Gold Nanoparticles Aggregation: Drastic Effect of Cooperative

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Gold Nanoparticles Aggregation: Drastic Effect of Cooperative Functionalities in a Single Molecular Conjugate Volodymyr Chegel,*,† Oleksandre Rachkov,‡ Andrii Lopatynskyi,† Shinsuke Ishihara,*,§ Igor Yanchuk,|| Yoshihiro Nemoto,§ Jonathan P. Hill,§,^ and Katsuhiko Ariga§,^ †

)

V. E. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences (NAS) of Ukraine, 41 Nauky Avenue, 03028 Kyiv, Ukraine ‡ Institute of Molecular Biology and Genetics, National Academy of Sciences (NAS) of Ukraine, 150 Academician Zabolotnyi Street, 03680 Kyiv, Ukraine § World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan Nano Medical Technologies LLC, 68 Gorkogo Street, 03150 Kyiv Ukraine ^ JST, CREST, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ABSTRACT: Aggregation of gold nanoparticles (AuNPs) can be utilized in chemical and biomolecular sensing as a sensitive and easyto-visualize process. However, interpretation of experimental results requires a clear understanding of physicochemical processes that take place upon multiple interactions between an analyte and AuNPs. In this article, interactions between citrate-stabilized AuNPs and organic compounds bearing various functional groups in an aqueous medium were experimentally and theoretically studied using spectrophotometry of the localized surface plasmon resonance (LSPR), transmission electron microscopy (TEM), conductometry, zeta potential measurements, and finite-difference time-domain (FDTD) modeling. As a result, it has been found that organic compounds containing both thiol and amine groups strongly promote the aggregation of AuNPs due to their cooperative functionalities. FDTD modeling has enabled consideration of the light extinction (i.e., LSPR response) properties of nanoparticle aggregates involving single, chain-like, and globular structures. Taking one billion distributions of differently structured aggregates into account, the theoretical light extinction was fitted to that of the experimental result with a root-mean-square deviation of 7%.

1. INTRODUCTION The unique physical and chemical properties of noble metal nanoparticles such as gold nanoparticles (AuNPs) open up a substantial field for investigations of new science.1
7 In comparison with bulk materials, nanoparticles possess a much higher concentration of electrons per surface unit. Therefore, attractive properties of AuNPs result from their coupling to an incident electromagnetic field (i.e., light) that appears as an enhancement of this field.8,9 Surface enhanced Raman spectroscopy,10 fluorometry11 and localized surface plasmon resonance (LSPR) spectroscopy12 utilize this feature and have made important contributions to the analytical sciences. A number of practical applications of AuNPs, such as highly effective solar cells,13 cancer therapy,14 integrated optics,15 and chemical and biological sensing,16
18 have been also developed. The LSPR phenomenon depends not only on the wave frequency and structural parameters (shape, size, and chemical nature) of nanoparticles but also on the distance between nanoparticles. Therefore, aggregation of nanoparticles induces variations in absorption spectra accompanied by significant color changes of solutions.19 Similar color changes can be observed on the addition of an analyte, r 2011 American Chemical Society

which initiates the aggregation of AuNPs, and this feature can be used for development of chemical and biological sensors for the detection of specific compounds.20
24 Research work toward the development of LSPR biosensors based on the phenomenon of AuNPs aggregation has been performed.23,25 Interpretation of the LSPR phenomenon requires a clear understanding of physicochemical processes that occur during interactions between an analyte and AuNPs. Although a number of factors that influence AuNPs aggregation, e.g., molecular size of modifying alkanethiols, have been investigated individually,26,27 the cooperative effect that takes place during multiple interactions between an analyte and AuNPs have not been fully discussed from a physical point-of-view. This makes understanding of aggregation mechanisms obscure and somewhat controversial, and it is not clear why aggregation occurs in some cases but not in others.28 AuNPs have strong binding affinities for thiols and amines,26,27,29
31 where modification of the AuNPs Received: September 25, 2011 Revised: December 22, 2011 Published: December 22, 2011 2683

