Porphyrins as SERRS spectral probes of chemically functionalized Agnanoparticles

K. Siskova a,b,1, B. Vlckova a,*, P.-Y. Turpin b, A. Thorel c, A. Grosjean c

a Department of Physical and Macromolecular Chemistry, Charles University in Prague, Hlavova 8/2030 Prague 2, 128 40, Czech Republicb BIOMoCeTi, Universite P. et M. Curie ParisVI, GENOPOLE Campus 1, Evry 91030, Francec Centre des Materiaux, Ecole des Mines de Paris, Evry 91030, France

Vibrational Spectroscopy 48 (2008) 44–52

A R T I C L E I N F O

Article history:

Received 16 July 2007

Received in revised form 2 April 2008

Accepted 9 April 2008

Available online 20 April 2008

Keywords:

Surface-enhanced resonance Raman

scattering (SERRS)

Citrate-modified Ag nanoparticles

Laser ablation

5,10,15,20-Tetrakis(1-methyl-4-

pyridiniumyl)porphine

5,10,15,20-Tetra(pyridyl)porphine

5,10,15,20-Tetrakis(4-

aminophenyl)porphine

A B S T R A C T

The results of surface-enhanced resonance Raman scattering (SERRS) spectral probing of citrate- and/or

citric acid-modified Ag nanoparticles by selected free-base porphyrins, namely a tetracationic

5,10,15,20-tetrakis(1-methyl-4-pyridiniumyl)porphine and neutral 5,10,15,20-tetra(pyridyl)porphine

(H2TPyP) and 5,10,15,20-tetrakis(4-aminophenyl)porphine (H2TAPP) are reported, along with a novel

procedure of the functionalized Ag nanoparticle hydrosols preparation by laser ablation of a Ag target in

aqueous sodium citrate and/or citric acid solutions of various concentrations. SERRS spectra obtained

from the Ag nanoparticle hydrosol/porphyrin system were analyzed using the spectral marker bands of

free-base, Ag metallated and diacid forms. In freshly prepared SERRS-active systems, adsorbed citrate

was found to function as an efficient molecular spacer for positively charged porphyrin species both in

the pH-neutral and in the acidic media, allowing for SERRS spectral detection of not only cationic, but also

additionally protonized neutral porphyrins in the native free-base (and/or, at low pH, in the diacid) form

without denaturation by Ag incorporation. Furthermore, a substantial increase of the SERRS signal

observed for H2TPyP and H2TAPP in systems with Ag nanoparticles prepared by laser ablation in

1 � 10�2 M citric acid solutions is attributed to both the electromagnetic enhancement increase

stemming from the presence of hot spots in compact aggregates of touching and intergrown Ag

nanoparticles (visualized by /HR/-TEM), and from the molecular resonance enhancement increase

originating from a close match between the Soret band of the diacid form (440 nm) and the 457.9 nm

excitation. For H2TAPP, the large SERRS enhancement manifests itself in the 1 � 10�10 M SERRS spectral

detection limit.

� 2008 Elsevier B.V. All rights reserved.

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1. Introduction

Surface-enhanced Raman scattering (SERS) spectroscopy,which has currently achieved the status of a well establishedspectroanalytical method [1], benefits from the coupled opticalresponses of plasmonic metal nanostructures and moleculeslocated at, or in a close proximity to their surfaces [2]. SERS-active systems constituted by Ag nanoparticles (NP) and porphyrinmolecules have been the subject of considerable research interest[3–11] chiefly due to a possibility to detect porphyrin species invery low concentrations, with an additional benefit of fluorescencequenching [12]. Recently, realistic possibilities to achieve a single

* Corresponding author. Tel.: +420 221951309; fax: +420 224919752.

E-mail address: vlc@natur.cuni.cz (B. Vlckova).1 Present address: Institute of Macromolecular Chemistry ASCR, Heyrovsky

Square 2, Prague 6, Czech Republic.

