and thus, the dominating factor for this toxicity
is bacterial contact with the nanosilver particles themselves. Furthermore, Kawata et
al. [15] also stated that the toxicity induced by nanosilver cannot be attributed solely
to the released Ag+ ions but rather to nanosilver particles, in agreement with Laban
et al. [7]. All these studies, however, employ commercially available nanosilver
[5,7,13-15] having limited, if any, control over Ag size, morphology and degree of
agglomeration. This makes difficult to draw universally accepted conclusions
regarding the toxicity mechanism of nanosilver. For example, it is not uncommon to
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have nanosilver flocculation in bacterial suspensions unless its surface is modified
with surfactants [16] that may alter again the antibacterial activity of nanosilver.
Here this potential drawback is overcome by immobilizing nanosilver on an
inert, nanostructured support (SiO2) upon its synthesis by flame aerosol technology
[17] that allows close control of product particle size and morphology [18]. The role
of this silica support with its corrugated texture is to prevent nanosilver particle
growth by sintering or coalescence during its characterization and to hinder
nanosilver agglomeration (flocculation) in bacterial suspensions. It should be noted,
however, that such composite nanoparticles can be made also by wet chemistry
methods [19]. The nanoparticle properties are measured systematically focusing,
first on the nanosilver size and exposed surface area qualitatively [20] and second,
on the Ag+ ion release as determined by ion analysis with an Ag+ ion selective
electrode [13] as a function of average nanosilver particle size (4-15 nm). So, the
antibacterial activity of nanosilver against Escherichia coli is correlated to particle
properties and its mechanism is elucidated by quantitatively distinguishing, perhaps
for the first time, between the role of Ag+ ions from that of nanosilver particles [21],
the “holy grail” in this field so-to-speak.
2.2 Materials and methods
2.2.1 Particle synthesis
Nanosilver on nanostructured silica was made in one step by flame spray
pyrolysis (FSP) of appropriate precursor solutions as described elsewhere [17]. Silver
acetate (Aldrich, purity >99%) and hexamethyldisiloxane (HMDSO, Aldrich,
purity >97%) were used as silver and silicon precursors, respectively. Appropriate
amounts of silver acetate were dissolved in a 1:1 mixture of 2-ethylhexanoic acid
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(Aldrich, purity >98%) and acetonitrile (Aldrich, purity >98%). The corresponding
amount of HMDSO for a given Ag-content product was added and stirred for a few
minutes just before that solution was fed into the FSP reactor. The total precursor
(HMDSO and Ag-acetate) concentration was 0.5 M. The precursor solution was fed
through the FSP capillary nozzle at 5 mL/min, dispersed to a fine spray by 5 L/min
oxygen (Pan Gas, purity >99%) and combusted to produce Ag/SiO2 nanoparticles
that were collected on a filter downstream. The Ag-content of product Ag/SiO2
particles ranged from x = 0 to 98 wt% and their composition corresponds to the
nominal Ag- and Si-content of the FSP precursor solution used here [4]. The
notation for such particles is: xAg/SiO2.
2.2.2 Particle characterization
High resolution transmission electron microscopy (HRTEM) was performed
on a CM30ST microscope (FEI; LaB6 cathode, at 300 kV, point resolution ~2 Å)
and scanning transmission electron microscopy (STEM) on a Tecnai F30 (FEI;
300 kV) with a high-angle annular dark field (HAADF) detector with bright Z
contrast and energy dispersive X-ray spectroscopy (EDXS; detector EDAX).
Product particles were dispersed in ethanol and deposited onto a perforated carbon
foil supported on a copper grid.
The crystallite size of silver was determined by XRD [17] using the TOPAS 3
software and fitting its (111) diffraction peak (2θ = 36°-40°) with the Inorganic
Crystal Database [ICSD Coll. Code.: 064995]. The exposed surface area of Ag
(AgSSAE) per unit mass of Ag was measured with O2 pulse chemisorption
(Micromeritics Autochem II 2920). The samples were reduced at 350 °C for 3 hours
under flowing H2 (20 mL/min) followed by 30 minutes flushing by He. Then the O2-
pulse chemisorption at 150 °C was performed, with 50 mL/min He [22] and 10
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pulses of 0.5 mL/min (5% O2 in He), assuming [20] a stoichiometric ratio (O:Ag) of
0.5. The Ag+ ion concentration was measured with an ion selective electrode and an
ion meter (Metrohm 781) [13]. The measurements were calibrated using silver
containing aqueous solutions (silver standard, Aldrich). Centrifugation (Rotina 35,
Hettich, 10000 rpm, 100 min) of aqueous solutions containing Ag/SiO2
nanoparticles along with UV/vis spectroscopy (Cary Varian 500) were employed to
ensure the removal of the Ag/SiO2 nanoparticles. Error bars correspond to the
standard deviation of, at least, 3 measurements. For diffuse reflectance
measurements, samples were diluted with barium sulfate (if needed) and placed in a
dry powder sample holder (Praying Mantis).
2.2.3 Antibacterial Activity
The antibacterial activity of the Ag/SiO2 nanoparticles was obtained by a
growth inhibition assay. E. coli JM101 bacteria synthesizing a green fluorescent
protein (GFP) from a plasmid-encoded gene were grown in Luria-Bertani (LB) broth
at 37 °C overnight [23]. The culture was subsequently diluted with LB to an optical
density (OD) of 0.05 at 600 nm, which corresponds to about 107 colony forming
units (CFU)/mL. The Ag/SiO2 nanoparticles were homogeneously dispersed in de-
ionized water by ultrasonication (Sonics vibra-cell) for 20 seconds at 75% amplitude
with a pulse configuration on/off of 0.5s/0.5s. For the assay, 50 μL of these
nanoparticle-containing solutions were added to 50 μL of the diluted cells. The
bacterial growth was monitored by the fluorescent signal of the GFP (Perkin Elmer
1420). The data were corrected for background fluorescence and normalized for the
control measurement. The error bars for each data point were the standard deviation
of 4 measurements.
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Figure 2.1: (a) STEM image of the 2Ag/SiO2 and the EDX spectra of silver-containing
(area 1) and pure SiO2 (area 2) locations (the Cu peaks arise from the carbon coated copper
grid). (b) Diffuse reflectance Uv/vis spectra of the xAg/SiO2 for x = 0, 1, 2 and 10 show the
plasmon absorption band of Ag metal at 410 nm in all Ag-containing samples with a TEM
image (inset) of 6Ag/SiO2 showing Ag nanoparticles (dark dots) dispersed on the
nanostructured silica (gray) support.
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2.3 Results and discussion
2.3.1 Nanosilver morphology and size
Figure 2.1a shows an STEM image and EDX spectra of two areas of a
sample with 2 wt% Ag (2Ag/SiO2). The nanosilver particles (bright contrast) are
dispersed on the SiO2 matrix, as verified also by EDX. In the spectra of area 1 from
Figure 2.1a which includes a bright spot, there is a clear peak of Ag confirming its
presence there along with Si and O which correspond to SiO2 [2,4]. In contrast, in
the spectra of area 2 that contains no bright spots, only peaks of Si and O are
present. The Cu peaks come from the carbon coated copper foil that was used to
obtain the electron microscopy images.
Figure 2.1b shows diffuse reflectance UV/vis spectra of the xAg/SiO2
particles for x = 0, 1, 2 and 10. Even for the lowest Ag-content particles (1Ag/SiO2),
the plasmon absorption band at ~410 nm [24] indicates metallic Ag, without
excluding, however, that its surface can be oxidized [2]. The inset of Figure 2.1b
shows a representative TEM image of FSP-made 6Ag/SiO2 sample where dark Ag
nanoparticles are dispersed on the gray nanostructured SiO2 particles exhibiting a
surface with corrugated texture.
The XRD patterns revealed the characteristic peaks attributed to silver metal,
for x ≥ 10 wt% [25]. For lower Ag-contents the crystal concentration was below the
XRD detection limit. No silver oxides were detected by XRD at all Ag-contents, x.
The obtained nanosilver particles had a unimodal size distribution [4] (Appendix A,
Figure A.1). Figure 2.2 shows the average nanosilver particle diameter as
determined by XRD (circles, dXRD) and electron microscopy (triangles, dS/TEM) while
Table A.1 (Appendix A) shows detailed particle counts and geometric standard
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deviations for each x. The two average diameters are in good agreement indicating
monocrystalline nanosilver. For an increasing Ag-content in the composite xAg/SiO2
particles the average nanosilver size increases monotonically so it can be closely
controlled from 4 to about 16 nm [25].
Ag‐content x in xAg/SiO2 particles, wt%
0 20 40 60 80 100
AgSSAE, m
2 /g of Ag
0
10
20
30
40
50
60
Ag particle diameter, d
S/TEM, d
XRD, nm
0
5
10
15
20
Figure 2.2: The exposed specific surface area (squares) of nanosilver per unit mass of Ag (as
determined by O2 chemisorption) AgSSAE, and average nanosilver particle diameter by
electron microscopy (triangles, dS/TEM) and X-ray diffraction (circles, dXRD) of the xAg/SiO2
composite nanoparticles as a function of their Ag-content, x. The close agreement between
microscopy and XRD indicates monocrystalline particles. In the inset, a TEM image of a
single nanosilver particle attached on amorphous SiO2.
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Figure 2.2 (left ordinate) shows also the exposed nanosilver surface area
(AgSSAE) per unit mass of Ag as a function of Ag-content, x. Lower Ag-content
particles have higher AgSSAE. By increasing x, the AgSSAE decreases, as larger
nanosilver particles are formed exposing less surface area per unit mass of Ag.
Additionally, a TEM image is presented as inset in Figure 2.2 showing a single
nanosilver particle attached on the amorphous SiO2, having a fraction of its surface
exposed. XRD spectra taken before and after O2 chemisorption were identical
indicating no bulk Ag oxidation by O2 chemisorption that took place solely on the
surface of nanosilver.
Figure 2.3: The Ag+ ion concentration (left ordinate) of suspensions with 20 mg/L of Ag with
xAg/SiO2 particles as a function of their Ag-content, x, along with the corresponding released
(leached) nanosilver mass fraction (right ordinate), before (filled symbols) and after the
nanosilver removal by centrifugation (open symbols). As inset, images of the corresponding
aqueous suspensions are presented before the removal of the Ag/SiO2 particles.
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2.3.2 Ag+ ion release
Figure 2.3 shows the Ag+ ion concentration (filled symbols) in aqueous
suspensions containing xAg/SiO2 (x = 1-98 wt%) particles at constant C = 20 mg/L
of Ag in solution as a function of x. All Ag+ ion concentrations were rapidly attained
(within few minutes upon dispersing) and were stable for at least 24 hours
(Appendix A, Figure A.2). Similarly, the stability of the dispersed Ag/SiO2
nanoparticles was monitored by dynamic light scattering (Appendix A, Figure A.3),
verifying that the suspensions were stable over 24 hours.
The inset of Figure 2.3 shows pictures of the corresponding suspensions
containing xAg/SiO2 particles. At low Ag-contents (x = 1 or 2 wt%), the suspensions
are nearly colorless indicating very few and small Ag metal nanoparticles. As x
increases, the suspension color darkens (Figure 2.3, inset) consistent with the
plasmonic behavior of nanosilver [24] that indicates here the presence of larger
silver particles.
In the right ordinate of Figure 2.3, the corresponding percentage of released
Ag+ ions over the total nanosilver mass is shown. For small Ag-contents
(x < 10 wt%) and particles, most of nanosilver is in the form of ions in solution, e.g.
for the 6Ag/SiO2, ~50% of nanosilver is as ions in solution. This is consistent with
Gunawan et al. [6] who reported 38% as Ag+ ions from their 5 at% Ag/TiO2 that
would correspond to 6.64Ag/SiO2 here. Such high Ag leaching and ion
concentrations have also been observed when employing rather fine Ag
nanoparticles [3]. High Ag-content (x > 95 wt%) composite Ag/SiO2 particles
contain rather large nanosilver particles (Figure 2.2, circles and triangles) that
hardly contribute to the exposed Ag surface area (Figure 2.2, squares) and rather
little on the released Ag+ ions in solution. This is also consistent with reported low
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Ag+ ion concentrations, when bigger particles (>50 nm) were dispersed in water by
ultrasonication [5,7], as in here.
Figure 2.3 also shows the Ag+ ion concentration after centrifugation of the
suspension and removal of the xAg/SiO2 particles (Ag+ ions only, open symbols).
The Ag+ ion concentration remains practically the same, indicating that by
centrifugation, only Ag/SiO2 particles are removed but not Ag+ ions. Furthermore,
after centrifugation all suspensions become colorless and transparent indicating
particle removal.
Figure 2.4: The UV/vis spectra of aqueous suspensions containing 2 (thin lines) and 20 mg/L
of Ag (bold lines) with 25Ag/SiO2 nanoparticles before (solid lines) and after centrifugation
(broken lines). In the inset, images of the suspensions of C = 2 mg/L is shown before and after
the removal of the 25Ag/SiO2 nanoparticles. Removing these nanoparticles converted the
yellowish suspension to a transparent one.
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Figure 2.5: The released Ag+ ion percentage as a function of the AgSSAE. For increasing
exposed nanosilver surface area, higher fraction of Ag is present as ions.
To verify that all xAg/SiO2 particles were removed from the suspensions,
their plasmon absorption band was monitored before and after centrifugation.
Figure 2.4 shows exemplarily the UV/vis spectra of aqueous solutions containing 2
(thin lines) and 20 mg/L of Ag (bold lines) of 25Ag/SiO2 particles, before (solid line)
and after centrifugation (broken line). In the inset of Figure 2.4, images before and
after removal of particles are presented for C = 2 mg/L of Ag. The peak intensity for
C = 20 mg/L of Ag is too high and has been cut in Figure 2.4 to distinguish also the
spectrum with C = 2 mg/L. Before centrifugation (solid lines), the characteristic
plasmon peak at ~410 nm attributed to Ag nanoparticles [24] exists for both
concentrations. After centrifugation (broken lines) this peak has been drastically
reduced for both concentrations, indicating that there are very few, if any, Ag
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nanoparticles in solution. Also, both yellow-colored suspensions become colorless
after centrifugation proving further the removal of Ag nanoparticles.
The fraction of released Ag+ ions (Figure 2.3) follows closely the
AgSSAE (Figure 2.2). In fact, it appears a linear relation (R2 = 0.96) between the
Ag+ ion fraction and AgSSAE (Figure 2.5), at constant Ag concentration in solution,
C. There is, however, significant scatter and quite likely a deviation from this at
smaller nanosilver sizes as discussed later on.
2.3.3 Antibacterial activity of nanosilver: Ag+ ions and Ag nanoparticles
The E. coli growth was investigated by monitoring the fluorescence intensity
of suspensions of E. coli cells at 37 °C which encode the green fluorescent protein
(GFP). Thus, the fluorescence intensity directly correlates with the E. coli population
[26] and the initial fluorescence corresponds to approximately 107 CFU/mL. Figure
2.6 shows the E. coli growth as a function of time up to 330 minutes in the absence
(control, stars) and presence of xAg/SiO2 nanoparticles for x = 1 (inverse triangles),
6 (squares), 10 (diamonds), 25 (circles), and 50 (triangles) wt% at constant Ag mass
concentration, C = 1 mg/L. The E. coli growth in the absence of Ag (stars) exhibited
the characteristic exponential bacterial growth [26]. The E. coli growth in the
presence of pure SiO2 (hexagons, normalized to the highest concentration of all Ag-
containing nanoparticles, specific surface area of 295 m2/g) is identical with the
control one (stars) indicating that SiO2 is indeed inert and does not influence the
process [27].
In the presence, however, of the smaller Ag-content and size particles (Figure
2.2), a much stronger antibacterial activity is observed than that of larger ones. Such
larger Ag-content Ag/SiO2 particles have lower exposed nanosilver specific surface
area and release less Ag+ ions (Figure 2.3) exhibiting lower antibacterial activity
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than smaller nanosilver particles. This result is in line with studies exhibiting also a
stronger antibacterial activity for smaller nanosilver particles [28]. Therefore, by
controlling the Ag-content in the Ag/SiO2 particles, the antibacterial activity of
nanosilver can be controlled also: From no or little E. coli growth for x = 1-10 wt%
Ag (Figure 2.6, inverse triangles, squares, diamonds), nearly maximum E. coli
growth is reached for x = 50 (triangles) at C = 1 mg/L of Ag (Figure 2.6).
Figure 2.6: The evolution of E. coli growth (fluorescence) for 330 minutes at 37 °C in the
presence of the xAg/SiO2 nanoparticles for x = 0-50 wt% for an Ag mass concentration of
C = 1 mg/L.
The toxicity of nanosilver is investigated against E. coli suspensions
containing equal Ag+ ion concentration in the presence and absence of nanosilver
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particles. Figure 2.7 shows the final E. coli population after 330 minutes before
(Ag+ ions and particles, filled squares) and after removal of xAg/SiO2 (Ag+ ions only,
open squares) by centrifugation. At C = 1 mg/L of Ag, the E. coli population is
identical in the presence and absence of nanosilver particles but always in the
presence of Ag+ ions that are not removed by centrifugation (Figure 2.3). For the
low Ag-content xAg/SiO2, very small silver particles (4 nm) are present that largely
dissociate into Ag+ ions dominating the toxicity of nanosilver, so particles alone do
not have a chance to play a significant role on toxicity against E. coli. This indicates
that the antibacterial activity is dictated by Ag+ ions alone. This result is consistent
with studies [13,14] reporting that Ag+ ions released from the nanosilver surface
play the most important role.
Since the higher Ag-content xAg/SiO2 particles (e.g. x > 40 wt%) do not
exhibit practically any E. coli growth inhibition for C = 1 mg/L of Ag and 330
minutes, their Ag mass concentration C in solution needs to be increased
substantially. So, at C = 20 mg/L of Ag (filled triangles) all the low Ag-content
(x ≤ 75 wt%) Ag/SiO2 particle suspensions exhibit strong antibacterial activity that
totally prevents E. coli growth for 330 minutes (Figure 2.7). At x = 95 and 98 wt%
Ag, however, the E. coli growth is only partially suppressed. For C = 30 mg/L (filled
circles), again all samples exhibit strong antibacterial activity and practically zero
E. coli growth.
When, however, the Ag/SiO2 particles are removed by centrifugation (open
symbols) and solutions become transparent, then significant E. coli growth takes
place for both Ag mass concentrations of 20 and 30 mg/L at x > 90 wt%. For
example, for x = 95 wt% and C = 20 mg/L when both Ag+ ions and particles are
present (filled triangles), the E. coli growth is inhibited by ~40%. When only Ag+
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ions are present (open triangles), this inhibition reaches half of it, ~20 %. At these
Ag concentrations and particle sizes, the antibacterial activity of Ag+ ions alone is
comparable to that in the presence of particles, indicating that the latter also play a
strong antibacterial role in agreement with recent studies [5,7,15], employing
relatively large (dp ~50 nm) nanosilver particles that had released also a minimal
fraction (~1%) of Ag+ ions [5]. This emphasizes the importance of the released Ag+
ions and particles in the mechanism of the antibacterial activity of such nanosilver
particles.
Figure 2.7: The final E. coli population after 330 minutes in the presence of 1 mg/L
(squares), 20 mg/L (triangles) and 30 mg/L (circles) of nanosilver before (Ag+ ions and
particles, filled symbols) and after removal of the nanosilver particles (Ag+ ions only, open
symbols). The E. coli growth of the control (no silver) is also displayed (star, broken line).
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2.4 Conclusions
All the above indicate that the mechanism of the antibacterial activity of
nanosilver particles seems to depend quite clearly on their size. When nanosilver
particles are small and release many Ag+ ions, the antibacterial activity is dominated
by these ions rather the nanosilver particles (Figure 2.7, yellow area I). This high
release rate of such small nanosilver particles could be related to their enhanced
curvature that facilitates mass transfer from their surface (Kelvin effect) and/or the
presence of an oxide layer on their surface. However, when relatively large (average
size larger than about 10 nm) nanosilver particles are employed with a low of Ag+
ion release, the particles themselves also influence the antibacterial activity of
nanosilver (Figure 2.7, blue area II). This result may explain seemingly
contradicting studies that support either the Ag+ ions [13,14] or the Ag particles
[5,7,15] as the dominant source of nanosilver toxicity. In fact, it appears that large
part of that the dispute in the literature may be traced to the lack of closely size-
controlled nanosilver or its actual state of agglomeration or flocculation. Finally, it
should be noted that these studies use as model biological systems other strains of
bacteria [5], algae [13,14], fish embryos [7] or human cells [15] corroborating the
validity of the present nanosilver toxicity mechanism to such systems beyond the
E. coli tested here.
2.5 References
[1] Chen, X. & Schluesener, H. J. Nanosilver: A nanoproduct in medical
application. Toxicol. Lett. 176, 1-12 (2008).
[2] Hannemann, S., Grunwaldt, J. D., Krumeich, F., Kappen, P. & Baiker, A.
Electron microscopy and EXAFS studies on oxide-supported gold-silver
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nanoparticles prepared by flame spray pyrolysis. Appl. Surf. Sci. 252, 7862-
7873 (2006).
[3] Benn, T. M. & Westerhoff, P. Nanoparticle silver released into water from
commercially available sock fabrics. Environ. Sci. Technol. 42, 4133-4139
(2008).
[4] Loher, S., Schneider, O. D., Maienfisch, T., Bokorny, S. & Stark, W. J.
Micro-organism-triggered release of silver nanoparticles from biodegradable
oxide carriers allows preparation of self-sterilizing polymer surfaces. Small 4,
824-832 (2008).
[5] Fabrega, J., Fawcett, S. R., Renshaw, J. C. & Lead, J. R. Silver nanoparticle
impact on bacterial growth: Effect of pH, concentration, and organic matter.
Environ. Sci. Technol. 43, 7285-7290 (2009).
[6] Gunawan, C., Teoh, W. Y., Marquis, C. P., Lifia, J. & Amal, R. Reversible
antimicrobial photoswitching in nanosilver. Small 5, 341-344 (2009).
[7] Laban, G., Nies, L. F., Turco, R. F., Bickham, J. W. & Sepulveda, M. S. The
effects of silver nanoparticles on fathead minnow (Pimephales promelas)
embryos. Ecotoxicology 19, 185-195 (2010).
[8] Lee, B. G., Griscom, S. B., Lee, J. S., Choi, H. J., Koh, C. H., Luoma, S. N.
& Fisher, N. S. Influences of dietary uptake and reactive sulfides on metal
bioavailability from aquatic sediments. Science 287, 282-284 (2000).
[9] Erickson, B. E. Nanosilver pesticides. Chem. Eng. News 87 (48), 25-26 (2009).
[10] Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M. V., Somasundaran,
P., Klaessig, F., Castranova, V. & Thompson, M. Understanding
biophysicochemical interactions at the nano-bio interface. Nature Mater. 8,
543-557 (2009).
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[11] Wijnhoven, S. W. P., Peijnenburg, W. J. G. M., Herberts, C. A., Hagens, W.
I., Oomen, A. G., Heugens, E. H. W., Roszek, B., Bisschops, J., Gosens, I.,
Van De Meent, D., Dekkers, S., De Jong, W. H., van Zijverden, M., Sips, A.
n. J. A. M. & Geertsma, R. E. Nano-silver - a review of available data and
knowledge gaps in human and environmental risk assessment. Nanotoxicology
3, 109 - 138 (2009).
[12] Tolaymat, T. M., El Badawy, A. M., Genaidy, A., Scheckel, K. G., Luxton,
T. P. & Suidan, M. An evidence-based environmental perspective of
manufactured silver nanoparticle in syntheses and applications: A systematic
review and critical appraisal of peer-reviewed scientific papers. Sci. Total
Environ. 408, 999-1006 (2010).
[13] Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N.,
Sigg, L. & Behra, R. Toxicity of silver nanoparticles to Chlamydomonas
reinhardtii. Environ. Sci. Technol. 42, 8959-8964 (2008).
[14] Miao, A. J., Schwehr, K. A., Xu, C., Zhang, S. J., Luo, Z. P., Quigg, A. &
Santschi, P. H. The algal toxicity of silver engineered nanoparticles and
detoxification by exopolymeric substances. Environ. Pollut. 157, 3034-3041
(2009).
[15] Kawata, K., Osawa, M. & Okabe, S. In vitro toxicity of silver nanoparticles
at noncytotoxic doses to HepG2 human hepatoma cells. Environ. Sci. Technol.
43, 6046-6051 (2009).
[16] Carlson, C., Hussain, S. M., Schrand, A. M., Braydich-Stolle, L. K., Hess, K.
L., Jones, R. L. & Schlager, J. J. Unique cellular interaction of silver
nanoparticles: size-dependent generation of reactive oxygen species. J. Phys.
Chem. B 112, 13608-13619 (2008).
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[17] Madler, L., Stark, W. J. & Pratsinis, S. E. Simultaneous deposition of Au
nanoparticles during flame synthesis of TiO2 and SiO2. J. Mater. Res. 18, 115-
120 (2003).
[18] Strobel, R. & Pratsinis, S. E. Flame aerosol synthesis of smart nanostructured
materials. J. Mater. Chem. 17, 4743-4756 (2007).
[19] Jackson, J. B. & Halas, N. J. Silver nanoshells: Variations in morphologies
and optical properties. J. Phys. Chem. B 105, 2743-2746 (2001).
[20] Czanderna, A. W. Adsorption of oxygen on silver. J. Phys. Chem. 68, 2765-
2771 (1964).
[21] Lubick, N. Nanosilver toxicity: ions, nanoparticles-or both? Environ. Sci.
Technol. 42, 8617-8617 (2008).
[22] Strobel, R., Madler, L., Piacentini, M., Maciejewski, M., Baiker, A. &
Pratsinis, S. E. Two-nozzle flame synthesis of Pt/Ba/Al2O3 for NOx storage.
Chem. Mater. 18, 2532-2537 (2006).
[23] Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual. (NY
Cold Spring Harbor Laboratory Press, 2001).
[24] Willets, K. A. & Van Duyne, R. P. Localized surface plasmon resonance
spectroscopy and sensing. Annu. Rev. Phys. Chem. 58, 267-297 (2007).
[25] Height, M. J., Pratsinis, S. E., Mekasuwandumrong, O. & Praserthdam, P.
Ag-ZnO catalysts for UV-photodegradation of methylene blue. Appl. Catal. B-
Environ. 63, 305-312 (2006).
[26] Gogoi, S. K., Gopinath, P., Paul, A., Ramesh, A., Ghosh, S. S. &
Chattopadhyay, A. Green fluorescent protein-expressing Escherichia coli as a
model system for investigating the antimicrobial activities of silver
nanoparticles. Langmuir 22, 9322-9328 (2006).
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[27] Brunner, T. J., Wick, P., Manser, P., Spohn, P., Grass, R. N., Limbach, L.
K., Bruinink, A. & Stark, W. J. In vitro cytotoxicity of oxide nanoparticles:
Comparison to asbestos, silica, and the effect of particle solubility. Environ.
Sci. Technol. 40, 4374-4381 (2006).
[28] Lok, C. N., Ho, C. M., Chen, R., He, Q. Y., Yu, W. Y., Sun, H., Tam, P. K.
H., Chiu, J. F. & Che, C. M. Silver nanoparticles: partial oxidation and
antibacterial activities. J. Biol. Inorg. Chem. 12, 527-534 (2007).
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CHAPTER 3
3. Nanosilver on Nanostructured Silica:
Antibacterial Activity and Ag Surface Area1
Abstract
Nanosilver immobilized on nanostructured silica facilitates investigations of
its antibacterial activity as the SiO2 support hinders the growth of nanosilver during
its synthesis and, most importantly, its flocculation in bacterial suspensions. Here,
such composite Ag/silica nanoparticles were made by flame spray pyrolysis of
appropriate solutions of Ag-acetate or Ag-nitrate and hexamethyldisiloxane or
tetraethylorthosilicate in ethanol, propanol, diethylene glucolmonobutyl ether,
acetonitrile or ethylhexanoic acid. The effect of solution composition on nanosilver
characteristics and antibacterial activity against the Gram negative Escherichia coli
was investigated by monitoring their recombinantly synthesized green fluorescent
protein. Suspensions with identical Ag mass concentration exhibited drastically
different antibacterial activity pointing out that the nanosilver surface area
concentration rather than its mass or molar or number concentration determine best
its antibacterial activity. Nanosilver made from Ag-acetate showed a unimodal size
distribution, while that made from inexpensive Ag-nitrate exhibited a bimodal one.
Regardless of precursor composition or nanosilver size distribution, the antibacterial
activity of nanosilver was correlated best with its surface area concentration in
solution.
1 Part of this chapter is published in Chem. Eng. J. 170, 547-554 (2011).
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3.1 Introduction
Nanosilver is used already in heterogeneous catalysis [1] and finds new
applications in textiles [2], biomedical devices [3], biodegradable polymer films for
food packaging [4], biological labeling [5], plasmon photonics and color [6],
optoelectronics and surface enhanced Raman scattering (SERS) [7]. At the same
time, the disposal of nanosilver raises concerns for its toxicity against aquatic micro-
organisms and therefore draws public attention [8]. In fact, nanosilver is one of the
first nanomaterials to be regarded toxic and petitions had been filed to the U. S.
Environmental Protection Agency (EPA) to regulate it as a pesticide [9]. Therefore
to safely employ nanosilver in commercial products, correct risk and dose relations
need to be determined [10-12].
The properties of nanosilver particles may change depending on their size
hindering, therefore, the correct assessment of such dose relations [10]. Smaller
nanosilver particles are more toxic than larger ones especially when oxidized
[13,14]. Even though metallic silver is practically insoluble in water [15], when
present in nanometer size range, Ag+ ions are released (leached) from its surface
[13,16,17]. The antibacterial activity of small (<10 nm) nanosilver particles is
dominated by Ag+ ions, while for larger ones (>15 nm) the antibacterial contribution
by Ag+ ions and particles is comparable [18]. Such a behavior implies a surface area
dependency of the antibacterial activity especially for small nanosilver sizes since
the Ag+ ion release is proportional to the exposed nanosilver surface area [18]. This
dependency could not be proven when evaluating [10] data of plasma-made
nanosilver and macrophages cells [19]. Toxicological studies, however, of other
engineered nanoparticles (e.g. TiO2) show this surface dependency [20]. So it has
been suggested that dose relations expressed in surface area concentration may
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reflect best the toxic activity of nanoparticles [21]. Furthermore, nanosilver particles
tend to agglomerate (flocculate) when dispersed in suspensions [22].
These limitations prevent a quantitative assessment of the antibacterial
activity of nanosilver [14] to determine correct dose relations [23]. To overcome
that, nanosilver particles with limited agglomeration and closely controlled size are
needed [24]. One way to address this is to immobilize nanosilver on a support
material [2,18]. That way, nanosilver is stabilized and retains its nanostructure since
it is anchored on an inert support. Several wet-chemistry based techniques have
been used for synthesis of composite Ag/SiO2 nanoparticles [25] or films [26] all of
them exhibiting a strong antibacterial activity attributed to the release of Ag+ ion
through the porous SiO2 matrix, typically made by such techniques. Alternatively,
gas-phase (aerosol) routes for synthesis of sophisticated nanoparticles including
Ag/SiO2 offer several advantages over wet-chemistry routes: no creation of liquid
by-products, easier collection of particles, fewer process steps, and the formation of
high-purity products [27]. For large scale gas-phase manufacture of nanoparticles
that involve the combustion of liquid precursors, for example flame-spray-pyrolysis
(FSP), inexpensive inorganic precursors are preferred over organometallic ones [28].
However, inorganic precursors often do not form homogeneous particles [29,30].
This inhomogeneity has been observed also for flame-made metal clusters on
ceramic particles (e.g. Pt-clusters supported on titania [31] or nanosilver on silica
[2]) resulting in often bimodal metal size distributions. Therefore, there is a need to
investigate the effect of the precursor composition on the morphology and
antibacterial activity of nanosilver made from inexpensive inorganic precursors (e.g.
Ag-nitrate) that are attractive for commercial synthesis of nanosilver [2,4].
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Here, the focus is on exploring the effect of precursor composition on the
characteristics of flame-made nanosilver-on-silica (Ag/SiO2) particles made from
inexpensive precursors that are typically used in industrial manufacture of
nanosilver products [2]. So particle properties are systematically measured by
S/TEM, EDX spectroscopy, XRD, N2 adsorption and UV/vis spectroscopy while
their antibacterial activity against the Gram-negative Escherichia coli (E. coli) is
investigated in aqueous suspensions monitoring the released Ag+ ion concentration.
The antibacterial activity of such Ag/SiO2 with bimodal Ag size distribution is
compared to that with unimodal distribution [18]. Finally, a systematic comparison
between Ag mass, number and surface area concentrations for the antibacterial
activity of nanosilver is made, investigating optimal dose-relations for risk
assessments of nanosilver.
3.2 Materials and methods
3.2.1 Particle synthesis and characterization
Composite Ag/SiO2 particles were made by FSP as described in detail
elsewhere [18]. Here, silver nitrate (Aldrich, purity > 99%) or silver acetate (Aldrich,
purity >99%) and hexamethyldisiloxane (HMDSO, Aldrich, purity > 97%) or
tetraethyl orthosilicate (TEOS, Aldrich, purity > 99%) were used as silver and
silicon precursors, respectively [2,4,18]. For all precursor solutions the total metal
(Ag + Si) concentration was 1 M. Silver nitrate and HMDSO were dissolved in 1:1
mixtures of ethanol (EtOH, Alcosuisse) and diethylene glycolmonobutyl ether
(DEGBE, Aldrich, purity >98%), silver nitrate and TEOS were dissolved in 2-
propanol (Aldrich, purity >98%) while silver acetate and HMDSO were dissolved in
2-ethylhexanoic acid (2-EHA, Aldrich, purity >98%) and acetonitrile (Aldrich,
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purity >98) in 1:1 ratio. All solutions were fed at 5 mL/min through the FSP nozzle
[18] and dispersed by 5 L/min oxygen (Pan Gas, purity >99%). The Ag-content of
the Ag/SiO2 product particles was x = 0 – 50 wt% and noted as xAg/SiO2. Their
composition corresponds to the nominal Ag- and Si- content of the FSP precursor
solution [4].
High resolution transmission electron microscopy (HRTEM) was performed
on a CM30ST microscope (FEI; LaB6 cathode, at 300 kV, point resolution ~2 Å)
and scanning transmission electron microscopy (STEM) on a Tecnai F30 (FEI;
300 kV). Product particles were dispersed in ethanol and deposited onto a perforated
carbon foil supported on a copper grid. Silver particle size distributions were
obtained by counting at least 165 particles from S/TEM images. Nanosilver particle
number and surface area concentrations in solution were calculated by multiplying
the nanosilver number concentration and surface area (both estimated by the size
distributions) with the nanosilver mass concentrations, respectively. X-ray
diffraction (XRD) patterns were recorded with a Bruker D8 advance diffractometer.
Crystallite sizes were obtained by refined Rietveld Analysis on the (111) peak of Ag.
The goodness-of-fit (GOF) was always below 1.2. Nitrogen adsorption-desorption
isotherms were determined at 77 K and the specific surface area was derived using
the Brunauer-Emmett-Teller (BET) method. The UV/vis optical absorption spectra
were obtained with a Cary Varian 500. The Ag+ ion concentration of aqueous
suspensions containing the xAg/SiO2 nanoparticles was measured with an ion
selective electrode and an ion meter (both Metrohm) [18].
3.2.2 Antibacterial activity
The antibacterial activity of the xAg/SiO2 nanoparticles was obtained by a
growth inhibition assay. Escherichia coli JM101 bacteria synthesizing a green
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fluorescent protein (GFP) from a plasmid-encoded gene were grown in Luria-
Bertani (LB) broth at 37 °C overnight [32]. The culture was subsequently diluted
with LB to an optical density (OD) of 0.05 at 600 nm, which corresponds to
approximately 107 colony forming units (CFU)/mL. The xAg/SiO2 nanoparticles
were dispersed in de-ionized water by ultrasonication (Sonics vibra-cell) for 20
seconds at 75% amplitude with a pulse configuration on/off of 0.5s/0.5s. The
nanoparticle suspensions were rather stable as determined by dynamic light
scattering (Malvern Zetasizer, Nanoseries) and by monitoring their color that
remained constant for more than a week and did not darken upon flocculation as it
commonly happens with nanosilver suspensions. For the assay, 50 μL of the
aqueous suspensions containing the dispersed Ag/SiO2 nanoparticles were added to
50 μL of the diluted cells. The bacterial growth was monitored by the fluorescent
signal of the GFP (Perkin Elmer 1420), corrected for background fluorescence and
normalized to the control measurement (no particles). The error bars for each data
point were the standard deviation of four measurements.
3.3 Results and discussion
3.3.1 Effect of precursor composition
Figure 3.1 shows a STEM image and EDX spectra of two selected areas of a
sample containing 1 wt% Ag (1Ag/SiO2) resulting from the Ag acetate/HMDSO
precursor. In the spectrum of area 1, comprising a relatively large bright spot as well
as the gray area surrounding it, there are signals of Si and O corresponding to SiO2
as well as of Ag confirming its presence there. In the spectrum of area 2 where the
electron beam is focused on a gray area, only peaks of Si and O are present
corresponding to amorphous SiO2 [1].
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Figure 3.1: STEM image of 1Ag/SiO2 nanoparticles and EDX spectra of the selected areas
(the C and Cu peaks arise from the carbon coated copper grid).