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The Journal of Physical Chemistry C surfaces can lead to improved stability of AuNPs dispersions permitting their industrial application in biosensing, immunological, and biochemical investigations.1,10,11 The effect of amine functionality on binding to the surface of AuNPs has been investigated.32 However, some authors have claimed that one type of amine group can readily bind to Au colloids, whereas others cannot.33 Moreover, effects of the cooperative action of thiol and amine on aggregation of AuNPs have not been well investigated although they commonly coexist in biological samples. In the research reported here, cooperative functionalities of amine and thiol groups for aggregation of AuNPs were experimentally and theoretically studied using spectrophotometry of localized surface plasmon resonance (LSPR), transmission electron microscopy (TEM), conductometry, zeta potential measurements, and finite-difference time-domain (FDTD) modeling. Compounds containing various functional groups including amine and thiol groups, sometimes connected within one molecule, were selected for the investigation of the effect of citratestabilized AuNPs (Figure 1). In addition, FDTD modeling was applied for the first time to estimate the light extinction of nanoparticle aggregates involving single, chain-like, and globular structures, which can be a powerful method to overcome the drawbacks of conventional theoretical methods applicable only for one structure of AuNP. This research reveals physicochemical processes involved in the aggregation of citrate-stabilized AuNPs caused by interactions with a variety of model organic compounds.

Figure 1. Chemical structures of organic compounds used for preparation of model solutions.

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By establishing the methodology described here, we hope to deepen the understanding of the mechanism of AuNPs-aggregation, which should have a significant impact on related technologies based on the LSPR method.

2. EXPERIMENTAL SECTION 2.1. Materials. 6-Mercapto-1-hexanol (MCH) was obtained from Fluka (Switzerland). HAuCl4, sodium citrate, NaCl, urea, thiourea, cysteamine hydrochloride (Cys), tris(hydroxymethyl)aminomethane (TRIS), ethanol amine (EtA), glutathione (GSH), and lipoic acid (LA) were purchased from Sigma-Aldrich. All compounds were used as received. Milli Q deionized water (type I, R = 18.2 MΩ cm) was used for preparation of all solutions. 2.2. Synthesis of AuNPs. Preparation of AuNPs was performed through reduction of tetrachloroaurate ions (AuCl4
) by boiling in aqueous sodium citrate solution.34

Au3þ ðaqÞ þ 3e
f AuðsÞ AuNPs obtained using this method appear as almost monodispersed globular structures with size of about 10
15 nm (Figure 2), which are stabilized by weakly bound citrate anions. AuNPs are characterized by the plasmonic absorption band at approximately 520 nm.35 2.3. Methods. Electroconductivity of aqueous solutions of the materials studied was measured using GMH 3430 conductometer (Greisinger, Germany) by mixing 5 mL of Milli Q deionized water (added successively) with 25, 75, and 100 μL from 200 mM stock solutions of respective compounds (for 1, 4, and 8 mM of final concentration). The electronic absorption spectra of AuNPs were measured using UV-3600 UV
vis-NIR spectrophotometer (Shimadzu, Japan) from the solutions contained in plastic cells with optical path of 1 cm and width of 4 mm. Zeta potential measurements were performed using Zetasizer Nano ZS (Malvern, UK) for 3.25 nM solution of AuNPs in 0.12 SSC buffer solution (pH 7.2). TEM measurements were performed using Hitachi S-4800 scanning electron microscope at an accelerating voltage of 30 kV. HR-TEM was performed using JEOL JEM-2100F at an accelerating voltage of 200 kV. Aqueous dispersions of Au nanoparticles (or mixture of Au nanoparticles and thiourea) were diluted by a factor of 10 with distilled water and then dropped onto a carbon-coated copper grid. Excess

Figure 2. (a) TEM imaging of AuNPs used in this study. (b) Histogram of distribution of measurements based on TEM (Figure 2b). Inset: High resolution TEM of single AuNP. 2684

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Figure 3. Conductivity dependency on concentration for solutions of the test compounds. Inset: concentration dependency of conductivity for solutions of low conductivity.

water was allowed to evaporate at room temperature, and the sample was dried in vacuum overnight.