0924-2031/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vibspec.2008.04.006

molecule level of porphyrin detection have also been demon-strated [11]. In Ag NP/porphyrin systems, the electromagnetic(EM) mechanism of SERS operates through enhancement of boththe incident and the Raman scattered light by resonance Miescattering of light by Ag nanoparticles [2]. Provided that, for aparticular porphyrin species and a selected wavelength of theexciting radiation, a molecular resonance condition is fulfilledsimultaneously with the Mie resonance condition, surface-enhanced resonance Raman scattering (SERRS) of the porphyrinis detected. In the case of free-base porphyrins, the first necessarystep in evaluation of the molecular resonance contribution to theoverall enhancement is identification of the actual porphyrinspecies providing the SE(R)RS signal [3–11]. The reason for that isthe free-base (f.b.) porphyrins are prone to metallation after theirdirect adsorption on weakly stabilized Ag nanoparticle surfaces,yielding the Ag metalloporphyrins [3–11]. Adsorption of f.b.porphyrins on the commonly available, oxidized Ag nanoparticlesurfaces mostly results into the formation of Ag metalloporphyrins

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–52 45

structurally nearly identical to their synthetically preparedanalogues [4–9,13]. On specifically chemically pretreated Agnanoparticle surfaces, some of the f.b. porphyrins can stabilizealso the reduced adsorption sites [7,8]. For practical spectro-analytical applications, such as SERRS spectral detection of f.b.porphyrins important for cancer diagnostics and treatment,preservation of the native, f.b. form of the porphyrin is of vitalimportance. This can be achieved by employment of a molecularspacer [4–6,10]. The molecular spacer is a chemical speciespossessing functional group(s) suitable for its attachment to Ag NPsurface as well as for a subsequent attachment of porphyrinmolecules to the adsorbed spacer which, in turn, results into theirlocalization in the vicinity of Ag NP surface.

In this paper, we report the results of SERRS spectral probing ofselected f.b. porphyrins bonding to surfaces of Ag NPs prepared bylaser ablation (LA) of a Ag target immersed into aqueous solutions

Fig. 1. (A) TEM image of water–Ag NP hydrosol; (B) TEM image of 1 � 10�5 M Na3citr–Ag

depiction of citrate adsorption of Ag NP surface (according to ref. [14]); (E) TEM image

hydrosol.

of sodium citrate or citric acid of various concentrations. Our goalwas first to evaluate the effect of these electrolytes presence and ofvariations of their concentration in the aqueous ablation ambienton size-distribution, aggregation state and stability of the resultinghydrosols prepared by LA under the specific conditions selected onthe basis of our previous systematic exploration of these factors[14–16]. Furthermore, our major goal was to establish, howvariations of surface coverage of Ag nanoparticles by adsorbedcitrate and of the protonation state of the adsorbed citrate canaffect the f.b. porphyrin – modified Ag NP surface interactions. Forevaluation of these interactions, we have adopted the model ofcitrate adsorption proposed by Munro et al. [17] (depicted asscheme D in Fig. 1).

For probing of the citrate and/or citric acid-functionalized AgNP surfaces, a tetracationic 5,10,15,20-tetrakis(1-methyl-4-pyr-idiniumyl)porphine (H2TMPyP) and two structurally different

NP hydrosol; (C) TEM image of 1 � 10�2 M Na3citr–Ag NP hydrosol; (D) schematic

of 1 � 10�5 M H3citr–Ag NP hydrosol; (F) TEM image of 1 � 10�2 M H3citr–Ag NP

Fig. 2. SERRS spectral probing of water–Ag NP hydrosol/porphyrin and of Na3citr–Ag NP hydrosol/porphyrin systems immediately after their preparation: (A) H2TMPyP; (B)

H2TPyP; (C) H2TAPP in (a) water–Ag NP hydrosol; (b) 1 � 10�5 M Na3citr–Ag NP hydrosol; (c) 1 � 10�4 M Na3citr–Ag NP hydrosol; (d) 1 � 10�3 M Na3citr–Ag NP hydrosol; (e)

1 � 10�2 M Na3citr–Ag NP hydrosol. Symbols �f or /f determine the numerical factor f, by which the particular spectrum had to be multiplied or divided, respectively, to

obtain spectra with comparable signal to noise ratio (a–e) within each of the spectral sets A, B, C. Marker bands of the f.b. and Ag metallated form of the particular porphyrin

are listed and marked on the wavenumber axis of each of the spectral set A, B, C.