So there are a few nanosilver particles (bright contrast) dispersed on an
amorphous SiO2 matrix (gray), characteristic morphology for flame-made noble-
metals supported on ceramics nanoparticles [33,34]. Similar morphology of
dispersed nanosilver particles homogeneously dispersed on an amorphous
nanostructured silica support with corrugated structure were obtained for the
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product particles from the three different precursors and such results are consistent
with such composite Ag/SiO2 nanoparticles made also by flame-spray-pyrolysis
[2,4,18].
The nanosilver particles are rather homogeneously dispersed on SiO2 as it
can be observed in Figure 3.2a where an STEM image of 10Ag/SiO2 coming from
FSP of Ag-acetate/HMDSO is presented. In its inset, the nanosilver number size
distribution (total 851 particles) is shown along with the number average nanosilver
particle diameter (dp) and the geometric standard deviation g). This distribution is
broader than other FSP-made noble metal clusters on ceramics [33]. This indicates
that Ag clusters may have grown by coagulation as this g = 1.51 is close to the self-
preserving distribution of FSP-made particles [35] rather than grown-on-support
particles [33].
Figure 3.2: STEM images of 10Ag/SiO2 resulting from the three different precursor
compositions (a: Ag-acetate/HMDSO, b: Ag-nitrate/HMDSO, c: Ag-nitrate/TEOS)
presenting the dispersed nanosilver particles on the amorphous nanostructured silica. In the
insets, the nanosilver particle number size distributions are shown with their average particle
diameter dp and geometric standard deviation g.
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Figure 3.2b shows a STEM image of the 10Ag/SiO2 nanoparticles from the
Ag-nitrate/HMDSO precursor. Apart from the finely dispersed nanosilver particles
(3-30 nm), there are also a few larger ones forming a second mode in nanosilver size
distribution (inset) at 30-150 nm [2]. Similar bimodal Ag size distributions are
obtained from FSP of Ag-nitrate/TEOS precursor solutions (Figure 3.2c) as with
flame-made bimodal Pt clusters on TiO2 [31]. The fine mode is attributed to
nanosilver made by gas-to-particle conversion while the larger one is made by
droplet-to-particle conversion, precipitation in precursor droplets prior to their full
evaporation and combustion [36].
Figure 3.3: (a) XRD patterns of the 25Ag/SiO2 composite nanoparticles resulting from FSP of
three different precursor compositions. (b) Optical absorption spectra of aqueous suspensions
containing the 25Ag/SiO2 nanoparticles made by FSP from all precursor solutions. The peak
position of the plasmon absorption band is red-shifted for larger nanosilver particles and the
bands are broader for nanosilver with bimodal size distributions made by FSP of inexpensive
Ag-nitrate precursors (dotted and broken lines).
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Figure 3.3a shows the XRD patterns of 25Ag/SiO2 made from the three
different precursor solutions (Ag-acetate/HMDSO, Ag-nitrate/HMDSO, Ag-
nitrate/TEOS). The peak positions of silver metal are also indicated. Even though
all samples contain the same mass fraction of nanosilver, the main diffraction peak
is broader for that coming from the Ag-acetate solution indicating smaller
nanosilver. For both samples that exhibit a bimodal nanosilver particle size
distribution (Figure 3.2b,c) the peaks are quite sharper since larger particles are also
detected. It should be noted that XRD is not suitable for detection of silver oxide
layers on the nanosilver surface. It is quite likely that such layers are there [1,13,18]
as has been shown very recently by EXAFS [37].
Table 3.1: XRD analysis of nanosilver made by FSP of the three different precursor
compositions with the distribution mode, average crystal size (dXRD) and corresponding weight
fraction (wt%) of the small (s) and large (l) mode of the Ag crystal size distribution.
Ag-nitrate/HMDSO in EtOH-DEGBE Ag-acetate/ HMDSO in Acetonitrile-2-EHA
Ag-nitrate/TEOS in 2-propanol
Ag-content x in xAg/SiO2
mode dXRD (nm) s:small, l:large wt% mode dXRD (nm) mode dXRD (nm)
10 bimodal s: 5.7 ± 1.6 70 ± 15 % unimodal 6.9 ± 0.9 bimodal by STEM s: undefined
l: 25.3 ± 2.0 30 ± 15 %
l: 20.9 ± 2.1
25 bimodal s: 7.1 ± 1.0 80 ± 2 % unimodal 8.1 ± 0.8 bimodal by STEM s: undefined
l: 53.6 ± 14.9 20 ± 2 %
l: 23.7 ± 1.8
50 bimodal s: 8.6 ± 1.2 82 ± 1 % unimodal 8.7 ± 0.6 not prepared
l: 40.6 ± 10.6 18 ± 1 %
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Table 3.1 shows that nanosilver crystals made from Ag-acetate/HMDSO are
unimodal, so one crystal size is estimated from the XRD spectra (Figure 3.3a). For
Ag-contents x < 10 wt% the crystal concentration was below the XRD detection
limit. For an increasing Ag-content x, larger nanosilver crystal sizes are formed [18].
For nanosilver made from Ag-nitrate/HMDSO, however, two average crystal sizes
can be estimated, with the mass fraction of the larger mode being approximately
20 %. Remarkably, when the samples resulting from FSP of the Ag-nitrate/TEOS
are fitted with two crystal sizes, only the larger mode can be estimated reliably as
the fine crystals were too small for detection by XRD. Since nanosilver made from
Ag-nitrate/TEOS could not be quantitatively distinguished, no further experiments
with this precursor composition were carried out.
The observed bimodality of nanosilver made from Ag-nitrate can also be
verified by the optical absorption spectra of the dispersed xAg/SiO2 nanoparticles
from all three precursor solutions. Figure 3.3b shows exemplarily these spectra of
aqueous suspensions containing the 25Ag/SiO2 nanoparticles. The characteristic Ag
plasmon absorption band at ~400 nm is normalized for all samples and can be
clearly distinguished [5,22]. The presence of larger nanosilver particles when made
from Ag-nitrate (dotted and broken lines) can be verified by the peak position of
their plasmon absorption bands [5]. These have been shifted to higher wavelengths
than those made from Ag-acetate (solid line). In addition, the presence of larger
particles in the tail of the nanosilver size distribution made from Ag-nitrate
(bimodal) can also be detected, since the spectra are wider than the one of
nanosilver made from Ag-acetate [5].
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Figure 3.4: (a) TEM image of the 10Ag/SiO2 made from FSP of Ag-acetate solutions showing
nanosilver (dark) particles dispersed on amorphous (gray) SiO2 support. (b) The N2
adsorption-desorption curve for the 2Ag/SiO2 nanoparticles. As inset the corresponding TEM
image is shown. (c-h) Counted Ag particle size distributions for all samples from S/TEM.
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Figure 3.4a shows a representative TEM image of the 10Ag/SiO2 from the
Ag-acetate/HMDSO precursor with the interdispersed nanosilver particles (dark) on
the amorphous silica (gray) support [18]. Figure 3.4b shows the N2 adsorption-
desorption isotherm of the 2Ag/SiO2 nanoparticles as representative example
showing the typical hysteresis curve for non-porous particles [38]. As inset, the
corresponding TEM image is shown. Figure 3.4c-h show the nanosilver particle size
distributions counting 165-851 Ag particles [18] as determined by the S/TEM
analysis having the characteristic lognormal shape of aerosol-made nanomaterials.
Such size distributions are employed to estimate an average nanosilver size,
especially for the lower Ag-contents x, which could not be detected by XRD (Table
3.1). The average nanosilver particle diameter increases with increasing Ag-content
x in the xAg/SiO2 particles from 4 to 9 nm [18]. This control over nanosilver size, in
addition their immobilization on SiO2, makes possible the investigation of their
antibacterial activity.
Figure 3.5 shows the specific surface area (SSA) of xAg/SiO2 particles as a
function of Ag-content x. For pure SiO2 (x = 0 wt%) the samples have 250-300 m2/g
which corresponds to an average silica primary particle diameter of ~9 to 11 nm, in
agreement with literature [39]. For increasing x (1-10 wt%), however, there is an
increase in the total SSA. This indicates that the presence of Ag in the product
particles affects the growth of SiO2, perhaps by formation of a solid solution at very
low Ag-contents, as it has been observed also with trace contents of SiO2-based
mixed oxides [39]. Another possibility is the presence of Ag atoms to act as seeds for
SiO2 nucleation and thus to affect the nucleation kinetics of SiO2 clusters. Increasing
x further, decreases the SSA monotonically, as the presence of Ag (being a higher
density material than SiO2) is significant. It should be noted that even for the highest
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Ag-contents here, the total SSA originates mostly from SiO2 as that attributed to
nanosilver as determined by TEM is only 0.26 - 4.5 % for x = 1 - 25 wt%. So, all
samples have high SSA regardless of precursor composition, a desired property [40]
when such particles are employed as fillers in textiles [2] or polymers [4].
Figure 3.5: The specific surface area (SSA) of composite xAg/SiO2 nanoparticles as a function
of the Ag-content x (wt%), for samples made from FSP of Ag-nitrate/TEOS (diamonds), Ag-
nitrate/HMDSO (triangles) and Ag-acetate/HMDSO (circles).
3.3.2 Antibacterial activity
The antibacterial activity of nanosilver is examined by monitoring the
fluorescence intensity of suspensions with E. coli cells (that correlates with their
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population) at 37 °C for 330 minutes which encode the green fluorescent protein
(GFP). Figure 3.6a shows the E. coli growth in the presence of 10Ag/SiO2 made from
Ag-acetate/HMDSO at various Ag mass concentration (C = 0-1.6 mg/L of Ag). The
experimental uncertainty of these E. coli growth data is within the error bar, which
was similar for all data points. The characteristic exponential E. coli growth
evolution in the absence of particles (stars) and presence of pure SiO2 (hexagons) is
identical proving that SiO2 alone does not influence bacterial growth. When
10Ag/SiO2 composite nanoparticles are present in suspension, the fluorescence was
reduced for increased nanosilver concentration (C = 0.4-1.6 mg/L) indicating its
antibacterial activity. Figure 3.6b shows E. coli growth curves in the presence of
xAg/SiO2 nanoparticles made from Ag-acetate/HMDSO at C = 0.1-2.5 mg/L of Ag
(all samples have 10 mg/L of the composite xAg/SiO2 nanoparticles). For both
Figure 3.6a,b the bacterial growth is inhibited for the highest Ag content and Ag
mass concentration. The released Ag+ ions dominate the antibacterial activity of
nanosilver for the employed sizes here in agreement with literature [13,41,42] and
showing a stronger antibacterial activity for higher Ag mass concentration.
Figure 3.7a shows the final (after 330 minutes) E. coli growth % as a function
of x for xAg/SiO2 particles made from bimodal Ag-nitrate/HMDSO (triangles) and
unimodal Ag-acetate/HMDSO (circles). The Ag mass concentration applied was
C = 1 (open symbols) and 2 mg/L (filled symbols), respectively. The 100 % E. coli
growth has been estimated as the value of the control (Figure 3.6, stars). The lower
Ag-content x samples exhibit stronger antibacterial activity with very similar E. coli
growth regardless of precursor composition. This indicates that, first, smaller
nanosilver particles are more active than larger ones because of their higher surface
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area [14,18] and second, the larger mode of bimodal nanosilver (made from Ag-
nitrate) does not really contribute much in its antibacterial activity.
Figure 3.6: Nanosilver toxicity against E. coli. The evolution of E. coli growth (fluorescence)
for 330 minutes at 37 °C in the presence of (a) 10Ag/SiO2 particles (C = 0-1.6 mg/L of Ag)
and (b) xAg/SiO2 for Ag-contents x = 0-25 wt% and Ag mass concentrations C = 0.1-
2.5 mg/L. The experimental uncertainty of the data is within the error bar, which was
similar for all final data points. The corresponding silver surface area concentration
(C·AgSSA) in these suspensions is also presented.
Figure 3.7b shows the Ag+ ion concentration [Ag+] of aqueous suspensions
containing xAg/SiO2 nanoparticles (20 mg/L of Ag) made from Ag-nitrate/HMDSO
(triangles) and Ag-acetate/HMDSO (circles, from Figure 2.3) as a function of the
Ag-content x. The top axis shows the concentration of xAg/SiO2 nanoparticles in
solution. For both samples the [Ag+] is similar and higher for the lower Ag-content x
samples. This is attributed to the smaller nanosilver size of these samples [18].
Nanosilver smaller than 10 nm (size range of the smaller mode, Figure 3.2b) releases
Ag+ ions from its surface that have much higher antibacterial activity than from
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direct bacterial contact with that surface [18]. Thus, when two modes are present,
the particles of the smaller one will release more Ag+ ions. As seen from XRD
(Table 3.1) of nanosilver made from Ag-nitrate/HMDSO solutions, the large Ag-
crystal mode consists of about 20 wt% of the total Ag. This fraction contributes
~5 % to surface area. Therefore, even though this nanosilver is bimodal its surface
area is dominated by the smaller particles so its antibacterial activity is similar to
that of unimodal nanosilver. This indicates that the nanosilver surface area plays
quite an important role for its antibacterial activity. It should be noted, however,
that if the mass fraction of the larger mode would be significantly larger, then the Ag
antibacterial activity would be influenced also by the larger particles [18].
Figure 3.7: (a) Final E. coli growth % for different Ag-content x of 1 (open symbols) and 2
mg/L (filled symbols) Ag mass concentration from bimodal (triangles) and unimodal (circles)
nanosilver. (b) The [Ag+] of suspensions (20 mg/L of Ag) of bimodal (triangles) and unimodal
(circles) nanosilver.
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Figure 3.8: The extent of E. coli growth of all data at 210, 270 and 330 minutes as a function
of the Ag (a) surface area concentration C·AgSSA, (b) mass concentration C in suspension and
(c) nanosilver particle number concentration.
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Figure 3.6 also shows the exposed nanosilver surface area concentration
(C·AgSSA) as calculated by the Ag mass concentration C and Ag SSA (obtained from
the Ag size distributions in Figure 3.4c-h). Now, if one selects the E. coli growth
curves from Figure 3.6a,b with similar nanosilver surface area concentration,
C·AgSSA : 21.6/15.2 (triangles), 43.2/40.2 (diamonds) and 86.4/90.0·10-3 m2/L
(squares), they overlap within their measurement error. In fact, when the E. coli
growth data in the presence of nanosilver made from Ag-acetate/HMDSO of Figure
3.6 and Figure 3.7a (Figure 3.8, open: 210, half-filled: 270, filled symbols: after 330
minutes) are normalized to the control growth curve (stars) and plotted as a function
of C·AgSSA (Figure 3.8a), they fall on a straight line (R2 = 0.90) excluding data with
minimal E. coli growth (< 10%) as their growth period becomes too uncertain. This
indicates that indeed the antibacterial activity of nanosilver depends on the exposed
surface area concentration and it is time independent as this holds for all three time
periods. This dependency originates from the released Ag+ ions from the nanosilver
surface [18]. Such a relation can be difficult to observe when nanosilver with
modified surface [19] is employed which may influence the Ag+ ion release as well
as the bacterial contact with its surface [18].
Now Figure 3.8b shows exactly the same E. coli data as a function of Ag
mass concentration, C. Clearly these data are much more scattered (R2 = 0.56, again
excluding from the regression data with E. coli growth < 10%). Most notably, for the
same Ag mass concentration of C = 1 mg/L (circles), the E. coli growth covers the
entire spectrum; from complete inhibition to nearly full E. coli growth. This
highlights best the limitations of using mass or molar concentrations to assess the
nanosilver dose relations for its antibacterial activity. When the nanosilver particle
number concentration is calculated (Figure 3.8c) the data are less scattered than for
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mass (Figure 3.8b) indicating that number is advantageous over mass concentration
for nanosilver. However, when the data are plotted as a function of the C (Figure
3.8a), they are the least scattered verifying that the dose relations for the
antibacterial activity of nanosilver particles are best reflected whith surface area
concentrations.
3.4 Conclusions
Nanosilver particles were made and immobilized on nanostructured SiO2 by
spray combustion (flame spray pyrolysis, FSP) of three different precursor solutions.
The effect of precursor composition on the characteristics of product Ag/SiO2
nanoparticles was investigated. Nanosilver made by FSP of Ag-acetate and HMDSO
showed a unimodal size distribution, while that made from Ag-nitrate and HMDSO
or TEOS precursor solutions exhibited a bimodal size distributions. The
antibacterial activity of these composite Ag/SiO2 nanoparticles was investigated
against E. coli bacteria. Nanosilver made from inexpensive Ag-nitrate had similar
antibacterial activity to that made from Ag-acetate as the fine mode of the
distribution dominated its bactericidal properties by releasing or leaching of silver
ions. Additionally, the nanosilver surface area concentration in suspension
correlates best with Ag antibacterial activity rather than with nanosilver mass/molar
or number concentration. This indicates that the nanosilver dose expressions in
toxicological studies might be most accurate when assessed in terms of surface area
concentrations of nanosilver.
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S. E. & Grunwaldt, J. D. Selective side-chain oxidation of alkyl aromatic
compounds catalyzed by cerium modified silver catalysts. J. Molec. Catal. A-
Chem. 331, 40-49 (2010).
[38] Schimmoeller, B., Hoxha, F., Mallat, T., Krumeich, F., Pratsinis, S. E. &
Baiker, A. Fine tuning the surface acid/base properties of single step flame-
made Pt/alumina. Appl. Catal. A-Gen. 374, 48-57 (2010).
[39] Schulz, H., Madler, L., Pratsinis, S. E., Burtscher, P. & Moszner, N.
Transparent nanocomposites of radiopaque, flame-made Ta2O5/SiO2 particles
in an acrylic matrix. Adv. Funct. Mater. 15, 830-837 (2005).
[40] Camenzind, A., Caseri, W. R. & Pratsinis, S. E. Flame-made nanoparticles
for nanocomposites. Nano Today 5, 48-65 (2010).
[41] Li, P., Li, J., Wu, C. Z. & Wu, Q. S. Synergistic antibacterial effects of beta-
lactam antibiotic combined with silver nanoparticles. Nanotechnology 16,
1912-1917 (2005).
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[42] Tiwari, D. K., Behari, J. & Sen, P. Time and dose-dependent antimicrobial
potential of Ag nanoparticles synthesized by top-down approach. Curr. Sci.
95, 647-655 (2008).
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CHAPTER 4
4. Quantifying the Origin of Nanosilver
Ions and their Antibacterial Activity
Abstract
Nanosilver (silver nanoparticles) is prominent in several nanotechnology
products. Concerns however about leached Ag ions from its surface into aquatic
environments have challenged its broad use. The origin of these ions is explored
here by conditioning the surface of nanosilver immobilized on nanostructured silica
made by both wet and dry processes. Such nanosilver is characterized by electron
microscopy, atomic absorption spectroscopy, X-ray diffraction and its Ag+ ion
release in de-ionized water is monitored over time. By comparing nanosilver size
distributions to their leached or dissolved Ag+ ion concentrations, the latter match
the dissolution of one to two surface silver oxide monolayers, depending on
nanosilver diameter, by a simple mass balance. So by washing nanosilver or
reducing it in H2 removes any oxide layers from its surface, drastically minimizing
Ag+ leaching and its uncontrolled antibacterial activity.
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4.1 Introduction
Among engineered nanomaterials in consumer products, silver nanoparticles
(nanosilver) is the most common one [1] for its antibacterial, plasmonic or
electronic properties. At the same time these properties raise concerns for aquatic
micro-organisms upon disposal [2]. In fact, not long ago, petitions had been filed to
U.S. E.P.A. to label nanosilver as pesticide [3]. Nanosilver formation from released
Ag+ ions can occur under relevant environmental conditions simulating the soil
sediments [4] that further emphasizes the environmental impact that the released
Ag+ ions may have. Even though nanosilver transforms to the less toxic silver
sulfide nanoparticles in sewage sludge [5], the latter can influence the silver uptake
and bioaccumulation into food chains [2] and could originate partly from released
or leached nanosilver particles from commercial products [6].
Even though the number of studies on nanosilver toxicity has grown
exponentially since 2006, an understanding of its antibacterial mechanism only now
emerges [7-10]. Nanosilver particles themselves can induce toxicity [11], however, a
major role is played by the released Ag+ ions from the nanosilver surface upon its
contact with water [9,10,12-14]. So the amount of released Ag+ ions strongly
depends on nanosilver particle size [10] since smaller particles release much more
Ag+ ions. In fact, size dictates whether Ag+ ions or particles are responsible for the
antibacterial activity [10], emphasizing the importance of Ag surface area
concentration, rather than mass or number concentrations, in nanosilver dose-
relations [8,15].
This Ag+ ion release is exploited in a variety of target applications and
products, so nanosilver particles, often supported on a ceramic support, are used as
antimicrobial fillers in textiles [16] and polymers for food-packaging [17] and
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biomedical applications [18], antimicrobial paints [19] or as drug delivery system
[8], Several factors influence the Ag+ ion release in aqueous solutions: surface
coatings, temperature, pH and dissolved oxygen concentration, presence of organic
matter and other ions [13,14,20,21], Most of Ag+ ion release occurs from oxidation
of metallic nanosilver with its interaction with dissolved oxygen and protons [22]. In
fact, oxidized silver has stronger antibacterial activity than metallic silver [8,12,22].
Here, the effect of nanosilver size and surface composition on Ag+ ion release
is investigated systematically by preparing composite Ag/SiO2 nanoparticles by wet
impregnation [23] and flame spray pyrolysis (FSP) [10]. The goal is to quantitatively
understand the characteristics of nanosilver that influence the Ag+ ion release and to
evaluate how their antibacterial performance is influenced after initial Ag
dissolution. This is done by monitoring the Ag+ ion release from composite Ag/SiO2
nanoparticles in aqueous solutions after washing (removal of the leached Ag+ ion
from solution) or reducing them under H2. The released Ag+ ions concentration is
quantitatively traced to the nanosilver surface layers by a mass balance. Finally, the
antibacterial activity of washed and reduced Ag/SiO2 nanoparticles is explored with
E. coli bacteria that produce a green fluorescent protein, focusing on nanosilver size
and surface treatment.
4.2 Materials and methods
Composite Ag/SiO2 nanoparticles were made either by FSP [10] or by
impregnation [23] of silver nitrate (Aldrich, purity > 99%) on nanostructured SiO2
(Evonik, A300). In brief, for the wet-made Ag/SiO2 nanoparticles, SiO2
nanoparticles were dispersed in deionized water and then appropriate amount of
silver nitrate was added. The mixture was stirred overnight and then dried at 85 °C
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for 24 hours. The dry powder was ground and annealed at 500 °C for 5 hours [23].
The mass fraction x of Ag in the flame-made Ag/SiO2 particles ranged from x = 0-
50 wt% (xAg/SiO2).
All particles were characterized by X-ray diffraction (XRD) on a Bruker AXS
D8 Advance spectrometer (Cu K, 40 kV, 40 mA). The crystallite sizes were
calculated using the Rietveld methods and the software TOPAS3. The actual Ag-
content x was determined by atomic absorption spectroscopy (AAS). For this,
appropriate amounts of xAg/SiO2 nanoparticles were digested in HNO3 (1 M) for 24
hours at room temperature and then centrifuged (14000 rpm, 4 minutes) to remove
the undissolved SiO2. The Ag-content of the supernatant was measured.
Transmission electron microscopy (TEM) was performed on a Tecnai F30 (FEI; 300
kV). Particles were dispersed in ethanol and deposited onto a perforated carbon foil
supported on a copper grid. Particle size distributions from the S/TEM analysis
were obtained by counting at least 200 particles with the software ImageJ [15]. The
Ag+ ion concentration of aqueous suspensions was determined by ion selective
electrode and an ion meter (both Metrohm) [10]. The washing of xAg/SiO2
nanoparticles was performed by centrifugation of their aqueous suspensions,
removal of supernatant containing the leached Ag+ ions and immediate redispersion
of nanoparticles in fresh de-ionized water (without drying them) by ultrasonication
(Sonics vibra-cell 600 W, 2 minutes, 0.5/0.5 s on/off pulse, 75% power) to avoid air
contact and possible reoxidation. With such a process, the as-prepared Ag/SiO2
nanoparticles are homogeneously dispersed in water, as after their centrifugation
they tend to form large flocs on the one side of the vial. By sonication, a
homogeneous nanoparticle dispersion is made.. The dissolved oxygen (DO)
concentration was constant at 8.5-8.9 ppm as measured in air-saturated aqueous
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suspensions by a DO meter (VWR International). All xAg/SiO2 samples were
reduced at 300 °C for 30 min under H2 (5% H2 in He) at 5 mL/min. The samples
were kept under He atmosphere until they cool down and return to ambient
conditions for suspension in de-ionized water.
The oxide surface layer fraction, R, was calculated over the entire size
distribution for one or two molecular layers of Ag2O (density = 7.14 g/cm3, Ag2O
molecular thickness of 0.46 nm) on the nanosilver surface as: ( )i i in s mR
N
, where
ni is the number of ith-sized by S/TEM nanosilver particles, si is the mass of the
surface mono- or bi-layer of the ith particle, mi is the mass of the ith particle and N is
the total number of nanosilver particles. The antibacterial activity of the xAg/SiO2
nanoparticles was obtained by a growth inhibition assay described in detail
elsewhere [10].
4.3 Results and discussion
4.3.1 Morphology and composition of flame- and wet-made Ag/SiO2 nanoparticles
Both flame- and wet-made Ag/SiO2 nanoparticles have nanosilver dispersed
homogeneously on the SiO2 support as shown by scanning transmission electron
microscopy (STEM) images in the insets of Figure 4.1. The bright particles
correspond to the higher atomic number silver, while the diffuse gray structure
corresponds to SiO2 for either flame- [10,24,25] or wet-made [23,24] Ag/SiO2
nanoparticles. Higher Ag-content x for the wet-made nanosilver (e.g. x = 10 wt%)
resulted in quite large nanosilver particles (dp > 30 nm), indicating that FSP might
offer a finer control of the nanosilver size than wet-methods [26].
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Figure 4.1: The measured Ag-content x in the composite flame- (circles) and wet-made
(triangle) xAg/SiO2 nanoparticles as a function of the nominal one. There is a good
agreement between the two indicating that the nanosilver particles are on the surface of the
nanostructured silica. At the insets, electron microscopy images of 6Ag/SiO2 nanoparticles
made by dry flame and wet-chemistry processes. The nanosilver particles are the dark dots.
Figure 4.1 shows the measured Ag-content x as a function of the nominal one
in composite xAg/SiO2 nanoparticles as determined by atomic absorption
spectroscopy (AAS) after acidic dissolution for both flame- (circles) and wet-made
(triangle) nanoparticles. These symbols are the average from three measurements
while the error bar is smaller than the symbol size. There is a good agreement
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between measured and nominal Ag-contents x = 1-10 wt%, indicating that most, if
not all, nanosilver is located on the surface of the nanostructured silica support for
both flame- [25] and wet-made [23] xAg/SiO2 nanoparticles. There is a small
difference for the flame-made 25Ag/SiO2 indicating that perhaps some nanosilver
particles are embedded [10,16] within the silica matrix (that prevents their
dissolution) for high Ag-contents x. Nonetheless, for x < 25 wt% employed here,
there is excellent agreement between the measured and nominal Ag-content as seen
also by AAS [17] and ICP-MS [25].
Nanosilver average diameter dp (nm)
4 6 8 10
Fraction of Ag as Ag+ ions (%
)
0
20
40
60
80
100
Ag+ ion concentration [Ag+] (m
g/L)
0
10
20
30
406Ag/SiO2-wet
xAg/SiO2-flame
Figure 4.2: The Ag+ ion concentration [Ag+] of aqueous suspensions after 28 days that contain
the as-prepared (open symbols) and washed (filled symbols) flame- (circles) and wet-made
(triangle) xAg/SiO2 nanoparticles as a function of average nanosilver size (bottom axis) or Ag-
content x (top axis) of the flame-made nanoparticles at Ag mass concentration of 40 mg/L.
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4.3.2 Ag+ ion release: Dissolution of the surface oxide layer by washing
Figure 4.2 shows the Ag+ concentration, [Ag+], after 28 days of aqueous
suspensions containing 40 mg/L of Ag of as-prepared (open symbols) flame-
(circles) and wet-made (triangle) xAg/SiO2 nanoparticles as a function of the
nanosilver average diameter (dp) determined by electron microscopy [15]. There is
clearly a size effect, since for smaller nanosilver more Ag+ ion leaching takes place
[8,10,22]. For example, upon dispersing 1Ag/SiO2 particles in water having 4 nm
nanosilver diameter, ~70% of Ag is leached into solution as ions, in agreement with
the release of similarly sized nanosilver [10,12,15]. This is why Ag+ ions
dominate the antibacterial activity of nanosilver in these sizes (dp = 4-12 nm) [10].
The nanosilver synthesis method does not seem to affect the Ag+ ion release as the
[Ag+] from wet-made 6Ag/SiO2 follows the same trend with that from flame-made
xAg/SiO2 for x = 1-50 wt% (Figure 4.2).
When the dispersed xAg/SiO2 nanoparticles are removed by centrifugation
[10] from these suspensions and re-dispersed in fresh deionized water (washed
xAg/SiO2, filled symbols), the [Ag+] is substantially lower, again regardless of
preparation method (Figure 4.2). This indicates that the release of Ag+ ions from
washed nanosilver is rather minimal. Silver oxide layer formation occurs during
processing for both flame- and wet-made nanosilver that involves high temperatures
and exposure to oxygen. The initial oxide layer on the surface of nanosilver [23,25]
dissolves upon dispersion of the as-prepared Ag/SiO2 nanoparticles releasing
Ag+ ions. As soon as, however, this oxide dissolution occurs, the nanosilver
particles hardly release more ions (Figure 4.2, filled symbols).
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Figure 4.3: (a) XRD patterns of as-prepared (bottom) and washed (top) flame-made
10Ag/SiO2 nanoparticles showing that the main diffraction peak of silver metal is similar for
both as-prepared and washed nanosilver. (b) The [Ag+] as a function of time for the as-
prepared (open symbols) and washed (filled symbols) wet- (triangles) and flame-made (circles)
6Ag/SiO2 nanoparticles. The time = 0 days corresponds to [Ag+] immediately before the
nanoparticle dispersion.
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Figure 4.3a shows the XRD patterns of the flame-made 10Ag/SiO2 before
(bottom) and after washing (top). The peak positions correspond to Ag metal while
the lack of flat baseline is attributed to the presence of amorphous silica support and
silver oxide. There is clearly metallic silver present in the as-prepared flame-made
10Ag/SiO2 nanoparticles with an average crystal size 6.9 nm. After washing, this
sample seems to release almost 50% of its Ag mass as ions (Figure 2). So after
washing its average crystal size should have decreased to 5.5 nm (assuming
monodispersed nanosilver). The main diffraction peak around 38°, however,
remains identical before and after washing of the nanoparticles. This indicates that
the Ag crystal size has not changed after washing and in fact, the average crystal
size is almost identical (7.1 nm). The practically identical nanosilver average crystal
sizes before and after washing further indicate that the dissolved Ag+ ions come
from the dissolution of amorphous silver oxide layer on the nanosilver surface [8]
rather than crystalline dissolved nanosilver. This layer cannot be detected by XRD
[15] but only by EXAFS [23-25] or XPS [28] as it has been shown for both flame-
[24,25] and wet-made [23,24] xAg/SiO2 nanoparticles. The XRD spectra of the
washed particles have a slightly better signal-to-noise ratio probably because their
amorphous oxide layer has been removed. This increase in the XRD signal-to-noise
ratio has been observed also for bare and amorphous silica-coated iron oxide
nanoparticles with similar sizes [27]. For x < 6 wt%, reliable XRD spectra could not
be obtained as the Ag-content x was too little in the composite xAg/SiO2
particles [10].
Figure 4.3b shows also the Ag+ ion release kinetics and that immediately
upon dispersing the nanosilver in solution, the [Ag+] reached a plateau that
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Figure 4.4: (a) XRD patterns of the as-prepared (bottom) and reduced under H2 (top) wet-
made 6Ag/SiO2 nanoparticles. The peak position of the silver metal is also indicated. After
reduction, a peak emerges at 2 = 38° corresponding to silver metal. (b) The [Ag+] as a
function of time for the as-prepared (open symbols) and reduced (filled symbols) wet-
(triangles) and flame-made (circles) 6Ag/SiO2 nanoparticles.
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remained stable for 28 days from both as-prepared and washed nanosilver
regardless of preparation route. This is in contrast to other dissolution studies of
nanosilver where it is observed that the [Ag+] initially increases reaching a plateau
after hours or days [13,14]. This is not observed here as the nanoparticles are
dispersed by ultrasonication which may have accelerated significantly the
dissolution. No change in the Ag+ ion release was observed for different
ultrasonication periods (20 s to 300 s), pulse (0.2 to 0.5 s) or power (50 to 85%) for
the as-prepared xAg/SiO2 (x = 1-50 wt%) and Ag mass concentration 40 mg/L.
Furthermore, perhaps more important is that the employed nanosilver in
other studies is coated by polyvinylpyrrolidone [13] and citrate [13,14] in order to
minimize nanosilver agglomeration. This coating, however, might influence the Ag+
ion release kinetics in aqueous suspensions. In fact such functionalized or coated
nanosilver might hinder its surface-dependent antibacterial activity [7,29]. The
nanosilver prepared here does not have any coating that could compromise the Ag+
ion release, as its agglomeration is inhibited by the presence of the SiO2 support
[10]. It should be noted that perhaps a small fraction of the dissolved nanosilver
comes from metal-dioxygen reaction after oxide dissolution [8]. This indicates that
the nanosilver surface is not oxidized further after the dissolution of the initial oxide
layer at the present conditions (dissolved oxygen concentration 8.5-8.9 ppm) or
little, if any, further oxidation occurs. Note that oxidation of nanosilver in solution
could occur for low pH values or high H2O2 concentrations present [14].
4.3.3 Ag+ ion release: Reduction under H2 of the surface oxide layer
By reduction under H2, the silver oxide layer on the nanosilver surface is
converted to metal one. Figure 4.4a shows the XRD spectra of before (as-prepared)
and after reduction under H2 of the 6Ag/SiO2 nanoparticles made by wet-
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chemistry [23]. Before H2 reduction, there are hardly any peaks corresponding to
silver metal or oxides though a peak seems to emerge at 38°, which however can be
surely detected after the reduction. This indicates that after reduction, the oxide
layer covering the core nanosilver surface becomes metallic and thus the metallic Ag
content increases, enabling its detection by XRD. Given that 60% of the wet-made
nanosilver here dissolves (Figure 4.2, open triangle), the metallic Ag mass increases
by 150% upon its reduction. No Ag enlargement is expected by the high temperature
H2 reduction (300 °C). The corrugated nanostructured SiO2 support [10] prevents
any nanosilver growth by surface diffusion and sintering, since even such lower
temperatures than the silver melting point (961 °C) can induce an increase in the
size of pristine nanosilver [30].
To further emphasize the importance of the oxide layer, Figure 4.4b shows
the [Ag+] for 28 days from solutions containing the as-prepared (open symbols) and
reduced under H2 (filled symbols) 6Ag/SiO2 nanoparticles made by flame- (circles)
and wet- (triangles) processes. The [Ag+] from reduced 6Ag/SiO2 nanoparticles is
much less than that of as-prepared ones. These [Ag+] values were also stable for
nearly a month (Figure 4.4b), indicating that the reduced nanosilver particles do not
oxidize further when dispersed in de-ionized water.
Therefore, the release of Ag+ ions from metallic nanosilver particles is much
less than that from nanosilver with oxidized surface [12]. This result also explains
why the antibacterial activity of metallic nanosilver is lower than that from oxidized
nanosilver [22], since for such small nanosilver sizes the antibacterial activity is
dominated by the released Ag+ ions [10]. It should also be noted that the [Ag+] from
the reduced xAg/SiO2 samples is not completely zero (Figure 4.4b), and actually
slightly higher than the washed nanoparticles (Figure 4.3b), indicating that a small
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release of Ag+ ions takes place immediately after their dispersion. This could be
attributed to the presence of a small fraction of oxidized Ag layer, as nanosilver may
be partially surface oxidized even at ambient conditions [31].
4.3.4 Ag oxide layer thickness and Ag+ ion concentration
Figure 4.5 shows the Ag mass fraction as Ag+ ions assuming one (squares,
dotted line) or two (diamonds, broken line) silver oxide monolayers on the surface
of nanosilver as calculated from the Ag particle size distributions from electron
microscopy [15] as a function of nanosilver average diameter, dp, (bottom axis or
abscissa) and Ag-content x in the flame-made xAg/SiO2 particles (top axis or
abscissa). Figure 4.5 also shows the mass fraction of Ag as ions in aqueous
suspensions from as-prepared flame- (circles) or wet-made (triangle) xAg/SiO2 from
Figure 4.2. For dp ≥ 8 nm the mass fraction of Ag as ions in suspension (solid line)
corresponds well to that of a single silver oxide monolayer (dotted line) on the
nanosilver surface. For dp < 5 nm, the Ag+ ion mass fraction (solid line) corresponds
asymptotically to the mass from two silver oxide surface layers. An intermediate
appears for 5 nm < dp < 8 nm. Therefore, the leached Ag+ ions in suspensions can
close the mass balance with 1-2 silver oxide layers from the nanosilver surface
depending on particle size.