3. RESULTS AND DISCUSSION 3.1. Experimental Study. First, we assumed that the level of ionization (protonation
deprotonation of functional groups) of potential modifying compounds may play some role in the process of AuNPs functionalization and consequently may affect the stability of their dispersions. On the basis of results of conductivity measurements of the aqueous solutions of some of the test compounds (Figure 3), they can be classified in 3 groups: (1) highly ionized, NaCl and Cys; (2) very weakly ionized, urea, thiourea, and MCH; (3) with some intermediate level of ionization, TRIS. Taking into account the values of urea pKa = ∼26, thiourea pKa = ∼20, MCH pKa = 16.8 (for OH-group) and 10.2 (for thiol group), and TRIS pKa = 8.95, the measured values of conductivity of the solutions are in good agreement with the degree of ionization of their functional groups (high conductivity of hydrochloride form of Cys can be associated with the presence of H+ and Cl
). UV
visible absorption spectroscopy is a conventional method to probe the stability, the surface chemistry, and the aggregation behavior of AuNPs. Initial characterization of AuNPs prepared by citrate reduction of HAuCl4 solution revealed the absorption maximum (λmax) of the surface plasmon resonance peak for single particles at ∼520 nm (Figure 4, curve 1). This value is in a good agreement with that reported for AuNPs with a diameter of 10
15 nm.34 An analysis of TEM imaging (Figure 2) shows that the prepared AuNPs are nearly monodisperse spheres with an average size of about 13 nm. The AuNPs’ concentration was estimated to be 8  1012 mL
1 that corresponds to about 13 nM in terms of molar concentration. This value was obtained taking into account that the entire mass of gold in HAuCl4 employed for colloidal dispersion preparation was fully transformed into nanoparticles. Highly dispersed Au nanoparticles (effectively considered as single particles) in solution should exhibit only a single peak, while linked Au particle pairs (or larger aggregates) show two light absorption maxima.26 The appearance of a second peak is attributed mainly to the change of interspacing distance between AuNPs but also depends on the quantity of AuNPs. As the interparticle spacing decreases, the first peak becomes weaker,

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Figure 4. Electronic absorption spectra of a AuNPs solution before (curve 1) and after the addition of thiourea up to 1 μM (curves 2) and up to 5 μM (curves 3). Time intervals between measurements for curves 3 are 1 min for period 0
4 min, 2 min for 4
10 min, and 5 min for 10
25 min.

while the second peak intensifies and shifts to longer wavelengths. The maximum peak shift is observed if the interparticle distance approaches zero at which point the electrodynamic interaction between the nanoparticles is at a maximum. Initially, highly conducting NaCl solutions were added to AuNPs colloids. According to DLVO theory,36 electrolyteinduced aggregation occurs when equilibrium between van der Waals attractive and electrostatic repulsive forces is disturbed. This phenomenon can be observed only at higher salt concentrations; additions of up to 10 mM NaCl did not lead to any shift of the surface plasmon resonance peak for single particles. No changes were observed during at least 0.5 h after salt addition (data not shown). According to some reports,32,33 the presence of two amine groups in urea could make it an efficient agent for nanoparticle modification. However, upon the addition of urea to AuNPs colloidal solution (as for the case of NaCl), neither a shift of the surface plasmon resonance peak for single particles at 520 nm nor an appearance of any other peaks were observed even for the relatively high concentration of 15 mM. These spectra also did not change over time. Thus, in spite of the presence of amine groups, no change of the citrate-stabilized AuNPs was registered (data not shown). This is probably because both amine groups of urea are not ionized under approximately physiological conditions and remain neutral, which is proven by the very low conductivity for the urea solutions (Figure 3). Unlike urea, an addition of only 1 μM thiourea to AuNPs gave some increase in light absorption in the range of 550
800 nm (Figure 4, curves 2), and 5 μM thiourea gave a strong decrease in peak intensity at 520 nm with a concurrent appearance of a broad peak in the range 660
680 nm (Figure 4, curves 3). This broad peak strongly overlaps and influences the shape and intensity of the surface plasmon resonance peak of the single particles. Just after the addition of thiourea, the intensity of the broad peak was greater than that of the transformed initial peak at 520 nm. Subsequently, the obtained spectra demonstrated essentially a decrease in intensity (arrow in Figure 4). This was due to formation of nanoparticle aggregates and their subsequent precipitation, which could be observed by the naked eye. The dramatic difference in behavior of solutions of these two compounds (i.e., urea and thiourea) in the presence of AuNPs can be ascribed to the characteristic tautomerism of thiourea in solution, namely, the transformation between the thione-form and the thiol-form. 2685