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–5246

Fig. 3. SERRS spectral probing of water–Ag NP hydrosol/porphyrin and of Na3citr–Ag NP hydrosol/porphyrin systems two days after their preparation: (A) H2TMPyP; (B)

H2TPyP; (C) H2TAPP in (a) water–Ag NP hydrosol; (b) 1 � 10�5 M Na3citr–Ag NP hydrosol; (c) 1 � 10�4 M Na3citr–Ag NP hydrosol; (d) 1 �10�3 M Na3citr–Ag NP hydrosol; (e)

1 � 10�2 M Na3citr–Ag NP hydrosol. Symbols �f or /f are used and porphyrin marker bands are presented as in Fig. 2.

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–52 47

neutral porphyrin species: 5,10,15,20-tetra(pyridyl)porphine(H2TPyP) and 5,10,15,20-tetrakis(4-aminophenyl)porphine(H2TAPP) were selected. Both neutral porphyrins selected forprobing possess strongly argentophilic groups containing lone

pairs on a nitrogen atom. Nevertheless, while in the case ofthe pyridyl peripheral substituent, this lone pair is rigidlyaligned with the plane of the pyridine aromatic ring, the aminegroup of the aminophenyl substituent can rotate and thus

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–5248

achieve a lone pair orientation favorable for bonding to the AgNP surface.

2. Experimental

2.1. Materials, preparation procedures and sample labeling

Analytical grade chemicals and deionized or redistilleddeionized water were used for all preparations: silver foil(99.99%, app. 1 mm thickness); Na3C6H5O7�2H2O (Na3citr, Aldrich),H3C6 H5O7 (H3citr, Lachema); 5,10,15,20-tetrakis(1-methyl-4-pyridiniumyl)porphine, tetra-p-tosylate salt (H2TMPyP, Aldrich);5,10,15,20-tetra(4-pyridyl)porphine (H2TPyP, Porphyrins Sys-tems), 5,10,15,20-tetrakis(4-aminophenyl)porphine (H2TAPP, Por-phyrins Systems); ethanol (Uvasol Merck).

Ag NP hydrosols modified by adsorbed citrate and/or citric acidwere prepared by LA of a ca. 1 mm thick Ag target immersed in aquarz cuvette containing 30 mL of pure water or sodium citrate orcitric acid aqueous solutions of various concentrations by using5 ns pulses of 1064 nm wavelength and of 300 mJ energy per pulseproduced by a Nd/YAG laser. The LA procedure was performed inthree irradiation steps of 5 + 5 + 10 min duration using pure waterand/or 1 � 10�5 M, 1 � 10�4 M, 1 � 10�3 M and/or 1 � 10�2 Msodium citrate and/or citric acid aqueous solutions as the ablationmedium. Samples of the resulting Ag NP hydrosols (ca. 4 mL) weredeposited on carbon-coated Cu grids for transmission electronmicroscopy (TEM) imaging.

Samples for SERRS spectral probing were prepared by adding1 mL of a 2 � 10�5 M stock solution of H2TMPyP and/or H2TPyPand/or H2TAPP in ethanol to 1 mL of the tested Ag NP hydrosol. Thefinal concentration of each of the porphyrins in the SERRS-activesystems was 2 � 10�8 M.

