Supplementary materials

Siderophore coated magnetic iron nanoparticles:

Rational designing of water soluble nanobiosensor for

visualizing Al3+ in live organism

M. Raju,† Sakshi Srivastava,† Ratish R. Nair,† Ishan H. Raval,‡

Soumya Haldar,*‡,§ and Pabitra B. Chatterjee*†,§

†Analytical Division and Centralized Instrument Facility,

CSIR-CSMCRI, Bhavnagar, Gujarat, India.

‡Marine Biotechnology and Ecology Division,

CSIR-CSMCRI, Bhavnagar, Gujarat, India.

§Academy of Scientific and Innovative Research,

CSIR-CSMCRI, Bhavnagar, Gujarat, India.

Corresponding author: pbchatterjee@csmcri.res.in

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1. Experimental details

1.1 Chemicals

2, 3-Dihydroxybenzoic acid, glycine ethyl ester, N,N'-dicyclohexylcarbodiimide (DCC),

iron(III) acetylacetonate Fe(acac)3, benzyl ether, oleylamine, potassium fluoride, different

metal perchlorate salts, and other analytical reagents were purchased from Aldrich and were

used as such. All other chemicals were purchased from local suppliers and were reagent

grade. Artemia cysts were procured from Tetra (Japan).

1.2 Instrumentation and physical measurements

Microanalysis (C, H, and N) were performed on an Elementar Vario MICRO CUBE analyser.

IR spectra in the FTIR-ATR mode (4000-400 cm-1) were recorded on Perkin-Elmer Spectra

GX 2000 spectrometer. TEM images of the nanoparticles were recorded using a transmission

electronic microscope (TEM; JEOL JEM 2100 microscope). For TEM, dispersed aqueous

sample was ultra-sonicated for 15 minutes and then the sample was deposited on to a carbon-

coated grid at room temperature and then allowed for air-dry (ca. 12 h). Laser-spectroscatter

201 (RiNA, GmbH, Germany) was used for the DLS experiments. Powder X-ray diffraction

(PXRD) of all samples were recorded using a diffractometer equipped with a CuKa X-ray

radiation at 40 kV and 30 mA. Diffraction patterns were collected over 2θ range 5-80 0 at a

scan rate 1 0/minute. Thermogravimetric analyses (TGA) were carried out on Netzch STA

409PC TG-DTA instrument from 30 0C through 800 0C with a scanning rate 5 0C/min in

presence of nitrogen flow. The UV-vis spectra were recorded by using Cary 500 scan UV-

vis-NIR spectrophotometer. For recording fluorescence spectra, Edinburgh instruments

Xe900 (μF 920H) spectrofluorimeter was used. Solutions pH were measured by Thermo

Scientific Orion Versastar Advanced Electrochemistry meter at 298 K. Estimation of metal

ion concentrations was carried out using an ICP-OES instrument from Parkin-Elmer, model

2000 dv. 1300 W RF power under argon gas flow (nebulizer, 0.86 L/min; auxiliary, 1.2

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L/min; plasma, 15 L/min) at 0.65 L/min sample uptake rate were used. Standard reference

solutions were purchased from Merck and were used for the calibration of the ICP-OES

instrument. Optical imaging of live organism was performed using an Olympus DP72 U-

TVO 63Xc microscope. Zeta potential measurements was performed using Zetasizer Nano

ZS light scattering apparatus (Malvern Instruments, UK) with a He/Ne laser (633 nm, 4 Mw).

1.3 General procedure for fluorescence experiments

Stock solution was prepared by dissolving 2 mg solid HL-FeNPs in 100 mL aqueous HEPES

buffer (50 mM, pH 7.4). Different metal perchlorate stock solutions of 50 μM (100 mL) were

prepared in H2O by dissolving solid metal salts in water in volumetric flasks. Final solutions

for fluorescence experiments were made by mixing 1 mL of the above 50 μM metal stock

solution and 1 mL of HL-FeNPs solution. The resolution of the fluorimeter was set at 1 nm.

The excitation was given at 340 nm and the emission spectra were recorded between 345 and

600 nm in all fluorescence experiments.