Silver oxide has higher solubility than silver metal in water [32]. As a result,
upon dispersion of nanosilver in water, its surface oxide monolayer dissolves quite
rapidly as ions (Figure 4.3) further indicating that the released Ag+ ions mostly
originate from the leaching (dissolution) of this oxidized layer [8,10]. The existence
of 1-2 layers of oxidized Ag on the surface of nanosilver for smaller particles and Ag
contents (Figure 4.5 at dp < 5 nm) is quite plausible given that smaller nanosilver
particles tend to oxidize more than larger ones [33].
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Figure 4.5: The fraction of Ag atoms as ions in the aqueous suspensions (flame- :circles and
wet-made: triangle, solid line) as a function of the nanosilver average diameter (bottom axis),
and Ag-content x in the composite xAg/SiO2 flame-made nanoparticles (top axis). The
estimated values [Ag+] from the nanosilver size distributions assuming 1 (squares, dotted line)
and 2 (diamonds, broken line) surface oxide layers.
The above result emphasizes the importance of the oxidation state of
nanosilver and shows that with flame- or wet-synthesis of nanosilver and subsequent
drying and calcinations the Ag surface is oxidized. In fact, it is remarkable that with
surface treatment (washing or H2 reduction), the Ag+ ion release was minimized.
This further indicates that by controlling the oxidation state of nanosilver surface,
the Ag+ ion release in aqueous solutions can be controlled [8,12,22].
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Figure 4.6: E. coli fluorescence (that corresponds to the E. coli population) in the presence of
as-prepared (open symbols) and washed (filled symbols) flame- (circles) and wet-made
(triangles) 6Ag/SiO2 nanoparticles for 2.5 mg/L Ag mass concentration. The initial Ag size
of the flame- and wet-made from the TEM analysis is 6.1 and 4.6, respectively. The E. coli
growth in the absence of any nanoparticles is also shown (control, stars). Error bars
correspond to the standard deviation of 4 measurements.
4.3.5 Antibacterial activity
Figure 4.6 shows the E. coli fluorescence that corresponds to their population
[10] for 330 minutes in the presence of as-prepared (open symbols) and washed
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(filled symbols) flame- (circles) and wet-made (triangles) 6Ag/SiO2 nanoparticles (Ag
mass concentration: 2.5 mg/L). The E. coli growth in the absence of any nanosilver
is also shown (control, stars) [10]. For both as-prepared, flame- and wet-made
nanosilver, E. coli growth is completely inhibited. This is attributed to the high
amount of Ag+ ions in solution from as-prepared samples (Figure 4.2). For the
employed nanosilver sizes here, the released Ag+ ions dominate their antibacterial
activity [10]. In the presence, however, of washed 6Ag/SiO2 nanoparticles, E. coli
growth is not significantly affected because the washed nanosilver does not release
many Ag+ ions (Figure 4.3b) and thus, these particles do not induce a strong
antibacterial activity for the employed concentration of 2.5 mg/L of Ag [10,15].
The Ag surface area concentration in the presence of washed nanosilver is
almost half that in the presence of as-prepared nanosilver (0.062 and 0.130 m2/L for
washed and as-prepared nanosilver, respectively, please see Appendix B). The E. coli
growth is not significantly influenced in the presence of the washed nanosilver
(Figure 4.6, filled symbols), even though for such surface area concentration an
E. coli growth of about 30% should have been observed during that period (Chapter
3, Figure 3.8). This further corroborates that the washed nanosilver does not release
Ag+ ions at the same rate to as-prepared nanosilver of that surface area
concentration, in agreement with Figure 4.2. Therefore, the antibacterial activity of
nanosilver can be controlled and even minimized when its surface is treated. Similar
results were obtained in the presence of reduced nanosilver, as they also have a
minimal Ag+ ion release (Figure 4.4b).
Figure 4.7 shows the final E. coli viability after 330 minutes (100%
corresponds to the control, stars from Figure 4.6) for the as-prepared (open symbols)
and washed (filled symbols) flame- (circles) and wet-made (triangles) 6Ag/SiO2
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nanoparticles for Ag mass concentrations of 2.5 to 20 mg/L. For all Ag
concentrations, the [Ag+] from the as-prepared nanoparticles is high enough to
completely inhibit the E. coli growth. The slightly negative values obtained for the
as-prepared nanosilver most probably originate from photobleaching of media
components. On the other hand, the E. coli viability of washed nanosilver is much
higher than that of as-prepared nanosilver because the former releases much less Ag+
ions (Figure 4.2). As, however, the Ag mass concentration is increased, even the
washed 6Ag/SiO2 nanoparticles start exhibiting some antibacterial activity, reducing
the cell viability almost completely for 20 mg/L of Ag. This antibacterial activity is
attributed mostly to the contact of the cells on the nanosilver surface, as these
particles hardly release any Ag+ ions (Figure 4.3b).
Figure 4.7: E. coli viability (shaded band at 100% corresponds to the control, stars in Figure
4.6) in the presence of as-prepared (open symbols) and washed (filled symbols) flame- (circles)
and wet-made (triangles) 6Ag/SiO2 nanoparticles for different Ag mass concentrations. The
error bars correspond to the standard deviation of 4 measurements.
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The effective [Ag+] of the washed flame- and wet-made nanosilver nanosilver
ranges from 0.0875 to 0.70 mg/L (please see Appendix B for detailed calculations).
Such low [Ag+] values can influence bacteria growth, and in fact the antibacterial
activity observed here for higher nominal Ag mass concentrations for the washed
nanosilver is attributed mainly to the released Ag+ ions [15].
It should be noted, however, that the actual Ag mass concentration of the
suspensions containing the washed xAg/SiO2 particles is not the nominal one, as a
large fraction of silver has been removed during washing: for example, ~60% for the
wet- and ~50% for the flame-made 6Ag/SiO2 nanoparticles (Figure 4.2). As a result,
the washed wet-made 6Ag/SiO2 nanoparticles (triangles) exhibit higher E. coli
viability than that of flame-made washed nanosilver (Figure 4.7) for the same
nominal Ag mass concentrations. More Ag dissolves from wet-made 6Ag/SiO2 that
are even smaller (dp,w = 4.6 nm) to start with than flame-made 6Ag/SiO2
nanoparticles (dp,f = 6.1 nm, Figure 4.2). So the actual Ag mass present in the wet-
made washed 6Ag/SiO2 nanoparticles (~40% of the nominal) is even lower than that
present in the flame-made washed 6Ag/SiO2 nanoparticles (~50% of the nominal).
So by washing nanosilver it might be possible to remove the oxide layer from
its surface and re-use the washed metallic nanosilver as an antibacterial agent that
would exhibit the desired bactericidal properties only by contact with its surface
with limited ion leaching. This, of course, would mean that higher Ag
concentrations of these washed nanosilver particles need to be used than the as-
prepared ones because the antibacterial activity of the latter is stronger than the
former for low Ag mass concentrations (e.g. 2.5 mg/L of Ag, Figure 4.7). The
antibacterial activity of the as-prepared nanosilver, however, may not last longer
than the first washing of a textile product, for example, as the oxide layer will be
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removed. If, however, nanosilver particles that have their surface oxide layers
removed are used at sufficiently high Ag concentrations, the antibacterial
performance could be maintained even after washing (e.g. for 20 mg/L in Figure
4.7) with potentially minimal impact to the environment by Ag+ ion leaching from
metallic nanosilver.
4.4 Conclusions
Composite Ag/SiO2 nanoparticles were made by flame aerosol technology
and wet-impregnation. In both cases, the nanosilver particles were homogeneously
dispersed on the amorphous SiO2 support and their size could be controlled by
varying the Ag-content x in the xAg/SiO2 particles. A finer nanosilver size control
could be obtained by flame technology and a good agreement between the nominal
and actual Ag-content x in the composite xAg/SiO2 nanoparticles was obtained. The
Ag+ ion release from these xAg/SiO2 nanoparticles was investigated in deionized
water. A higher Ag+ ion concentration was obtained for smaller nanosilver particles.
When, however, these particles were washed and re-dispersed in water, the Ag+ ion
concentration was minimal. Additionally, the Ag+ ion release could be controlled by
surface treatment (reduction) of the nanosilver emphasizing the significance of the
oxidation state of its surface. It is shown, for the first time to our knowledge, that
the release of Ag+ ions from nanosilver in aqueous solutions corresponds to the mass
leached or dissolved of one or two oxidized monolayers from its surface depending
on nanosilver size. The antibacterial activity of small (< 10 nm) nanosilver particles
that have their surface silver oxide layer either removed by dissolution or reduced to
metallic silver is significantly lower than that of as-prepared nanosilver particles. So
the use of metallic nanosilver in nanotechnology products can reduce the release of
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toxic Ag+ ions and facilitate its broader use, for example as fillers in textiles or
polymers for biomedical uses (e.g. catheters).
4.5 References
[1] Project on Emerging Nanotechnologies. www.nanotechproject.org.
[2] Lee, B. G., Griscom, S. B., Lee, J. S., Choi, H. J., Koh, C. H., Luoma, S. N.
& Fisher, N. S. Influences of dietary uptake and reactive sulfides on metal
bioavailability from aquatic sediments. Science 287, 282-284 (2000).
[3] Erickson, B. E. Nanosilver pesticides. Chem. Eng. News 87 (48), 25-26 (2009).
[4] Akaighe, N., MacCuspie, R. I., Navarro, D. A., Aga, D. S., Banerjee, S.,
Sohn, M. & Sharma, V. K. Humic acid-induced silver nanoparticle formation
under environmentally relevant conditions. Environ. Sci. Technol. 45, 3895-
3901 (2011).
[5] Kim, B., Park, C. S., Murayama, M. & Hochella, M. F. Discovery and
characterization of silver sulfide nanoparticles in final sewage sludge
products. Environ. Sci. Technol. 44, 7509-7514 (2010).
[6] Nowack, B. Nanosilver revisited downstream. Science 330, 1054-1055 (2010).
[7] Auffan, M., Rose, J., Bottero, J.-Y., Lowry, G. V., Jolivet, J.-P. & Wiesner,
M. R. Towards a definition of inorganic nanoparticles from an
environmental, health and safety perspective. Nature Nanotechnol. 4, 634-641
(2009).
[8] Liu, J. Y., Sonshine, D. A., Shervani, S. & Hurt, R. H. Controlled release of
biologically active silver from nanosilver surfaces. ACS Nano 4, 6903-6913
(2010).
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[9] Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N.,
Sigg, L. & Behra, R. Toxicity of silver nanoparticles to Chlamydomonas
reinhardtii. Environ. Sci. Technol. 42, 8959-8964 (2008).
[10] Sotiriou, G. A. & Pratsinis, S. E. Antibacterial activity of nanosilver ions and
particles. Environ. Sci. Technol. 44, 5649-5654 (2010).
[11] Morones, J. R., Elechiguerra, J. L., Camacho, A., Holt, K., Kouri, J. B.,
Ramirez, J. T. & Yacaman, M. J. The bactericidal effect of silver
nanoparticles. Nanotechnology 16, 2346-2353 (2005).
[12] Gunawan, C., Teoh, W. Y., Marquis, C. P., Lifia, J. & Amal, R. Reversible
antimicrobial photoswitching in nanosilver. Small 5, 341-344 (2009).
[13] Kittler, S., Greulich, C., Diendorf, J., Koller, M. & Epple, M. Toxicity of
silver nanoparticles increases during storage because of slow dissolution
under release of silver ions. Chem. Mater. 22, 4548-4554 (2010).
[14] Liu, J. Y. & Hurt, R. H. Ion release kinetics and particle persistence in
aqueous nano-silver colloids. Environ. Sci. Technol. 44, 2169-2175 (2010).
[15] Sotiriou, G. A., Teleki, A., Camenzind, A., Krumeich, F., Meyer, A., Panke,
S. & Pratsinis, S. E. Nanosilver on nanostructured silica: Antibacterial
activity and Ag surface area. Chem. Eng. J. 170, 547-554 (2011).
[16] Height, M. J. & Pratsinis, S. E. Antimicrobial and antifungal powders made
by flame spray pyrolysis. Eur. Patent, EP1846327 (A1) (2007).
[17] Loher, S., Schneider, O. D., Maienfisch, T., Bokorny, S. & Stark, W. J.
Micro-organism-triggered release of silver nanoparticles from biodegradable
oxide carriers allows preparation of self-sterilizing polymer surfaces. Small 4,
824-832 (2008).
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[18] Kumar, R. & Munstedt, H. Silver ion release from antimicrobial
polyamide/silver composites. Biomaterials 26, 2081-2088 (2005).
[19] Kumar, A., Vemula, P. K., Ajayan, P. M. & John, G. Silver-nanoparticle-
embedded antimicrobial paints based on vegetable oil. Nature Mater. 7, 236-
241 (2008).
[20] Sotiriou, G. A., Hirt, A. M., Lozach, P. Y., Teleki, A., Krumeich, F. &
Pratsinis, S. E. Hybrid, silica-coated, Janus-like plasmonic-magnetic
nanoparticles. Chem. Mater. 23, 1985-1992 (2011).
[21] Sotiriou, G. A., Sannomiya, T., Teleki, A., Krumeich, F., Vörös, J. &
Pratsinis, S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors.
Adv. Funct. Mater. 20, 4250-4257 (2010).
[22] Lok, C. N., Ho, C. M., Chen, R., He, Q. Y., Yu, W. Y., Sun, H., Tam, P. K.
H., Chiu, J. F. & Che, C. M. Silver nanoparticles: partial oxidation and
antibacterial activities. J. Biol. Inorg. Chem. 12, 527-534 (2007).
[23] Shimizu, K., Miyamoto, Y. & Satsuma, A. Silica-supported silver
nanoparticles with surface oxygen species as a reusable catalyst for alkylation
of arenes. ChemCatChem 2, 84-91 (2010).
[24] Beier, M. J., Schimmoeller, B., Hansen, T. W., Andersen, J. E. T., Pratsinis,
S. E. & Grunwaldt, J. D. Selective side-chain oxidation of alkyl aromatic
compounds catalyzed by cerium modified silver catalysts. J. Molec. Catal. A-
Chem. 331, 40-49 (2010).
[25] Hannemann, S., Grunwaldt, J. D., Krumeich, F., Kappen, P. & Baiker, A.
Electron microscopy and EXAFS studies on oxide-supported gold-silver
nanoparticles prepared by flame spray pyrolysis. Appl. Surf. Sci. 252, 7862-
7873 (2006).
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[26] Height, M. J., Pratsinis, S. E., Mekasuwandumrong, O. & Praserthdam, P.
Ag-ZnO catalysts for UV-photodegradation of methylene blue. Appl. Catal. B-
Environ. 63, 305-312 (2006).
[27] Teleki, A., Suter, M., Kidambi, P. R., Ergeneman, O., Krumeich, F., Nelson,
B. J. & Pratsinis, S. E. Hermetically coated superparamagnetic Fe2O3
particles with SiO2 nanofilms. Chem. Mater. 21, 2094-2100 (2009).
[28] Fragala, M. E., Compagnini, G., Malandrino, G., Spinella, C. & Puglisi, O.
Silver nanoparticles dispersed in polyimide thin film matrix. Eur. Phys. J. D 9,
631-633 (1999).
[29] Carlson, C., Hussain, S. M., Schrand, A. M., Braydich-Stolle, L. K., Hess, K.
L., Jones, R. L. & Schlager, J. J. Unique cellular interaction of silver
nanoparticles: size-dependent generation of reactive oxygen species. J. Phys.
Chem. B 112, 13608-13619 (2008).
[30] Lan, Y., Wang, H., Chen, X., Wang, D., Chen, G. & Ren, Z.
Nanothermometer using single crystal silver nanospheres. Adv. Mater. 21,
4839-4844 (2009).
[31] Schmidt, M., Masson, A. & Bréchignac, C. Oxygen and silver clusters:
Transition from chemisorption to oxidation. Phys. Rev. Lett. 91, 243401
(2003).
[32] Lide, D. R. CRC Handbook of Chemistry and Physics. (CRC Press/Taylor and
Francis, 2010).
[33] Ivanova, O. S. & Zamborini, F. P. Size-dependent electrochemical oxidation
of silver nanoparticles. J. Am. Ceram. Soc. 132, 70-72 (2010).
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CHAPTER 5
5. Non-toxic Dry-coated Nanosilver for
Plasmonic Biosensors1
Abstract
The plasmonic properties of noble metal nanoparticles facilitate their use in
novel in vivo bio-applications such as targeted drug delivery and cancer cell therapy.
Nanosilver is best suited for such applications as it has the lowest plasmonic losses
among all such materials in the UV-visible spectrum. Its toxicity, however, can
destroy the surrounding healthy tissue and thus, hinders its safe employment. Here,
that toxicity against a model biological system (Escherichia coli) is “cured” by coating
nanosilver hermetically with a thin SiO2 layer by a scalable flame aerosol method
without reducing its plasmonic performance. This creates the opportunity to safely
use powerful nanosilver for intracellular bio-applications. The label-free biosensing
and surface bio-functionalization of these ready-to-use, non-toxic (benign)
nanosilver particles is presented here by measuring the adsorption of bovine serum
albumin (BSA) in a model sensing experiment. Furthermore, the silica coating
around nanosilver prevents its agglomeration or flocculation and thus, enhances its
biosensitivity.
1 Part of this chapter is published in Adv. Funct. Mater. 20, 4250-4257 (2010).
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5.1 Introduction
Noble metal (e.g. gold or silver) nanoparticles possess plasmonic properties
that are attractive in novel biological sensing applications [1]. These unique optical
properties originate from collective oscillations of conduction electrons, the so-
called localized surface plasmons [2]. These properties do not degrade over time and
depend on nanoparticle shape and size as well as on the refractive index of their
surroundings [3]. For label-free biosensing, protein molecules which have a higher
refractive index than aqueous solutions cause a red shift of the plasmon absorption
band [2]. The latter dependency can be exploited to detect biomolecules such as
proteins [4,5].
Certain diseases such as bacterial infections or cancer are accompanied by a
higher concentration of specific analytes. Such target analytes are known to bind
specifically to the corresponding capture biomolecules (e.g. antibodies) [2]. Thus, by
anchoring the latter on the surface of plasmonic biosensors (bio-functionalization),
their detection is possible by the local change in the refractive index. In fact, this has
been exploited by multi-step synthesis of plasmonic sensors including rods [6] or
disks [7] that exhibit promising ultra-sensitive biodetection performance, bringing
plasmonic biosensors close to detection limits achieved by other techniques [8].
Moreover, plasmonic nanoparticles strongly scatter and absorb light [9] enabling
their detection under dark field illumination [9]. So they have been used as
intracellular in vivo biomarkers [10,11] and as diagnostic or even therapeutic tools
[12] for targeted drug delivery or cancer cell treatment [13].
Among noble metal nanoparticles, nanosilver is ideal as it has the lowest
plasmonic losses in the UV-visible spectrum [14]. There is, however, concern
regarding toxicity and environmental impact of nanosilver [15] that blocks its use in
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bio-applications. In fact, nanosilver is the first nanomaterial to draw the attention of
the U.S. Environmental Protection Agency (EPA) [16]. Nanosilver is toxic to
biological systems by its direct contact with cells [17] and/or release of toxic Ag+
ions from its surface [18]. Such toxicity remains even after modification of the
nanosilver surface with a biocompatible layer of polysaccharides [19]. If the toxicity
of nanosilver would be controlled and essentially “cured”, new opportunities would
be created in biosensing and bio-imaging [9].
A potent way to achieve this is by applying hermetically a thin, transparent
and inert silica-coating around the nanosilver surface. The role of such silica shell is
triple (Figure 5.1a): a) inhibits the toxicity of nanosilver by preventing the direct
contact of cells with its surface, b) blocks the release of toxic Ag+ ions and c)
facilitates the colloidal dispersion of nanosilver particles that otherwise flocculate
and exhibit limited biosensitivity [3]. Additionally, it facilitates surface
functionalization of nanosilver with bio-molecules since the surface chemistry of
silica is reasonably well understood [20]. Such nanosilver-silica core-shell particles
have been made already by employing silane coupling agents [20], sol-gel [21] and
reverse microemulsion [22]. Silica coating by such wet-methods has been applied
also to quantum dot core nanoparticles resulting in fluorescent materials with
reduced toxicity [23]. Such wet-coated nanosilver, however, retains its toxicity, most
probably because such SiO2 shells tend to be porous [24] enabling toxic Ag+ ion
transport, and thus hindering the use of nanosilver as in vivo biomarker [19].
Here, encapsulation of nanosilver with silica is made in one-step by a dry,
scalable [25] flame aerosol method [26]. Figure 5.1b illustrates the in-flight SiO2-
coating on freshly-formed nanosilver core particles similar to hermetic coating of
photocatalytic [26] TiO2 and superparamagnetic [27] Fe2O3 nanoparticles. The
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influence of this coating on nanosilver toxicity is investigated here against a model
biological system, the Gram-negative bacterium Escherichia coli (E. coli). The effect of
SiO2 coating on the stability of the suspensions and the plasmonic properties of
nanosilver is measured and finally, the feasibility of these core-shell particles as
biosensors is demonstrated in the presence of adsorbed bovine serum albumin (BSA)
which serves as a model protein.
Figure 5.1: (a) Bare nanosilver particles are toxic and tend to flocculate. By applying on them
a hermetic SiO2 coating, both flocculation and toxicity of nanosilver are prevented enabling
synthesis of powerful and non-toxic nanosilver biosensors. (b) Schematic of the enclosed flame
aerosol reactor process for synthesis of hermetically SiO2-coated nanosilver.
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5.2 Materials and methods
5.2.1 Nanosilver particle production and characterization
Nanosilver particle production and in situ SiO2 coating was achieved in a
modified flame aerosol reactor (Figure 5.1b) which is described in detail
elsewhere [26]. In brief, a precursor solution containing Ag-benzoate (0.5 M, Sigma
Aldrich, purity 99%) dissolved in 2-ethylhexanoic acid (Sigma Aldrich, purity ≥99%)
and benzonitrile (Sigma Aldrich, purity ≥ 99%) (volume ratio 1:1) was fed through a
capillary (feed rate 5 mL/min) and dispersed by oxygen (PanGas, purity >99.9%,
flow rate: 5 L/min) and combusted forming the Ag nanoparticles. The freshly-
formed core Ag nanoparticles were coated in-flight by swirl injection of
hexamethyldisiloxane (HMDSO, Sigma Aldrich, purity ≥ 99%) vapor along with
nitrogen (15 L/min, PanGas, purity >99.9%) at room temperature through a
metallic ring with 16 equidistant openings (Figure 5.1b). The ring was placed on top
of a quartz glass tube (20-30 cm long). The reactor was terminated by a quartz glass
tube (25 cm). The HMDSO vapor was supplied by bubbling nitrogen through
approximately liquid HMDSO (350 mL) in a glass flask (max. 500 mL). The SiO2
content in the product particles was calculated at full saturation [26] and by varying
the bubbler temperature (5-7 °C) and nitrogen flow rate (0.05-0.3 L/min). The N2
stream carrying HMDSO vapor to the coating ring outlet is fully saturated with
HMDSO up to 0.8 L/min N2 flow rate through the HMDSO bubbler [26]. The full
conversion of HMDSO to SiO2 has been proven by DC plasma optical emission
spectroscopy for SiO2-coated TiO2 nanoparticles [26], produced with the same
enclosed flame aerosol reactor.
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Figure 5.2: Tuning the nanosilver crystallite size by a) the SiO2 content of the final product
nanosilver particles and b) the injection height of the SiO2 precursor vapor at 30 (circles) 25
(triangles) and 20 cm (squares) above the flame spray burner. Increasing the SiO2-content
prevents Ag-nanoparticle agglomeration and crystal growth. The injection of HMDSO (the
SiO2 precursor) at lower heights cools the flame aerosol and prevents also further Ag crystal
growth [26].
The nanosilver size can be tuned by varying the silica content and/or the
distance between the flame spray burner and the metallic ring from which the SiO2
precursor (HMDSO) is injected. Figure 5.2 shows the nanosilver crystal size as a
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function of SiO2 content, for three different HMDSO injection heights. The error
bars correspond to the standard deviation of 3 measurements and are similar for all
data. Lowering the injection height and/or increasing the silica-content, reduces the
nanosilver size. Additionally, increasing SiO2 content prevents also nanosilver
agglomeration [27]. Pure (0 wt% SiO2) nanosilver particles were sintered on the
filter and their further processing was not possible, as their aggregate size was rather
big (several microns) and they did not exhibit any plasmonic performance.
High resolution transmission electron microscopy (HRTEM) was performed
with a CM30ST microscope or a Tecnai F30 microscope (both: FEI; LaB6 cathode,
operated at 300 kV, point resolution ~2 Å). Product particles were dispersed in
ethanol and deposited onto a perforated carbon foil supported on a copper grid. X-
ray diffraction (XRD) patterns were obtained with a Bruker AXS D8 Advance
spectrometer (Cu Kα, 40 kV, 40 mA). The crystallite size of silver was determined
using the TOPAS 3 software and fitting only the (111) main diffraction peak
(2θ = 36°-40°) with the Inorganic Crystal Database [ICSD Coll. Code.: 064995].
The error bar for each data point was taken as the standard deviation of 3
measurements. The Ag+ ion concentrations were measured electrochemically with
an ion selective electrode and an ion meter (Metrohm). Particles were dispersed in
pure water at a concentration 20 mg/L of Ag.
5.2.2 Toxicity evaluation
A growth inhibition assay was performed to examine the toxicity of the
nanosilver particles. Therefore, E. coli JM101 bacteria synthesizing green fluorescent
protein (GFP) from a plasmid-encoded gene were grown in Luria-Bertani broth (LB)
[28] at 37 °C overnight. The culture was subsequently diluted with LB by mixing
25 L of the culture in 5 mL LB. The nanosilver particle concentration was
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normalized with respect to their surface area, calculated from the crystal size that is
close (within 3%) to number average Ag particle diameter from TEM. All
measurements have an equal Ag surface concentration of ~2.3·10-4 m2/mL
(corresponding to 20 mg/L of Ag of the 1.4 SiO2 wt% sample) The nanosilver
particles were dispersed in de-ionized water by ultrasonication (Sonics vibra-cell) for
20 seconds at 75% amplitude, with a pulse configuration on/off of 0.5s/0.5s, at
200 mg/L of Ag. The corresponding measured concentrations were made by
diluting these batch suspensions. After ultrasonication, the nanoparticles were
homogeneously dispersed and stable in the aqueous suspension. For the assay,
50 μL of the aqueous solutions containing the dispersed nanosilver particles were
added to 50 μL of the diluted cells. The growth of E. coli JM101 was investigated by
monitoring the fluorescent signal of the GFP (Perkin Elmer 1420) corrected for
background fluorescence. The growth percentages were calculated by assuming
100% growth for the control measurements (no silver). The error bars for each data
point were obtained as the standard deviation of 4 measurements.
5.2.3 Biosensor performance
The biosensing of nanosilver particles was assessed using bovine serum
albumin (BSA) as model protein. Oxygen-plasma-cleaned (Harrick Plasma
PDC32G, 18W, 2min) glass slides were coated by poly(L-lysine), PLL, by
incubating the substrate surface with PLL solution (100 mg/L). Nanosilver particles
were then deposited from the aqueous suspension onto the PLL-coated glass
substrate. The positively charged PLL layer immobilizes the negatively charged
nanoparticles by electrostatic force (both silver and silica are negatively charged at
pH 7). The substrate was then set in a flow cell which was mounted on a UV/vis
spectrometer (Cary Varian 500) and the Ag plasmon absorption peak positions were
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monitored during BSA adsorption. The measurement was started in a buffer
solution consisting of 10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid
(HEPES) with a pH adjusted to 7.4 using NaOH and supplemented with 150 mM
NaCl. The BSA solution (100 mg/L in HEPES) was then injected followed by
rinsing with HEPES buffer solution to confirm the adsorption signal. The peak
position was determined by parabolic fitting of the absorption bands. Scanning
electron microscope (SEM) images from the deposited particles on the glass
substrates were taken by a Zeiss Supra 50 VP.
5.3 Results and discussion
5.3.1 Hermetic SiO2 coating: ‘Curing’ nanosilver’s toxicity
Figure 5.3 shows the E. coli growth at 37 °C in the absence (control, stars)
and presence of pure SiO2 (hexagons) as well as various SiO2-coated nanosilver
samples for up to 390 minutes. The error bars correspond to the standard deviation
of 4 measurements and are similar for all data. The E. coli growth in the presence of
SiO2 is identical with the control, therefore SiO2 does not influence the process.
Most importantly, the nanosilver samples coated at higher SiO2-contents, 7.8
(circles) and 9.5 wt% SiO2 (diamonds) do not influence E. coli growth that nearly
overlaps with that in the absence of nanosilver (stars, hexagons). At lower SiO2-
contents (triangles, squares), however, E. coli growth decreases, pointing out the
toxic activity of nanosilver. This indicates that the higher SiO2-content nanosilver is
non-toxic and fully-coated by SiO2 as with similarly coated TiO2 and Fe2O3
nanoparticles [26,27]. In contrast, the lower SiO2-content nanosilver particles are
partially-coated, and thus a fraction of the bare nanosilver surface is exposed,
reducing the E. coli growth. It should be noted that the nanosilver particles
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employed here are rather large and their Ag+ ion release is minimal (<1 %) [29] as
determined electrochemically. Therefore, the observed toxicity against E. coli of the
uncoated nanosilver is mostly attributed to direct contact of the cells to the
nanosilver surface.
Figure 5.3: The E. coli fluorescence monitored at 37 °C in the absence (stars) and presence of
pure SiO2 (hexagons) and nanosilver coated by 1.4 (squares), 1.9 (triangles), 7.8 (circles) and
9.5 wt% SiO2 (diamonds). The SiO2-coating does not inhibit E. coli growth. For a decreasing
SiO2-content, however, significant E. coli growth inhibition is observed indicating the
exposure of nanosilver surface to E. coli. Coating nanosilver, however, with 7.8 and 9.5 wt%
SiO2 blocks its toxicity resulting in nanosilver that can be used as plasmonic biosensor.
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Figure 5.4: TEM images of the 1.4 wt% (a,b) and 7.8 wt% (c,d) SiO2 coated nanosilver.
Patchy coatings of nanosilver with bare surface as well as with very thin (<1 nm), non-
continuous amorphous SiO2-coating are observable (b). The nanosilver core can be
distinguished from its amorphous silica coating (c). The distance of the crystal planes (ca. 2.35
Å) corresponds to the (111) plane of Ag metal (d).
Figure 5.4 shows TEM images of the 1.4 (a,b) and 7.8 wt% (c,d) SiO2-coated
nanosilver. The surface of the low SiO2-content nanosilver particles (Figure 5.4a,b)
is indeed bare or coated with a very thin, non-continuous, amorphous, “patchy”
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SiO2 layer (< 1 nm) [26]. The TEM images of the 7.8 wt% SiO2 sample (Figure
5.4c,d) show that a thin amorphous SiO2 layer surrounds the crystal core nanosilver
particles, which largely prevents the toxic action of the latter and thus may have
“cured” their toxicity. The amorphous SiO2 shell is approximately 2 nm thick, while
crystal planes of the nanosilver core can be distinguished. The distance between
crystal planes (ca. 2.35 Å) corresponds to the (111) plane of Ag metal. Furthermore,
a close observation of high silica-content nanosilver particles shows that these
particles are partially aggregated as the silica coating bridges 5-6 particles there
(Figure 5.4c).
Figure 5.5: Sintering of nanosilver: Its core crystal size as a function of annealing temperature
for 4 hours. Partially-coated nanosilver particles grow above 400 °C while fully-coated ones
(9.5 wt% SiO2) keep their size up to 500 °C.
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The SiO2 coating achieved here hermetically encapsulates the nanosilver core
particles diminishing, if not eliminating, their toxicity and making them safe
biomaterials to be used as diagnostic and therapeutic tools [1,12]. To further
evaluate the extent of coating of nanosilver, its sintering behavior is investigated.
Figure 5.5 shows the average nanosilver core crystal size as a function of annealing
temperature. Initial nanosilver crystal sizes differ as SiO2 on its surface hinders Ag
particle coalescence or sintering as seen with TiO2 or Fe2O3 core nanoparticles [27].
All SiO2-containing nanosilver particles are stable till 400 °C. At 500 °C, however,
the crystal size of partially-coated nanosilver (broken lines) increases substantially,
while for fully-coated nanosilver it remains constant. This indicates that, partially-
coated nanosilver undergoes further sintering during annealing at 500 °C. In
contrast, no sintering or crystal growth takes place with nanosilver fully-coated by a
thin SiO2 layer.
This is further verified by electron microscopy. Figure 5.6 shows TEM
images of the partially-coated 1.4, 1.8 wt% SiO2 (a,b and d,e, respectively) and fully-
coated 9.5 wt% SiO2 (c,f), as-prepared (a,b,c) and after their sintering for 4 hours at
500 °C (d,e,f). Sinter necks cannot be distinguished in all as-prepared, fully- or
partially-coated nanosilver (a,b,c). The fully-coated nanosilver annealed at 500 °C
also does not show significant growth (f), indicating that the SiO2 coating prevents
its crystal or particle growth. In contrast, in the images of the partially-coated
nanosilver after annealing (d,e), large sinter necks appear [30] verifying the crystal
growth of nanosilver particles (note the different magnification in the images d,e). It
should be noted that some slightly larger silver particles in Figure 5.6f might arise
from the few escaping the coating process as has been determined by computational
fluid dynamic analysis of these reactors [31]. These particles probably are
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responsible for the limited antibacterial activity of the 7.8 and 9.5 wt% SiO2-coated
nanosilver that result in slightly lower bacterial growth than that of the control in
Figure 5.3 (stars, hexagons).
Figure 5.6: TEM images of the 1.4, 1.9 and 9.5 wt% SiO2-coated nanosilver as-prepared
(a,b,c) and after their sintering or annealing for 4 hours at 500 °C (d,e,f). Please note the
difference in magnification (d,e). Partially-coated nanosilver with SiO2 (a,b) grows
substantially (d,e). In contrast, fully-coated nanosilver (c) hardly grows upon annealing (f)
consistent with Figure 5.5.
5.3.2 Optical properties of SiO2-coated nanosilver: Agglomeration
Figure 5.7a shows the optical absorption spectra of fully- (9.5 and
7.8 wt% SiO2) and partially-coated (1.9 and 1.4 wt% SiO2) nanosilver films on glass
slides. All spectra have been normalized to the peak of the plasmon absorption band
(~400 nm). Even though all samples have their peak around 400 nm in wavelength,
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the absorption bands of the fully-coated nanosilver are narrower than those of the
partially-coated. This is attributed to agglomeration of nanosilver [3], and can be
visually detected by the color of their aqueous suspensions and films on glass slides
(Figure 5.7a, inset). The fully-coated nanosilver suspensions are yellowish
corresponding to the typical plasmonic color of spherical (~30 nm) Ag colloid
particles (Figure 5.5). The color of the aqueous suspensions becomes redish-brown
and the absorption band broadens as the SiO2 wt% on nanosilver particles decreases.
The SiO2 shell surrounding the nanosilver core particles limits agglomeration
of fully-coated nanosilver. In contrast, the surface of partially-coated nanosilver is
exposed leading to agglomeration or flocculation resulting in broadening of the
plasmon absorption band and change in the color of the suspensions (Figure 5.7a,
inset) [3]. This is further verified by scanning electron microscopy (SEM). Figure 5.7
shows SEM images of b) fully- (9.5 wt% SiO2) and c) partially-coated (1.4 wt% SiO2)
nanosilver films on glass slides. While the aggregates of the fully-coated nanosilver
are rather homogeneously dispersed with a low degree of agglomeration, the
partially-coated nanosilver exhibits much bigger agglomerates or flocs (~1-2 m)
that are highlighted in red circles. These agglomerates were formed within the
aqueous suspensions and not during nanosilver deposition, since the optical
absorption spectra of both aqueous suspensions (Figure 5.7a: lines) and those on
glass slides (Figure 5.7a: symbols) are identical. Large agglomerates or flocs of the
partially-coated nanosilver are undesirable as plasmonic bio-probes for bio-imaging
since they have similar size to the examined cells. In contrast, the fully-coated
nanosilver particles, apart from the reduced toxicity, are also much less
agglomerated requiring no post-treatment. The smaller degree of agglomeration or
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flocculation of such particles facilitates further their employment in bio-imaging
applications.
Figure 5.7: (a) The optical absorption spectra of deposited nanosilver particles on glass slides.
In the inset, pictures of the dispersed nanosilver in aqueous suspensions and on glass slides are
shown. Partially-coated nanosilver particles (1.4 and 1.9 wt% SiO2) form agglomerates which
broaden the plasmon absorption band (broken lines). The SiO2 layer, however, surrounding
the fully-coated nanosilver particles (7.8 and 9.5 wt% SiO2) prevents their agglomeration or
flocculation and narrows their plasmon absorption band (solid lines). This occurs in the
suspensions, since their spectra are identical to the ones from the glass slides (symbols). SEM
images of the (b) fully-coated (9.5 wt%) and (c) partially-coated (1.4 wt% SiO2) nanosilver
verifying the absorption spectra. The partially-coated nanosilver results in bigger
agglomerated or flocculated structures (highlighted in red circles) in suspensions.
5.3.3 Label-free biosensor performance
The performance of plasmonic nanostructures for label-free biosensing is
typically investigated by their response when biomolecules (e.g proteins) are
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adsorbed on their surface [6,7]. Besides label-free biosensing, such surface
functionalization is also an essential process for employment of plasmonic
nanostructures as optical agents in bio-imaging, as most often specific proteins are
attached on the agent’s surface which bind selectively on the desired analyte [9].