Finally, the following abbreviations are used for description ofthe parent Ag NP hydrosols and the SERRS-active systemscomposition: Ag NP hydrosols are labeled by using the composi-tion of the ablation medium in which they were prepared as aprefix. Labels of SERRS-active systems are constituted by theparticular Ag NP hydrosol label and by the acronym of the

Fig. 4. Schematic depiction of H2TMPyP, H2TPyP and H2TAPP bonding to citrate-modifi

particular porphyrin. Example: 1 � 10�2 M H3citr–Ag NP hydrosol/H2TAPP system.

2.2. Instrumentation

TEM and HR-TEM imaging was performed by using a JEOL-JEM200 CX and a Tecnai F 20 ST transmission electron microscope atvarious magnifications.

Electronic absorption spectra of the f.b. porphyrins solutionswere recorded with a Cary j1Ej UV–visible spectrophotometer in4 mm cuvette and found to match those reported earlier in [18,19].The Soret band maxima of the porphyrins are located at 423 nm forH2TMPyP, 414 nm for H2TPyP and 425 nm for H2TAPP.

SERRS spectra of the Ag NP hydrosol/porphyrin systems weremeasured with a Jobin Yvon CCD Raman spectrometer using457.9 nm excitation provided by a Spectra Physics 2017 Ar ionlaser and 3 min accumulation time (with exception of the1 � 10�2 M H3citr–Ag NP hydrosol/H2TAPP system measurement,for which 4 s accumulation time was used).

2.3. Reference spectra and calculations

The characteristic Raman spectral marker bands of the f.b. andthe Ag metallated porphyrin species were obtained in thefollowing manner: H2TMPyP and AgTMPyP spectral markers wereadopted from the pure component SERRS spectra obtained byfactor analysis (FA) [8] as well as from the resonance Ramanscattering (RRS) spectra [13]. Marker bands of H2TPyP were takenfrom the RRS and SERRS spectra reported in [20,21], those ofAgTPyP from the SERRS spectra reported in [14,21]. Markersof H2TAPP were extracted from the RRS spectra [14,19] and thoseof AgTAPP from the SERRS spectra presented in [14]. For eachporphyrin, the marker bands of its f.b. and Ag metallated form arepresented and marked on wavenumber axis of the appropriatespectral set in Figs. 2, 3 and 5.

A Matlab program and pKa = 5.1 value of pyridine were used forcalculation of the fraction of the protonized pyridyl groups atvarious pH values (listed further in Table 1).

ed Ag NP surfaces and details of the porphyrin peripheral substituent structures.

Fig. 5. SERRS spectral probing of water–Ag NP hydrosol/porphyrin and of H3citr–Ag NP hydrosol/porphyrin systems immediately after their preparation: (A) H2TMPyP; (B)

H2TPyP; (C) H2TAPP in (a) water–Ag NP hydrosol; (b) 1 � 10�5 M H3citr–Ag NP hydrosol; (c) 1 � 10�4 M H3citr–Ag NP hydrosol; (d) 1 � 10�3 M H3citr–Ag NP hydrosol; (e)

1 � 10�2 M H3citr–Ag NP hydrosol. Symbols �f or /f are used and porphyrin marker bands are presented as in Fig. 2.

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–52 49

Table 1Measured pH values of H3citr–Ag NP hydrosols

Ag NP hydrosol pH

H3citr(1 � 10�5 M)–Ag hydrosol 5.6

H3citr(1 � 10�4 M)–Ag hydrosol 4.1

H3citr(1 � 10�3 M)–Ag hydrosol 3.3

H3citr(1 � 10�2 M)–Ag hydrosol 2.7

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–5250

3. Results and discussion

3.1. Morphological characteristics of ‘‘bare’’ and functionalized Ag NPs

TEM images of Ag NP hydrosols obtained by LA in pure water,1 � 10�5 M and in 1 � 10�2 M sodium citrate and citric acidaqueous solutions are mutually compared in Fig. 1. The resultsindicate that the presence of sodium citrate in the aqueousablation medium leads to reduction of Ag NP sizes (comparison ofFig. 1A with B and C), most probably due to an efficientstabilization of the NPs by adsorption of the negatively chargedcitrate ions in the course of LA (Fig. 1D). In contrast to that, theincrease of citric acid concentration in the ablation medium,accompanied by the decrease of the pH, causes Ag nanoparticlefusion into small, very compact aggregates, as demonstrated byFig. 1E and F.