1.4 General procedure for fluorescence titrations

The above mentioned stock solutions were also used for fluorescence titration studies. The

air-equilibrated stock solutions of aluminium perchlorate salt were prepared by increasing its

concentrations from 0 μM to 250 μM in water. Final solutions for titration experiments were

prepared by mixing 1 mL each of HL-FeNPs and aluminium salt solutions of different

concentration (0 to 250 μM). For each measurement, excitation was given at 340 nm and all

experiments were carried out in triplicates.

1.5 Calculation of binding constant between Al3+ and HL-FeNPs

The binding constant between Al3+ and HL-FeNPs was estimated from the emission titration

data. This was calculated from the following linear expression of Benesi-Hildebrand (Benesi

and Hildebrand, 1949; Wang et al., 2010).

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I 0

I −I 0=

I 0

[ L ]+

I 0

[ L ] K1

[ M ]

I0 is the fluorescence intensity of HL-FeNPs in the absence of Al3+. I is the fluorescence

intensity of HL-FeNPs at 437 nm in presence of Al3+. K is the binding constant. [L] and [M]

are the concentrations of HL-FeNPs and Al3+, respectively. By plotting 1/(I-I0) versus

1/[Al3+], the binding constant can be calculated out.

1.6 Determination of LOD from fluorescence titration

The lower limit of detection (LOD) of HL-FeNPs for Al3+ was calculated from the equation

LOD = (3σ/S), wherein σ is the standard deviation and S is the slope of the calibration curve.

Upon plotting fluorescence intensities against concentrations of Al3+, the LOD value was

found to be 20 nM.

1.7 Al3+removal experiment using HL-Fe3O4 NPs and external magnet

Different amounts of HL-Fe3O4 NPs (0, 5, 10, 20, 30, 40, 50 and 60 mg) were dissolved in

aqueous solution of Al3+ (8.5 ppm, 50 mL). Different mixtures were stirred at room

temperature for ca. 2 h and the dispersed substances was separated by the use of an external

magnet. Finally, residual concentrations of Al3+ were measured by ICP experiments. Other

metal ions removal experiments were also performed as per the above mentioned procedure.

1.8 Al3+ enrichment experiment using natural water source

The Al3+ enrichment experiment in Artemia was also carried out with coastal water collected

from Alang ship breaking yard (World biggest ship breaking yard) where estimated Al3+

concentration is less than 1 μM (i.e. not detectable). 1L water sample was collected in

polypropylene bottle and were transported to the laboratory in cold condition.

1.9 Toxicity study of HL-FeNPs

To examine the toxic nature of the nanobiosensor HL-FeNPs, Artemia mortality count was

also performed. In 500 mL autoclaved sea water, Artemia cysts were allowed to harvest

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overnight under strong aeration. After complete harvesting, approximately 50 Artemia were

added in each tube (containing 10 mL aqueous HEPES buffer at pH 7.4). Three sets of tubes

were used in this study. Artemia were allowed to grow in the absence (control) and presence

of HL-FeNPs. Finally, by mortality count, the toxicity of HL-FeNPs was evaluated.

1.10 Synthesis and characterization of siderophore (H3L)

Siderophore 2,3-dihydroxybenzoylglycine (H3L) was synthesized following the procedure

described earlier in the literature (Soulere et al., 2002). Yield: 56 %. Anal. Calcd for

C9H9NO5: C, 51.19; H, 4.30, and N, 6.63. Found: C, 52.04; H, 4.25, and N, 6.51. ESI-MS

(+ive, m/z): 234.19 [H3L + Na+]. 1H NMR (CD3OD, 500 MHz) δ = 7.34 (m, 1H), 6.99 (m,

1H), 6.73 (m, 1H), 3.53 (d, j=7.8 Hz, 2H) ppm.

Fig. S1. Room temperature (25 °C) 600 MHz 1H NMR spectrum of H3L, recorded in CD3OD.

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Fig. S2. (a) TEM image of bare FeNPs; (b) histogram of bare FeNPs prepared from TEM results; (c) histogram of HL-FeNPs made from TEM of HL-FeNPs; and (d) Selected Area Electron Diffraction (SEAD) pattern of HL-FeNPs.

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Fig. S3. FTIR-ATR spectra of H3L (bottom) and HL-FeNPs (top).