Here, the feasibility of the label-free biosensing (also bio-functionalization) of silica-
coated nanosilver biosensors is demonstrated by monitoring their spectral response
on the adsorption of a model protein, bovine serum albumin (BSA), on their surface.
Figure 5.8: (a) The optical absorption spectra before (solid line) and after (broken line)
adsorption of bovine serum albumin (BSA, 100 mg/L). The inset shows the shift of the peak
position which corresponds to the sensor response. (b) The sensor response as a function
of time for the fully-coated (circles, diamonds) and partially-coated (squares, triangles)
nanosilver. The fully-coated nanosilver outperforms the partially-coated one, because of the
less agglomeration of the former (Figure 5.7b,c).
High concentration of BSA was employed to compare the response of
various SiO2-coated nanosilver particles with full BSA coverage. The aqueous
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solution around the deposited nanosilver is controlled through a flow cell and its
optical properties are monitored in situ [5]. Thus, any change in the refractive index
of the surroundings of nanosilver (e.g. by BSA adsorption) is reflected on its
absorbance spectrum with a shift of the peak position of the Ag plasmon
absorption band [2], which is translated as the sensor response. Since BSA has a
higher refractive index than the buffer solution, a red shift of the peak is
expected [2].
Figure 5.8a shows such a response, from ~403 to ~406 nm by the
9.5 wt% SiO2-coated nanosilver biosensor before (solid line) and after (broken line)
injection of 100 mg/L or 1.44 M BSA into the flow cell. In the inset of Figure 5.8a,
the same spectra are magnified at 390 – 420 nm. This shift remains after the rinsing
of the flow cell with buffer solution indicating the presence of a stable physisorbed
layer of BSA on nanosilver.
Figure 5.8b presents the adsorption kinetics of nanosilver coated at various
SiO2 contents (1.4 – 9.5 wt%). The error bars correspond to the standard deviation
of 3 measurements and are similar for all data. As soon as the sensor response
stabilizes in the buffer solution (t = 0 min), BSA is added. When the adsorption of
BSA gives a saturated signal at about t = 20 min, the buffer solution is injected again
for rinsing to exclude the effect of bulk medium change. In the adsorption kinetics,
BSA uptake was observed followed by the signal saturation for all nanosilver-SiO2
contents. The non-toxic, fully-coated biosensors, however, outperform the partially-
coated ones, reaching a sensor response of ~3 nm, which is almost twice as large as
the response of the partially-coated (1.4 wt% SiO2) nanosilver. This is attributed to
the reduced effective surface area of partially-coated nanosilver by flocculation or
agglomeration, as seen in Figure 5.7a by the broader plasmon absorption band and
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the images in Figure 5.7b,c. This facilitates higher BSA adsorption on the nanosilver
surface. Therefore, the SiO2 coating, apart from “curing” nanosilver toxicity, also
enhances its biosensor performance by preventing agglomeration or flocculation of
nanosilver and facilitating its use in biosensing.
Apart from the obvious advantages of these particles as agents for bio-
imaging, they can also be used as label-free biosensors, although the sensor response
is lower than lithographically fabricated nanostructures [32]. Even though the
present measurement system is not yet optimized, the wavelength shift as well as the
peak sharpness is similar to the plasmonic gold nanostructures made by multiple-
step nanofabrication techniques [6,7] (e.g. ~3 nm for 100 mg/L BSA [7]). While the
advantage of lithography lies in the possibility to control the geometry, such
nanofabrication and the use of gold are typically quite costly. Employment of silver
as plasmonic material is practically more desired in terms of the fabrication cost and
the optical loss, provided that silver can be chemically stabilized with proper coating
by a simple fabrication method. The flame aerosol technology presented here
allowed one-step synthesis of nanosilver for plasmonic biosensors having
comparable response to the biosensors made by elaborate nanofabrication processes.
5.4 Conclusions
The toxicity of nanosilver can be effectively eliminated by a thin, hermetic
coating on its surface. This results in a safe biomaterial ready to be employed as
diagnostic and/or therapeutic agent, for example, in targeted cancer cell treatment
[12], without inducing any damage to the surrounding healthy tissue. The hermetic
SiO2 layer prevents the direct contact of cells with the nanosilver surface and blocks
the release of toxic Ag+ ions. This was demonstrated here by one-step, in situ
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synthesis of SiO2-coated nanosilver by a dry scalable flame aerosol technology
resulting in core-shell nanostructures.
For the first time, hermetically SiO2-coated nanosilver particles exhibited
limited, if any, toxicity, while partially-coated ones partly inhibited E. coli bacterial
growth indicating their toxicity. Partially-coated nanosilver particles formed large
agglomerates or flocs when dispersed in aqueous suspensions while the SiO2 layer
on the fully-coated nanosilver largely prevented its agglomeration or flocculation.
The feasibility of the surface bio-functionalization and label-free biosensing of these
silica-coated nanosilver was shown here by monitoring the shift of its plasmon
absorption band in the presence of bovine serum albumin (BSA) on the SiO2-coated
nanosilver surface.
The prevention of nanosilver agglomeration resulted in increased surface
area for BSA adsorption, since the sensor response of the fully-coated nanosilver
was higher than that of partially-coated or uncoated ones. The observed sensor
response was comparable to that obtained by plasmonic biosensors manufactured by
multiple-step nanofabrication techniques. The fast, one-step silica encapsulation of
nanosilver here resulted in non-toxic plasmonic biosensors easy to disperse in
aqueous suspensions and to bio-functionalize, enabling their use in bio-imaging.
5.5 References
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[2] Willets, K. A. & Van Duyne, R. P. Localized surface plasmon resonance
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[3] Evanoff, D. D. & Chumanov, G. Synthesis and optical properties of silver
nanoparticles and arrays. ChemPhysChem 6, 1221-1231 (2005).
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optical sensors with zeptomole sensitivity. Nano Lett. 3, 1057-1062 (2003).
[5] Sannomiya, T., Hafner, C. & Voros, J. In situ sensing of single binding events
by localized surface plasmon resonance. Nano Lett. 8, 3450-3455 (2008).
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Atkinson, R., Pollard, R., Podolskiy, V. A. & Zayats, A. V. Plasmonic
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an extremely low limit-of-detection for bacterial and cancer diagnostics.
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of live cells using gold plasmonic nanoparticles. Laser & Photonics Rev. 3, 146-
158 (2009).
[10] Lee, K. J., Nallathamby, P. D., Browning, L. M., Osgood, C. J. & Xu, X. H.
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[11] Kumar, S., Harrison, N., Richards-Kortum, R. & Sokolov, K. Plasmonic
nanosensors for imaging intracellular biomarkers in live cells. Nano Lett. 7,
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[12] Jain, P. K., Huang, X., El-Sayed, I. H. & El-Sayad, M. A. Review of some
interesting surface plasmon resonance-enhanced properties of noble metal
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(2007).
[13] Loo, C., Lowery, A., Halas, N., West, J. & Drezek, R. Immunotargeted
nanoshells for integrated cancer imaging and therapy. Nano Lett. 5, 709-711
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[14] Barnes, W. L., Dereux, A. & Ebbesen, T. W. Surface plasmon subwavelength
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[15] Wijnhoven, S. W. P., Peijnenburg, W. J. G. M., Herberts, C. A., Hagens, W.
I., Oomen, A. G., Heugens, E. H. W., Roszek, B., Bisschops, J., Gosens, I.,
Van De Meent, D., Dekkers, S., De Jong, W. H., van Zijverden, M., Sips, A.
n. J. A. M. & Geertsma, R. E. Nano-silver - a review of available data and
knowledge gaps in human and environmental risk assessment. Nanotoxicology
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[16] Erickson, B. E. Nanosilver pesticides. Chem. Eng. News 87 (48), 25-26 (2009).
[17] Morones, J. R., Elechiguerra, J. L., Camacho, A., Holt, K., Kouri, J. B.,
Ramirez, J. T. & Yacaman, M. J. The bactericidal effect of silver
nanoparticles. Nanotechnology 16, 2346-2353 (2005).
[18] Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N.,
Sigg, L. & Behra, R. Toxicity of silver nanoparticles to Chlamydomonas
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[19] Schrand, A. M., Braydich-Stolle, L. K., Schlager, J. J., Dai, L. M. &
Hussain, S. M. Can silver nanoparticles be useful as potential biological
labels? Nanotechnology 19, 235104-235117 (2008).
[20] Liz-Marzan, L. M., Giersig, M. & Mulvaney, P. Synthesis of nanosized gold-
silica core-shell particles. Langmuir 12, 4329-4335 (1996).
[21] Kobayashi, Y., Katakami, H., Mine, E., Nagao, D., Konno, M. & Liz-
Marzan, L. M. Silica coating of silver nanoparticles using a modified Stober
method. J. Colloid Interface Sci. 283, 392-396 (2005).
[22] Han, Y., Jiang, J., Lee, S. S. & Ying, J. Y. Reverse microemulsion-mediated
synthesis of silica-coated gold and silver nanoparticles. Langmuir 24, 5842-
5848 (2008).
[23] Selvan, S. T., Tan, T. T. & Ying, J. Y. Robust, non-cytotoxic, silica-coated
CdSe quantum dots with efficient photoluminescence. Adv. Mater. 17, 1620-
1625 (2005).
[24] Xu, K., Wang, J. X., Kang, X. L. & Chen, J. F. Fabrication of antibacterial
monodispersed Ag-SiO2 core-shell nanoparticles with high concentration.
Mater. Lett. 63, 31-33 (2009).
[25] Mueller, R., Madler, L. & Pratsinis, S. E. Nanoparticle synthesis at high
production rates by flame spray pyrolysis. Chem. Eng. Sci. 58, 1969-1976
(2003).
[26] Teleki, A., Heine, M. C., Krumeich, F., Akhtar, M. K. & Pratsinis, S. E. In
situ coating of flame-made TiO2 particles with nanothin SiO2 films. Langmuir
24, 12553-12558 (2008).
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[27] Teleki, A., Suter, M., Kidambi, P. R., Ergeneman, O., Krumeich, F., Nelson,
B. J. & Pratsinis, S. E. Hermetically coated superparamagnetic Fe2O3
particles with SiO2 nanofilms. Chem. Mater. 21, 2094-2100 (2009).
[28] Sambrook, J. & Russell, D. W. Molecular Cloning: A Laboratory Manual. (NY
Cold Spring Harbor Laboratory Press, 2001).
[29] Sotiriou, G. A. & Pratsinis, S. E. Antibacterial activity of nanosilver ions and
particles. Environ. Sci. Technol. 44, 5649-5654 (2010).
[30] Teleki, A., Wengeler, R., Wengeler, L., Nirschl, H. & Pratsinis, S. E.
Distinguishing between aggregates and agglomerates of flame-made TiO2 by
high-pressure dispersion. Powd. Technol. 181, 292-300 (2008).
[31] Teleki, A., Buesser, B., Heine, M. C., Krumeich, F., Akhtar, M. K. &
Pratsinis, S. E. Role of gas-aerosol mixing during in situ coating of flame-
made titania particles. Ind. Eng. Chem. Res. 48, 85-92 (2009).
[32] Haes, A. J., Chang, L., Klein, W. L. & Van Duyne, R. P. Detection of a
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CHAPTER 6
6. Hybrid, Silica-coated, Janus-like
Plasmonic-Magnetic Nanoparticles1
Abstract
Hybrid plasmonic-magnetic nanoparticles possess properties that are
attractive in bioimaging, targeted drug delivery, in vivo diagnosis and therapy. The
stability and toxicity, however, of such nanoparticles challenge their safe use today.
Here, biocompatible, SiO2-coated, Janus-like Ag/Fe2O3 nanoparticles are prepared
by one-step, scalable flame aerosol technology. A nanothin SiO2 shell around these
multifunctional nanoparticles leaves intact their morphology, magnetic and
plasmonic properties but minimizes the release of toxic Ag+ ions from the nanosilver
surface and its direct contact with live cells. Furthermore, this silica shell hinders
flocculation and allows for easy dispersion of such nanoparticles in aqueous and
biological buffer (PBS) solutions. As a result, these hybrid particles exhibited no
cytotoxicity during bioimaging and remained stable in suspension with no signs of
agglomeration and sedimentation or settling. Their performance as biomarkers was
explored by selectively binding them with live tagged Raji and HeLa cells enabling
their detection under dark-field illumination. Therefore, these SiO2-coated Ag/Fe2O3
nanoparticles do not exhibit the limiting physical properties of each individual
component but retain their desired functionalities facilitating thus, the safe use of
such hybrid nanoparticles in bio-applications.
1 Part of this chapter is published in Chem. Mater. 23, 1985-1992 (2011).
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6.1 Introduction
Plasmonic (Au or Ag) nanoparticles are superior markers for cell monitoring
in bioimaging, diagnosis and therapy [1]. Such nanoparticles can be readily detected
and traced by optical techniques such as light scattering [2], dark-field illumination
[3], two-photon fluorescence imaging [4] and photon illumination confocal
microscopy [5]. Alternatives to plasmonic nanoparticles for bioimaging are the
commonly used fluorescent organic dyes and semiconducting nanoparticles [6]. The
former, however, exhibit the so-called photobleaching and degrade during
bioimaging [7]. The latter, on the other hand, may exhibit optical blinking [8] while
concerns arise for their toxicity as most contain cadmium or lead [6]. Even though
plasmonic nanoparticles also induce toxicity [9], they are functionally advantageous
over fluorescent organic dyes and semiconducting nanoparticles because they have
superior photostability [10] and can be used also as photothermal therapeutic agents
(e.g. tumor treatment) [5], offering an extra functionality in bio-applications.
When plasmonic nanoparticles are combined with another material, e.g. a
magnetic component, multifunctional nanostructured materials [1] are created, that
can be detected and guided by multiple imaging and control [11] techniques.
Magnetic resonance imaging (MRI) is an example of a traditional technique, with
which magnetic particles can be used as contrast agents [12] and for targeted drug
delivery by directing them to organs, tissues or tumors using an external magnetic
field or for magnetically-assisted cell sorting and separation [11]. Furthermore, a
SiO2 film on the surface of such magnetic nanoparticles facilitates their surface bio-
functionalization and minimizes their magnetic interactions [13] and flocculation
[14] or agglomeration.
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Composite plasmonic-magnetic nanostructures are typically made by multi-
step wet methods. Depending on synthesis route, core/shell [15] or heterodimer
(Janus-like) [4] plasmonic-magnetic materials are formed. These “as-prepared”
nanoparticles are often hydrophobic requiring a surface modification to stably
suspend [4] them in aqueous solutions. Typically, the magnetic material is iron
oxide (-Fe2O3 or Fe3O4) for its high spontaneous magnetization [11] in a
superparamagnetic state. Live macrophage cells bound on labeled Ag/Fe3O4
nanoparticles have been imaged and manipulated [4] by an external magnetic field.
There are limitations, however, that hinder the use of such materials in bio-
applications. First and foremost, their toxicity needs to be addressed before they can
be employed [16]. Even though Ag has the lowest optical plasmonic losses in the
UV-visible spectrum [17], the more expensive Au nanoparticles are preferred for
bioimaging because of their lower cytotoxicity [9]. The release of toxic Ag+ ions [18]
when Ag nanoparticles are dispersed in aqueous solutions blocks their safe use [19]
in bio-applications. By coating Ag nanoparticles, however, with a nanothin shell,
this toxicity can be eliminated while their surface bio-functionalization can be
enhanced by hindering also their flocculation [14] that poses another limitation in
the use of plasmonic-magnetic nanomaterials [11,20] when dispersed in aqueous
solutions. Overcoming these particle-particle interactions including magnetic ones
requires typically additional surface modification of these particles [1].
Here, one-step synthesis of hybrid, silica-coated, plasmonic-magnetic
nanomaterials is explored by scalable [21] flame aerosol technology. The
morphology of these composite nanoparticles and the influence of their SiO2 shell
on the release of Ag+ ions (Ag leaching) is investigated along with their cytotoxicity
against HeLa cells and biocompatibility. The magnetic hysteresis of these
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nanoparticles is examined and the effect of SiO2-coating on their stability in aqueous
and phosphate buffer saline (PBS) solutions is explored. The plasmonic properties of
these hybrid nanoparticles are investigated and their magnetic guiding is
demonstrated. Finally, the feasibility of these multifunctional materials in
bioimaging is studied by selectively labeling live Raji and HeLa cells and monitoring
them under dark-field illumination.
6.2 Materials and methods
6.2.1 Hybrid SiO2-coated Ag/Fe2O3 nanoparticle synthesis
Silica-coated Ag/Fe2O3 particles were made in one-step with an enclosed
flame aerosol reactor, described in detail elsewhere [14]. In brief, the composite core
Ag/Fe2O3 nanoparticles were made by flame spray pyrolysis (FSP) of precursor
solutions containing iron (III) acetylacetonate (Sigma Aldrich, purity ≥ 97%) and
silver acetate (Sigma Aldrich, purity ≥ 99%) dissolved in 2-ethylhexanoic acid and
acetonitrile (both Sigma Aldrich, purity ≥ 97%, volume ratio 1:1, stirring 100 °C for
30 minutes). The precursor solutions were fed at 5 mL/min to the FSP reactor and
dispersed by 5 L/min O2 (all gases Pan Gas, purity >99%) forming a spray (pressure
drop = 1.5 bar at the nozzle tip) that was ignited by a ring-shaped, premixed
methane/oxygen flame (1.5/3.2 L/min) and sheathed by 40 L/min O2. The Fe
precursor concentration was kept constant at 0.5 M, while corresponding amounts
of silver acetate were added to reach the nominal Ag wt%, which was defined as
x = mAg/(mAg + mFe2O3), and labeled as xAg/Fe2O3.
The freshly-formed composite xAg/Fe2O3 particles were coated in-flight by
swirl injection of hexamethyldisiloxane (HMDSO, Sigma Aldrich, purity ≥ 99%)
vapor with 15 L/min nitrogen (PanGas, purity > 99.9%) at room temperature
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through a metallic ring with 16 equidistant openings. The ring was placed on top of
a 20 cm long quartz glass tube followed by another 30 cm long such tube. The
HMDSO vapor was supplied by bubbling nitrogen through approximately 350 mL
liquid HMDSO in a 500 mL glass flask. The SiO2 amount was kept constant in the
product particles and was calculated at saturation conditions [22] (bubbler
temperature 10 °C and 0.5 L/min N2) corresponding to 23 wt% for pure Fe2O3 core
particles [13] (SiO2 wt% = mSiO2/(mSiO2 + mFe2O3)). Silica-coated pure core Ag or
Fe2O3 particles were made at identical conditions in the absence, however, of the
corresponding precursor (Fe or Ag precursor, respectively).
6.2.2 Particle characterization
High resolution transmission electron microscopy (HRTEM) and scanning
transmission electron microscopy (STEM) was performed on a Tecnai F30 (FEI;
field emission gun, operated at 300 kV). The STEM images were recorded with a
high-angle annular dark field (HAADF) detector revealing the Ag particles with
bright Z contrast. Product particles were dispersed in ethanol and deposited onto a
perforated carbon foil supported on a copper grid. X-ray diffraction (XRD) patterns
were obtained [13,14] with a Bruker AXS D8 Advance spectrometer (Cu Kα, 40 kV,
40 mA). The crystallite size of silver and iron oxide was determined using the
TOPAS 3 software and fitting only the main diffraction peaks.
The release of Ag+ ions was measured by monitoring the Ag+ ion
concentration of aqueous suspensions containing the composite particles [23].
Particles were dispersed by ultrasonication (Sonics vibra-cell, 5 minutes at 75%
amplitude with a pulse configuration on/off of 0.2s/0.2s) in de-ionized water (Milli-
Q) and their Ag+ ion concentration was monitored with an ion selective electrode
and an ion meter (Metrohm, 867 module) [18]. The measurements were calibrated
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using silver containing aqueous solution (silver standard, Aldrich) with a calibration
slope of 59.3 mV/log [Ag+]. Diffusive gradients in thin film (DGT) measurements
were performed and the Ag concentration was measured with an ICP-MS [18]. The
optical properties of the composite particles were monitored with UV/vis
spectroscopy (Cary Varian 500) of their aqueous suspensions. Particles were
dispersed in de-ionized water and in phosphate PBS solution by ultrasonication
(Sonics vibra cell, 5 minutes, power 50%, pulse on/off 0.2 s/0.2 s). Size
distributions of particles in aqueous and PBS solutions and -potential
measurements of aqueous suspensions [24] were obtained by dynamic light
scattering (DLS, Zeta-sizer, Malvern Instruments). Magnetic measurements were
made on a Princeton Measurements Corporation vibrating sample magnetometer
(VSM).
6.2.3 Biocompatibility of the hybrid biomarkers
The cytotoxicity of uncoated and SiO2-coated 35Ag/Fe2O3 nanoparticles was
investigated with the human cell line HeLa. The cell viability was monitored by
using the LIVE/DEAD fixable dead cell stain kit (Invitrogen) and flow cytometry
(~5’000 cells counted) [25]. Suspensions of nanoparticles in PBS were prepared by
ultrasonication as above and added on HeLa cells (2·105 cells at a volume ratio of
1:1). The following three incubation conditions were examined: with composite
Ag/Fe2O3 particle concentration of 5 mg/L (Ag concentration of 1.45 mg/L) i) at
4 °C for 1 hour (identical to the conditions during particle binding with cells for
bioimaging) and at Ag concentrations of 5, 2.5, 1.25 and 0.625 mg/L incubated at
37 °C for ii) 1 and iii) 24 hours. As a negative control to assess viability, pure PBS
was added instead of the particle suspension. Staurosporin (20 μM, Sigma) was used
as a positive control. This drug induces strong cytotoxicity and consequently
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apoptosis, leading to cell death. Error bars correspond to the standard deviation of
three measurements.
6.2.4 Bioimaging
For bioimaging, 5 mg/L of SiO2-coated 35Ag/Fe2O3 nanoparticles were
dispersed by ultrasonication as above in sterile PBS. Purified mouse monoclonal
antibody against hDC-SIGN (MAB1621, R&D Systems) was reconstituted in water
with a concentration 1 mg/mL. Then 100 L of this antibody-containing solution
were added in 5 mL of the particle suspension for surface biofunctionalization. After
its incubation for 1 hour at room temperature, the bio-functionalized particles were
washed with PBS three times. HeLa and Raji cells expressing or not the protein
hDC-SIGN were washed three times with PBS. The biofunctionalized particles were
added to cells (2x105 and 5x105 cells for HeLa and Raji cells respectively) in a 1:1
ratio (v/v) reaching a final volume of 2 mL and then incubated for 1 hour at 4 °C.
As controls, cells which do not express hDC-SIGN were incubated [26] at 4 °C in
the presence or absence of biofunctionalized particles. The optical detection was
performed with a microscope equipped with a dark-field condenser (Olympus MM)
and a black-and-white CCD camera. The biofunctionalization was verified by
monitoring the shift of the plasmon absorption band of the nanosilver [14] with a
UV/vis spectrometer (Varian Cary 500).
6.3 Results and discussion
6.3.1 Morphology
Uncoated and SiO2-coated xAg/Fe2O3 nanoparticles were made with varying
Ag-content x (wt%). Figure 6.1a shows an STEM image of the uncoated 50Ag/Fe2O3.
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Figure 6.1: A HAADF-STEM (Z contrast) image of the uncoated 50Ag/Fe2O3 sample (a) and
TEM images of the SiO2-coated 10Ag/Fe2O3 (b) and the SiO2-coated 35Ag/Fe2O3 sample (c).
As insets, a schematic drawing of the uncoated and SiO2-coated particles is presented, for
which the red, gray and blue colors correspond to iron oxide, silver and silica particles,
respectively.
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There are Ag nanoparticles (bright spots) attached onto Fe2O3 particles (gray)
forming dumbbell- or Janus-like particles. Figure 6.1b shows a TEM image of SiO2-
coated 10Ag/Fe2O3 nanoparticles where Ag particles (dark spots) are attached on the
Fe2O3 particles (gray) as in Figure 6.1a, but there is an amorphous (light gray) SiO2
shell or film encapsulating the core Ag/Fe2O3 particles. Figure 6.1c shows this more
clearly with an image of a SiO2-coated 35Ag/Fe2O3 nanoparticle at higher
magnification. The iron oxide and the silver particles are completely coated by a
smooth, amorphous SiO2 shell or film of a couple nm thickness [13]. All STEM and
TEM images indicate that there is no significant trace of individual or separate Ag
or Fe2O3 particles, a pre-requisite for their bio-application. The average crystal size
of the -Fe2O3 is approximately 15 nm and independent on the presence of Ag or
SiO2, even though there is a small presence of -Fe2O3 within the samples [13]. The
-Fe2O3 content was slightly increased at Ag-content x = 50 wt%. This could be
attributed to the higher combustion enthalpy of the employed precursor solutions for
the largest Ag-content, and thus higher temperatures within the enclosed flame
aerosol reactor [13]. The average Ag crystal size increases with increasing Ag-
content [23] (from ~10 nm for the 20Ag/Fe2O3 to ~20 nm for the 50Ag/Fe2O3) and
was also rather independent from the presence of the SiO2 shell.
6.3.2 Magnetic and plasmonic performance
Figure 6.2 shows the magnetization of all SiO2-coated particles normalized to
their Fe2O3 mass at various Ag-contents x at room temperature. All particles show
some hysteresis, which indicates that are magnetically blocked during the
measurement. The coercive force, however, is very low (inset of Figure 6.2) while
the SiO2-coating does not influence [13] their magnetic properties. The highest
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magnetization (Ms = 39.4-46.1 emu/g) of the lower Ag-content particles (x = 6-
35 wt%) is similar to pure Fe2O3 core (38.4 emu/g). These Ms values are lower than
that of bulk -Fe2O3 (63.6 emu/g) [27], as smaller crystallites [13] are employed here
that contain [27] -Fe2O3.
Figure 6.2: Magnetization curves of the SiO2-coated xAg/Fe2O3 samples. The magnetization
values have been normalized for an equal mass of Fe2O3. The inset shows a magnification at
low magnetic fields highlighting the coercivity and remanence of the particles.
Figure 6.3a shows the UV/vis absorbance of the SiO2-coated hybrid particles
for x = 10 (purple), 20 (yellow) 35 (green) and 50 wt% (blue) here in aqueous
suspensions before (B = 0, solid lines) and after application (B >0, broken lines) of
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an external magnetic field by a permanent Ni-Cu-Ni magnet (inset). At B = 0 for the
10Ag/Fe2O3 (purple line) the Ag-content is rather low and the Ag-metal plasmon
absorption band is not distinguishable as the spectrum is dominated by the Fe2O3
absorption. However, for an increasing Ag-content x > 10 wt% the Ag plasmon band
clearly emerges [20] at ~400 nm. This indicates that the optical properties of Ag
nanoparticles are not influenced [28] by the presence of Fe2O3 nor SiO2. At B > 0,
however, all absorption spectra (from Fe2O3 and Ag) disappeared completely and
the suspension is transparent, since all particles have been attracted to the cuvette
bottom (Figure 6.3a, inset) by the magnet. This further verifies that there are no
individual Ag nanoparticles present in the as-prepared sample, but only composite
Ag/Fe2O3 (Figure 6.1) that were moved by a magnetic field. Therefore, these hybrid
nanoparticles could be employed in bio-applications where both magnetic
manipulation and optical monitoring are desired.
As a control experiment, separate pure nanosilver [14] and iron oxide [13]
nanoparticles, both SiO2-coated were made by FSP and mechanically mixed (for Ag-
contents x = 35 and 50 wt%) in water as in Figure 6.3a. These suspensions also
exhibit the plasmon absorption band of Ag nanoparticles (Figure 6.3b). However,
after applying an external magnetic field (B > 0) the plasmon band is still
distinguishable at 400 nm (broken lines) as only the SiO2-coated Fe2O3 particles
were attracted to the cuvette bottom by the magnet. The reduced intensity of the
plasmon absorption band at B > 0 is attributed to the absence of absorption by the
Fe2O3 particles. The SiO2-coated pure Ag particles, however, remained dispersed in
solution (Figure 6.3b, inset, B > 0) exhibiting the characteristic yellowish color that
is attributed to that nanosilver size [14] and concentration.
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Figure 6.3: (a) Optical absorption spectra of the SiO2-coated xAg/Fe2O3 before (solid lines)
and after their removal with the application of an external magnetic field (broken lines). In
the inset, a photograph of the aqueous suspensions before and after the magnetic field of the
SiO2-coated 35Ag/Fe2O3 sample. (b) Optical absorption spectra of mechanically mixed SiO2-
coated Ag and Fe2O3 nanoparticles, before (solid lines) and after (broken lines) the application
of an external magnetic field.
6.3.3 Stability in aqueous suspensions and buffer solutions
When multifunctional (hybrid) nanoparticles are employed in bioimaging,
they have to be stable in solution. Figure 6.4 shows the hydrodynamic size
distributions of the SiO2-coated (a) and uncoated (b) 35Ag/Fe2O3 particles in water
(solid lines) and in PBS (broken lines) immediately after their dispersion (t = 0
hours). The SiO2-coated particles (a) have quite a low degree of agglomeration in
both water and PBS (dp = 50-500 nm). The uncoated samples (b), however, have a
bimodal distribution and much larger agglomerate diameters (500-7’000 nm),
reaching the limit of the particle size measurement. That is attributed to the absence
of SiO2 which acts as an anti-flocculation agent [14], as the SiO2-coated particles are
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stable when dispersed in water and PBS (Figure 6.4c). In contrast, the formation of
the larger agglomerates of uncoated particles results in their sedimentation within
both aqueous and PBS suspensions (after 2 hours) as seen in Figure 6.4d.
This stability is especially desired for bio-applications otherwise the resulting
agglomerates (flocs) would be similarly sized to the target biological systems (e.g.
Raji or HeLa cells) prohibiting their use for cell imaging. Additionally, these silica-
coated particles are hydrophilic [22] as-prepared and therefore readily dispersible
without requiring an extra functionalization step. The exhibited stability of the SiO2-
coated hybrid nanoparticles is further verified by -potential measurements in water.
For all Ag-contents (x = 0 – 50 wt%) the -potential of the SiO2-coated particles
varies from -42 to -48 mV, in agreement with other SiO2-coated nanoparticles made
by flame- [24] or wet-chemistry [29]. For the uncoated Ag/Fe2O3 particles, the -
potential lies from -0.5 to -5.0 mV, also in agreement with those of wet-made Fe2O3
[30] or Ag [31] nanoparticles.
6.3.4 Biocompatibility of SiO2-coated hybrid plasmonic-magnetic biomarkers
To evaluate the effect of the SiO2 shell or film on the core Ag/Fe2O3 particles
on the Ag+ ion release (leaching), the Ag+ ion concentration, [Ag+] (Figure 6.5a),
was monitored in aqueous suspensions of silica-coated and uncoated Ag/Fe2O3 by
ion selective electrode (ISE, open symbols) [23] and diffusive gradients in thin film
(DGT, filled symbols) [18] measurements. Figure 6.5a shows this [Ag+] as a
function of Ag-content x (in the xAg/Fe2O3 particles) for the uncoated (triangles) and
SiO2-coated (circles) nanoparticles, for an equal Ag mass concentration of 50 mg/L.
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Figure 6.4: Agglomerate SiO2-coated (a) and uncoated (b) 35Ag/Fe2O3 particle size
distributions as determined by dynamic light scattering (hydrodynamic diameter) in water
(solid lines) and in phosphate buffer saline (PBS) solution (broken lines) immediately after
their dispersion (t = 0 hours). Images of dispersion of the SiO2-coated (c) and uncoated
35Ag/Fe2O3 (d) nanoparticles immediately after their dispersion (t = 0 hours) and after 2
hours in water (top) and PBS (bottom). The hybrid SiO2-coated Ag/Fe2O3 nanoparticles
remain stable while the uncoated ones flocculate (agglomerate) and settle after a few hours.
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The [Ag+] was attained immediately upon dispersion and was constant over a
period of, at least, 24 hours [23]. The value obtained when de-ionized water was
measured by ISE are also shown (gray broken line). There was a good agreement
between ISE and DGT for the uncoated 35Ag/Fe2O3: the silver mass dissolved as
silver ions was 7 ± 1.7 % by ISE and 8.6 ± 0.5% by DGT. For the SiO2-coated there
was a small difference, the silver dissolved fraction was 2.8 ± 1.4% by ISE, and 5.4
± 0.5% by DGT.
Uncoated particles release more Ag+ ions for lower Ag-content xAg/Fe2O3
nanoparticles [23] that contain smaller Ag nanoparticles. As the Ag-content x
increases, Ag nanoparticles grow bigger releasing less Ag+ ions [23] from their
surface. Silica-coated xAg/Fe2O3 release much less [Ag+] especially for the fine Ag
nanoparticles (dp < 20 nm at x = 6-50 wt%). As the Ag-content x increases, a
minimal Ag+ ion release for x = 50 wt% occurs, which is comparable to the SiO2-
coated Ag/Fe2O3 nanoparticles. Nevertheless, the uncoated 50Ag/Fe2O3 particles
have the lowest magnetization and poor stability in aqueous suspensions (Figure
6.4d). For the lower Ag-content x (6-35 wt%), however, the inert silica shell
minimizes nanosilver leaching significantly, but not completely. Few Janus-like
particles have been partially coated by incomplete mixing [32,33] of HMDSO vapor
with the core Ag/Fe2O3 nanoparticles.
In order to assess the cytotoxicity and biocompatibility of these particles as
biomarkers, they were incubated for 24 hours at 37 °C with HeLa cells. Figure 6.5b
shows the fraction of dead HeLa cells for different biomarker particle concentrations
(5, 2.5, 1.25, 0.625 mg/L of Ag), as well as for the positive and negative controls. In
the absence of toxic agents (negative control), there is no significant fraction of
apoptotic cells (only the standard, ~10%) [34].
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Figure 6.5: (a) The Ag+ ion concentration of suspensions containing the dispersed uncoated
(red triangles) and SiO2-coated (blue circles) Ag/Fe2O3 particles as determined from ISE (open
symbols) and DGT (filled symbols). The ISE values of the de-ionized water are also shown
(gray broken line). (b) The cytotoxicity evaluation of the uncoated and SiO2-coated
35Ag/Fe2O3 biomarker incubated at 37 °C for 24 hours at various concentrations. The
positive (toxic drug inducing apoptosis, Staurosporin) and negative controls are also shown.
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The SiO2-coated nanoparticles did not induce any toxicity for this incubation
period (24 hours), further indicating their biocompatibility with HeLa cells. The
toxicity evaluation of the uncoated plasmonic-magnetic nanoparticles was not
possible, as these particles formed large agglomerates and settled quite fast (Figure
6.4d), thus not giving reliable toxicity results. This emphasizes the limitations of
toxicological tests of agglomerated nanoparticles, as their sedimentation inhibits
their correct evaluation. It should be noted that for incubation conditions of 1 hour
at 4 °C and 37 °C with the SiO2-coated Ag/Fe2O3 nanoparticles similar results were
obtained and no apoptotic cells were detected.
6.3.5 Bioimaging
The potential of these multifunctional nanoparticles as biomarkers is
explored by selectively binding them on tagged living cells. So the surface of SiO2-
coated 35Ag/Fe2O3 nanoparticles was labeled (biofunctionalized) with an antibody
against the human form of DC-SIGN (hDC-SIGN). Figure 6.6a shows a shift
= ~10 nm of the Ag-metal normalized plasmon absorption band of these
particles after antibody adsorption [14,20] on their surface. The higher refractive
index of the antibody over that of PBS causes a red shift [14,20] of the Ag plasmon
absorption band. This shift remains even after washing the nanoparticles with PBS,
excluding thus the effect of medium change.
Figure 6.6b shows a dark-field image of Raji cells that do not express hDC-
SIGN on their surface (untagged Raji cells) in the absence of labeled particles. The
characteristic scattering from the cell membrane as well as from intracellular
components can be barely observed [3]. With the exception of very few residual
scatterings, probably originating from unwashed labeled particles, identical results
were obtained when such cells were incubated with labeled particles (Figure 6.6c).
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This suggests that the SiO2-coated 35Ag/Fe2O3 particles labeled with the antibody
against hDC-SIGN do not unspecifically bind to the cell surface.
Figure 6.6: (a) The plasmon band of SiO2‐coated 35Ag/Fe2O3 particles before (solid red line)
and after (broken green line) their antibody labeling. Dark‐field images of the Raji cells that
do not express the tag (untagged) and (b) with labeled particles (c), Raji cells that express the
tag (tagged) with labeled particles (d) are shown. Similarly, dark-field images of HeLa cells
that express the tag (e) and incubated with the labeled biomarkers (f) show significant
differences enabling their readily detection.
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In contrast, when Raji cells, that express hDC-SIGN on their surface (tagged
cells), are incubated with the labeled particles, it can be seen that the surface of the
cells is completely covered by the present hybrid particles (Figure 6.6d) that strongly
scatter light and appear very bright (Ag) under dark-field-illumination [3]. This
indicates that by labeling the surface of these nanoparticles with an antibody, they
can specifically bind to cells that express the corresponding ligand biomolecule.
Additionally, the magnetic manipulation of such cells was possible in the presence
of an external magnetic field (permanent magnet, not shown).