3.2. SERRS spectral probing of citrate-modified Ag NPs

SERRS spectral probing of the Ag NP surface stabilized byvarious citrate ion coverages (during LA performed in sodiumcitrate solutions) by the tetracationic porphyrin H2TMPyPperformed immediately after the SERRS-active system preparationhas shown that the presence of adsorbed citrate ions fully inhibitsmetallation of the porphyrin, as witnessed by observation of thecharacteristic marker bands of the native, free-base form of theporphyrin in the spectra b–d in Fig. 2A. In contrast to that, thespectral bands of the Ag metallated porphyrin are clearly observedin spectrum a in Fig. 2A, which indicates that, on the surfaces of AgNPs weakly stabilized mostly by OH� ions and prepared by LA inpure water, a partial metallation of the porphyrin proceedsimmediately after the SERRS-active system preparation. SERRSspectral measurements of the same systems carried out two daysafter their preparation (Fig. 3A) indicate that although theefficiency of the porphyrin metallation inhibition by adsorbedcitrate decreases at longer times, the presence of adsorbed citrateat high surface coverages has a favorable effect on the porphyrinSERRS signal intensities. This effect can be attributed to stabiliza-tion of the SERRS-active system (against a too fast and extensiveaggregation and aggregate sedimentation) by residual charges onthe Ag NPs originating likely from the presence of the excess citrateions (i.e. those the charges of which were not compensated byattachment of the cationic porphyrin).

On the other hand, in the case of the neutral porphyrin H2TPyP,the SERRS spectra measured immediately after the SERRS-activepreparation show no indications of the porphyrin metallation, bothon the surfaces of Ag NP weakly stabilized by OH� ions and onthose modified by adsorbed citrate, since entirely the markerbands of the f.b. form of the porphyrin are observed in spectra a–ein Fig. 2B. This result indicates that H2TPyP on Ag NP surfaces mostprobably adopts the edge-on adsorption geometry, being attachedto the surface by one or two lone pair(s) on the nitrogen atom(s) ofthe pyridyl group(s), which is the same adsorption geometry asthat encountered on Au NP surfaces [20]. Nevertheless, within twodays after the SERRS-active system preparation, this porphyrinbecomes completely metallated on OH� stabilized surfaces, while

in system with the low surface coverages of Ag NPs by adsorbedcitrate, metallation is at least slightly hindered (Fig. 3B). Further-more, the decrease of the SERRS signal of the porphyrin with theincreasing coverage by the negatively charged adsorbed citrateions observed in the SERRS spectral sets obtained both immedi-ately and two days after the SERRS-active system preparation(Figs. 2B and 3B) can be tentatively attributed to the increasinghindrance of Ag NP aggregation caused by the increasing negativecharges on their surfaces. Nevertheless, the decrease of theporphyrin SERRS signal can also originate from the increasingoccupation of the available adsorption sites by citrate ions whichhinder the edge-on adsorption of the porphyrin.

Finally, the other neutral porphyrin investigated, i.e. H2TAPP,becomes partially metallated both on the OH� and citratestabilized Ag NP surfaces immediately after the SERRS-activesystem preparation, as witnessed by the presence of the markers ofboth the Ag metallated and the residual free-base form in SERRSspectra a–e in Fig. 2C. Apparently, modification of Ag NP surfacesby citrate ions has no effect on prevention of the porphyrinmetallation, neither immediately (Fig. 2C), nor after the ageing ofthe SERRS-active system (Fig. 3C). Furthermore, a decrease of theporphyrin SERRS signal observed in both spectral sets in Figs. 2Cand 3C is analogous to that encountered in the SERRS spectral setswith the H2TPyP spectral probe (Figs 2B and 3B) discussed above,and can be tentatively explained in the same manner.