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Figure 5. Electronic absorption spectra of a AuNPs solution before (curve 1) and after the addition of GSH up to 20 μM (curve 2). Time intervals between measurements were 5 min for each subsequent spectrum. Inset: detail of the spectra.

Figure 6. Electronic absorption spectra of a AuNPs solution before (curve 1) and after the addition of TRIS up to 3 mM (curves 2). Time intervals between measurements are 2 min for the second and 5 min for each subsequent spectrum.

Obviously, stable chemical bonds between the thiol group and gold atoms formed at the thiourea addition allow these molecules efficiently to replace citrate ions at the surface of AuNPs. In addition, amine as well as imine groups (CdNH) due to tautomeric processes should attenuate the negative charge at the surface of AuNPs with a resulting collapse of colloidal stability followed by aggregation. Similarly to the case for thiourea, the addition of 5 μM positively charged sulfur-containing Cys to AuNPs solutions led to a decrease in peak intensity at 520 nm and an appearance of a broad aggregative peak, the maximum of which shifted from 640 to 676 nm during 25 min (data not shown). All spectral changes as well as the processes of aggregation and precipitation of nanoparticles were characterized by somewhat faster kinetics than that of thiourea addition, which would be due to the absence of tautomerism to provide thiol groups. The addition of 5 μM uncharged sulfur-containing MCH to AuNPs also led to a reduction in peak intensity at 520 nm and an appearance of an aggregative peak (data not shown). Compared with the previous cases, the maximum of the broad aggregative peak shifted only from 623 to 640 nm (red-shift is not so large as for thiourea and Cys). Meanwhile, no visible precipitate was observed. Obviously, MCH reacts with AuNPs. However, the kinetics of the aggregation process is slower and can probably be associated with the lower acidity of OH-groups of MCH molecules: efficient replacement of citrate ions on the surface of AuNPs does not lead to such a rapid decrease of surface negative charge. Consequently, the stability of AuNPs modified by MCH is somewhat higher. Of the sulfur-containing compounds, the addition of those bearing negative charge was also investigated. Addition of 5
200 μM GSH solutions to AuNPs did not yield any evidence of aggregative behavior. However, some changes in absorption spectra were apparent: the maximum of the surface plasmon resonance peak for single particles shifted from 520.2 to 521.7 nm for 20 μM GSH (Figure 5). Upon further increase of GSH concentration a larger shift was observed. This shift can be explained by the presence of the thiol group, which reacts with gold, and by the overall negative charge of GSH. In other words, replacement of citrate ions most probably takes place, but the amount of negative charge on the surface of nanoparticles does not decrease significantly. In fact, the negative charge probably becomes greater. As a result, the AuNPs colloid remains stable and does