These biofunctionalized biomarkers were also used with human HeLa cells
that also express the hDC-SIGN. Similarly to the Raji cells, untagged HeLa cells
show limited scattering (Figure 6.6e). When, however, cells that express on their
surface the hDC-SIGN are incubated with particles labeled with antibodies against
DC-SIGN (Figure 6.6f), there is a strong scattering originating from the plasmonic
silver, and enabling their detailed imaging and fast detection.
6.4 Conclusions
Hybrid, Janus- or dumbbell-like Ag/Fe2O3 nanoparticles are made and
coated with a nanothin SiO2 shell or film by one-step, scalable flame aerosol
technology. These “as-prepared” nanoparticles were dispersible in aqueous and
buffer solutions without any surface treatment. The plasmon absorption band of Ag
appeared clearly for an increasing Ag-content and size, while the near
superparamagnetic Fe2O3 component allowed for the magnetic manipulation of the
Ag/Fe2O3 nanoparticles in aqueous suspensions. The SiO2 coating reduced
drastically the release of Ag+ ions when nanoparticles were dispersed in aqueous
suspensions, essentially “curing” their cytotoxicity and enabling them as
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biocompatible multifunctional probes for bioimaging. Their agglomeration
(flocculation) in aqueous suspensions was minimized by SiO2 coating, in contrast to
uncoated Ag/Fe2O3 particles which flocculated and settled within few hours.
The potential of these superior hybrid plasmonic-magnetic nanoparticles as
bioprobes was explored by successfully labeling their surface and specifically
binding them on the membrane of tagged Raji and HeLa cells. Their detection under
dark-field illumination was achieved. The hybrid nanocomposite SiO2-coated
Ag/Fe2O3 particles prevented the individual limitations of Fe2O3 (poor particle
stability in suspensions) and of Ag (toxicity) nanoparticles, while retaining the
desired magnetic properties of Fe2O3, the inert surface of SiO2 and the plasmonic
optical properties of Ag at the nanoscale.
6.5 References
[1] Zeng, H. & Sun, S. H. Syntheses, properties and potential applications of
multicomponent magnetic nanoparticles. Adv. Funct. Mater. 18, 391-400
(2008).
[2] El-Sayed, I. H., Huang, X. H. & El-Sayed, M. A. Surface plasmon resonance
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nanoparticles in cancer diagnostics: Applications in oral cancer. Nano Lett. 5,
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[3] Aaron, J., Travis, K., Harrison, N. & Sokolov, K. Dynamic imaging of
molecular assemblies in live cells based on nanoparticle plasmon resonance
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[4] Jiang, J., Gu, H. W., Shao, H. L., Devlin, E., Papaefthymiou, G. C. & Ying,
J. Y. Manipulation bifunctional Fe3O4-Ag heterodimer nanoparticles for two-
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photon fluorescence imaging and magnetic manipulation. Adv. Mater. 20,
4403-4407 (2008).
[5] Wang, C. G., Chen, J., Talavage, T. & Irudayaraj, J. Gold nanorod/Fe3O4
nanoparticle "Nano-pearl-necklaces" for simultaneous targeting, dual-mode
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48, 2759-2763 (2009).
[6] Medintz, I. L., Uyeda, H. T., Goldman, E. R. & Mattoussi, H. Quantum dot
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(2005).
[7] Das, G. K. & Tan, T. T. Y. Rare-earth-doped and codoped Y2O3
nanomaterials as potential bioimaging probes. J. Phys. Chem. C 112, 11211-
11217 (2008).
[8] Nirmal, M., Dabbousi, B. O., Bawendi, M. G., Macklin, J. J., Trautman, J.
K., Harris, T. D. & Brus, L. E. Fluorescence intermittency in single cadmium
selenide nanocrystals. Nature 383, 802-804 (1996).
[9] Singh, S., D'Britto, V., Prabhune, A. A., Ramana, C. V., Dhawan, A. &
Prasad, B. L. V. Cytotoxic and genotoxic assessment of glycolipid-reduced
and -capped gold and silver nanoparticles. New J. Chem. 34, 294-301 (2010).
[10] Lee, K. J., Nallathamby, P. D., Browning, L. M., Osgood, C. J. & Xu, X. H.
N. In vivo imaging of transport and biocompatibility of single silver
nanoparticles in early development of zebrafish embryos. ACS Nano 1, 133-
143 (2007).
[11] Lu, A. H., Salabas, E. L. & Schuth, F. Magnetic nanoparticles: Synthesis,
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[12] Weissleder, R., Elizondo, G., Wittenberg, J., Rabito, C. A., Bengele, H. H. &
Josephson, L. Ultrasmall superparamagnetic iron-oxide - Characterization of
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[13] Teleki, A., Suter, M., Kidambi, P. R., Ergeneman, O., Krumeich, F., Nelson,
B. J. & Pratsinis, S. E. Hermetically coated superparamagnetic Fe2O3
particles with SiO2 nanofilms. Chem. Mater. 21, 2094-2100 (2009).
[14] Sotiriou, G. A., Sannomiya, T., Teleki, A., Krumeich, F., Vörös, J. &
Pratsinis, S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors.
Adv. Funct. Mater. 20, 4250-4257 (2010).
[15] Lim, J., Eggeman, A., Lanni, F., Tilton, R. D. & Majetich, S. A. Synthesis
and single-particle optical detection of low-polydispersity plasmonic-
superparamagnetic nanoparticles. Adv. Mater. 20, 1721-1726 (2008).
[16] Anker, J. N., Hall, W. P., Lyandres, O., Shah, N. C., Zhao, J. & Van Duyne,
R. P. Biosensing with plasmonic nanosensors. Nature Mater. 7, 442-453
(2008).
[17] Barnes, W. L., Dereux, A. & Ebbesen, T. W. Surface plasmon subwavelength
optics. Nature 424, 824-830 (2003).
[18] Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N.,
Sigg, L. & Behra, R. Toxicity of silver nanoparticles to Chlamydomonas
reinhardtii. Environ. Sci. Technol. 42, 8959-8964 (2008).
[19] Schrand, A. M., Braydich-Stolle, L. K., Schlager, J. J., Dai, L. M. &
Hussain, S. M. Can silver nanoparticles be useful as potential biological
labels? Nanotechnology 19, 235104-235117 (2008).
[20] Willets, K. A. & Van Duyne, R. P. Localized surface plasmon resonance
spectroscopy and sensing. Annu. Rev. Phys. Chem. 58, 267-297 (2007).
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[21] Mueller, R., Madler, L. & Pratsinis, S. E. Nanoparticle synthesis at high
production rates by flame spray pyrolysis. Chem. Eng. Sci. 58, 1969-1976
(2003).
[22] Teleki, A., Heine, M. C., Krumeich, F., Akhtar, M. K. & Pratsinis, S. E. In
situ coating of flame-made TiO2 particles with nanothin SiO2 films. Langmuir
24, 12553-12558 (2008).
[23] Sotiriou, G. A. & Pratsinis, S. E. Antibacterial activity of nanosilver ions and
particles. Environ. Sci. Technol. 44, 5649-5654 (2010).
[24] Teleki, A., Akhtar, M. K. & Pratsinis, S. E. The quality of SiO2-coatings on
flame-made TiO2-based nanoparticles. J. Mater. Chem. 18, 3547-3555 (2008).
[25] Ammar, D. A., Noecker, R. J. & Kahook, M. Y. Effects of benzalkonium
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glaucoma medications on human ocular epithelial cells. Adv. Ther. 27, 837-
845 (2010).
[26] Lozach, P. Y., Lortat-Jacob, H., de Lavalette, A. D., Staropoli, I., Foung, S.,
Amara, A., Houles, C., Fieschi, F., Schwartz, O., Virelizier, J. L., Arenzana-
Seisdedos, F. & Altmeyer, R. DC-SIGN and L-SIGN are high affinity
binding receptors for hepatitis C virus glycoprotein E2. J. Biol. Chem. 278,
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[27] Peters, C. & Dekkers, M. J. Selected room temperature magnetic parameters
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[28] Gole, A., Agarwal, N., Nagaria, P., Wyatt, M. D. & Murphy, C. J. One-pot
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[29] Zhai, J., Tao, X., Pu, Y. A., Zeng, X. F. & Chen, J. F. Core/shell structured
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nanoparticles using spinning disk reactor. AIChE J. 54, 445-452 (2008).
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Pratsinis, S. E. Role of gas-aerosol mixing during in situ coating of flame-
made titania particles. Ind. Eng. Chem. Res. 48, 85-92 (2009).
[33] Buesser, B. & Pratsinis, S. E. Design of gas-phase synthesis of core-shell
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[34] Massich, M. D., Giljohann, D. A., Schmucker, A. L., Patel, P. C. & Mirkin,
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CHAPTER 7
7. Outlook and Research Recommendations
In this thesis a fundamental understanding of nanosilver toxicity was
obtained. This facilitated its synthesis to maintain desired (e.g. plasmonic)
properties, and at the same time to prevent adverse effects (e.g. toxicity,
flocculation). Here the toxicity of nanosilver was controlled by applying a dense,
nanothin inert coating on its surface. This facilitated its employment in biomedical
applications as plasmonic biosensors and for bioimaging. This understanding does
not only help to synthesize safer nanosilver for these specific applications, but it can
be extended to a number of others. After this initial proof-of-concept, tailored-made
nanosilver particles could be employed wherever needed, always taking into account
the target application and what potential adverse effects need to be avoided.
In order to establish the link between the physicochemical properties of
nanosilver particles and the biological outcomes after their exposure to humans,
however, a detailed toxicological characterization is essential. High-throughput
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cytotoxicity assays against a number of different biological systems need to be
developed in order to directly compare the effects of nanosilver particles with
different morphology [1]. Additionally, long term toxicity data need to be gathered
to assure that nanosilver can be employed safely to biomedical applications.
The potential of nanosilver in biosensing should also be explored in more
detail. Nanosilver, being the material with the lowest plasmonic losses, is not used
widely as biosensor because of process limitations [2]. Therefore, solutions for the
easy, scalable and sustainable synthesis of nanosilver structures for biosensing need
to be developed. One technique with promising scalability and compatibility with
electronic devices is flame aerosol synthesis and deposition of nanomaterials [3].
Nanosilver made by this method can be easily deposited on substrates without
utilizing elaborate lithographic or nanofabrication techniques. These nanosilver
structures can be further integrated in a biosensing assay. The unique control over
the nanosilver size and surface coating obtained by flame-synthesis facilitates the
tailored production of biosensors with high reproducibility and performance. These
biosensors could then be tested in “real” conditions, detecting diseases such as
cancer [4] or Alzheimer’s [5].
However, in order to achieve the above, further studies are needed to identify
the optimum characteristics that a nanosilver structure ought to have with superior
plasmonic performance. The size, shape, surface coatings are only a few of the
important parameters that can influence it. Therefore, a systematic investigation of
these parameters needs to be performed, ideally with a system that allows the
control over these properties, such as flame-synthesis. More specifically, the
influence of the material surrounding the nanosilver particles on their plasmonic
properties, either as support or surface coating, could be investigated in detail.
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Materials with a higher refractive index than SiO2 (e.g. ZnO, TiO2, Al2O3, HfO2,
WO3) could be used as the nanosilver particles support. This should lead to a shift of
the nanosilver plasmon absorption band to higher wavelengths [2] and perhaps
improve the biosensing performance.
In this work, the toxicity of nanosilver was minimized against bacteria and
human cells by applying a nanothin, dense SiO2 coating on its surface. This coating
inhibited the Ag+ ion release and the direct contact of the nanosilver surface with
the biological systems. The SiO2 surface was then further functionalized by the
physical adsorption of biomolecules. However, further biofunctionalization with a
desired orientation could be explored. Specific biomolecules could graft on the SiO2
coating by covalent bonding and thus obtain a desired orientation, preferably with
the active site on the outer side. This will facilitate their attachment on the target
sites and optimize their performance. In that way, nanosilver particles could attach
more efficiently on the surface of specific cells also when administered in vivo.
Apart from the employment of nanosilver particles in diagnosis, their
therapeutic potential is worth exploring, too. Because of its plasmonic properties,
nanosilver particles can convert a tissue-permeable infrared irradiation to heat,
acting as tiny heaters. When these particles are attached on cancerous cells in vivo
and the infrared irradiation is directed towards the tumor site, the heat induced by
the nanosilver particles could destroy the tumor. This opens an opportunity for a
non-invasive cancer treatment. Of course several tests and optimization studies need
to be performed before such a treatment is ready to be used in patients. Therefore,
overcoming the adverse toxicity to the healthy tissue that the nanosilver particles
could exhibit through their Ag+ ion release brings this one step closer.
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Furthermore, the synthesis of multicomponent particles containing
nanosilver could be explored, beyond the magnetic functionality. Wet and dry
synthetic routes open a number of possibilities for the manufacturing of smart
multifunctional materials that include the plasmonic nanosilver. With such
methods, several functionalities could be combined in a single nanostructure
targeting a number of novel devices and applications. Finally, when such
nanostructures are made by a simple and scalable way, their incorporation into
existing technologies could be performed. In that way, the potential benefits of
nanotechnological products would be relished.
7.1 References
[1] Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M. V., Somasundaran,
P., Klaessig, F., Castranova, V. & Thompson, M. Understanding
biophysicochemical interactions at the nano-bio interface. Nature Mater. 8,
543-557 (2009).
[2] Willets, K. A. & Van Duyne, R. P. Localized surface plasmon resonance
spectroscopy and sensing. Annu. Rev. Phys. Chem. 58, 267-297 (2007).
[3] Strobel, R. & Pratsinis, S. E. Flame aerosol synthesis of smart nanostructured
materials. J. Mater. Chem. 17, 4743-4756 (2007).
[4] Zhou, W., Ma, Y. Y., Yang, H. A., Ding, Y. & Luo, X. G. A label-free
biosensor based on silver nanoparticles array for clinical detection of serum
p53 in head and neck squamous cell carcinoma. Int. J. Nanomed. 6, 381-386
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[5] Haes, A. J., Hall, W. P., Chang, L., Klein, W. L. & Van Duyne, R. P. A
localized surface plasmon resonance biosensor: First steps toward an assay
for Alzheimer's disease. Nano Lett. 4, 1029-1034 (2004).
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APPENDIX A
A Supplementary Information: Antibacterial
Activity of Nanosilver Ions and Particles
A.1 Morphology of flame-made nanosilver particles
Figure A.1 shows an exemplary STEM image of 10Ag/SiO2 and the
nanosilver particle size distribution (inset) along with its average size, geometric
standard deviation and total number of nanosilver particles counted. The nanosilver
particles are homogeneously dispersed on amorphous SiO2 and exhibit a unimodal
size distribution.
Table A.1 shows the summary of the XRD and S/TEM analysis of
nanosilver of all composite xAg/SiO2 nanoparticles for x = 1-98 wt%. The average
Ag crystal size dXRD is obtained from the X-ray diffraction spectra. Values are shown
for x = 10-98 wt% as for smaller x the XRD could not determine reliably the Ag
crystal content. Additionally, the average nanosilver particle diameter dS/TEM from
S/TEM and its standard deviation along with the geometric standard deviation g of
the distribution and the total number of counted particles N is presented in Table
A.1 for all x. Good agreement is obtained between Ag dXRD and dS/TEM indicating
that nanosilver immobilized on SiO2 by FSP consists of monocrystalline Ag.
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Particle diameter dp, (nm)
1 10 100
(Nin
i/N
ini)/(lndp,i)
0.01
0.1
1
Figure A.1: STEM of 10Ag/SiO2 with a unimodal nanosilver size distribution. The number
average particle diameters dp and geometric standard deviations g and the number of
nanosilver particles N are shown also.
Table A.1: Average Ag crystal diameter, dXRD, and particle diameter, dS/TEM, along with its
standard deviation and geometric standard deviation, g, and with the total number of
counted Ag nanoparticles N in composite xAg/SiO2 particles made by flame spray pyrolysis
(FSP).
Ag content x wt% dXRD (nm) dS/TEM (nm) g N
1 - 4.0 ± 2.0 1.6 203
2 - 4.3 ± 3.2 1.45 445
6 - 6.1 ± 3.1 1.7 165
10 6.9 ± 0.9 6.7 ± 4.1 1.61 851
25 8.1 ± 0.8 8.2 ± 3.4 1.42 326
50 8.7 ± 0.6 8.9 ± 3.5 1.45 744
75 10.8 ± 0.2 12.1 ± 4.0 1.4 544
95 14.6 ± 0.4 15.2 ± 4.4 1.33 608
98 15.1 ± 0.6 16.6 ± 3.8 1.35 178
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A.2 Ag+ ion release and stability in suspensions
Figure A.2 shows the Ag+ ion concentration evolution in aqueous
suspensions containing xAg/SiO2 (x = 2-75 wt%) particles at constant C = 20 mg/L
of Ag in solution. The time t = 0 corresponds to dispersion of xAg/SiO2 particles by
ultrasonication in water. At all x, the equilibrium Ag+ ion concentration is attained
within a few minutes. The Ag+ ion concentration decreases with increasing Ag-
content x, but each concentration is constant over time, within experimental
uncertainty, regardless of x. This indicates that high Ag-content particles release
much less Ag+ ions upon dispersion in solution.
Time, hours
0 1 2 3 4 23 25
Ag+ ion concentration, m
g/L
0
5
10
15
xAg/SiO2
20 mg/L of Ag in H2O
x = 2
x = 6
x = 10
x = 50
x = 75
Figure A.2: Ag+ ion concentration of aqueous suspensions containing 20 mg/L Ag over time,
with time t = 0 corresponding to that immediately after their dispersion.
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Figure A.3 shows the particle size distribution of 50Ag/SiO2 composite
nanoparticles measured by dynamic light scattering (DLS) in water immediately
after dispersion and after 24 hours. It can be seen that the particle size distribution
has practically remained the same over that period, indicating that the Ag/SiO2
nanoparticles are stable in suspension during their antibacterial evaluation.
Agglomerate diameter, nm
10 100 1000
Volume fraction, %
0
20
40
60
0 hours
24 hours
50Ag/SiO2
Figure A.3: Dynamic light scattering of aqueous suspension containing the 50Ag/SiO2
sample immediately after its dispersion and after 24 hours, indicating its stability.
Figure A.4 shows the agglomerate volume size distributions of composite
xAg/SiO2 nanoparticles for x = 1 – 95 wt% as determined by DLS in water. For an
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increasing Ag content x, the agglomerate size becomes smaller as the content of the
fractal-like silica support is reduced [1].
Agglomerate diameter of xAg/SiO2, nm
20 40 60 80 200 400 600 80010 100 1000
Volume fraction, %
0
10
20
30
40
50x = 1 wt%
2 "
6 "
10 "
50 "
95 "
Figure A.4: The agglomerate volume size distributions of composite xAg/SiO2 particles for
x = 1-95 wt%.
A.3 Reference
[1] Kammler, H. K., Beaucage, G., Mueller, R. & Pratsinis, S. E. Structure of
flame-made silica nanoparticles by ultra-small-angle X-ray scattering.
Langmuir 20, 1915-1921 (2004).
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APPENDIX B
B Comparison of the Antibacterial Activity of as-
prepared and washed Nanosilver Particles
B.1 Introduction
The antibacterial activity of small (<10 nm) nanosilver particles is dominated
by the released Ag+ ions from the nanosilver surface. These ions originate from the
dissolution of one or two silver oxide layers on the surface of these nanosilver
particles, and thus making the dose relations for as-prepared small nanosilver best
assessed by surface area concentrations, as seen in chapter 3. Here, the dose
relations of the antibacterial activity of nanosilver that has its surface oxide layer
removed are explored. The toxicity of washed nanosilver particles of various sizes is
monitored against E. coli and compared to the one caused by as-prepared nanosilver.
The antibacterial activity of washed nanosilver particles is mainly attributed to their
surface contact with the cell, revealing also a surface area concentration. However,
the toxicity rate against E. coli of the washed nanosilver is two orders of magnitude
lower than the one of the as-prepared nanosilver, emphasizing the drastically faster
effect of the released Ag+ ions when compared to the particles.
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B.2 Materials and methods
Nanosilver on nanostructured silica was prepared by flame spray pyrolysis as
described in chapter 2. Surface area concentrations of the washed nanosilver
particles were calculated by assuming single, monodisperse spherical nanosilver
particles and their average nanosilver diameter determined by electron microscopy
(Figure 3.4), and by removing one oxide layer for sizes > 6 nm and two oxide layers
for sizes < 5 nm (Figure 4.5).
B.3 Results and discussion
B.3.1 Comparison of antibacterial activities
Figure B.5 shows the E. coli viability % (100% viability corresponds to the
E. coli growth of the control, in the absence of any nanosilver) as a function of the
(a) Ag mass and (b) surface area concentration, for various sized washed nanosilver
particles. When the Ag mass concentration is used, it can be seen that the data are
scattered, and no clear trend can be detected. In contrast, when the data are
expressed as a function of the surface area concentration, a clear linear relation can
be seen. Therefore, when nanosilver has its surface oxide layers removed by
washing, surface area concentration reflects better than the Ag mass concentration
its antibacterial activity.
This surface area-based dose relation is in agreement with the one of the as-
prepared nanosilver of rather small size (< 10 nm) as seen in Figure 3.7. It should be
noted, however, that there are two different reasons for these surface area
concentration dependencies. The small as-prepared nanosilver particles exhibit this
dependency because the released Ag+ ions that dominate the antibacterial activity
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originate from the dissolution of one or two silver oxide surface layers. The washed
nanosilver particles exhibit this dependency because of the direct contact of the cells
with the nanosilver surface. In fact, the surface area dependency of the washed
nanosilver is not only limited to the small (< 10 nm) size range as for as-prepared
nanosilver (Figure 3.8).
Figure B.5: E. coli viability % (100% is the control) in the presence of various sized washed
nanosilver particles as a function of (a) Ag mass and (b) nanosilver surface area concentration.
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Two ways to characterize the antibacterial activity of a compound is the
concentration that is needed in order to cause 50% lethality (LC50) and the toxicity
rate that causes a decrease in the bacteria population per unit of the compound.
When a single compound is used, typically this unit is its mass. In the case of
nanosilver particles of various sizes, however, the surface area concentration should
be used, as seen above. Now, when the antibacterial activity of the as-prepared
nanosilver is compared to the one of the washed nanosilver (in Figure B.6, as
obtained by Figure 3.7 and Figure B.5b) it can be seen that the former caused by the
released Ag+ ions is much stronger than the latter. In fact, the LC50 obtained by the
linear fit of the small as-prepared nanosilver is ~45·10-3 m2/L, while the one of the
washed nanosilver particles is ~1250·10-3 m2/L, clearly showing that the small as-
prepared nanosilver particles that release a large amount of Ag+ ions need much
smaller concentrations to exhibit toxicity.
Additionally, when the E. coli viability is converted to colony forming units
per mL (CFU/mL, a measure of viable bacteria concentration), the toxicity rate is
calculated by the slope of the linear fit. The toxicity rate (tr) per square meter of
nanosilver present in solution of the washed particles is much lower than the one of
the as-prepared nanosilver (as-prepared: 2·1012 CFU/m2, washed: 6·1010 CFU/m2).
This further verifies that the antibacterial activity exhibited by the released Ag+ ions
of the small as-prepared nanosilver is much stronger than the one cause by the
contact of the cell with the surface of the washed nanosilver particles.
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Figure B.6: E. coli viability as a function of the nanosilver surface area concentration in the
presence of as-prepared (open circles) small (< 10 nm) and washed (filled circles) nanosilver
particles (4-16 nm).
B.3.2 Effective Ag+ ion concentration on E. coli viability
In order to calculate the effective Ag+ ion concentrations for the washed
nanosilver: the Ag+ ion release from both flame- and wet-made washed 6Ag/SiO2 is
1.4 mg/L (Fig. 3b: filled symbols). As the total Ag concentration in solution is 40
mg/L of Ag (Fig. 3b: inset top-right), the fraction of Ag ions is 1.4/40 = 3.5%.
Taking this into account, the effective [Ag+] for the washed nanosilver in Fig. 7
ranges from [Ag+] = 0.0875 mg/L for nominal Ag mass concentration 2.5 mg/L
(3.5% x 2.5 mg/L = 0.0875 mg/L), and [Ag+] = 0.35 mg/L for nominal Ag mass
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concentration 10 mg/L (3.5% x 10 mg/L = 0.35 mg/L), to [Ag+] = 0.70 mg/L for
nominal Ag mass concentration 20 mg/L (3.5% x 20 mg/L = 0.70 mg/L).
We will now compare the antibacterial activity of these [Ag+] from the
washed nanosilver, to that of similar [Ag+] from as-prepared nanosilver. Let’s
consider, for example, first the washed nanosilver with nominal Ag mass
concentration of 10 mg/L (Fig. 7: filled circles). This has effective [Ag+] = 0.35
mg/L, as shown above. The E. coli viability of this nanosilver was 58±15%. Second,
the Ag+ ion release of flame-made as-prepared nanosilver with size 8.2 nm
(25Ag/SiO2) was 27±5% [10: Fig. 3, filled circles, right axis]. Since the Ag mass
concentration was 1 mg/L and this sample released 27±5%, this would correspond
to [Ag+] = 0.27±0.05 mg/L. The E. coli viability (or growth) of the released Ag+ ions
from this nanosilver was 52±5% [10: Fig. 7, open squares: in the presence of only
Ag+ ions]. Therefore, for similar [Ag+] (0.35 and 0.27±0.05 mg/L), the washed
nanosilver exhibits similar antibacterial activity to as-prepared nanosilver (58±15%
vs. 52±5%). This indicates that the antibacterial activity of the washed nanosilver is
indeed induced mostly by the released Ag+ ions.
This is the case also for higher [Ag+] from the washed nanosilver (e.g. [Ag+]
= 0.70 mg/L for nominal Ag mass concentration 20 mg/L from Fig. 7: filled circle)
the E. coli viability is 12±15%. As-prepared nanosilver with size 4 nm (1Ag/SiO2)
releases 68±10% Ag+ ions [10: Fig. 3, filled circles, right axis]. For Ag mass
concentration 1 mg/L, the [Ag+] is 0.68±0.10 mg/L (Fig. A.1, open squares)
[10:Fig. 7] and its E. coli viability is 9.4%.
B.3.3 Calculation of Ag mass concentration
For example, the 2.5 mg/L of Ag was prepared as follows: 13.33 mg of the
composite 6Ag/SiO2 nanoparticles (corresponding to 13.33mg·6% = 0.8 mg of Ag
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mass) were dispersed in 20 mL of de-ionized water, forming a batch solution of Ag
mass concentration 0.8/20 mg/mL = 40 mg/L. This batch solution was diluted with
de-ionized water 4 times, reaching therefore, a Ag mass concentration of 40/24 =
2.5 mg/L.
B.3.4 Calculation of Ag surface area concentration
In order to calculate the surface area concentration of washed nanosilver,
one needs to take into account two things. First, that the actual nanosilver size after
its washing is smaller, since a fraction has been dissolved (one or two layers). That
means that nanosilver < 4 nm would be dissolved completely. Second, because of
this dissolution the actual Ag mass concentration of the washed nanosilver is lower
than the nominal one (e.g. ~50 wt% with the flame-made 6Ag/SiO2).
For example: the Ag mass concentration C of the washed flame-made
6Ag/SiO2 (Fig. 2, x = 6 on top abscissa) nanosilver for nominal Ag mass
concentration 2.5 mg/L (after 4 dilutions of initial 40 mg/L in Fig. 2) is 50 % of
this: 1.25 mg/L. The specific surface area SSA obtained from the nanosilver size
distributions assuming the dissolution of one layer for dp > 5 nm and two layers for
dp < 5 nm is 49 m2/g. Therefore, the Ag surface area concentration of washed
nanosilver is:
C · SSA = 1.25 mg/L · 49 m2/g = 0.062 m2/L
in contrast to the C · SSA = 2.5 mg/L · 52 m2/g =0.130 m2/L for the as-prepared
nanosilver.
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APPENDIX C
C Hermetically Silica-coated Nanosilver:
Optimizing the Coating Reactor
Abstract
Nanosilver particles can be coated by a nanothin silica layer in one-step by
an enclosed flame spray pyrolysis reactor. However, a small fraction “escapes” the
coating by incomplete mixing between the Si-precursor vapor and the core hot
aerosol, resulting in nanosilver that is not 100% hermetically coated. Here, the
process parameters of the above mentioned system are systematically explored in
order to achieve a fully hermetic coating on nanosilver particles. The nanosilver
crystal size is controlled and kept to small values (< 5 nm) by co-oxidizing silver and
Si-precursors simultaneously. The silica coating is applied further downstream in-
flight on the freshly prepared composite Ag/SiO2 nanoparticles. The effect of the
process parameters and the reactor geometry (single and double coating ring) on the
silica coating and its efficiency are investigated by monitoring the Ag+ ion release of
the nanosilver when dispersed in aqueous solutions.
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C.1 Introduction
A nanothin silica layer on the surface of freshly made nanoparticles was
recently achieved in one-step by using a modified enclosed flame spray pyrolysis
(FSP) reactor [1]. With this, Si-precursor (hexamethyldisiloxane, HMDSO) vapor is
injected through a metal torus ring which is placed above the flame. Therefore, the
HMDSO vapor is mixed with the freshly-made hot aerosol and forms a smooth
nanothin coating on the surface of the core particles [2]. This in situ coating has
been successfully applied previously on TiO2 [1] and Fe2O3 [3] nanoparticles and
was evaluated by isopropanol chemisorption [3,4].
In chapter 5, nanosilver particles were also fully encapsulated by nanothin
coating with this reactor, and these particles exhibited limited antibacterial activity
when compared to the partially-coated ones (Figure 5.2). However, it is difficult to
evaluate the efficiency of the silica coating on these particles: because of their large
size they do not release a high fraction of Ag+ ions. In chapter 6, however, the
nanosilver crystal size was controlled by the co-oxidation of Fe- and Ag-precursors,
forming Janus-like particles. The nanosilver size was controlled by the presence of
Fe2O3 particles, since this ceramic inhibited further silver crystal growth. The
smaller nanosilver sizes obtained by this method facilitated the direct comparison of
Ag+ ion release of coated and uncoated particles, in order to evaluate the coating
efficiency. The SiO2-coating significantly inhibited the Ag+ ion release, but it was
not completely zero (about 5%, Figure 6.5a).
Here, the process parameters of this coating reactor are systematically
investigated, focusing on the mixing improvement between hot core particle aerosol,
and HMDSO vapor [2,5]. The nanosilver crystal growth is controlled by its co-
oxidation with SiO2. The mixing is investigated by varying the gas flow rates in the
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reactor, as well as its set-up with a single ring or double coating ring. The coating
efficiency of the core nanosilver particles is evaluated by monitoring the Ag+ ion
release in aqueous solutions.
Figure C.1: The nanosilver crystal sizes as a function of the burner-ring-distance for different
Ag-contents x and precursor molarities. The N2 mixing flow rate is 15 L/min and the O2
sheath flow rate is 40 L/min.
C.2 Materials and methods
The enclosed FSP reactor is described in detail in chapter 5. The core freshly-
formed nanoparticles are coated in situ by swirl injection of HMDSO vapor
downstream. The precursor solution for the composite core Ag/SiO2 nanoparticles
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(Ag-content x = 10-95 wt%) is described in detail in chapter 2. The reactor is
enclosed by a quartz glass tube (20-40 cm) on top of which the HMDSO vapor is
supplied through a metallic torus ring with 16 equidistant openings. The double ring
reactor setup was made by placing a second ring (32 equidistant openings, distance
between rings, 5 cm) on top of the first ring, and also HMDSO vapor was supplied
through it. The reactor was terminated by placing a 40 cm quartz glass tube on top
of the rings. The precursor solution was fed through the FSP-capillary (5 mL/min)
and dispersed by O2 (5 L/min) forming a spray which was ignited by a
methane/oxygen (1.5/3.2 L/min) premixed flame. The flame was sheathed by O2
(15-40 L/min). The particle characteristics (XRD, BET) and the Ag+ ion release
measurements are described in detail in chapter 2.
C.3 Results and discussion
C.3.1 Control of nanosilver size
The nanosilver crystal size is controlled by the co-oxidation of Ag and Si-
precursors forming composite xAg/SiO2 nanoparticles. Figure C.1 shows the
average nanosilver crystal size as a function of the burner-ring-distance (BRD) of a
single ring. Through that ring N2 gas with a flow rate of 15 L/min is injected. The
precursor molarity varies from 0.1 to 0.5 M and the O2 sheath gas is kept constant at
40 L/min. For higher Ag-content x > 70 wt% and precursor molarity 0.5 M, there is
an increase in the nanosilver crystal size for increasing BRD, indicating that the
temperature profile is high enough to cause nanosilver growth. By decreasing,
however, the Ag-content to x = 50 wt% (open triangles), the crystal size growth is
inhibited significantly from about 11 nm to 15 nm. Now, if the precursor molarity
decreases to 0.25 M (filled triangles), the nanosilver crystal growth is further
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decreased and maintained from ~9 to 11 nm. It should be noted that for Ag-content
x = 10 wt% and precursor molarity 0.25 (filled hexagon) and 0.1 M (semi-filled
hexagon), the average nanosilver crystal size was just 5.3 and 3.9 nm for
BRD = 20 cm. Therefore, for Ag-contents x < 50 wt% and precursor molarities
< 0.5 M, the presence of SiO2 drastically inhibits the nanosilver crystal growth.
Figure C.2: The Ag+ ion release as a percentage over the total Ag mass as a function of the
nanosilver average crystal size for the composite Ag/SiO2 made from the open (open circles)
and enclosed FSP (filled circles).
C.3.2 Ag+ ion release of composite Ag/SiO2
In order to evaluate the coating efficiency, nanosilver particles with average
crystal size < 10 nm should be synthesized that release high fractions of Ag+ ions
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(Figure 2.3). Figure C.2 shows the fraction of Ag+ ions that are released of the total
Ag present in solution as a function of the average nanosilver crystal size of the co-
oxidized samples obtained by the open (chapter 2) and the enclosed FSP-reactor.
The Ag+ ion release of the composite Ag/SiO2 nanoparticles made in the enclosed
reactor is lower than the one obtained by the nanosilver made in the open reactor,
even when their average nanosilver crystal size is similar. This could be attributed to
the larger residence time at high temperatures that occur within the enclosed
reactor. This temperature profile facilitates the silica growth, and thus, allowing
more nanosilver surface to be embedded in silica. This can be further verified by the
specific surface area values of these particles which are lower than the SSA values of
the co-oxidized samples (~350-400 m2/g for the open, and ~250 m2/g for the
enclosed reactor), clearly indicating silica growth in the enclosed reactor.
C.3.3 Single coating ring
Since nanosilver with average crystal size ~4 nm on nanostructured silica
releases a significant amount of Ag+ ions (~25%), the SiO2-coating process
parameters were explored with these particles. Figure C.3 shows the Ag+ ion
concentration [Ag+] of this nanosilver size for a constant Ag mass concentration of
200 mg/L as a function of the N2 mixing gas flow intensity. HMDSO vapor (for an
equivalent theoretical SiO2-coating thickness (calculated by the SSA of the samples)
of ~2 nm: open symbols, and ~3.5 nm: filled symbols) was injected through the
single torus ring at BRD = 20 cm resulting in SiO2-coated nanosilver. The [Ag+] that
corresponds to the uncoated Ag/SiO2 sample and the values obtained only by water
are also shown.
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Figure C.3: Ag+ ion concentration of ~4 nm nanosilver dispersed in water as a function of the
N2 mixing gas flow rate and different theoretical coating thicknesses (2 and 3.5 nm). The
values of the uncoated nanosilver and of water are also shown.
The [Ag+] decreases with increasing N2 mixing flow rate for a theoretical
coating thickness of ~2 nm and O2 sheath gas flow rate of 40 L/min (open circles) in
agreement with literature [2,5]. The lowest Ag+ ion release is reached at 22 L/min
of N2 mixing. When the theoretical coating thickness increases to 3.5 nm, the [Ag+]
decreases, reaching almost 7.5% (see right axis). This indicates that perhaps 2 nm
theoretical coating thickness may not be enough for a complete coating on the
composite Ag/SiO2 nanoparticles. The remaining [Ag+] concentration may originate
from incomplete mixing between the freshly-formed hot aerosol and the HMDSO
vapor.
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One way to optimize this mixing is to increase the N2 mixing flow rate
further [2,5]. This, however, may result in incomplete HMDSO to SiO2 conversion
since the temperature after an increased N2 mixing flow rate may not be high
enough (it will quench the aerosol) and additionally the residence time after the
mixing will become shorter. Another way to optimize the mixing is to decrease the
O2 sheath gas flow rate. By doing so there are two advantages. First, there will be a
better mixing since the freshly formed aerosol has a lower velocity and second, the
residence time after the HMDSO injection will be longer than before.
Figure C.4: The [Ag+] of aqueous suspensions containing Ag/SiO2 nanoparticles as a function
of different O2 sheath gas flow rates. The N2 mixing flow rate is 22 L/min and the theoretical
coating thickness is 3.5 nm.
In Figure C.4, the [Ag+] for different O2 sheath gas flow rates are shown for a
theoretical coating thickness of 3.5 nm and N2 mixing flow rate of 22 L/min. The
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[Ag+] decreases with decreasing O2 sheath gas flow rate reaching an Ag+ ion release
of just 3.9% at 20 L/min O2. However, the [Ag+] is again higher when reducing O2
further down to 15 L/min. This could be attributed to the higher temperature after
HMDSO vapor injection that exists for the lowest sheath gas flow rate. This may
result in the formation of segregated SiO2 nanoparticles from the HMDSO vapor,
rather than a smooth homogeneous coating on the surface of the core particles [4]. It
seems however, that still some composite nanosilver particles may escape from the
coating process even at optimized conditions, perhaps through the space between
two equidistant openings of the ring.