In summation, the results of the SERRS spectral probing haveshown that by modification of Ag NPs by adsorbed citrate, f.b.porphyrin metallation can be substantially hindered, but notpermanently prevented. The hindrance operates differently foreach of the testing porphyrins and is crucially dependent on theperipheral substituent charge and structure, which, in turn, governthe possibilities of the porphyrin–surface interaction. For thetetracationic H2TMPyP, adsorbed citrate works as an excellentmolecular spacer (in systems probed immediately after prepara-tion) through attractive electrostatic interaction between thenegatively charged adsorbed citrate ions and the cationicperipheral substituents of the porphyrin macrocycle (Fig. 4).Nevertheless, the efficiency of the spacer is temporally limited. Theneutral porphyrin H2TPyP appears to be initially adsorbed on Ag NPsurface through one or two lone pair(s) on pyridine nitrogenatom(s) (Fig. 4). The rigid alignment of the lone pair with thearomatic system excludes a possibility of the porphyrin to be bondto the surface by its peripheral substituent(s) while adopting aparallel orientation of the porphyrin macrocycle with respect tothe surface which, in turn, is required for the porphyrinmetallation. H2TPyP can thus be metallated only after reorienta-tion of the porphyrin macrocycle with respect to the surface, whichappears to be a kinetically slow (in terms of days), butthermodynamically favored process. Finally, in the case of H2TAPP,the presence of citrate does not hinder porphyrin metallation at all,most probably due to a unique ability of this porphyrin to be bondto Ag NP surface by all four lone pairs on aminophenyl-groupnitrogen atoms adopting thus immediately upon adsorption theparallel orientation with respect to the surface required for theporphyrin metallation.

We can conclude that SERRS spectral probing by all the threeporphyrin species has demonstrated that the strength of theadsorbed citrate bonding to Ag NP surface cannot compete withthe stabilization energy produced by the porphyrin metallationand that the citrate is finally desorbed from the surface to bereplaced by a Ag metalloporphyrin. The rate of this process issufficiently slow to enable SERRS spectral detection of H2TMPyPand H2TPyP in their native, f.b forms in systems investigatedimmediately after their preparation. By contrast, for detection off.b. H2TAPP, employment of a molecular spacer with a higher

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–52 51

surface bonding energy than the adsorbed citrate would berequired.

3.3. SERRS spectral probing of citrate/citric acid-modified Ag NPs

Ag NP hydrosols prepared by LA/NF in citric acid solutions(H3citr–Ag hydrosols) represent substantially more complex sub-strates than those stabilized by citrate anions during LA/NF inneutral sodium citrate solutions (Na3citr–Ag hydrosols). First of all,the H3citr–Ag hydrosols contain compact aggregates of touching andintergrown Ag NPs, the fraction of which increases with theincreasing citric acid concentration in the ablation medium (Fig. 1Eand F). The corresponding decrease of pH in H3citr–Ag hydrosols isdemonstrated by the measured pH values listed in Table 1.Consequently, we have to consider the presence of an increasingfraction of protonated carboxylate groups of the adsorbed citrate/citric acid on Ag NP surfaces. In addition to that, one has to considerthe structural changes undergone by the testing porphyrins insystems with the decreasing pH values, in particular protonation ofthe peripheral pyridyl and aminophenyl substituents of H2TPyP andH2TAPP, respectively, as well as, for all three testing porphyrins, thepossibilities of the diacid form formation.