Fig. S4. (a) Histogram of bare FeNPs prepared from the DLS data and (b) histogram of HL-FeNPs made from the DLS results.

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Fig. S5. TG graphs of (a) H3L, (b) HL-FeNPs, and (c) bare FeNPs.

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Fig. S6. Powder XRD pattern of bare FeNPs.

Fig. S7. Photographs of aqueous solutions of HL-FeNPs in absence and presence of different

metal ions under (observed under UV light at 360 nm).

Fig. S8. Bar diagram of the emission peak maxima of FeNPs and HL-FeNPs with and

without Al3+.

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Fig. S9. pH titration profile of aqueous solutions of HL-FeNPs performed in presence and in

absence of Al3+.

Fig. S10. Fluorescence titration profiles of HL-FeNPs upon addition of increasing

concentrations of Al3+ to the aqueous HEPES buffer solutions of the probe at pH 7.4 (at 25

°C). Excitation was given at 340 nm.

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Fig. S11. (a) Benesii-Hildebrand plot from emission titration data of HL-FeNPs in presence

of Al3+. (b) LOD determination plot of HL-FeNPs (λemi=437 nm) as a function of Al3+

concentrations. Error bars indicates the standard deviations. Each data point is the average of

three values.

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Fig. S12. (a) Reversibility experiment showing alternate fluorescence enhancement and

quenching of HL-FeNPs upon sequential addition of Al3+ and KF to its solution. (b)

Illustration of the reusability of HL-FeNPs for the detection of Al3+ using KF.

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Fig. S13. Plot of fluorescence emission maxima against time after the addition of Al3+. The

data points represent the average of three values and the error bars indicate the standard

deviations.

Fig. S14. (a) TEM image of HL-FeNPs in presence of Al3+ showing aggregation of the

nanosensor. (b) Hydrodynamic diameter measurement of HL-FeNPs after the addition of

Al3+ using dynamic light scattering technique.

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Fig. S15. Powder XRD of HL-FeNPs in presence of Al3+.

Fig. S16. Fluorescence microscopy image of live Artemia which was exposed to 10 μM

Al3+ followed by the addition of HL-FeNPs. Arrow indicates the position of the GI-

tract.

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Fig. S17. Fluorescence microscopy image of live Artemia which was exposed to the natural

seawater sample as described below in Table S3. Arrow indicates the position of the GI-

tract.

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Table S1. Summary of optical properties of different water soluble small-molecule based Al3+-selective chemosensors.

1

Receptor Binding constant

(M-1)

LOD Interferencea Solvent

system

Bio-imaging Excitation

(nm)

Ref

Salicylimineprobe L 5.5 x 104 5.2 nM Cr3+, Cu2+, and Ca2+ Water HeLa cells 366 Wang et al., 2015

Receptor 1 1.13 x 103 6.4 μM Cr3+, Cu2+, Fe2+, and Fe3+ Water HeLa cells 380 Choi et al., 2016

Salpn-ONPS NA 1.25 μM Cu2+ and Cr3+ Water Staphylococcus

Aureus bacteria

365 Aguilar et al., 2016

Sensor L 9.5 x 105 66.5 nM Also selective for Zn2+ Water NA 335 Yan et al., 2015

Lsen NA 1.5 ppb No interference Water NA 374 Vallejos et al., 2015

Chemo sensor 1 4.2 x 103 336 nM Cu2+ interferes Water NA 334 Yan et al., 2015

PMD2 6.22 x 106 1.49 nM NA Water NA 440 Kumar et al., 2013

ANTPY NA 320 nM No interference Water NA 340 Zhou et al., 2013

Quinoline functionalized

Schiff base (L)

NA 2.1 M Cr3+ and Fe3+ Water HeLa cells 296 Samanta et al., 2015

Metal−organic framework 1 NA 2.5 M NA Water NA 283 Singha et al., 2015

Dansyl-SEE 1.84 x 104 230 nM Cu2+, Hg2+, Cr3+, and Fe3+ Water NA 380 In et al., 2016