C.3.4 Double coating ring
One way to improve the mixing between HMDSO vapor and core aerosol, is
to insert a second coating ring downstream which would “capture” the particles that
have escaped the coating process from the first ring. The flow rate of the second ring
does not have to be too high, as it targets the particles that travel near the wall of the
glass tube [5]. So the second ring is placed 5 cm above the first one, and has 32
equidistant openings through which the HMDSO vapor will be injected along with
5 L/min of N2. The total HMDSO flow rate through the two rings corresponds to
the theoretical coating thickness of 3.5 nm, while the N2 mixing through the first
ring is 20 L/min.
Figure C.5 shows the [Ag+] as a function of HMDSO fraction that is injected
through the first, bottom coating ring (the rest is injected through the second, upper
ring). The lowest Ag+ ion release occurs when 25% of the HMDSO is injected
through the bottom ring and the rest 75% through the upper ring, reaching ~2% Ag+
ion release. For an increasing fraction of HMDSO through the bottom ring, the Ag+
ion release becomes slightly larger, but is still lower than the best results obtained by
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a single coating ring. It should be noted that when fractions of HMDSO <25% were
injected through the bottom ring (>75% through the upper ring) the HMDSO was
clearly not fully converted to SiO2, as the as-prepared particles were hydrophobic,
and thus their dispersion in water was not possible.
Figure C.5: The [Ag+] of aqueous suspensions containing the SiO2-coated Ag/SiO2
nanoparticles made by double coating ring, as a function of HMDSO fraction injected through
the bottom ring.
C.4 References
[1] Teleki, A., Heine, M. C., Krumeich, F., Akhtar, M. K. & Pratsinis, S. E. In
situ coating of flame-made TiO2 particles with nanothin SiO2 films. Langmuir
24, 12553-12558 (2008).
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[2] Teleki, A., Buesser, B., Heine, M. C., Krumeich, F., Akhtar, M. K. &
Pratsinis, S. E. Role of gas-aerosol mixing during in situ coating of flame-
made titania particles. Ind. & Eng. Chem. Res. 48, 85-92 (2009).
[3] Teleki, A., Suter, M., Kidambi, P. R., Ergeneman, O., Krumeich, F., Nelson,
B. J. & Pratsinis, S. E. Hermetically coated superparamagnetic Fe2O3
particles with SiO2 nanofilms. Chem. Mater. 21, 2094-2100 (2009).
[4] Teleki, A., Akhtar, M. K. & Pratsinis, S. E. The quality of SiO2-coatings on
flame-made TiO2-based nanoparticles. J. Mater. Chem. 18, 3547-3555 (2008).
[5] Buesser, B. & Pratsinis, S. E. Design of gas-phase synthesis of core-shell
particles by computational fluid–aerosol dynamics. AIChE J. in press, (2011).
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APPENDIX D
D Color-tunable Nanophosphors by Co-doping
Flame-made Y2O3 with Tb and Eu1
Abstract
Rare-earth phosphors with tunable optical properties are used in display
panels and fluorescent lamps and have potential applications in lasers and bio-
imaging. Here, non-aggregated Y2O3 nanocrystals either doped with Tb3+ (1-5 at%)
or co-doped with Tb3+ (2 at%) and Eu3+ (0.1-2 at%) ions are made in one-step by
scalable flame spray pyrolysis. The morphology of these nanophosphors is
investigated by X-ray diffraction, electron microscopy and N2 adsorption while their
optical properties are monitored by photoluminescent spectroscopy. When yttria
nanocrystals are doped with terbium, a bright green emission is obtained at an
optimum Tb-content of 2 at%. When, however, europium is added, the emission
color of these Tb-doped yttria nanophosphors can be tuned precisely from green to
red depending on the Tb/Eu ratio. Furthermore, energy-transfer from Tb3+ to Eu3+ is
observed, thus allowing the control of the excitation spectra of the co-doped
nanophosphors.
1 Part of this chapter is published in J. Phys. Chem. C 115, 1084-1089 (2011).
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D.1 Introduction
Rare-earth phosphors are light emitting materials that find a wide variety of
applications [1]. These materials are multicomponent: one material being the host
matrix and the rare-earth doping element being responsible for the radiation [2]. The
emission wavelength of such materials depends mostly on the chosen doping rare-
earth element. Phosphors are commonly used in fluorescent lamps and luminescent
displays [1] and have potential applications in lasers [3] and X-ray imaging [4].
Nanosized phosphors (nanophosphors) improve the resolution of displays [5] and
have promising applications as bioimaging probes [6]. The superior photostability
(no blinking or fading) and biocompatibility [7] of nanophosphors makes them
advantageous over traditionally used organic dyes and semiconducting
nanoparticles. Organic fluorescent dyes photobleach during bio-imaging while
semiconducting nanoparticles exhibit optical blinking [8] and can be toxic [9].
Furthermore, the nano size of dispersible nanophosphors facilitates their
employment in nanocomposite materials such as flexible displays [5].
One of the most studied ceramics as host matrix for phosphors is yttrium
oxide (Y2O3). Several studies address synthesis of nanosized, Y2O3-based phosphors
doped with Eu3+ ions for their bright red emission. Such phosphor nanoparticles are
typically made by elaborate wet methods [7,10,11] while it is not unusual that
further annealing process steps are required to obtain the desired crystallinity
[12,13]. Post heat-treatment of such nanoparticles, however, results in aggregates
with strong sinter necks that may hinder their dispersion in host polymers or liquid
suspensions [14]. An alternative to wet-chemistry methods is flame aerosol synthesis
[15-18] which is a scalable process [19] that allows for fine tuning of the crystallinity
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of the resulting particles in one-step without any post heat-treatment [17]. This
results in mostly non-aggregated nanoparticles that are attractive in bio-imaging [6].
The color tunability of Y2O3-based nanophosphors can be achieved by doping
with rare-earth elements [1]. For example, while Eu3+ doping of Y2O3 results in a red
emission, doping it with Tb3+ results in a green emission [10]. In fact, this color
tunability is exploited already by combining three different colored phosphors (blue,
green, red) for white-light lamps [1]. For all rare-earth phosphors, there is an
optimum concentration of the doping material [20]. This concentration, however,
depends on several parameters, such as synthetic route or even the size of the
phosphors [12].
Even though there are many studies investigating synthesis and optical
properties of Y2O3-based nanophosphors doped with a rare-earth element, there are
only a few that explore the co-doping of two or more rare-earth elements in the
same crystal host matrix. Co-doping enables the fine tuning of the excitation and
emission spectra of phosphors [21]. When Y2O3 is co-doped with Eu and Tb, for
example, a strong energy transfer occurs from Tb3+ to Eu3+ ions [21], while a back
energy-transfer from Eu3+ to Tb3+ is not significant [13]. In fact, there is an optimum
ratio (Eu/Tb = 8) for maximum luminescent efficiency [22]. Such energy transfer
has been observed also for other rare-earth doped up-converting nanophosphors (e.g.
Yb-Er) [7].
Here, the one-step synthesis of Y2O3:Tb3+ and co-doped Y2O3:Tb3+/Eu3+
nanoparticles is explored by flame aerosol technology [17]. The morphology and
particle characteristics are investigated by EDX spectroscopy, TEM analysis, X-ray
diffraction and N2 adsorption (BET). The optical properties of the as-prepared
nanophosphors are explored by photoluminescent spectroscopy. The optimum Tb-
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content is investigated by monitoring the photoluminescent intensity of the
Y2O3:Tb3+ nanophosphors. The emission wavelength of the co-doped
nanophosphors is tuned by the ratio of the doping rare-earth elements. A strong
energy transfer is observed from Tb3+ to Eu3+ which allows for the characteristic red
emission of Eu3+ at higher excitation wavelengths.
D.2 Materials and methods
Multi-component nanophosphors were produced by flame spray pyrolysis
(FSP) of appropriate precursor solutions as described in detail elsewhere [17].
Yttrium nitrate (Aldrich, 99.9%) was dissolved in a 1:1 by volume mixture of 2-
ethylhexanoic acid (EHA, Riedel-de Haen, 99%) and ethanol (Alcosuisse) to form
the precursor solution. The molarity was kept constant at 0.5 M for Y metal. The
terbium and/or europium doping was achieved by adding 1-5 at% terbium nitrate
(Aldrich, 99.9%) or 0.1-2 at% europium nitrate (Fluka, 95%) to the above solution.
The atomic fraction (at%) of dopants was defined with respect to the total metal ion
concentration. The precursor solution was fed to the FSP nozzle at a constant feed
rate (11.6 mL/min) provided by a syringe pump (Inotech) and dispersed to a fine
spray by 3 L/min oxygen (PanGas, purity >99.9%). The pressure drop at the nozzle
tip was kept constant at 1.5 bar. Subsequently, the spray was ignited by a premixed
methane/oxygen (1.5/3.2 L/min, PanGas, purity >99.9%) flame leading to
formation of nanophosphor particles that were collected on a glass microfiber filter
(Whatman GF6, 257 mm diameter) with the aid of a vacuum pump (Busch, Seco SV
1040C).
X-ray diffraction (XRD) patterns were recorded by a Bruker AXS D8
Advance diffractometer (40 kV, 40 mA, Cu Kα radiation) from 2θ=20-70° with a
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step size of 0.03°. The obtained spectra were fitted using the TOPAS 3 software
(Bruker) and the Rietveld fundamental parameter refinement [17]. The specific
surface area (SSA) was obtained according to Brunauer-Emmet-Teller (BET) by five-
point N2 adsorption at 77 K (Micrometrics Tristar 3000). Prior to that, samples were
degassed in N2 for at least 1h at 150 °C. High resolution transmission electron
microscopy (HRTEM) was performed with a CM30ST microscope (FEI; LaB6
cathode, operated at 300 kV, point resolution ~2 Å). The electron beam could be set
to selected areas to determine material composition by energy dispersive X-ray
spectroscopy (EDXS). Product particles were dispersed in ethanol and deposited
onto a perforated carbon foil supported on a copper grid. The photoluminescence of
the produced particles was characterized at room temperature using a fluorescence
spectrophotometer (Varian Cary Eclipse) containing a Xe flash lamp with tunable
emission wavelength. Samples of 30 mg were filled in a cylindrical substrate holder
of 10 mm diameter and pressed towards a quartz glass front window. Emission
spectra were recorded from 450 – 650 nm, excitation spectra from 200 – 400 nm
with a step size of 0.5 nm. Additionally, photoluminescence decay curves were
recorded at a resolution of 0.03 ms. The obtained data were fitted by the Cary
Eclipse software (Varian) and thus first order exponential decay time constants were
extracted.
D.3 Results and discussion
D.3.1 Phosphor morphology
Energy-dispersive X-ray (EDX) spectroscopy over a large area of the 2 at%
Tb-doped Y2O3 sample (Figure D.1a) reveals the presence of both Y and Tb. The C
and Cu peaks come from the carbon-coated copper grid which was used to obtain
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the TEM images. Figure D.1b shows a TEM image of the same nanoparticles that
are non-aggregated [17]. A high resolution TEM image showing a single crystalline
nanoparticle is shown in Figure D.1c. The distance between their crystal planes is
3.03 Å corresponding to the (222) crystal plane [13] of cubic Y2O3.
Figure D.1: Energy dispersive X-ray (EDX) spectrum of the 2 at% Tb-doped Y2O3 with its
corresponding Tb and Y peaks. The C and Cu peaks come from the TEM carbon coated
copper grid TEM. (b) A TEM image showing the non-aggregated structure of the 2 at% Tb-
doped Y2O3 nanoparticles. (c) A high resolution TEM image showing the crystal planes of a
single nanoparticle. The distance between crystal planes (3.03 Å) corresponds to the (222)
crystal plane of Y2O3.
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Figure D.2: X-ray diffraction patterns of flame-made pure Y2O3 nanoparticles. All peaks
correspond to cubic Y2O3. As inset, the cubic Y2O3 mass fraction as a function of the Tb-
content is presented. The presence of Tb does not significantly influence the crystallinity of the
particles.
Figure D.2 shows the X-ray diffraction spectra of as-prepared pure Y2O3
nanoparticles that correspond to mostly cubic [5] phase of Y2O3, without involving
post heat-treatment [17]. The long residence time in high temperatures allows for
control of the crystalline structure from the monoclinic phase for low combustion
enthalpy flames to the cubic phase for high combustion enthalpy flames, as
here [17]. All XRD spectra in the presence of terbium doping (1-5 at%) are identical
(not shown). The cubic mass fraction as estimated by Rietveld analysis is ~90%
while the rest ~10% corresponds to the monoclinic phase. The presence of terbium
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doping does not influence significantly this crystallinity, as seen in the inset of
Figure D.2. Perhaps there is a slight decrease with an increasing Tb-content. This is
consistent with flame-made Y2O3 doped with other rare earth ions (e.g. Eu) [5,17]
where a slight decrease of the Y2O3 cubic mass fraction was also observed for an
increasing Eu-doping concentration.
Figure D.3: The average cubic (dXRD,cubic, circles) and monoclinic (dXRD,monoclinic, squares)
Y2O3 crystal sizes and the average particle or grain diameter as determined by N2 adsorption
(dBET, triangles) of the as-prepared flame-made nanophosphors as a function of the Tb-content
that has hardly any influence on them.
The average crystal sizes of cubic (dXRD,c, circles) and monoclinic (dXRD,m,
squares) Y2O3 as determined by XRD are presented in Figure D.3. The average
particle or grain diameter as determined from N2 adsorption (dBET, triangles) is also
shown in Figure D.3. The monoclinic Y2O3 particles are smaller than the cubic ones
consistent with the literature [17]. The slightly lower dBET values than dXRD,c originate
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from the small fraction of the monoclinic Y2O3 (~10 wt%). The presence of Tb does
not influence significantly the morphology of the nanophosphors, consistent with
prior flame-made Y2O3-based nanophosphors [5,15-17] as well as with rare-earth
doped Y2O3-based nanophosphors made by wet chemistry [7,12,13]. The good
agreement between dXRD,c and dBET indicates that the particles are monocrystalline
and non-aggregated, a desirable feature for bio-imaging applications [7].
Figure D.4: (a) The excitation spectrum of the 2 at% Tb-doped Y2O3 nanoparticles monitored
at 545 nm. The two bands around 280 and 310 nm are attributed to Tb3+ transitions. (b)
Emission spectrum of the same sample under 276 nm excitation. The appearing peaks
correspond to Tb3+ ion transitions, with most dominant being the one at 545 nm attributed to
the 5D4 → 7F5 transition.
D.3.2 Photoluminescence of Y2O3:Tb3+ nanophosphors
Figure D.4a shows the excitation spectrum of the 2 at% Tb-doped Y2O3
monitored at 545 nm. It consists of two broad bands at ~280 and 310 nm
corresponding to 4f → 5d transition [13] of Tb3+. The emission spectrum of the same
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sample (2 at% Tb) under 276 nm excitation is shown in Figure D.4b. There are the
characteristic emission peaks attributed to Tb3+ ions[7] with the most intense one at
545 nm (green) corresponding to the 5D4 → 7F5 transition [7,13].
Figure D.5: The maximum phosphorescence intensity monitored at 545 nm under excitation
of 276 nm (circles, left axis) and the decay time constant (triangles, right axis) as a function of
Tb-content. The optimum Tb-concentration is 2 at%.
The optimum Tb doping concentration was investigated by varying the Tb
content from 1 to 5 at%. Figure D.5 shows the phosphorescence maximum intensity
of the Y2O3:Tb3+ nanophosphors (at 545 nm) under 276 nm excitation as a function
of the Tb-content (circles). The error bars correspond to the standard deviation of
three different samples. The highest intensity is obtained for 2 at% Tb. At higher Tb
contents, the intensity decreases. The enhanced photoluminescence (PL) for higher
doping up to a critical concentration (here 2 at%) results from the increasing number
of luminescent centers. The PL reduction for higher doping, the so-called
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“quenching”, is a characteristic behavior and inherent to all phosphors [20]. It is
based on energy transfer between adjacent luminescent centers [23]. As the energy
levels of similar lanthanide ions match perfectly, this energy transfer is highly
efficient and will be favored instead of the light emitting decay [13]. At high doping
concentrations, this probability is enhanced [12]. Even though Y2O3:Tb3+
nanoparticles have never been made by flame spray pyrolysis before, the present
optimum dopant concentration is within the reported ones from different synthetic
routes [12,13].
Figure D.6: Emission spectra of the co-doped Y2O3:Tb3+/Eu3+ nanophosphors under 276 nm
excitation. For an increasing Eu-content, the phosphorescence peaks attributed to Eu3+
(dominating one at 612 nm) are also increasing.
The PL-decay time constant is shown in Figure D.5 (triangles) also. For the
nanophosphor with the highest emission intensity (2 at% Tb) it is 2.1 ms. For an
increasing doping concentration, however, it monotonically decreases, reaching 1.25
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ms for the 5 at% Tb, values similar to Y2O3:Tb3+ nanophosphors made by wet
chemistry [13,24]. Such a reduction of the decay time for an increasing rare-earth
ion concentration has been observed for Y2O3:Tb3+ at similar Tb-contents (0.005-
5 at%) [25] as well as for flame-made Y2O3:Eu3+ nanophosphors [5,26].
Figure D.7: Images of the co-doped Y2O3:Tb3+/Eu3+ nanophosphors dispersed in ethanol
under 254 nm excitation. The color of the nanophosphors can be fine tuned by selecting the
atomic ratio of these rare earth ions.
D.3.3 Co-doped Y2O3:Tb/Eu
Co-doping the nanophosphors with Eu and Tb did not alter the morphology
and crystallinity of flame-made Y2O3 based nanophosphors (Figure D.1). Figure D.6
shows the emission spectra of the co-doped Y2O3:Tb/Eu under 276 nm excitation
(the wavelength with maximum intensity attributed to Tb3+ transitions, Figure D.4).
The Tb-content was kept constant at 2 at% while the Eu content varied from 0 to
2 at%. For comparison, also a sample in the absence of Tb was made (with
1 at% Eu) [26]. Even with a minimal Eu content of 0.1 at%, the characteristic
emission peak at 612 nm attributed to the 5D0 → 7F2 transition within Eu3+ ions [7] is
detectable. As the Eu-content increases, the corresponding intensity peak (at
612 nm) increases, as more Eu3+ luminescent centers are created. For the
nanophosphor with 2 at% Tb and 0.5 at% Eu the emission peaks at 545 and 612 nm
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have similar intensities (for excitation at 276 nm). This is consistent with such co-
doped nanophosphors made by wet-chemistry [13] employing, however, higher
dopant concentrations (6 at% Tb and 4 at% Eu).
Figure D.7 shows suspensions of the as-prepared nanophosphors in ethanol
under 254 nm excitation (from a commercial UV lamp). The change of the ratio
between the two doping ions results in different colors. Therefore, by adjusting the
ratio between the doping rare earth ions in the Y2O3 host matrix, a fine tuning of the
emitted color can be achieved: from light green-blue for 2 at% Tb and yellow for
2 at% Tb/ 0.25 at% Eu, to red for 2 at% Tb/ 1 at% Eu.
Figure D.8: Excitation spectra of the co-doped Y2O3:Tb3+/Eu3+ nanophosphors monitored at
545 nm (a) and 612 nm (b). The existence of the broad bands at 280 and 310 nm when
monitoring the Eu3+ emission peak (612 nm) indicates an energy transfer from Tb3+ to Eu3+
ions.
A close examination of Figure D.6 shows that the intensity of the Tb3+
emission peaks decreases for an increasing Eu-content, although the Tb-content
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remains constant. This is seen also in Figure D.8a where the excitation spectra
monitoring the 545 nm emission peak at an increasing Eu-content are presented.
This indicates that there is no radiative [27] energy transfer [13] from Eu3+ to Tb3+.
In contrast, the intensity of the Eu3+ peak at 612 nm for 1 at% Eu content is higher
for the sample co-doped with Tb (Figure D.6). The excitation wavelength of 276 nm
with which the emission spectra of Figure D.6 are recorded corresponds to the
4f → 5d transition of Tb3+ (Figure D.4). This indicates that there is a strong energy
transfer [13,22] from Tb3+ to Eu3+. This energy transfer is further verified from the
excitation spectra of the co-doped nanophosphors monitoring the 612 nm Eu peak
(Figure D.8). In the absence of Tb (top line), the excitation band centered around
230 nm is typical for Y2O3:Eu3+ nanophosphors [7]. In the presence of Tb, however,
there is Eu3+ emission at 612 nm after exciting at wavelengths corresponding to the
Tb3+ ions transitions (bands at 280 and 310 nm). It should be noted, however, that
the emitted color of each co-doped nanophosphor (such as the ones observed in
Figure D.7) depends on the excitation wavelength. Figure D.8 also shows that when
the excitation wavelength is <250 nm, only red emission at 612 nm occurs
(e.g. 2 Tb/0.5 Eu), with no green emission at 545 nm. Therefore, it is possible to
control the excitation spectrum, as well as the emission color of the co-doped
Y2O3:Tb3+/Eu3+ nanophosphors through the energy transfer from Tb3+ to Eu3+.
D.4 Conclusions
Multicomponent nanophosphors have been made in one-step by flame spray
pyrolysis. Rare earth Tb3+ and Eu3+ ions have been incorporated in nanocrystalline
Y2O3 (cubic phase). The resulting nanocrystals (dXRD = 33 nm) were non-aggregated
and no further post heat-treatment was needed. Photoluminescent examination
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revealed bright green phosphorescence for Y2O3:Tb3+ nanocrystals in agreement with
the literature. When these nanophosphors were co-doped with Eu3+, their emission
color could be controlled from bright green to bright red. A strong energy transfer
occurs from Tb3+ to Eu3+ ions, which results in the precise tuning of the excitation
spectrum of these light emitting nanophosphors. This fine tuning along with the
scalable process employed here for synthesis of these nanophosphors may facilitate
their employment in several applications, such as fluorescent lamps, display panels
and bio-imaging given their photostability and biocompatibility.
D.5 References
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[13] Mukherjee, S., Sudarsan, V., Vatsa, R. K., Godbole, S. V., Kadam, R. M.,
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step fabrication of nonaggregated, size-controlled and cubic nanocrystalline
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APPENDIX E
E Flame Synthesis and Characterization of
Silica-coated Y2O3:Tb3+ Nanophosphors
Abstract
Silica-coated and uncoated Tb-activated (1-5 at% Tb) Y2O3 nanophosphors
were made by flame aerosol technology with controlled crystal phase (cubic and
monoclinic) and morphology. The as prepared nanophosphors are characterized by
X-ray diffraction, N2 adsorption, high resolution electron microscopy and
photoluminescence spectroscopy. The monoclinic crystal structure of Y2O3:Tb3+
nanophosphors favors the electric dipole 5D4 → 7F5 transitions responsible for their
green phosphorescence. Even though the phosphorescence of the SiO2-coated
monoclinic Y2O3:Tb3+ is lower than the uncoated monoclinic one, for the first time
the synthesis of non-toxic silica-coated biocompatible nanophosphors can be
achieved in a single-step process. Finally, an annealing study is performed on the
uncoated and SiO2-coated monoclinic nanophosphors where the decrease in the
phosphorescence intensity is observed for the Y2O3:Tb3+ crystal transformation from
monoclinic to cubic. This further indicats the superior performance of the
monoclinic crystal phase for the electric dipole transitions of the Tb3+.
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E.1 Introduction
Luminescent light-emitting nanoparticles have lately attracted a lot of
attention with potential applications in high-definition displays [1], lasers [2] and
bioimaging [3]. Among these particles, rare-earth phosphors doped with lanthanide
ions have advantageous optical properties because of their superior photostability
[4,5]. Yttrium oxide (Y2O3) is one of the most studied ceramics as host matrix,
especially when doped with europium (Eu3+) ions resulting in a bright red emission
[6]. Furthermore, the emission color depends on the chosen dopant element [7]. For
example, when Y2O3 is doped with terbium (Tb3+) ions, a bright green emission
occurs [6,8]. In fact, the color of nanosized Y2O3 nanocrystals can be finely tuned
from bright green to red by codoping them with Tb3+ and Eu3+ ions during their
flame synthesis preserving thus their crystal and particle sizes [9].
Such nanocrystals are particularly attractive for application in bioimaging as
they do not degrade during analysis (photobleaching) compared to organic dyes and
are relatively non-toxic when compared to semiconducting (e.g. CdSe, PbS)
nanoparticles (quantum dots) [3,5]. For such bioapplications, the nanoparticle
surface often needs to be modified to increase their biocompatibility. Recently, it
was shown that a dense inert nanothin silica layer [10,11] can minimize the toxicity
of plasmonic nanosilver as it prevented the release of toxic ions and the direct
contact with the cell surface [12] In fact, such a layer can facilitate the surface
biofunctionalization with molecules for specific binding [10].
The crystal structure of the host matrix (e.g. Y2O3) as well as the particle size
can influence the luminescent properties of the nanophosphors [13]. Many studies
have investigated the luminescence of cubic [13-16] Y2O3 nanoparticles doped with
Eu3+. The monoclinic Y2O3 phase can also be obtained, especially when processes
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with high temperatures and fast cooling rates are employed [13,17]. In cubic Y2O3
there are two sites where the rare earth ions can substitute the Y3+ ions. Responsible
for the electric dipole radiative transitions are the ones with C2 symmetry and
without any inversion center (75% of the sites). The rest sites have S6 symmetry with
inversion center and there the electric dipole transitions are forbidden [18,19]. In
monoclinic Y2O3 all sites have Cs symmetry without any inversion center [19] and it
has been suggested that Tb3+ ions should be embedded in lattice sites without
inversion symmetry [20]. However, because the red emission of monoclinic
Y2O3:Eu3+ is less intense than the one obtained by its cubic crystal structure [13]
there are only a few studies that investigate the luminescent properties of
monoclinic[13,17,21,22] Y2O3:Eu3+ nanoparticles and even fewer that involve other
lanthanide ions such as Tb3+ [19,22].
Here, the focus lies on the synthesis and detailed characterization of
uncoated and silica-coated Y2O3:Tb3+ nanophosphors made by flame aerosol
technology that allows for fine control on the nanophosphor size and crystal
structure (cubic and monoclinic) [13]. The Tb-content ranges from 1-5 at%. The as
prepared nanoparticles are characterized by X-ray diffraction, N2 adsorption and
high-resolution electron microscopy and their optical properties by
photoluminescence spectroscopy. The effect of the crystallinity and the silica coating
on luminescence of Y2O3:Tb3+ nanophosphors is investigated. An annealing study is
performed to the monoclinic Y2O3 nanophosphors to monitor the influence of the
resulting phase transition from monoclinic to cubic on their phosphorescence.
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E.2 Materials and methods
SiO2-coated monoclinic Y2O3:Tb3+ nanophosphors were made by using a
modified enclosed FSP reactor [23]. In brief, yttrium nitrate (Aldrich, 99.9%) was
dissolved in a 1:1 by volume mixture of 2-ethylhexanoic acid (EHA, Riedel-de
Haen, 99%) and ethanol (Alcosuisse) to form the precursor solution. The molarity
was kept constant at 0.5 M for Y metal. The Tb doping was achieved by adding 1-
5 at% Tb nitrate (Aldrich, 99.9%) to the above solution. The Tb atomic fraction
(at%) was defined with respect to the total metal ion concentration. The precursor
solution was fed at the FSP nozzle (5 mL/min) and dispersed by 5 L/min oxygen
(PanGas, purity >99.9%) and sheathed by 40 L/min oxygen. The freshly-formed
core Y2O3:Tb3+ particles were coated in-flight by swirl injection of
hexamethyldisiloxane (HMDSO, Sigma Aldrich, purity ≥ 99%) vapor with 15
L/min nitrogen (PanGas, purity > 99.9%) at room temperature through a metallic
ring with 16 equidistant openings. The ring was placed on top of a 20 cm long
quartz glass tube (inner diameter 4.5 cm) followed by another 30 cm long such tube.
The HMDSO vapor was supplied by bubbling nitrogen through approximately
350 mL liquid HMDSO in a 500 mL glass flask. The SiO2 amount was kept constant
in the product particles and was calculated at saturation conditions (bubbler
temperature 9 °C and 0.5 L/min N2) corresponding to 16.6 wt%. The as-prepared
nanophosphor particles were collected on a glass microfiber filter (Whatman GF6,
257 mm diameter). Uncoated monoclinic Y2O3:Tb3+ particles were made at identical
conditions as above in the absence, however, of the HMDSO vapor. Uncoated cubic
Y2O3:Tb3+ nanophosphors were made as described in detail elsewhere [9]. Shortly,
the precursor solution was fed to the FSP nozzle at a feed rate of 11.6 mL/min and
dispersed to fine spray by 3 L/min oxygen.
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X-ray diffraction (XRD) patterns were recorded by a Bruker AXS D8
Advance diffractometer (40 kV, 40 mA, Cu Kα radiation) from 2θ=20-70° with a
step size of 0.03°. The obtained spectra were fitted using the TOPAS 3 software
(Bruker) and the Rietveld fundamental parameter refinement [13]. High resolution
transmission electron microscopy (HR-TEM) was performed with a CM30ST
microscope (FEI; LaB6 cathode, operated at 300 kV, point resolution ~2 Å).
Product particles were dispersed in ethanol and deposited onto a perforated carbon
foil supported on a copper grid. The photoluminescence of the particles was
characterized at room temperature using a fluorescence spectrophotometer (Varian
Cary Eclipse) containing a Xe flash lamp with tunable emission wavelength.
Samples of 30 mg were filled in a cylindrical substrate holder of 10 mm diameter
and pressed towards a quartz glass front window. Emission spectra were recorded
from 450 – 650 nm, excitation spectra from 200 – 400 nm with a step size of 0.5 nm.
Annealing of the samples was performed at 750, 850, 900, 1100 °C for 10 hours.
E.3 Results and discussion
E.3.1 Morphology and crystallinity of Y2O3:Tb3+ nanoparticles
Figure E.1 shows HR-TEM images of uncoated Y2O3:Tb3+ (2 at% Tb)
nanoparticles made by open (a,d) and enclosed (b,e) FSP, in which the crystal
planes of the Y2O3 matrix can be seen [9]. For the uncoated Y2O3 from the open FSP
(a,d) there are both spherical and rhomdohedrally shaped particles [13], while for
the Y2O3 made by the enclosed FSP (b,e), there are mostly spherical. For the SiO2-
coated nanoparticles made by enclosed FSP (c,f) a smooth amorphous SiO2-layer of
about 2 nm encapsulates the crystal core, as has also been observed for similarly
SiO2-coated TiO2 [23], Fe2O3 [24] and Ag [11] nanoparticles.
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Figure E.1: High-resolution transmission electron microscopy images from the open uncoated
(a,d), enclosed uncoated (b,e) and enclosed SiO2-coated (c,d) Y2O3:Tb3+ (2 at%).
Figure E.2 shows the XRD spectra of the Y2O3:Tb3+ (2 at%) nanoparticles
made by open FSP and enclosed FSP. The as prepared nanophosphors by open FSP
are mostly cubic (89 %), in agreement with the literature for these flame synthesis
conditions (11.6/3 precursor feed rate over dispersion gas flow rate ratio) and
precursor solutions (Y-nitrate in EHA/ethanol) [9,25]. The Y2O3 nanoparticles
made by enclosed FSP reactor, however, have only a minimal cubic phase fraction
(<10 %) and exhibit the characteristic pattern of the monoclinic phase [26].
Additionally, the presence of SiO2 coating does not seem to significantly influence
nanoparticle crystallinity [24] as the XRD patterns are almost identical for the
uncoated and SiO2-coated nanoparticles. The average crystal sizes (Figure E.2,
inset) of nanoparticles made by open FSP (from now on: cubic Y2O3) and the
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enclosed FSP (from now on: monoclinic Y2O3) are similar for all three conditions,
which facilitates their further evaluation regarding the luminescent properties as
shown later on.
Figure E.2: X-ray diffraction patterns of open uncoated (red line), enclosed uncoated (green
line) and enclosed SiO2-coated (blue line) Y2O3:Tb3+ (2 at%). The open FSP made Y2O3:Tb3+
nanoparticles exhibit the characteristic cubic crystal structure, while both enclosed FSP made
nanoparticles exhibit the monoclinic Y2O3 crystal structure. As inset, the average crystal size of
the cubic and monoclinic phases is shown.
E.3.2 Phosphorescence of cubic and monoclinic Y2O3:Tb3+ nanoparticles
Figure E.3a shows the excitation spectra of the uncoated cubic [9] (solid
line), uncoated monoclinic (broken line) and SiO2-coated monoclinic (dotted line)
Y2O3:Tb3+ (2 at% Tb) nanoparticles monitoring their emission at 545 nm. The cubic
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Y2O3:Tb3+ nanoparticles have a dominant band centered around 280 nm and a
secondary band centered around 310 nm [5]. The monoclinic Y2O3:Tb3+ have the
dominant band centered also around 280 nm, however, the secondary excitation
band is centered around 265 nm. These bands are assigned for transitions of the Tb3+
ions within the nanocrystals [27]. This is the first indication that the crystal phase of
the Y2O3 host matrix influences the luminescent properties.
Emission wavelength, nm
450 500 550 600 650
Pho
spho
resc
ence
inte
nsity
, a.u
.
Excitation wavelength, nm
250 300 350
Pho
spho
resc
ence
inte
nsity
, a.u
.
Figure E.3: (a) The excitation spectra of the 2 at% Tb-doped Y2O3 cubic (red line), monoclinic
(green line) and monoclinic SiO2-coated (blue line) nanoparticles monitored at 545 nm. The
two bands around 280 and 310 nm are attributed to Tb3+ transitions. (b) Emission spectra of
the same samples under 276 nm excitation. The appearing peaks correspond to Tb3+ ion
transitions, with most dominant being the one at 545 nm attributed to the 5D4 → 7F5
transition.
This can be verified in the emission spectra (Figure E.3b) of these
nanoparticles when excited at 276 nm [5]. The strongest emission band from the
electric dipole transitions of Tb3+ ions is typically located around 550 nm
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(5D4 → 7F5) [5,28], (a major peak at ~545 nm and a secondary peak at ~555 nm)
corresponding to green color, and one at around 490 nm (5D4 → 7F6) corresponding
to blue color [5,20,28]. The strongest emission of the monoclinic Y2O3:Tb3+ is
shifted to 547 nm (broken and dotted lines, Figure E.3b). This shift could be
attributed to symmetry changes at the cationic sites where the Tb3+ in Y2O3 can
substitute in the two different crystal phases [6]. Additionally, the intensity of the
secondary peak at ~555 nm has decreased significantly when compared to the
dominant one at 547 nm. Such spectrum characteristics have been obtained also
with monoclinic Gd2O3:Tb3+ phosphors [19]. The intensity of the band at ~490 nm
has also decreased when compared to the intensity at 547 nm. The reduction of the
band located at ~490 nm has been related to cross relaxation phenomena [28,29] in
Y2O3:Tb3+ nanocrystals and perhaps the monoclinic crystal phase also facilitates
this. When the three emission spectra of Figure E.3b are compared, the intensity
around 545 nm of the monoclinic Y2O3 (broken line) is quite higher than the one of
the cubic (solid line).
Figure E.4 shows the maximum phosphorescence intensities of the three
samples as a function of Tb-content. The uncoated monoclinic (open triangles)
Y2O3:Tb3+ has higher phosphorescence for all Tb-contents than both SiO2-coated
monoclinic (filled triangles) and uncoated cubic [9] (open circles) Y2O3
nanophosphors. The uncoated monoclinic nanophosphors have stronger
phosphorescence than the SiO2-coated ones most probably because of the light
absorption and scattering [30] of amorphous SiO2 that encapsulates the core
Y2O3:Tb3+ nanoparticles and therefore, the intensity of the excitation irradiation is
reduced. However, the SiO2-coated monoclinic Y2O3:Tb3+ nanoparticles still
outperform the uncoated cubic ones. This enables such biocompatible nanoparticles
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to be employed safely in bioapplications without any adverse toxic effects, common
with other light emitting nanoparticles that contain heavy metals (quantum dots).
The radiative time decay of the monoclinic Y2O3:Tb3+ (1-5 at%) nanophosphors
decreased for an increasing Tb-content from 2.72 to 1.66 ms and was slightly larger
than the ones from the cubic Y2O3:Tb3+ (1-5 at%): from 2.67 to 1.09 ms.
Tb-content, at%
1 2 3 4 5
Max
imum
inte
nsity
, a.u
.
0
5
10
Uncoated monoclinic
SiO2‐coated monoclinic
Uncoated cubic [Ref. 9]
Emission at 545 nm
Figure E.4: The maximum phosphorescence intensity monitored at 545 nm under excitation
of 276 nm for the cubic (red line), monoclinic (green line) and monoclinic SiO2-coated (blue
line) nanoparticles as a function of Tb-content.
For both uncoated nanophosphors (monoclinic and cubic), however, the
maximum intensity achieved by the monoclinic (broken line, open triangles) Y2O3
was ~3 times higher than that achieved by the cubic (solid line, open circles). This
suggests that the monoclinic Y2O3 phase favors the green color emission from the
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Tb3+ ions. This is in contrast to the red color emission from the Eu3+ activated Y2O3
(5D0 → 7F2) [13] for which the cubic structure is favorable and emphasizes the effect
of the chosen dopant element on the final luminescent properties of the
nanophosphors. Furthermore, Y2O3:Tb3+ nanoparticles made by microemulsion [22]
of a diameter ~20-40 nm show an increase in the phosphorescence intensity from
the monoclinic to cubic Y2O3 crystal phase (by annealing), however, this was
attributed to the removal of impurities that were formed during their wet-synthesis.