SERRS spectral probing of H3citr–Ag NP hydrosols by H2TMPyPperformed immediately after the SERRS-active system preparationreveals that the testing porphyrin is detected in its native, f.b. form inall H3citr–Ag hydrosols (Fig. 5A, spectra b–e). The SERRS spectra ofH2TMPyP in Fig. 5 (b–e) are virtually identical with those obtained

Fig. 6. Spectrum representing the SERRS spectral detection limit of H2TAPP obtained from

the TEM and HR-TEM images of Ag NP aggregates deposited from this system.

fromthesystems withNa3citr–Aghydrosol (Fig.2A,spectrab–e),andcontain no spectral features of the diacid form of the porphyrin. Thisresult indicates that citrate works as an efficient molecular spacer forH2TMPyP both in the neutral and the acidic ambient, regardless apartial protonation of the adsorbed citrate and the consequentdecrease of the negative charge density on Ag NP surface.

In the case of H2TPyP, our calculations have shown that theextent of the peripheral pyridyl group protonation to thepyridiniumyl group is ca. 27%, 92%, 98% and 99% at the decreasingpH values provided in Table 1. In accord with that, the 1637 cm�1

band attributable to pyridiniumyl group dominates the 1600–1700 cm�1 spectral region of the H2TPyP SERRS spectra obtainedfrom all the H3citr–Ag hydrosols (Fig. 5B spectra b–e). Further-more, while the spectra b–d in Fig. 5B are governed by the spectralfeatures of the f.b. form of H2TPyP and contain only very weakcontributions of those of the diacid form, the latter spectralfeatures, in particular the 988 and 1372 cm�1 markers of the diacidform, are clearly observable in spectrum e obtained from thesystem with pH 2.7. No spectral markers of the AgTPyP have beenobserved. All these results indicate that H2TPyP becomes a cationicporphyrin species in the acidic media (with the average charge perone porphyrin molecule increasing with the decreasing pH), and isattached to Ag NP surface through a citrate/citric acid molecularspacer. Furthermore, an intensity increase of the SERRS signal ofthe porphyrin becomes significant particularly in spectrum e(divided by a factor f = 14 to fit within the spectral set a–e inFig. 5B). At least two contributions to the observed signal increase

the 1 � 10�2 M H3citr–Ag NP hydrosol/1 � 10�10 M H2TAPP system together with

K. Siskova et al. / Vibrational Spectroscopy 48 (2008) 44–5252

in this particular system can be envisaged: (i) the presence of alarge fraction of compact aggregates in the parent 1 � 10�2 MH3citr–Ag NP hydrosol and (ii) the presence of a small fraction ofthe porphyrin diacid form. In the latter case, the significant factorcan be an additional molecular resonance contribution stemmingfrom a close match between Soret band of the diacid form withmaximum at 440 nm [22] with the 457.9 nm excitation wave-length.

In the case of H2TAPP, the extent of the peripheral aminophenylgroup protonation is estimated to be only slightly lower than that ofthe pyridyl group of H2TPyP, since the pKa = 4.6 of aniline is only of aslightly lower value than the pKa = 5.1 of pyridine. SERRS spectralprobing of the H3citr–Ag NP hydrosols by H2TAPP (Fig. 5C, spectra b–e) shows, together with marker bands of the f.b. form of theporphyrin, also distinct spectral features of the diacid form, namelythe 988, 1324 and 1370 cm�1 spectral bands, the relative intensitiesof which increase with the decreasing pH in the sequence of spectrab–e. From these indications we can deduce that H2TAPP, analogouslyto H2TPyP, is protonated in the acidic media on the peripheralsubstituents. The extent of protonation increases with the decreas-ing pH, and, at low pH values, involves also the porphyrinmacrocycle. The cationic porphyrin species is bond to the Ag NPsurface via the citrate/citric spacer. Furthermore, a progressiveincrease of the porphyrin SERRS signal within the spectral set a–e inFig. 5C has been observed and an additional enhancement by morethan two orders of magnitude has been detected in the spectrum e.In parallel with the case of H2TPyP discussed above, the increase ofthe SERRS signal of H2TAPP can stem from both the EM mechanismcontribution likely originating from the presence of numerous hotspots in compact aggregates of touching and intergrown Ag NP, andfrom the molecular resonance contribution originating from a closematch between the Soret band of the diacid form at ca. 440 nm [22]and the 457.9 nm excitation wavelength. It should be noted thatboth the extent of compact aggregate formation and the relativecontribution of the SERRS signal of the H2TAPP diacid formprogressively increase with the increasing citric acid concentrationin H3citr–Ag NP hydrosol/H2TAPP system. Furthermore, to evaluatethe impact of the large SERRS signal observation on the possibilitiesof porphyrin detection, the concentration value of the SERRS spectraldetection limit of H2TAPP in the 1� 10�2 M H3citr–Ag NP hydrosol/H2TAPP has been determined and found to be 1 � 10�10 M fordetection of a complete porphyrin SERRS spectrum with a full set ofthe f.b. and diacid form markers (Fig. 6). TEM images of the deposited1 � 10�2 M H3citr–Ag NP hydrosol/H2TAPP system and HR-TEMimages of the selected aggregates (Fig. 6) show the presence ofisolated small compact aggregates (dimers, trimers) of touchingand/or interpenetrating Ag NP, i.e. of the particular morphologies forwhich very large EM mechanism enhancement have been theore-tically predicted [23].