not aggregate. A similar result was obtained during the addition of another negatively charged sulfur-containing compound, LA (data not shown). Of the compounds containing amine group(s) but lacking sulfur atom (apart from the already mentioned urea), TRIS and EtA were also investigated. Similarly to the case of urea, on the addition of TRIS to AuNPs at micromolar concentrations, neither a shift of the surface plasmon resonance peak for single particles at 520 nm nor an appearance of any other peaks could be observed. Changes in the spectrum of AuNPs appeared only at much higher TRIS concentrations (3 mM instead of 5 μM for sulfur-containing compounds) (Figure 6). Taking into account that the overall concentration of citrate ions in the AuNPs colloid was also about 3 mM, we can assume that an almost equimolar quantity of TRIS is needed to influence the properties of the AuNPs. Even so, the aggregation process is significantly slower, and the aggregates formed seem to be relatively stable. TRIS molecules probably form a diffuse layer near the AuNP surface without replacement of citrate ions and only partially neutralize their negative charge. Hydroxyl groups of TRIS afford more hydrophilic properties and stability of such AuNPs complexes, which is revealed by the limited aggregation without essential precipitation. The next compound, which contains an amine group but lacks a sulfur atom, EtA, had an even less pronounced effect on AuNPs. The structure, size, and presence of amine group in molecules of EtA make it very similar to Cys. The only difference between them is that in place of a thiol group, EtA contains an OH-group. However, unlike Cys, micromolar concentrations of EtA do not change the absorption spectrum of AuNPs. Only millimolar concentrations of EtA evoked the appearance of a low intensity and very broad shoulder in the range of 550
800 nm. It seems that in the absence of a thiol group, EtA cannot bind covalently with the surfaces of AuNPs. Replacement of citrate ions and reduction of negative charge at the surface of AuNP does not take place (as it does on Cys addition), and at only relatively large concentrations does EtA (like TRIS) very slowly form a diffuse layer near the AuNP surface partially neutralizing their negative charge. To further investigate the above-mentioned observations, measurements of zeta potential during interactions of AuNPs with the test compounds were performed. It should be noted that 2686

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Figure 7. Zeta potential dependence on concentration for the differently charged compounds. Black line, Cys; red line, MCH; blue line, GSH; and pink line, EtA.

in order to obtain reliable and reproducible results, the initial AuNPs colloid was diluted by a factor of 4 with a buffer solution to a final concentration of 3.25 nM AuNPs and 0.12 SSC (pH 7.2). As shown in Figure 7, the addition of 0.5
2.0 mM positively charged sulfur-containing Cys leads to a sharp decrease of negative charge on the surface of AuNPs. At the concentration of Cys around 5 mM, zeta potential even becomes positive. When MCH with a neutral hydroxyl group is added, the weakening of negative potential on the surface of AuNPs occurs somewhat slowly. Upon the addition of GSH (containing thiol, amine, and two carboxyl groups and carrying overall negative charge), the negative zeta potential of AuNPs, as expected, does not fall and even increases. Lastly, the addition of EtA (positively charged, similar to Cys but without thiol group) leads to a very slow, gradual weakening of the negative zeta potential of AuNPs. Thus, the addition of sulfur-containing compounds, which are able to bind covalently to gold, actively influences AuNPs mainly as a charge-dependent process. The positively charged components, which cannot create S
Au bonds, influence the stability of citrate-stabilized AuNPs by formation of a diffuse layer near the AuNP surface (partially neutralizing their negative charge) at much higher concentration. It should be noted that the zeta potential and electronic absorption spectra measurements were performed using different dilutions of AuNPs. Therefore, for these two methods, the different ranges of concentrations for all tested compounds could demonstrate their influence on AuNPs. Thus, the data obtained by both methods are in good agreement and confirm our previous assertions. To estimate the influence of the compounds when added to AuNPs dispersion, at least four processes should be considered: (I) the diffusion of test molecules to the AuNPs, (II) surface displacement of adsorbed anions by test molecules, (III) aggregation of modified AuNPs, and (IV) precipitation of the aggregates.26 For diffusion, from our observations, an intense mixing of the test compounds with the AuNPs dispersions led to a very limited influence by this process. For efficient surface displacement of citrate ions the presence of sulfur atoms capable of forming stable chemical bonds with gold atoms in the test molecule is necessary. As nitrogen atoms cannot form such a stable bond with gold, only compounds with highly protonated amine groups (and at a higher concentration) can neutralize the negative charge of the citrate-stabilized AuNPs without real chemical modification.