Additionally, the highest intensity is obtained for Tb-content 2-4 at% regardless of
Y2O3 crystal phase or SiO2-coating, resulting from the increasing number of
luminescent centers [31]. Above 4 at% Tb-content energy transfer between adjacent
luminescent centers occurs which leads to phosphorescence quenching [32].
E.3.3 Annealing of uncoated and SiO2-coated monoclinic Y2O3:Tb3+ nanoparticles
In order to further evaluate the effect of the crystal structure on the
luminescence of Y2O3:Tb3+ nanophosphors, an annealing study of the monoclinic
Y2O3 nanoparticles was performed. Figure E.5a shows the XRD patterns of the
uncoated monoclinic Y2O3:Tb3+ (2 at% Tb) for different annealing temperatures (at
750, 850, 900 and 1100 °C for 10 hours). The as prepared monoclinic phase
transforms completely to the cubic one above 850 °C [22,26,33]. With increasing
temperature the cubic phase is obtained while the estimated cubic crystal size
increases from ~36 to ~90 nm [26].
In contrast, the SiO2-coated samples (Figure E.5b) retain their mostly
monoclinic phase till 850 °C as well as their crystal sizes (dXRD = 22.5-25 nm). This
indicates that the SiO2-shell inhibits the core crystal growth [11]. The fact that the
monoclinic phase does not transform to cubic at 850 °C for the SiO2-coated samples
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Diffraction angle 2,°
20 30 40 50 60 70
Inte
nsity
, a.u
.
1100 °C
900 °C
850 °C
750 °C
as prepared
11%
10%
9%
cubic massfraction
Diffraction angle 2,°
20 30 40 50 60 70
Inte
nsity
, a.u
.
1100 °C
900 °C
850 °C
750 °C
as prepared
100%
99%
95%
22%
7%
cubic massfraction
Figure E.5: XRD patterns of the monoclinic (a) and SiO2-coated monoclinic (b) Y2O3:Tb3+ (2
at% Tb) for different annealing temperatures (750, 850, 900 and 1100 °C) for 10 hours. The
stars indicate peaks that correspond to yttrium-silicates.
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(in contrast to the uncoated Y2O3, Figure E.5a) also indicates that the Y2O3 crystal
phase strongly depends on the particle size [34]. Above 900 °C, however, a
significant change in the XRD spectrum is observed. Apart from the formation of
the cubic phase, also a few peaks that correspond neither to monoclinic nor cubic
Y2O3 emerge (Figure E.5b, stars). These peaks most probably correspond to yttrium-
silicates [35] that were formed by the interaction of the SiO2 shell with the core
crystal Y2O3 particles at this high temperature. This inhibits also the accurate
estimation of the cubic and monoclinic crystal sizes and their mass fractions from
the XRD-spectra for temperatures above 850 °C.
Figure E.6: (a) The specific surface area (SSA) as a function of the annealing
temperature for the uncoated (open triangles) and SiO2-coated monoclinic (filled
triangles) Y2O3:Tb3+ nanophosphors. TEM images of the uncoated (b) and SiO2-coated
Y2O3:Tb3+ after 1100 °C annealing.
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Figure E.6a shows the specific surface area (SSA) as determined by N2
adsorption as a function of the annealing temperature for the uncoated (open
triangles) and SiO2-coated monoclinic Y2O3:Tb3+ (filled triangles) for 2 at% Tb-
content, respectively. For the uncoated sample, the SSA monotonically decreases
with annealing temperature with the formation of sinter necks [26] (Figure E.6b)
and coalescence of particles, in agreement with the increase of the crystal size from
the XRD (Figure E.5a). The SSA for the SiO2-coated sample remains fairly constant
up to 850 °C so there is no significant increase in the grain size. This indicates that
the SiO2 coating inhibits the core crystal growth and encapsulates fully the Y2O3
core crystals. Furthermore, the SSA of the as prepared uncoated and SiO2-coated
nanoparticles is practically the same indicating that there is no separate SiO2
nanoparticles formed that would lead to larger SSA values. For temperatures above
850 °C, however, the SSA decreases significantly and formation of sinter necks
occurs (Figure E.6c). No amorphous phase is detected by TEM, verifying the
formation of the larger cubic Y2O3 crystal phase and the yttrium-silicates, in
agreement to the XRD analysis (Figure E.5b).
Figure E.7a and b shows the excitation and emission spectra, respectively, of
the uncoated monoclinic Y2O3:Tb3+ (2 at% Tb) nanophosphors annealed at different
temperatures. For increasing annealing temperature there is a phase transformation
from monoclinic to cubic (Figure E.5a) and formation of larger sizes (Figure E.5a
and E.6a). Therefore, both excitation and emission spectra shift from the ones
corresponding to the monoclinic to those of the cubic phase. Figure E.7b also shows
that the emission phosphorescence intensity decreases with increasing annealing
temperature and phase transformation, further indicating that the monoclinic Y2O3
crystal phase favors the green phosphorescence of Tb3+ ions. Such a decrease in the
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phosphorescence for higher annealing temperatures has also been observed for other
systems (SiO2:Tb3+) and has been attributed to the reduction of effective Tb3+
luminescent centers because of the formation of optically inactive Tb clusters [29]. It
should be noted that this behavior is in contrast to the Y2O3:Eu3+ system [26] that
shows stronger phosphorescence intensity for increasing annealing temperature and
larger sizes. In that case, however, the phosphorescence of the cubic Y2O3 crystal
phase outperforms the one from the monoclinic phase. Therefore, the increase in the
phosphorescence in that system is mainly attributed to the elimination of crystal
defects that exist in the cubic as prepared samples [26].
It is not clear why the green phosphorescence of the Tb3+ ions is stronger in
the monoclinic Y2O3 crystal phase than the cubic one. Perhaps there is a better
distribution of Tb3+ ions in the monoclinic Y2O3 crystal host matrix because of its
smaller average crystal size, facilitating their radiative transitions [29]. Furthermore,
the probability of the 5D4 → 7F5 electric-dipole transition of the Tb3+ ions contains
contributions from the linear and third order terms of the crystal lattice [20]. Some
of the crystal point groups in the cubic Y2O3 phase have no linear term, and
therefore, a larger transition probability may be realized by embedding the Tb3+ ions
in lattice sites with crystal field having linear terms [20], such as the monoclinic
Y2O3 (with corresponding crystal point group Cs) [19].
The excitation spectra of the SiO2-coated monoclinic Y2O3:Tb3+ (2 at% Tb)
for an increasing annealing temperature (Figure E.7c) exhibit the characteristic
bands related to the monoclinic phase till 850 °C. This indicates that the SiO2-
coating prevents the phase transformation, in agreement with the XRD results
(Figure E.5b). However, for annealing temperatures above 850 °C, an excitation
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Figure E.7: The excitation monitored at 545 nm (a,c) and emission under 276 nm excitation
(b,d) spectra of the 2 at% Tb-doped Y2O3 monoclinic (a,b) and monoclinic SiO2-coated (c,d)
nanoparticles for the different annealing temperatures (750, 850, 900 and 1100 °C). The
phosphorescence intensity decreases for the transition from monoclinic to cubic crystal
structure.
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band arises at quite low wavelength (< 250 nm), which becomes the dominating one
at 900 and 1100 °C. This band is most probably attributed to the interaction of the
SiO2-coating with the core Y2O3:Tb3+ and the formation of yttrium-silicates and
could be related to the excitation of the 7D energy level in the Tb3+ ion [29].
Nonetheless, at 1100 °C where there is cubic Y2O3:Tb3+ (Figure E.5b) the excitation
bands related to the cubic phase are also present.
This transformation from monoclinic to cubic can also be observed in the
emission spectra of the SiO2-coated nanophosphors (Figure E.7d), in which the
reduction of the dominant peak occurs for the highest annealing temperature.
Furthermore, the highest intensity at ~545 nm is obtained at 850 °C. At this
temperature the monoclinic phase (Figure E.5b) and core size (dXRD = ~25 nm) are
retained. Most probably the annealing temperature has improved the core Y2O3:Tb3+
crystallinity and eliminated any Y2O3 crystal defects [22] leading to an increase in
the phosphorescence intensity [26,36]. This further indicates that the monoclinic
phase of Y2O3 is favorable for the Tb3+ phosphorescence.
The above results are summarized in Figure E.8 that shows the maximum
phosphorescence intensity of the uncoated (a) and SiO2-coated (b) monoclinic
Y2O3:Tb3+ (2 at% Tb) nanophosphors when excited at 276 nm for the different
annealing temperatures. For the uncoated monoclinic nanophosphors the graph is
divided in two areas: one ≤ 750 °C in which there is no significant change, and one
> 750 °C in which core crystal growth and phase transformation from monoclinic to
cubic occurs and thus, the maximum phosphorescence decreases. For the SiO2-
coated nanophosphors, the phosphorescence increases for temperatures up to 850 °C
because there is an improvement of the monoclinic phase and possibly elimination
of any crystal defects while the SiO2 coating prevents the crystal size increase and
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phase transformation. As soon as, however, the temperature exceeds 850 °C, a
dramatic decrease in the phosphorescence occurs attributed to the formation of the
Y2O3 cubic phase and yttrium-silicates.
Figure E.8: The maximum phosphorescence intensity of the uncoated (a) and SiO2-coated (b)
monoclinic Y2O3:Tb3+ (2 at% Tb) nanophosphors when excited at 276 nm for the different
annealing temperatures.
E.4 Conclusions
Uncoated and SiO2-coated Y2O3:Tb3+ nanocrystals with controlled
crystallinity (cubic and monoclinic) were made by flame spray pyrolysis (FSP) for
Tb-contents of 1-5 at%. The optimum Tb-content was 2-4 at% for both cubic and
monoclinic Y2O3 nanophosphors. For the first time, to the best of our knowledge, it
is experimentally demonstrated that the phosphorescence intensity is higher for the
Y2O3:Tb3+ with the monoclinic crystal structure rather than with the cubic one.
Furthermore, biocompatible SiO2-coated Y2O3:Tb3+ nanophosphors are made in
one-step facilitating their employment in bioapplications. By annealing the
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monoclinic nanophosphors the phosphorescence decreased for the crystal
transformation from monoclinic to cubic structure. Additionally, the SiO2-coating
on the monoclinic core particles prevented their growth and phase transformation
till 850 °C, while their crystallinity was improved that resulted in an increase in the
phosphorescence. Therefore, the monoclinic structure of Y2O3 favors the green
phosphorescence of Tb3+ ions, in contrast to the phosphorescence of the well-studied
Eu3+ ions where there is higher phosphorescence for the cubic crystal structure. This
understanding may facilitate the synthesis of bright green nanophosphors with a
biocompatible coating suitable for lighting applications such as displays and
bioimaging.
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[31] Blasse, G. The physics of new luminescent materials. Mater. Chem. Phys. 16,
201-236 (1987).
[32] Blasse, G. & Grabmaier, B. C. Luminescent Materials. (Springer, 1994).
[33] Zhang, P., Navrotsky, A., Guo, B., Kennedy, I., Clark, A. N., Lesher, C. &
Liu, Q. Y. Energetics of cubic and monoclinic yttrium oxide polymorphs:
Phase transitions, surface enthalpies, and stability at the nanoscale. J. Phys.
Chem. C 112, 932-938 (2008).
[34] Guo, B., Harvey, A., Risbud, S. H. & Kennedy, I. M. The formation of
cubic and monoclinic Y2O3 nanoparticles in a gas-phase flame process.
Philos. Mag. Lett. 86, 457-467 (2006).
[35] Rakov, N., Amaral, D. F., Guimaraes, R. B. & Maciel, G. S. Spectroscopic
analysis of Eu3+-and Eu3+:Yb3+-doped yttrium silicate crystalline powders
prepared by combustion synthesis. J. Appl. Phys. 108, 073501 (2010).
[36] Pradhan, A. K., Zhang, K., Mohanty, S., Dadson, J., Hunter, D., Loutts, G.
B., Roy, U. N., Cui, Y., Burger, A. & Wilkerson, A. L. Luminescence and
spectroscopic behavior of Eu3+-doped Y2O3 and Lu2O3 epitaxial films grown
by pulsed-laser deposition. J. Appl. Phys. 97, 023513 (2005).
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APPENDIX F
F A Novel Platform for Pulmonary and
Cardiovascular Toxicological Characterization of
Inhaled Engineered Nanomaterials
Abstract
A novel method is presented which is suitable for assessing in vivo the link
between the physicochemical properties of engineered nanomaterials (ENMs) and
their biological outcomes. The ability of the technique to generate a variety of
industry-relevant, property-controlled ENM exposure atmospheres for inhalation
studies was systematically investigated. The primary particle size for Fe2O3, SiO2,
Ag and Ag/SiO2 was controlled from 4 to 25 nm, while the corresponding
agglomerate mobility diameter of the aerosol was also controlled and varied from 40
to 120 nm. The suitability of the technique to characterize the pulmonary and
cardiovascular effects of inhaled ENMs in intact animal models is also
demonstrated using in vivo chemiluminescence (IVCL). The IVCL technique is a
highly sensitive method for identifying cardiopulmonary responses to inhaled ENMs
under relatively small doses and acute exposures. It is shown that moderate and
acute exposures to inhaled nanostructured Fe2O3 can cause both pulmonary and
cardiovascular effects.
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F.1 Introduction
The use of engineered nanomaterials (ENM) in household products, textiles,
industrial processes, medical devices and therapeutics is widespread and increasing
exponentially. Environmental and occupational exposures are considered by experts
to be “inevitable” [1-3]. Preliminary evidence demonstrates the potential for ENM
to cause adverse biological effects [4-6]. Indeed, the potential of nanoparticles and
nanofibers to translocate through the air-blood barriers, and thus to reach the
pulmonary connective tissues, lymphatic system, or even to reach the circulating
blood and thus have access to other critical organs, is of concern. Nano-sized
particles (NPs) may enter cells, and be more biologically active than their micro-
sized counterparts due to their small size and large surface to volume ratio [7-14]
and increased release of ions [15].
Many in vitro assays and novel aerosol generation methods suitable for
inhalation studies have been developed and contributed significantly to improving
our understanding of biological mechanisms related to atmospheric particle health
effects [16-18]. However, these currently available particle exposure methods can
not be applied in the nanotoxicology field, primarily because of the unique
properties of ENM. For in vivo ENM inhalation studies, an important limiting
factor is the difficulty in aerosolizing and dispersing commercially available
nanopowder ENM down to the nano-size level, as they would exist in many
relevant inhalation exposures [19]. Some common aerosol generators used in the
past to disperse nanopowders for inhalation studies, including nebulizers, fluidized
beds, and other venturi aspirator type systems, have limited applicability for
nanotoxicology [19]. For example, such nanopowder aerosol generation systems
have difficulty producing consistent nanosized aerosol distributions and more
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importantly, fail to control physicochemical properties in order to elucidate the link
between properties and toxic biological responses [19,20].
As a result of the challenges and limitations, the majority of nanotoxicity
research has focused on in vitro cellular or in vivo instillation studies using
commercially available ENM nanopowders and nanopowder liquid suspensions;
however, there is a consensus among scientists that data will require verification
from animal inhalation experiments [2,19]. There are also serious shortcomings
related to the physiological relevance of this particle to cell delivery approach using
liquid ENM suspensions. The shortcomings of these in vitro and instillation
approaches are twofold: First, commercial ENM are limited in diversity of
physicochemical and morphological properties – usually to a few sizes for a given
composition – making it impossible to perform comparative parametric toxicological
studies of ENM properties (size, surface, composition, shape, charge, etc.). Second,
nanoparticles suspended in culture media flocculate or dissolve, and interact with
serum components [21-23], which can alter their biological properties and effects.
Furthermore, comparison of particle doses delivered in suspension to those
administered by inhalation is difficult, which can result in large differences in
effective dosage between in vivo and in vitro studies. These limitations may explain
some of the disparities reported in the literature between in vivo and in vitro ENM
studies [20,21,24]. It is apparent that new methods and systems suitable for both in
vitro and in vivo inhalation toxicological characterization need to be developed.
Here, a novel technique is presented which is suitable for both ENM in vivo
inhalation and in vitro toxicological characterization studies. The ability of this
technique to generate a variety of industry-relevant, property-controlled exposure
atmospheres for inhalation studies was systematically investigated. The suitability of
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the technique to characterize the pulmonary and cardiovascular effects of inhaled
ENM in intact animal models was also demonstrated in an in vivo study involving
Sprague-Dawley rats, using freshly generated nano-iron oxide (Fe2O3) as a test
aerosol. We demonstrated both pulmonary and systemic toxicity using in vivo
chemiluminescence of heart and lung [25-31]. This novel platform will make it
possible for toxicologists to link physicochemical properties of inhaled ENMs to
biological outcomes and help the industry to develop safer ENM.
F.2 Materials and methods
F.2.1 Versatile engineered nanomaterials generation system (VENGES)
Figure F.1 illustrates the Versatile Engineered Nanomaterial Generation
System (VENGES) [32]. The system consists of four main components: ENM
synthesis; particle sampling and collection system; animal exposure system; and
exposure monitoring system.
ENM synthesis: The synthesis of ENMs is based on the industry-relevant
flame spray pyrolysis (FSP) method, as most of today’s ENMs are made by gas-
phase techniques [33]. An inflammable liquid precursor, containing dissolved
organometallic compounds, is pumped through a stainless-steel capillary tube (ID
0.41 mm; OD 0.71 mm). Immediately surrounding the capillary tube is a narrow
annular gap of adjustable cross-sectional area that supplies oxygen, which is used to
disperse the liquid precursor into fine droplets. A small pilot flamelet (premixed CH4
and O2 at 1 and 2 L/min, respectively) ignites the droplets so that the organic parts
of the precursor combust, while the metals are oxidized, and form particles that
grow by coagulation and sintering in the flame. The precursor liquid feed is metered
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by a mass rate-controlled syringe pump, and all gas flows are controlled by mass
flow controllers [34,35].
Figure F.1: Overview of VENGES. Engineered nanomaterials (ENM) are generated by flame
synthesis. The nanoparticles are collected on a filter for further ex-situ characterization.
Directly above the flame, in situ sampling of the test aerosol is performed (QS). Immediately
after, the ENM aerosol stream can be further diluted with HEPA filtered room air (QD) and
directed into the exposure animal chambers (QA) for in vivo toxicological testing.
Simultaneously, the test ENM aerosol is in situ real-time characterized (QP). The total
particle number concentration is measured, as well as the mobility particle size distribution.
Finally, the CO2, CO, NO2, relative humidity (RH) and temperature (T2) are continuously
monitored.
Sampling and off line characterization of generated ENM: The flame-
generated particles are collected using a water-cooled, stainless-steel filter housing
supporting a glass fiber filter (Whatman GF6, 25.7 cm in diameter, USA), with
exhaust gases drawn by a vacuum pump (Busch, Seco SV 1040C, Netherlands).
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Collected particles can be extracted from the filter as nanopowder, and used for off-
line physicochemical and morphological characterization, as well as for in vitro or
in vivo instillation studies. Off-line ENM characterization included N2 adsorption
(5-point isotherm, Micromeritic Tristar, Switzerland). ENM Samples were degassed
under N2 at 150 ºC for 1.5 hours. The average primary particle diameter dBET was
calculated from their specific surface area SSA: dBET = 6000/(·SSA), where is the
materials density. X-ray diffraction (XRD, Bruker D8 Advance, Cu K, Switzerland)
was also performed over scan range 2 = 20 – 70º. Morphology of the generated
ENM was also obtained using transmission electron microscopy and scanning
transmission electron microscopy (STEM, instrument for both: Tecnai F30,
Switzerland). The samples were dispersed in ethanol and then placed on a carbon
coated copper grid.
Animal exposure system: The continuously produced (in the gas phase)
nanoparticles are sampled in situ over the flame (QS, Figure F.1), and transferred to
inhalation chambers for animal studies through a 1 L aerosol container to stabilize
the flow rate. The sampled aerosol can be further diluted with high-efficiency-
particulate (HEPA) -filtered dry air (QD). The ENM remain airborne, with a very
low level of agglomeration, and are drawn directly through the animal exposure
chambers (QR, Figure F.1). For this study, each animal was housed in its own whole
body exposure chamber (10 cm in diameter, 18 cm in length). Chambers were made
of clear polycarbonate to allow visualization of the animals during the exposure,
and used a modified rubber stopper as a cap. Flow through each chamber was
maintained constant at 1.5 L/min.
Exposure monitoring system: The animal exposure atmosphere was
monitored continuously, using a number of real time instruments. Exposure
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parameters monitored included total particle counts (P-Track, TSI, USA), aerosol
size distribution (FMPS, TSI, USA), NO2, CO, CO2, temperature, and relative
humidity (Figure F.1).
F.2.2 Performance characterization experiments
The versatility and ability of VENGES to generate an array of industrially
relevant ENM while controlling important nanoparticle properties (i.e. primary
particle size, agglomeration state, surface properties, shape) as well as its suitability
for in vivo inhalation studies was demonstrated in a number of performance
characterization experiments, as follows:
ENM generated nanopanel: Four different ENM, silicon dioxide (silica,
SiO2), iron oxide (Fe2O3), silver (Ag, nanosilver), and composite nanosilver
supported on silica particles (Ag/SiO2) were generated here. Table F.1 summarizes
the ENM used in these experiments and their respective liquid precursor
characteristics. It is worth pointing out that the FSP synthesis method used in
VENGES allows for generation of a large variety of industrially relevant ENM [36].
Table F.1: Synthesized ENM, their corresponding precursors and solvent, as well as their
process parameters (precursor molarity and x/y ratio).
ENM Metal precursor Solvent Precursor
molarity (M)Liquid feed rate (x)/ O2 dispersion flow rate (y)
SiO2 Hexamethyl disiloxane Ethanol 0.1-1 2/7 - 6/4
Fe2O3 Iron acetyloacetonate Acetonitrile/2-
ethylhexanoic acid (1:1) 0.01-0.5 2/7 - 6/4
Ag Silver nitrate Ethanol/diethylene glycol
monobutyl ether (1:1) 0.06-0.31 2/7
Ag/SiO2 Silver
nitrate/Hexamethyl disiloxane
Ethanol/diethylene glycol monobutyl ether (1:1)
0.1 2/7
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Controlling both the aerosol size (airborne phase) and primary particle
size: In order to investigate whether VENGES can successfully vary both the size of
the generated aerosol as well as the primary particle size, a study was performed by
varying two important VENGES process parameters: i) liquid precursor feed rate
(from now on: x) over the O2 dispersion flow rate (from now on: y) ratio (x/y); ii)
liquid precursor molarity, M (mol/L). Table F.1 summarizes for each ENM the x/y
ratios and the variable molarity experiments, used in the experiments. For all
experiments, the properties of the ENM VENGES generated aerosol were
characterized in situ in real-time. In addition, the physicochemical and
morphological characterization of the ex-situ collected nanoparticles was also
performed off line as previously described.
Reproducibility of the generated test aerosols over time: For inhalation
toxicological studies, it is important to: a) generate in a reproducible manner test
aerosols with constant concentration and size distribution for the duration of the
exposure (hours); and b) for dose/response toxicological studies, the number
concentration should be able to be controlled. The ability of VENGES to fulfill both
requirements was explored by varying the ratio of the sampling to the dilution flow
rate (QS/QD). ENM were synthesized at constant precursor molarity and x/y ratio
was kept constant. The QS/QD ratio varied in a step-wise manner over a period of
more than 5 hours of continuous ENM aerosol generation. The mass concentrations
were calculated from the FMPS data assuming Fe2O3 density of 5.24 g/cm3 for the
test aerosol and 1 g/cm3 for indoor particles.
ENM surface modification experiment: The VENGES system also allows
for generation of surface modified test aerosols in the gas phase [37]. At the
nanoscale level, surface properties and chemistry may affect the biological
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properties of ENM and are among the important parameters for toxicological
investigation. Surface modification is also one of the emerging methods for safer
nanomaterial formulation. Surface modification may result in a less toxic ENM
while its important electrical or structural properties of interest remain unchanged.
Here, VENGES was used to generate SiO2 nanoparticles and their surface was
modified in situ by adding nanosilver particles on their surface [32]. The total
particle number concentration and size distributions of both the basic SiO2 and the
surface modified Ag/SiO2 aerosol properties were measured in situ. Furthermore,
ex-situ characterization of the collected ENM was also performed as described
above.
F.2.3 Acute Pulmonary and cardiovascular effects of inhaled nanostructured Fe2O3 using the
VENGES platform and IVCL assay
The suitability of VENGES platform for in vivo animal inhalation studies
was demonstrated here. The pulmonary and cardiovascular effects of inhaled Fe2O3
nanoparticles were assessed. Fe2O3 nanoparticles have been used widely in a number
of applications including drug delivery, bio-imaging and nutrition [38-40]. The
toxicological assay used to assess health outcomes in this study was in vivo
chemiluminescence (IVCL) of the lung and heart surface immediately after
exposure; this technique has been widely used in our laboratory for ambient particle
toxicity studies and is a highly sensitive method for identifying pulmonary and
cardiovascular responses to inhaled particles under relatively small doses and acute
exposures [29,41].
Protocol: Male Sprague-Dawley rats (200-250 g) were obtained from Taconic
Laboratories (Rensselae, NY), housed, and managed according to the NIH
guidelines for the care and use of laboratory animals. Upon arrival, animals were
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assigned a unique identification number, which determined the exposure date and
exposure group (Aerosol or Filtered Air) for the animal. Rats were allowed to
acclimate to the animal facility for 4-5 days prior to start of experiments. The
Harvard Medical Area’s Animal Use Committee approved the animal protocols
used in this study. Each day two animals were exposed to Fe2O3 test aerosol and
two animals to filtered air (sham) in individual exposure chambers for 5 hours. Four
repetitions (days) with a new set of animals each day were carried out. At the end of
each five-hour exposure, two animals from each group had IVCL [42].
F.3 Results and discussion
F.3.1 Performance characterization experiments
Effect of x/y ratio on both the aerosol size (airborne phase) and primary
particle size: Figure F.2a illustrates the particle number concentration as a function
of mobility diameter for pure SiO2. It is worth mentioning that the mobility diameter
is correlated to the aerodynamic diameter and volume equivalent (thermodynamic)
diameter [43-45]. Particles that are sized based on their mobility in an electric field
are considered for practical purposes classified by their thermodynamic diameter
[46,47]. Thermodynamic diameter is an important determinant of nanoparticle lung
deposition [48]. As shown in the figure, the VENGES generated aerosol has a
unimodal and narrow size distribution. As inset in Figure F.2a, the mode, mean,
median diameters and the geometric standard deviations are presented showing the
statistical analysis of the obtained particle size distributions [49]. Additionally, the
modal diameter increases with increasing x/y ratios, as the SiO2 aerosol
concentration increases. It is clear that VENGES not only has the ability to generate
aerosols with unimodal size distributions but also has the ability to adjust the
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aerosol size. This can be seen more clearly in Figure F.2b, where greater modal
diameters (circles, left axis) were indicated for larger x/y ratios. This is attributed to
the higher particle concentration with higher x/y ratio during their flame formation
that leads to larger aggregates/agglomerates. It is noteworthy that all particle size
distributions are stable over time, as it will be discussed later on (see Figure F.5a
and Figure F.6b).
Figure F.2b compares the average primary particle diameter of the collected
ENM as obtained by ex-situ N2 adsorption with their airborne phase modal
diameter. The average SiO2 primary particle size of the collected ENM decreases
with increasing x/y ratios [32,34]. However, there is a difference between the
primary particle size of the collected ENM determined by N2 adsorption and the
modal diameter of the same particles in their airborne phase. This discordance
between the mobility (airborne phase) and the primary particle diameter
(nanopowder form) indicates, that the latter is not necessarily a good predictor of
the ENM behavior when it becomes airborne. In other words, the nanopowder-
derived primary particle diameter is not a good predictor of its fate, transport and
deposition in the environment and in human or animal lungs. This finding may
explain some of the discrepancies of the toxicological results reported in the
literature between in vivo and in vitro ENM studies [20,21,24]. The in vivo
validation of in vitro (cellular) studies is considered an important element of the
proposed Environmental Health and Safety strategy recently released by the
National Nanotechnology Initiative (NNI).
Similarly, Figure F.2d shows the effect of x/y ratio on the primary particle
and the mobility diameter of Fe2O3. Figure F.2c also shows the XRD patterns of the
ex-situ collected and characterized nanoparticles as a function of the x/y ratio.
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Figure F.2: (a) Mobility size distributions of SiO2 for a varying x/y ratio and the mode,
median, mean diameters and the geometric standard deviation are shown. (b) The mode
mobility (circles, left axis) and primary particle diameter (triangles, right axis) as a function
of the x/y ratio. (c) XRD patterns of Fe2O3 nanoparticles for different x/y ratios. (d) Mode
mobility (circles, left axis), primary particle (open triangles, right axis) and crystal (filled
triangles, right axis) diameters as a function of the x/y ratio. The precursor molarity was
0.5 M and the total particle number concentration (CT) 2·105 #/cm3 for both SiO2 and Fe2O3
samples.
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This indicates that larger Fe2O3 particles are formed with higher x/y ratio, in
agreement with literature [32,40]. This is due to their larger residence time in higher
temperature zones that result in larger crystals. It is worth pointing out that the
XRD-estimated average crystal particle sizes (filled triangles, Figure F.2d) are
consistent with their average primary particle sizes (open triangles, Figure F.2d),
with the latter having slightly larger values indicating polycrystalline or aggregated
nanoparticles. The modal mobility diameter for the Fe2O3 aerosol (Figure F.2d,
circles, left axis) is not significantly affected by the x/y ratio, as for most ratios it is
fairly constant. Furthermore, as it is shown above in the case of SiO2, there is a
difference between mobility diameter (airborne phase) and the primary particle
diameter (nanopowder form).
Effect of precursor molarity on both the aerosol size and primary
nanopowder particle size: Figure F.3 shows the effect of another important process
parameter of VENGES, the metal precursor molarity, on both the aerosol size
(mobility diameter) and primary nanopowder particle size for SiO2, Fe2O3 and Ag
ENM. Figure F.3a shows for the SiO2 aerosol, the particle number concentration as
a function of mobility diameter (for a constant 2/7 ratio) for different metal
precursor molarities with their corresponding diameters and geometric standard
deviation as inset. It can be seen that the mobility size distribution shifts to larger
sizes with an increased precursor molarity. It is worth mentioning that there is an
overlapping among the different particle size distributions presented in the figure.
However, the aerosol size distributions were randomly selected to prove the point
that aerosol properties can be adjusted. Whether this size distribution differences are
clinically relevant, it remains to be seen in future toxicological studies.
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Figure F.3: Mobility size distributions of SiO2 (a), Fe2O3 (c) and Ag (f), for different precursor
molarities with the mode, median, mean diameters and the g. (b,d) Mode mobility size
distributions (circles, left axis) primary particle (open triangles, right axis) and crystal (filled
triangles, right axis) diameters for SiO2 (b) and Fe2O3 (d). (e) TEM image of Fe2O3
nanoparticles for precursor molarity 0.5 M and 2/7 x/y ratio.
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Similarly, this pattern can be seen also in Figure F.3b, where the mode
mobility diameter (circles, left axis) is also increasing for higher precursor molarity.
This could be attributed to the higher concentration of the particles at an early stage
during their synthesis that would lead to formation of larger agglomerates. In
contrast to the behavior of the SiO2 particles in airborne phase, the average primary
particle diameter (dBET, triangles, right axis) does not significantly vary with
precursor molarity [50] as sintering rather than coagulation determines the primary
particle size in contrast to mobility diameter that is determined by coagulation and
particle concentration levels.
For Fe2O3 nanoparticles the mobility size distributions shift to larger sizes for
higher precursor molarities (Figure F.3c and Figure F.3d). The average primary
particle (Figure F.3d, dBET, open triangles, right axis) and the crystal (Figure F.3d,
dXRD, filled triangles, right axis) diameter seem to slightly increase for higher
precursor molarity, forming polycrystalline nanoparticles, as with SiO2. The TEM
image of Fe2O3 nanoparticles shown in Figure F.3e shows that there are many
agglomerates/aggregates composed of smaller primary particles, in agreement with
the results obtained by XRD, N2 adsorption and mobility size distribution analysis.
Finally, Figure F.3f indicates that the mobility size distribution of nanosilver (Ag)
can be also shifted to larger values for higher precursor molarity.
Control of concentration/Reproducibility of the generated test aerosols
over time: We have demonstrated above that VENGES has the ability to control
both the aerosol or agglomerate size (airborne phase) and the primary particle or
crystal size (nanopowder form), in a highly reproducible way. However, for
inhalation toxicological studies, aerosol generation methods need to be able to i)
adjust the concentration level (dose/response studies); and ii) deliver test aerosols
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with constant properties for hours. Figure F.4a shows the total particle number
concentration CT (solid line) and the modal (broken line) and median (dotted line)
mobility diameters of the nanosilver aerosol (2/7, 0.02 M precursor concentration)
as a function of exposure time. The dilution of the sampled aerosol (Qs/QD, Figure
F.1) was periodically adjusted in a stepwise manner during the experiment to
control the concentration level. As expected, for higher QS/QD ratio, higher CT is
reached. Most importantly, when a constant QS/QD ratio is used, both the aerosol
total particle concentration (CT) and the size distribution indicated by the modal and
median (dotted line) mobility diameters remain constant. This is clearly an
indication that VENGES can control the aerosol concentration levels and deliver
test aerosols with constant size distributions over time.
Figure F.4: Controlling total particle number concentration and mobility size distribution by
dilution. Total particle number concentration (CT, solid lines, left axis), mode (broken lines,
right axis) and median (dotted lines, right axis) mobility diameters of nanosilver (a) and
Fe2O3 (b) nanoparticles for different QS/QD ratios.
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It is noteworthy that when the QS/QD ratio is adjusted back to its initial
value, the aerosol size distribution returns to its initial one, a clear indication of the
reproducibility of the method. Similarly, for Fe2O3, Figure F.4b shows this constant
aerosol particle concentration and size distribution over time for different QS/QD
ratios.
Figure F.5: (a) Mobility size distributions of SiO2 (triangles) and composite Ag/SiO2
nanoparticles (circles, Ag-content 10 wt%) for 0.10 M precursor molarity and 2/7 ratio for a
total particle number concentration CT = 2·105 #/cm3. The size distribution remains the
same, as seen also in the inset, where the mode, median, mean diameters and the geometric
standard deviation are also shown. The error bars correspond to the standard deviation of the
particle concentration corresponding to each particle size over time. (b) STEM image of the
Ag/SiO2 nanoparticles. The nanosilver particles (bright spots) are homogeneously dispersed
on the amorphous SiO2 matrix (diffuse gray).
ENM surface modification: The surface of ENMs can be modified in order
to add desired attributes such as dispersibility [35] (e.g. SiO2-coated Ag) [51] or
antibacterial activity [15] (e.g. SiO2 supported nanosilver). It may also be a useful
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concept for the formulation of safer ENM [32,51]. Recently, it was shown that
doping ZnO nanoparticles with Fe can reduce the release of Zn ions [52]. Figure
F.5a illustrates the particle number concentration as a function of mobility diameter
for both pure SiO2 and for the surface modified nanocomposite Ag/SiO2 (Ag-
content 10 wt%). Both size distributions are almost identical (Figure F.5a) as
verified also by the mode, median, mean diameters and the geometric standard
deviations (inset). This indicates that VENGES has the ability to generate surface
modified nanoparticles while maintaining the intended size distribution. This will be
useful when studying the comparative toxicity of surface modified nanoparticles in
vivo. By controlling the size, we can also influence control the particle deposition in
the lungs. In addition, ENMs can also be used as a “vehicle” for delivery of drugs in
vivo. Figure F.5b shows a STEM image of the composite Ag/SiO2 nanoparticles.
The nanosilver particles (bright spots) are homogeneously dispersed on the
amorphous SiO2 (diffuse gray) surface [15,32].
F.3.2 Pulmonary and cardiovascular effects of inhaled nanostructured Fe2O3 using the
VENGES platform
Figure F.6a indicates the total particle number concentration as a function of
time for the 5 hour exposure period for the Fe2O3 aerosol (2/7 ratio, 0.02 M
precursor molarity). As shown in this figure, the concentration levels remained
fairly constant for the whole animal exposure (the 5 minute stops indicated in the
figure were necessary in order to replace the syringe with liquid precursor). For this
exposure scenario, VENGES was tuned to generate a test aerosol of a total number
concentration of 2-3·105 particles/cm3 (this would correspond to approximately to
100-200 μg/m3), approximately 10-20 times higher concentration of indoor
conditions (10 g/m3). The temperature, relative humidity, CO2, CO and NO2
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concentrations of the test aerosol were identical to the room conditions, ensuring no
interference with the in vivo toxicological results. Additionally, Figure F.6b shows
the averaged mobility size distribution of the test aerosol. The error bars correspond
to the standard deviation of the particle concentration for each particle size, over the
exposure period. This clearly shows that VENGES can generate a highly
reproducible exposure aerosol with constant properties (size, size distribution and
concentration) for toxicological inhalation studies.