4. Conclusions

1. LA of an Ag target in sodium citrate and/or citric acid aqueous

solutions of various concentrations has been established as anew pathway to preparation of stable hydrosols of citrate-modified Ag NPs. The morphologies of Ag NP in the resultinghydrosols are affected by the particular agent presence andconcentration in the ablation medium.

2. H

ydrosols prepared by LA in 1 � 10�5 M–1 � 10�2 M sodiumcitrate solutions are constituted by small Ag NPs well stabilizedby negatively charged adsorbed citrate. SERRS spectral probingof the citrate-modified Ag NP surfaces by the selected f.b.porphyrins has shown that the energy of adsorbed citratebonding to the surface cannot compete with the stabilizationenergy of porphyrin metallation. As a result, porphyrin

metallation on citrate-modified Ag NP surfaces cannot bepermanently prevented, but can be hindered kinetically, and theefficiency of this hindrance is critically dependent on the chargeand structure of the peripheral substituents of a particularporphyrin.

3. H

ydrosols prepared by LA in 1 � 10�5 M–1 � 10�2 M citric acidsolutions contain compact aggregates of touching and inter-grown Ag NPs, the fraction of which increases with theincreasing citric acid concentration. The partially protonizedadsorbed citrate works as an efficient molecular spacer for boththe cationic porphyrin H2TMPyP, and for the originally neutralH2TPyP and H2TAPP, which, in the acidic media, becomepositively charged due to protonization of their peripheralsubstituents and/or the diacid form formation. A substantialincrease of the SERRS signal observed for H2TPyP and H2TAPP insystems with Ag NPs prepared by LA in 1 � 10�2 M citric acid isattributed to contributions from both the EM mechanismenhancement stemming from the presence of hot spots in thecompact aggregates of touching and intergrown Ag nanopar-ticles and from the molecular resonance enhancement originat-ing from a close match between the Soret band of the diacidform (440 nm) and the 457.9 nm excitation. For H2TAPP, thesignal increase manifests itself further in the 1 � 10�10 M SERRSspectral detection limit.

4. A

g NP hydrosols prepared by LA in 1 � 10�2 M citric acid thusemerge as versatile substrates for SERRS of porphyrins allowingfor detection of f.b. porphyrins without their denaturation byincorporation of Ag and in very low concentrations.

Acknowledgments

The authors thank Mrs Jirina Hromadkova for her excellenttechnical assistance. Financial support by the 203/07/0717 grantawarded by GACR and by the MSM 0021620857 long-termresearch project awarded by MSMT CR is gratefully acknowledged.The authors also thank the French Ministry of Foreign Affairs andService Culturel de l’Institut Francais de Prague for the support ofthe co-tutoring of PhD thesis of Karolina Siskova.

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