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From these points-of-view, cooperative functionalities of thiol and amine groups are a crucial factor for highly enhanced nanoparticle aggregation. That is, the concentration of TRIS needs to be of millimolar order to cause the aggregation of AuNPs, whereas micromolar concentration is sufficient for thiourea to cause the aggregation. It should be noted that similar absorption spectra for the addition of thiourea and Cys to AuNPs were obtained in spite of the significant difference in the conductivity of their solutions, whereas the addition of NaCl or Cys (both solutions with high conductivity) to AuNPs led to completely different spectra. The same is true for urea and thiourea. These results prove that the assumption about the absence of a simple and direct relationship between electroconductivity of the aqueous solutions of the test compounds and behavior of citrate-stabilized AuNPs is correct. However, it was shown by zeta potential measurements that the sign of charge carried by the actively substituting molecules is of great importance. If modification does not noticeably change the zeta potential of AuNPs, interparticle distance also does not change, and consequently, no aggregation is observed. In contrast, if the interparticle spacing decreases, aggregation occurs, and peak shifts to longer wavelengths are observed in the electronic absorption spectra. 3.2. Theoretical Modeling. A theoretical study was carried out to clarify the experimental results and elucidate the variation in the electronic absorption spectrum of an Au colloidal solution during the nanoparticle aggregation process. As mentioned previously, the localized surface plasmon resonance (LSPR) phenomenon in noble metal nanoparticles, which is responsible for their light extinction properties, is size- and shape-dependent. Thereby, the absorption spectrum of an aggregated Au colloid should reflect both increases in the average AuNP size due to nanoparticle agglomeration and changes in nanoparticle shape from spherical to a variety of aggregate forms, which may include spherically symmetric and chain-like configurations. The analysis of TEM images allows the size of the nanoparticles to be defined, and in this case, it lies in the range of 10
15 nm (the average value is about 13 nm). A model of citrate-stabilized Au colloid comprised of AuNPs with a diameter of 13 nm34 covered with 1-nm-thick citrate ion shell,37 immersed in water, was developed and shown to fit an experimental spectrum for an unaggregated colloid by means of Mie theory for a spherical core
shell nanoparticle.38 It takes into account sizedependent gold optical constants through eqs 1
3 and treats the citrate ion shell surrounding the nanoparticles by means of symmetrical Bruggeman effective medium theory (eqs 4
5).38   Vf
1
1 τef ðRÞ ¼ τbulk þ A ð1Þ R ε1 ðω, RÞ ¼ ε1bulk ðωÞ þ

ε2 ðω, RÞ ¼ ε2bulk ðωÞ þ

ωp 2 ω2 þ

1


τbulk

2

ωp 2 ω2 þ

1 τef

ð2Þ

2 ðRÞ

  ωp 2 τef ðRÞ τbulk
ω ω2 τef 2 ðRÞ þ 1 ω2 τbulk 2 þ 1

ð3Þ where τbulk = 9.3  10
15 s39 is the electron relaxation time for bulk Au, Vf = 1.4  106 m/s40 is the Fermi velocity for Au, R is the nanoparticle radius, A is a parameter, which for spherical 2687

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Figure 8. (a) Normalized total absorption spectra for a single citratestabilized AuNP and a close-packed spherically symmetric aggregate consisting of 13 citrate-stabilized AuNPs, immersed in water. (b) Total absorption spectra for a single citrate-stabilized AuNP and linear aggregates consisting of 2 to 6 citrate-stabilized AuNPs immersed in water.