Chemosensor 1 4 x 103 2.01 M Cu2+, Fe2+, Fe3+, Co2+, Ni2+

and Cr3+

Water HeLa cells 410 Jo et al., 2016

Chemosensor 1 NA 5 x 10-11

M

Zn2+, Fe3+, and Cu2+ Water Zebrafish 317 Wang et al., 2014

Schiff base-type fluorescent

chemosensor H2L

7.8 x 10-4 0.17 M No interference Water HeLa cells 340 Guo et al., 2013

ARS NA 40 nM No interference Water NA 420 Wang et al., 2011

SA1 4.05 x 103 M-1/2 2.73 nM Fe3+ and Cu2+ Water LLC Cells 419 Neeraj et al., 2014

Sensor L 1.19 x 108 0.1 μM No interference Water NAb 320 Liu et al., 2014

aInterference study in presence of other competing metal ions. bData is not reported. NA stands for information not available.

Table S2. Comparison of optical properties of HL-Fe3O4 NPs with different water soluble Al3+ -specific nanosensors.

Receptor Binding

constant (M-1)

LOD Interferencea Solvent

system

Bio-imaging system Method Ref

HL-FeNPs 0.4 x 104 M-1 20 nM No interference Water Live brine shrimp Artemia FL This study

Citrate capped AuNPs NA 1 M Cr3+ Water NA UV-vis Chen et al., 2012

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C-AuNPs NA 0.2 M NA Water HeLa cells UV-vis Li et al., 2010

A-AgNPs NA 0.09 M NA Water HeLa cells UV-vis Zhang et al., 2012

A-AuNPs NA 0.46 M NA Water HeLa cells UV-vis Zhang et al., 2012

TTP–AuNP NA 18 nM No interference Water NA UV-vis Chen et al., 2013

MMT-AuNPs NA 0.53 M NA Water NA UV-vis Xue et al., 2014

GSH-AgNPs NA 0.16 M NA Water NA UV-vis Yang et al., 2014

Fe3O4 NPs conjugate 1c 3.2 x 107 M-1 0.3 ppb No interference Water Hela cells FL Zhi et al., 2013

Conjugate polymer NPs NA 35 ppb NA water Vero cells FL Liu et al., 2013

Zw-AuNPs NA 10 M Also selective for

Cr3+ and Fe3+

Water NA UV-vis Zheng et al., 2016

HNAET–AuNPs NA 0.29 M No interference Water NA UV-vis Huang et al., 2016

Au NC NA 0.3 M Also selective for

Fe3+

Water NA FL Mu et al., 2014

PCA-DTC-Au NPs NA 38 nM No interference Water NAb UV-vis Mehta et al., 2015

aInterference study in presence of other competing metal ions. bData is not reported. NA stands for information not available.

Table S3. Quantitative determination (via ICP-OES) of Al3+ in natural seawater after the removal of Al3+ by the magnetic nanosensor HL-FeNPs

in presence of external magnet. The concentrations of other selected metal ions present in the natural marine water sample collected from Alang

ship breaking yards are as follows:

Zn2+ (11 ppm); Fe3+ (8 ppm); Pb2+ (5 ppm); Mn2+ (2 ppm); and Co2+ (4 ppm).

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Al3+ found (ppm)

Al3+ added (ppm)

After removal of Al3+ by adding 60 mg HL-FeNPs

(ppm)

After removal of Al3+ by adding 100 mg HL-FeNPs

(ppm)ND 4.92 1.8 0.18

ND: Not detectable

Table S4. Toxicity results of HL-FeNPs

% mortality with respect to time

2 hours 4 hours 6 hours

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Experiment Set 1 Set 2 Set 3 mean Std. Dev.

Set 1 Set 2 Set 3 mean Std. Dev.

Set 1

Set 2

Set 3 mean Std. Dev.

ctrl 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HL-FeNPs

HL-FeNPs (10 μg iron per mL)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HL-FeNPs (at 10 μg iron per mL) plus Al3+

6 µM Al3 0 0 0 0 0 0 0 0 0 0 0 033 0 0 0

8 µM Al3 0 0 0 0 0 0 0 0 0 0 0 0 1 0.333 0.577

10 µM Al3 0 0 0 0 0 0 0 1 0.333 0.577 1 2 2 1.666 0.577

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