Figure F.6: (a) Total particle number concentration (CT) of the test ENM aerosol containing
Fe2O3 nanoparticles monitored continuously over the whole period of exposure for in vivo
toxicological characterization. The CT remains constant at approximately 2-3·105 #/cm3.
Every 25 minutes there was a 5 minutes break during the system was filled up (gray area). (b)
Mobility size distribution averaged over the periods that the CT was constant. The mode,
median, mean diameters and the geometric standard deviation are also shown. The error bars
correspond to the standard deviation of the particle concentration corresponding to each
particle size over time.
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Figure F.7 shows the in vivo chemiluminescence (IVCL) difference (counts
per second-cps/cm2) of lungs and hearts, which corresponds to the relative reactive
oxygen species (ROS) concentration in those organs of both exposed (red) and
unexposed (blue) groups of animals. The error bars correspond to the standard
deviation of the data points and the significance level of the measured data is
P < 0.001. The IVCL measurements in the lungs of the exposed animals were about
60 times higher than for the unexposed animals, indicating that the Fe2O3 test
aerosol increased ROS in the lungs. In addition, oxidative stress was present in the
heart of the animals (11-fold increase in the chemiluminescence of the heart).
Figure F.7: In vivo chemiluminescence of the lungs and hearts of the exposed to the test
aerosol for 5 hours rats (red) and of the ones to filtered room air (blue). The error bars
correspond to the standard deviation of the data and the significance level P is also shown.
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A promising technological platform suitable for both in vitro and in vivo
toxicological characterization of engineered nanomaterials, with emphasis on the
cardiovascular and pulmonary effects of inhaled ENM, is presented in this study.
ENM are produced continuously in the gas phase allowing their continuous transfer
to inhalation chambers, with minimal alterations in their state of agglomeration.
Defining properties of the generated aerosols (i.e. primary and aerosol particle size,
concentration, shape, state of agglomeration, surface chemistry) can be easily
modified by adjusting simple process parameters allowing for both in vitro and in
vivo investigations of toxicity. The ability of the developed technique to generate a
variety of industry relevant, property controlled exposure atmospheres for inhalation
studies was systematically investigated and documented in the previous section.
The suitability of the technique to characterize the pulmonary and
cardiovascular effects of inhaled ENM in intact animal models was also
demonstrated here using the highly sensitive IVCL assay. IVCL measures the
reactive oxygen species generation (ROS). ROS and free radical generation is
considered one of the primary mechanisms of nanoparticle toxicity; it was shown in
many ambient particle health effect studies that ROS generation may result in
oxidative stress, inflammation, and damage to proteins, membranes and DNA [25-
31]. ROS generation has been also found in many ENMs including carbon based
ENMs (fullerenes, carbon nanotubes) and metal oxides [53]. Our results indicate
that moderate acute exposures to inhaled nanostructured Fe2O3 can generate ROS
and oxidative stress and cause both pulmonary and cardiovascular effects. The
IVCL measurements in the lungs of the exposed animals were about 60 times higher
than for the unexposed animals, indicating that the Fe2O3 test aerosol increased
ROS in the lungs (Figure F.7). This oxidative stress was also present in the heart of
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the animals showing that the inhalation of ENM influences not only the respiratory
but also the cardiovascular system with an 11-fold increase in the
chemiluminescence of the heart. This substantial effect was found with a moderate
mass exposure concentration of 200 μg/m3, a concentration approximately 20 times
the fine particle concentration of room air. The substantial IVCL response observed
in our study, indicates that these particles reached deep in the lung and evoked a
toxicological effect. The increased IVCL response of the heart may indicate a direct
effect on the heart [41] but also may be a manifestation of indirect effects via the
autonomic nervous system [54,55]. It should be noted, however, that the proposed
platform is not only limited to the evaluation of pulmonary and cardiovascular
effects. It can also be used to assess other biological outcomes related to inhaled
ENM and it can be a powerful tool to understand the link between certain ENM
properties and their bioavailability and toxicity.
F.4 Conclusions
In conclusion, this novel approach enables us to generate industry-relevant,
property controlled ENM exposure atmospheres suitable for inhalation toxicological
studies and assess the link between ENM physicochemical properties and specific
biological outcomes. In addition, the documented in the study ability of the
technique to alter in situ ENM surface properties can be one of the ways to further
explore the formulation of safer ENM. Furthermore, this technological platform can
be a powerful tool for validation of in vitro screening assays with adverse biological
effects in intact animals, an important element of the strategy recently proposed by
the National Nanotechnology Initiative (NNI) on Environmental Health and Safety
of Engineered nanomaterials. Its future use will help to assess the cardiovascular,
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pulmonary and other toxicological effects of inhaled ENM and improve our
understanding on our central hypothesis that physical and chemical characteristics
of ENM determine their bioavailability, redistribution, and toxicity in the lungs and
elsewhere.
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APPENDIX G
G Multi-layer Polymer Nanocomposite Films
Abstract
The synthesis of multi-layer polymer nanocomposite films with
superparamagnetic filler particles was achieved by flame aerosol deposition and
subsequent spin-coating of the polymer solution. The filler film thickness was
controlled by the deposition time. The as-deposited filler films were mechanically
stabilized by in situ annealing. The synthesis of multi-layer films can be achieved by
repeating the process. Free-standing microstructures can be made by depositing the
polymer nanocomposite films on a sacrificial layer. The filler film stays intact after
the addition of the polymer, resulting in high-filler-fraction polymer nanocomposites
with uniquely homogeneous distribution. The polymer nanocomposite films
exhibited superparamagnetic behavior and their magnetic actuation was
demonstrated. When the deposition of different materials (e.g. plasmonic,
phosphorescent) was performed, multi-functional nanocomposites were synthesized.
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G.1 Introduction
Polymer nanocomposite materials are advantageous because they combine
the desired mechanical properties of the polymer and the functionality of the filler
used. Several methods have been employed for the synthesis of sophisticated
polymer nanocomposites such as magnetic actuators [1], dichroic films [2],
luminescent polymers [3], to name a few. In most methods, the dispersion of the
filler particles within the polymer matrix cannot be easily achieved. Therefore, often
an extra process step is performed for the surface modification of the filler
material [4]. However, even when such extra process steps are used, a high
(> 5 vol%) filler fraction cannot be achieved within the nanocomposite with a
homogeneous distribution [5].
Here, a novel, versatile method is developed with which the synthesis of
high-volume-fraction and homogeneous polymer nanocomposites are made. The
process is composed of two steps: first the filler material is directly deposited on a
substrate and stabilized by flame aerosol deposition [6], and second the polymer
solution is spin-coated on the as-prepared filler film. The mechanical stabilization of
the filler film is crucial, as it helps retain its structure and not collapse after the spin-
coating of the polymer solution. The process allows for a precise control over the
filler-content in the nanocomposite by adjusting the filler deposition time and the
polymer solution spin-coating speed. Additionally, the synthesis of multi-layered
nanocomposite structures is also possible, by repeating the process numerous times.
G.2 Materials and methods
This has been realized here by synthesizing superparamagnetic
nanocomposite films. Nanosized (dp = 10 nm) superparamagnetic iron oxide
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nanoparticles [7] were deposited on polymer-coated glass or silicon substrates by
flame aerosol deposition. The resulting filler film was in situ annealed and
mechanically stabilized [6]. Its thickness was controlled by adjusting the deposition
time. The final polymer nanocomposite was made by spin-coating the polymer
solution (PMMA in anisole, 10 wt%) and its thickness was adjusted by the spin-
coating speed. The solvent was then let to evaporate, leaving behind a thin polymer
film. Multi-layered films were made by repeating the above process.
G.3 Results and discussion
Figure G.1a shows the assembly of the multilayer nanocomposite films.
Initially, a thin polymer film (e.g. PMMA) is deposited by spin-coating on a
substrate (e.g. silicon, metal, glass). The initial polymer film thickness can be easily
tuned by the spin-coating speed and/or the viscosity of the polymer solution. The
nanostructured filler particle porous film is then deposited on the polymer film and
in situ stabilized. With this last step its porosity can be tuned from 60-98% that
affects the final filler loading. The inorganic/polymer nanocomposite is made by
subsequently spin-coating the polymer solution on the nanostructured porous
particle film. The liquid polymer solution fills the porous particle film, and by
varying the spin-coating conditions the inorganic loading in the nanocomposite is
also controlled (Figure G.1b). By repeating the particle film deposition and polymer
spin-coating a number of times, multiple layers can be made, either of the same or
different material. It should be noted that flame aerosol synthesis allows for a wide
selection of product nanomaterials, broadening therefore, the potential target
applications of such inorganic/polymer nanocomposites. By choosing appropriate
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precursors, the synthesis of multilayer and multifunctional polymer nanocomposites
(e.g. magnetic-plasmonic, magnetic phosphorescent) is therefore feasible.
Figure G.1: The assembly procedure of single- and multi-layer nanocomposites. (a) The
substrate is first coated by a thin polymer film on a sacrificial layer, then follows the porous
film deposition and stabilization and the polymer solution spin-coating. If the above is
repeated numerous times, multi-layer films can be made, that can be further released from the
substrate forming free-standing nanocomposites.
One main advantage of the current process is the ability to synthesize free-
standing nanocomposite films. By first depositing a sacrificial layer on the substrate,
it is possible to release the inorganic/polymer nanocomposite and obtain a free-
standing multi-functional film (Figure G.1c) with thickness from tens of nanometers
to a few microns. This indicates the compatibility of this process with existing
synthesis of magnetic cantilevers that are integrated in microelectromechanical
systems (MEMS) for biosensing. Currently, similar processes for synthesis of
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multilayer films include the layer-by-layer (LbL) assembly technique. The
nanocomposite assembly technique presented here, however, offers several
advantages: (i) there is a broad selection of inorganic filler material that does not
have to be charged in order to be deposited, (ii) it involves fewer process steps than
the LbL technique, since the nanoparticle synthesis and their deposition occurs in a
single-step, (iii) there is practically no limitation on the chosen polymer provided
that it can be dissolved in a solvent or the corresponding monomers are in the liquid
phase, in contrast to the polymers used by LbL that ought to be charged, (iv) the
technique employed here allows for the synthesis also of relatively thick films in the
range of 1-10 m and is scalable and CMOS wafer-level compatible. Nanocomposite
films with such a thickness made by LbL would require numerous alternate
immersions in the opposite charged solutions since with each step the film would
grow approximately 10-200 nm.
Figure G.2a shows the film coating density, which is equivalent to film
thickness for a given porosity, of aerosol generated iron oxide nanoparticles
deposited mostly by thermophoresis on the water-cooled substrate. The main crystal
phase of the flame-made iron oxide nanoparticles is maghemite (-Fe2O3) with an
average crystal size of about 9 nm and is superparamagnetic in room temperature
with a saturation magnetization of Ms = 37 emu/g [7]. The as prepared film is
highly porous, about 97-98% (Figures G.2b,d), as typically obtained by flame
aerosol deposition [6]. The coating density clearly increases with longer deposition
time allowing therefore, the fine-tuning of the as prepared nanoparticle film
thickness. After the in situ stabilization, however, the particle films are significantly
compacted regardless of deposition time, decreasing therefore their porosity to 60-
70% (Figure G.2c,e). In fact this fine tuning on the particle film porosity offers the
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possibility to tune the loading of the inorganic particles in the final nanocomposite
while the film homogeneity and distribution on the substrate in the planar
dimensions is retained.
Deposition time, s0 50 100 150
Coating den
sity, m
g/cm
2
0.00
0.05
0.10
0.15
10 μm
d
e
a
cb as prepared particle film
stabilized particle film
as prepared particle film
stabilized particle film
porosity 98% porosity 60%
porosity 98%
porosity 60%
Figure G.2: (a) The coating density (corresponding to particle film thickness) as a function of
the deposition time. The particle film thickness can be finely tuned by varying the deposition
time.
The distribution of the inorganic filler film within the polymer
nanocomposite is strongly influenced by the stability of the as deposited inorganic
nanoparticle film. Figure G.3a shows a cross-sectional SEM image of a
nanocomposite film after the spin-coating of the polymer on a not stabilized iron
oxide particle film (Figure G.2b,d). The forces of the polymer solution during the
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spin-coating are strong enough to compact the loosely connected as-deposited
nanostructured film. Even though, however, the particle film collapses, the
homogeneity in the planar dimensions is retained. In fact, by repeating the polymer
solution spin-coating and the particle deposition, multilayer films can be made by
precisely tuning the distance between the particle films by the spin-coating
conditions (Figure G.3b). Figure G.3e shows a multilayer iron oxide/polymer
nanocomposite film consisting of six particle films embedded in the polymer, having
on top a highly porous film. All of the particle films have identical deposition time,
including the top one. The initial particle film thickness with porosity 97% is about
8 m (top particle film) [6], however, after its compaction is reduced to about
0.5 m as shown in Figure G.3f. Therefore, by tuning the process parameters the
formation of precisely spaced particle films embedded within a polymer is possible,
emphasizing the versatility of the employed nanocomposite assembly technique.
When, however, the as-deposited particle films are stabilized by in-situ
annealing (Figure G.2c,e), no structural change occurs after the spin-coating of the
polymer solution (Figure G.3c). This is attributed to the higher adhesion and
cohesion of the stabilized films which is sufficient to withstand the forces from the
polymer deposition. Furthermore, one major difference between the as-deposited
and stabilized particle films is their porosity and consequently the filler loading
(wt%) of their corresponding inorganic/polymer nanocomposites. The lower
porosity of the stabilized film enables their nanocomposites to obtain high loadings
up to 30-40 vol%, but still preserving their homogeneity throughout the whole film.
This is a major advantage of this assembly technique, as it overcomes the
nanoparticle agglomeration that increases with a higher filler content, without the
need of any extra process-step of surface functionalization. This enables such
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nanocomposites to exhibit indeed superior filler-specific functionality (e.g. high
magnetization) without compromising the film homogeneity and surface
smoothness.
Figure G.3: Cross-sectional SEM images of nanocompsoites consisting of: a single-layer (a)
and three-layer (b) not mechanically stabilized particle films, single-layer (c) and three-layer
(d) mechanically stabilized film, 6-layer with the deposited seventh layer before the polymer
solution spin-coating (e) and a ten-layer nanocomposite of not mechanically stabilized film.
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When this procedure is repeated a number of times, the formation of
multilayer nanocomposites with stabilized particle filler films is also possible.
Figure G.3f shows a cross-sectional SEM image of a three-layer iron oxide/PMMA
nanocomposite. The stabilized dense particle films are completely embedded in the
PMMA and are separated by a tunable spacing by adjusting the speed during the
polymer deposition by spin-coating. Such structures could improve the final
nanocomposite performance or even include multiple functionalities by changing
the deposited filler material as it will be shown later on.
The morphology of the iron oxide/PMMA nanocomposites also influences
the final magnetic perofmance. Figure G.4a shows the hysteresis curves of three iron
oxide/PMMA nanocomposites: a three-layer composite with its filler particle film
not stabilized (green line), a single-layer nanocomposite with stabilized particle film
(red line), and a three-layer nanocomposite with stabilized particle films (blue line).
The single-layer nanocomposite with stabilized particle film has the highest
magnetization (Ms = 6.1 emu/g of composite). It is the highest reported value, to
the best of our knowledge, for a composite with homogeneous inorganic filler
distribution and controlled surface smoothness, as there is no large agglomerate
formation that would otherwise occur for higher filler loadings [5]. The three-layer
nanocomposite has magnetization Ms = 4.3 emu/g and this lower value is most
probably attributed the lower filler loading (there is more polymer present between
the layers) than the single-layer one. However, the Ms of the three-layer
nanocomposite of the not stabilized iron oxide particle film is significantly lower
(Ms = 2.5 emu/g) when compared to the magnetization values obtained by the
nanocomposites of the stabilized iron oxide particle films. This clearly shows the
advantage of the in-situ stabilization of the particle films, that not only helps the
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inorganic film retain its homogeneity after the spin-coating of the polymer solution,
but also facilitates the synthesis of high filler-content of inorganic/polymer
nanocomposites.
External Field, mT‐1000 ‐500 0 500 1000
Magnetization, emu/g
‐6
‐4
‐2
0
2
4
6 1 yes
3 yes
3 no
‐20 ‐10 0 10 20‐2
‐1
0
1
2
# of layersParticle layerstabilized
Figure G.4: Magnetization hysteresis curves for single-layer (red line) and three-layer (blue
and green lines) of mechanically stabilized particle films (red and blue lines) and not stabilized
(green line). (b,c) Images of the single-layer stabilized films in the absence (b) and presence of
a magnetic field (c) induced by a permanent magnet.
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These nanocomposites exhibit a highly superparamagnetic behavior, since
there is no significant hysteresis in their magnetization curves, as shown in the inset
of Figure G.4a. This indicates that the particles have retained their
superparamagnetic properties and that the presence of the polymer does not
influence these properties, forming therefore, polymer nanocomposites that can be
actuated in the presence of a magnetic field. The magnetic response of the single-
layer nanocomposite of a stabilized iron oxide particle film that exhibited the
highest magnetization (red line) is shown in Figure G.4 in the absence (b) and
presence (c) of a magnetic field. Clearly, when a permanent magnet is approached
the 3.5 mm thin polymer nanocomposite is actuated. Therefore, the manufacture
approach here allows for the realization of superparamagnetic inorganic/polymer
nanocomposites with high filler-content and superior performance using
nanotechnology with proven scalability.
As mentioned already before, flame aerosol technology allows for synthesis
of a broad range of smart and sophisticated nanomaterials with a fine control on
their morphology, crystallinity and primary particle size. The novel manufacture
technique presented here capitalizes on this unique control over the nanoparticle
physico-chemical properties. After the initial formation of a single-layer
superparamagnetic nanocomposite, a second particle film can be deposited
consisting of another material, for example, plasmonic. Subsequently, the PMMA
solution can be spin-coated forming, therefore, multi-functional nanocomposites.
Figure G.5 shows a SEM image of such a nanocomposite film consisting of a
stabilized iron oxide particle film on the bottom and having a second particle layer
on top of composite silver/silica nanoparticles (50 wt% Ag, dp,Ag = 9 nm), spaced by
PMMA with controllable thickness. The higher-proton number silver appears
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brighter in these SEM image, clearly showing the particle film homogeneity and
further verifying that this homogeneity can be retained not only for iron oxide but
also for other materials.
Wavelength, nm300 400 500 600 700
Extinction, a.u.
0
1
2
3
4Plasmonic‐Superparamagnetic NC
Superparamagnetic NC
External Field, mT‐1000 ‐500 0 500 1000
Magnetization, emu/g
‐3
‐2
‐1
0
1
2
3
‐15 ‐10 ‐5 0 5 10 15‐1.0
‐0.5
0.0
0.5
1.0
Figure G.5: (a) A double-layer nanocomposite consisting of magnetic iron oxide layer and a
plasmonic nanosilver layer. This hybrid nanocomposite exhibits both the superparamagnetic
performance (b) and the plasmonic performance (c).
The magnetic behavior of this hybrid nanocomposite is shown in
Figure G.5b. Clearly, this nanocomposite retains the superparamgnetic
performance, as there is no hysteresis, reaching a saturation magnetization of Ms =
2.4 emu/g. This Ms value is lower than the single-layer iron oxide/PMMA
nanocomposite and this is attributed to the presence of the magnetically inactive
silver/silica nanoparticles. Such Ms is comparable to the ones achieved by other
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manufacture techniques of nanocomposites containing only iron oxide nanoparticles
[5]. The presence of the plasmonic silver nanoparticles enables this hybrid
nanocomposite to be employed in applications such as biosensing or
optoelectronics. Figure G.5c shows the extinction spectra of a single-layer
superparamagnetic nanocomposite (red line) and a hybrid plasmonic-
superparamagnetic (green line). The hybrid plasmonic-superparamagnetic
nanocomposite exhibits a strong plasmon peak around 400 nm, characteristic for
silver nanoparticles. This plasmonic properties of such hybrid nanocomposite here
can facilitate its employment in applications where both the magnetic and
plasmonic performance is required. It should be noted that with the manufacture
technique presented here, the particle filler nanoparticles can consist of multiple
materials, thus exhibiting the desired properties, for example composite silver/iron
oxide nanoparticles.
Figure G.6: (a) Phosphorescence of a stabilized Y2O3:Eu3+ particle film (blue line) and its
corresponding polymer nanocomposite (red line), both showing an intense emission around
612 nm, characteristic for the Eu3+ transitions. (b) An SEM image of a hybrid magnetic-
phosphorescent nanocomposite, that exhibits the magnetic actuation only in the presence of a
magnetic field (c,d) and the red color emission under UV irradiation (e).
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Furthermore, the novel nanomanufacture technique here allows the synthesis
of multi-layer nanocomposites without any polymer between the two inorganic filler
materials. Figure G.6a shows a SEM image of a multi-layer nanocomposite
consisting of an initial iron oxide film, followed by a phosphorescent yttrium oxide
doped with europium particle film and a PMMA layer. In this case, after the initial
iron oxide deposition and stabilization, the phosphorescent film deposition followed
immediately without any PMMA layer in between. Again the higher proton-number
yttrium enables the Y2O3:Eu3+ film brighter than the iron oxide. After the deposition
of the second material, a final PMMA layer is applied forming therefore, hybrid
phosphorescent-superparamagnetic nanocomposites.
Figure G.6b shows the emission phosphorescent spectra of an as-prepared
Y2O3:Eu3+ film (blue solid line, no PMMA) and of a phosphorescent
Y2O3:Eu3+/PMMA nanocomposite (red broken line) for excitation 254 nm. The
characteristic emission peak at 612 nm attributed to the Eu3+ ions transitions is
present in both those cases. The emission intensity is slightly higher for the
Y2O3:Eu3+ particle film than the polymer nanocomposite. This is attributed to the
absorption of the excitation irradiation by the PMMA film, and therefore, the Eu3+
ions are excited with less power. Nonetheless, with such a method, it is possible not
only to obtain phosphorescent polymer nanocomposite but to include an extra
functionality such as magnetic. In fact, such multi-functional nanocomposite can be
also actuated by an external magnetic field (Figure G.6c,d) and simultaneously
exhibit the characteristic phosphorescent properties when excited with UV
irradiation (Figure G.6e).
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G.4 Conclusions
Summing up, a scalable flame aerosol technology was utilized here to
produce high-filler-content polymer nanocomposites with a uniquely homogeneous
dispersion within the polymer film. This was achieved by depositing the filler film
on a substrate and mechanically stabilizing it by in situ annealing. The polymer
solution was then spin-coated on the filler film. A precise control over the filler
fraction and the total film thickness can be easily obtained by tuning the process
parameters. The synthesis of multi-layered nanocomposite films was also
demonstrated by repeating the process numerous times. Even though here this was
realized for superparamagnetic polymer (PMMA) nanocomposite with
unprecedented maximum magnetization (6.1 emu/g of composite) and high-filler-
contents, this process is not limited only to these materials. Filler particles can be
most metal oxides that can be made by flame-spray-pyrolysis, including
nanophosphors for luminescent polymer nanocomposites, high-dielectric-constant
materials for dielectric applications, plasmonic materials for dichroic films, UV
absorbing fillers to name a few. Additionally, the polymer chosen is not limited to
PMMA as used here, but expands to most thermoplasts, silicones, polyethylene and
others.
G.5 References
[1] Hua, F., Cui, T. & Lvov, Y. M. Ultrathin cantilevers based on polymer-
ceramic nanocomposite assembled through layer-by-layer adsorption. Nano
Lett. 4, 823-825 (2004).
[2] Caseri, W. R. Nanocomposites of polymers and inorganic particles:
preparation, structure and properties. Mater. Sci. Technol. 22, 807-817 (2006).
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[3] Bao, Y., Luu, Q. A. N., Lin, C., Schloss, J. M., May, P. S. & Jiang, C.
Layer-by-layer assembly of freestanding thin films with homogeneously
distributed upconversion nanocrystals. J. Mater. Chem. 20, 8356-8361 (2010).
[4] Camenzind, A., Caseri, W. R. & Pratsinis, S. E. Flame-made nanoparticles
for nanocomposites. Nano Today 5, 48-65 (2010).
[5] Suter, M., Ergeneman, O., Zurcher, J., Schmid, S., Camenzind, A., Nelson,
B. J. & Hierold, C. Superparamagnetic photocurable nanocomposite for the
fabrication of microcantilevers. J. Micromech. Microeng. 21, 025023 (2011).
[6] Tricoli, A., Graf, M., Mayer, F., Kuhne, S., Hierlemann, A. & Pratsinis, S.
E. Micropatterning layers by flame aerosol deposition-annealing. Adv. Mater.
20, 3005-3010 (2008).
[7] Li, D., Teoh, W. Y., Selomulya, C., Woodward, R. C., Amal, R. & Rosche,
B. Flame-sprayed superparamagnetic bare and silica-coated maghemite
nanoparticles: Synthesis, characterization, and protein adsorption-desorption.
Chem. Mater. 18, 6403-6413 (2006).
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Curriculum Vitae
Georgios A. Sotiriou Born 25th of July, 1983 in Athens, Greece
Education
2011 PhD in Mechanical & Process Engineering, ETH Zurich, Switzerland, Title: “Understanding the toxicity of nanosilver for synthesis of biocompatible plasmonic-superparamagnetic nanocomposites” at the Particle Technology Laboratory. Advisor: Prof. S.E. Pratsinis.
2008 M.Sc. in Micro- and Nanosystems, ETH Zurich, Switzerland. Thesis:
“Flame-made fillers for dielectric nanocomposites” at the Particle Technology Laboratory. Advisor: Prof. S. E. Pratsinis.
2006 Diploma in Applied Physics, School of Applied Mathematical and
Physical Sciences, National Technical University of Athens (NTUA), Greece.
Experience
2008 - Research Associate & Teaching Assistant, Institute of Process Engineering, Particle Technology Laboratory, ETH Zurich.
2010 - 2011 Graduate Research Internship (part-time), Harvard School of Public
Health, Harvard University, Boston, USA, Advisor: Prof. P. Demokritou.
9 - 12/2007 Industrial Internship, PFISTERER SEFAG AG, Malters, Switzerland. 10 - 12/2005 Research Internship, National Hellenic Research Foundation, Athens,
Greece. 6 - 8/2001 Industrial Internship, Phaedra ltd. Piraeus, Greece.
Award
2011 Bionanotechnology Graduate Student Award, 1st place, American Institute of Chemical Engineers (AIChE) 2011 Annual Meeting, Minneapolis, Minnesota, USA.
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Publications and Presentations
A. Refereed Publications
1. Sotiriou, G. A. & Pratsinis, S. E. Antibacterial activity of nanosilver ions and particles. Environ. Sci. Technol. 44, 5649-5654 (2010).
2. Sotiriou, G. A., Sannomiya, T., Teleki, A., Krumeich, F., Vörös, J. & Pratsinis, S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors. Adv. Funct. Mater. 20, 4250-4257 (2010). Frontispiece of that issue #24, published 21 December 2010.
3. Sotiriou, G. A., Schneider, M. & Pratsinis, S. E. Color-tunable nanophosphors by codoping flame-made Y2O3 with Tb and Eu. J. Phys. Chem. C 115, 1084-1089 (2011).
4. Dahlin, A. B., Sannomiya, T., Zahn, R., Sotiriou, G. A. & Voros, J. Electrochemical crystallization of plasmonic nanostructures. Nano Lett. 11, 1337-1343 (2011).
5. Sotiriou, G. A., Hirt, A. M., Lozach, P. Y., Teleki, A., Krumeich, F. & Pratsinis, S. E. Hybrid, silica-coated, Janus-like plasmonic-magnetic nanoparticles. Chem. Mater. 23, 1985-1992 (2011).
6. Sotiriou, G. A., Teleki, A., Camenzind, A., Krumeich, F., Meyer, A., Panke, S. & Pratsinis, S. E. Nanosilver on nanostructured silica: Antibacterial activity and Ag surface area. Chem. Eng. J. 170, 547-554 (2011).
7. Sotiriou, G. A., Diaz, E., Long, M. S., Godleski, J., Brain, J., Pratsinis, S. E. & Demokritou, P. A novel platform for pulmonary and cardiovascular toxicological characterization of inhaled engineered nanomaterials. Nanotoxicology in press, DOI: 10.3109/17435390.2011.604439 (2011).
8. Sotiriou, G. A. & Pratsinis, S. E. Engineering nanosilver as an antibacterial, biosensor and bioimaging material. Curr. Opin. Chem. Eng. 1, 3-10 (2011).
Sotiriou, G. A., Meyer, A., Knijnenburg, J. T. N., Panke, S. & Pratsinis, S. E. Quantifying the origin of nanosilver ions and their antibacterial activity. Environ. Sci. Technol. in review (2011).
Hirt, A. M., Sotiriou, G. A., Kidambi, P. & Teleki, A. Evaluating magnetic properties of iron oxide/silica nanoparticles with FORC diagrams. J. Mater. Chem. in review (2011).
Sotiriou, G. A., Schneider, M. & Pratsinis, S. E. Flame synthesis and characterization of silica-coated Y2O3:Tb3+ nanophosphors. in preparation (2011).
Sotiriou, G. A., Franco, D., Ferrari, A., Poulikakos, D. & Pratsinis, S. E. Optically stable biocompatible nanophosphors for cancer cell imaging. in preparation (2011).
Sotiriou, G. A., Blattmann, C. O. & Pratsinis, S. E. Manufacture of multi-functional superparamagnetic nanocomposite films. in preparation (2011).
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B. Patents
Sotiriou, G. A., C. O. Blattmann & S. E. Pratsinis. Method for the generation of nanoparticle composite films and films made using such a method. European Patent 11005058.0 (2011).
C. Conference Presentations
1. Oral Presentations
Sotiriou, G. A., A. Camenzind, A. Meyer, S. Panke, S. E. Pratsinis, “Antibacterial activity of flame-made Ag/SiO2 nanoparticles” EAC, Karlsruhe, Germany (6-11/11/2009).
Sotiriou, G. A., A. Camenzind, A. Meyer, F. Krumeich, S. Panke, S. E. Pratsinis, “Universal correlation and mechanism for the antibacterial activity of silver nanoparticles” AAAR, Minnesota, USA (26-30/10/2009).
Sotiriou, G. A., A. Camenzind, A. Meyer, F. Krumeich, S. Panke, S. E. Pratsinis, “Universal correlation and mechanism for the antibacterial activity of silver nanoparticles” AIChE Annual Meeting, Nashville, USA (8-13/11/2009).
Sotiriou, G. A., A. Camenzind, A. Meyer, F. Krumeich, S. Panke, S. E. Pratsinis, “Universal correlation and mechanism for the antibacterial activity of silver nanoparticles” MRS 2009, Boston, Massachusetts, USA (30/11-4/12/2009).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Silica-coated Ag nanoparticles by flame spray pyrolysis” World Congress on Particle Technology 2010 (WCPT10), Nurenberg, Germany (26-29/4/2010).
Sotiriou, G. A., A. Camenzind, A. Meyer, S. Panke, S. E. Pratsinis, “Antibacterial activity of flame-made Ag/SiO2 nanoparticles” NSTI NanoTech 2010, Anaheim, California, USA (21-25/6/2010).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Plasmonic biosensors with silica-coated nanosilver particles” NSTI NanoTech 2010, Anaheim, California, USA (21-25/6/2010).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Non-toxic plasmonic biosensors with nanosilver” ISMANAM 2010, Zurich, Switzerland (5-9/7/2010).
Sotiriou, G. A., A. M. Hirt, P-Y. Lozach, F. Krumeich, A. Teleki, A. Helenius, S. E. Pratsinis, “Hybrid magnetic/plasmonic nanoparticles for biomarkers” AIChE Annual Meeting 2010, Salt Lake city, Utah, USA (7-12/11/2010).
Sotiriou, G. A., S. E. Pratsinis, “Antibacterial activity of nanosilver ions and particles” AIChE Annual Meeting 2010, Salt Lake city, Utah, USA (7-12/11/2010).
Sotiriou, G. A., S. E. Pratsinis, “Antibacterial activity by nanosilver ions and particles” MRS 2010, Boston, Massachusetts, USA (29/11-3/12/2010).
Sotiriou, G. A., A. M. Hirt, P-Y. Lozach, F. Krumeich, A. Teleki, A. Helenius, S. E. Pratsinis, “Hybrid silica-coated plasmonic/magnetic biomarkers” MRS 2010, Boston, Massachusetts, USA (29/11-3/12/2010).
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Sotiriou, G. A., A. M. Hirt, P-Y. Lozach, A. Teleki, F. Krumeich, S. E. Pratsinis, “Hybrid Silica-coated plasmonic-magnetic biomarkers” NSTI NanoTech 2011, Boston, Massachusetts, USA (13-19/6/2011).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Plasmonic biosensors with biocompatible nanosilver” NSTI NanoTech 2011, Boston, Massachusetts, USA (13-19/6/2011).
Sotiriou, G. A., A. Teleki, A. Camenzind, F. Krumeich, A. Meyer, S. Panke, S. E. Pratsinis, “Quantifying the origin of nanosilver’s antibacterial activity” AAAR 2011, Orlando, Florida, USA (3-7/10/2011).
Sotiriou, G. A., A. M. Hirt, P-Y. Lozach, A. Teleki, F. Krumeich, S. E. Pratsinis, “Hybrid, Janus-like, silica-coated plasmonic-magnetic nanoparticles for cell imaging & treatment” AAAR 2011, Orlando, Florida, USA (3-7/10/2011).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Flame-synthesis of SiO2-coated color tunable nanophosphors” AAAR 2011, Orlando, Florida, USA (3-7/10/2011).
Sotiriou, G. A., A. Meyer, J. Knijnenburg, S. Panke, S. E. Pratsinis, “Quantifying the origin of nanosilver’s antibacterial activity” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
Sotiriou, G. A., A. M. Hirt, P-Y. Lozach, A. Teleki, F. Krumeich, S. E. Pratsinis, “Hybrid, silica-coated, Janus-like, plasmonic-magnetic nanoparticles for cell imaging & treatment” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Plasmonic biosensors with biocompatible nanosilver” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
Sotiriou, G. A., E. Diaz, M. Long, J. Godleski, J. Brain, S. E. Pratsinis, P. Demokritou, “A novel technique for in vivo toxicological characterization of nanomaterials” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
2. Poster Presentations
Sotiriou, G. A., A. Camenzind, S. E. Pratsinis, “Silica-coated Ag nanoparticles by scalable flame technology” NSTI NanoTech 2009, Houston, USA, (3-7/5/2009).
Sotiriou, G. A., A. Camenzind, A. Meyer, S. Panke, S. E. Pratsinis, “Antibacterial activity of flame-made Ag/SiO2 nanoparticles” NSTI NanoTech 2009, Houston, USA, (3-7/5/2009).
Sotiriou, G. A., S. E. Pratsinis, “Silica-coated silver nanoparticles” MRS, Boston, USA, (30/11-4/12/2009).
Sotiriou, G. A., A. Camenzind, A. Meyer, F. Krumeich, S. Panke, S. E. Pratsinis, “Universal correlation and mechanism for the antibacterial activity of silver nanoparticles” World Congress on Particle Technology 2010 (WCPT), Nurenberg, Germany, (26-29/4/2010).
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Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Voros, S. E. Pratsinis, “Non-toxic plasmonic biosensors with nanosilver” Material Research Center (MRC), Zurich, Switzerland, (10/5/2010).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Non-toxic nanosilver for plasmonic biosensors” AAAR 2010, Portland, Oregon, USA (25-29/10/2010).
Demokritou, P., G. A. Sotiriou, E. Diaz, J. Godleski, S. E. Pratsinis, “Methods and systems for in vivo toxicological characterization of engineered nanomaterials” AAAR 2010, Portland, Oregon, USA (25-29/10/2010).
Sotiriou, G. A., T. Sannomiya, A. Teleki, J. Vörös, S. E. Pratsinis, “Benign nanosilver for plasmonic biosensors” AIChE Annual Meeting 2010, Salt Lake city, Utah, USA (7-12/11/2010).
Sotiriou, G. A., A. Camenzind, A. Meyer, S. Panke, S. E. Pratsinis, “Nanosilver: exposed surface area and antibacterial activity” AIChE Annual Meeting 2010, Salt Lake city, Utah, USA (7-12/11/2010).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Color tunable nanophosphors” MRS 2010, Boston, Massachusetts, USA (29/11-3/12/2010).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Silica-coated nanophosphors” MRS 2010, Boston, Massachusetts, USA (29/11-3/12/2010).
Sotiriou, G. A., D. Franco, A. Ferrari, D. Poulikakos, S. E. Pratsinis, “Biocompatible nanophosphors as biomarkers for cancer cell imaging” NSTI NanoTech 2011, Boston, Massachusetts, USA (13-19/6/2011).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Color tunable nanophosphors” NSTI NanoTech 2011, Boston, Massachusetts, USA (13-19/6/2011).
Sotiriou, G. A., D. Franco, A. Ferrari, D. Poulikakos, S. E. Pratsinis, “Silica-coated nanophosphors for bioimaging” NSTI NanoTech 2011, Boston, Massachusetts, USA (13-19/6/2011).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Flame-made silica-coated nanophosphors” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
Sotiriou, G. A., M. Schneider, S. E. Pratsinis, “Color tunable nanophosphors by codoping Y2O3 with Tb and Eu” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
Sotiriou, G. A., “Biological interactions of nanosilver particles and biomedical applications” AIChE Annual Meeting 2011, Minneapolis, Minnessota, USA (16-21/10/2011).