nanoparticles considered here with isotropic electron scattering is selected as equal to 1, ε1 and ε2 denote the real and imaginary parts of the dielectric function of Au, ω is the light angular frequency, and ωp =1.37  1016 rad/s41 is the bulk Au plasma frequency. f

ncitrate 2
nef f 2 nwater 2
nef f 2 þ ð1
f Þ ¼0 ncitrate 2 þ 2nef f 2 nwater 2 þ 2nef f 2

 d 2 πd R þ 1 2 f ðR, dÞ ¼ 3 2 ðR þ dÞ
R 3

ð4Þ



ð5Þ

where f is a shell filling factor, ncitrate = 1.57542 is the refractive index of citrate ions, neff denotes the effective refractive index of the citrate ion shell, nwater = 1.33 is the refractive index of water, and d is the shell thickness. To study the variation of the electronic absorption spectrum upon the increase of average Au nanoparticle size, Mie theory for a spherical core
shell configuration was used initially. It was shown that the two peaks in the absorption spectrum, corresponding to dipolar and quadrupolar LSPR excitations, are produced near to experimental positions only for nanoparticle diameters exceeding 120 nm, implying that aggregates should be composed of hundreds of nanoparticles, which differs from previously published work.26,27,43,44 In addition, Mie theory in

Figure 9. (a) TEM imaging of thiourea-covered AuNPs aggregate with a structure similar to that used in the simulation procedure. (b) Normalized absorption spectrum of AuNPs solution after the addition of thiourea up to 3 μM (black line) and a theoretical best fit, which was obtained using the total absorption spectra of all studied aggregate geometries (linear (1
6 mer) and spherical aggregates (13 mer) shown in Figure 8a,b) (red line). Inset: histogram of distribution of nanoparticle number in an aggregate obtained as a result of fitting procedure.

applied approximation did not allow a taking into account of the interaction between adjacent nanoparticles in an aggregate, which is known also to influence the absorption spectrum. Therefore, we used the finite-difference time-domain (FDTD) method for investigation of both effects influencing the absorption spectrum during nanoparticle aggregation and consideration of electromagnetic interactions between nanoparticles. The aggregate geometries studied included close-packed spherically symmetric aggregates consisting of 13 nanoparticles and linear aggregates consisting of 2 to 6 nanoparticles. Distance between gold cores of citrate-capped nanoparticles in the linear aggregate and between central and surrounding nanoparticles in the spherical aggregate was fixed at 0.5 nm, which is close to the size of a small organic molecule.26 To simulate the aggregated colloidal system response to unpolarized light, we estimated the total absorption spectrum for each of the aggregate geometries studied by summarizing the spectra calculated for a complete range of angles of incidence of linearly polarized light on the individual nanoparticle aggregate with a step of 10. To the best of our knowledge, this approach for studying extinction properties of aggregated Au colloids has not been applied previously. It was found that the total spectrum for a spherical aggregate (Figure 8a) exhibits only one distinct peak (at 549 nm) in the wavelength range 450
800 nm, where experimental peaks are located. In contrast, total absorption spectra for linear aggregates 2688

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contain two peaks in the above-mentioned region, magnitudes and positions of which are dependent on aggregate length (Figure 8b). However, both experimental absorption peaks for an aggregated colloid are red-shifted with respect to the peak for an unaggregated colloid, while simulated absorption peaks behave differently: for a spherical aggregate, it is red-shifted, but for all of linear aggregates, one of the peaks is blue-shifted, and the other is red-shifted. This implies that the optical response of an aggregated Au colloid should be due to close to spherical and chain-like aggregates present in solution, which is consistent with published data.26,27,43
45 Simulated spectra for different aggregate geometries were also applied to fit an experimental spectrum in order to reveal the character of aggregate formation. The fitting procedure was carried out under the assumption that differently shaped nanoparticle aggregates act as independent light absorbers and contribute to total absorption in a linear manner in proportion to their concentrations. The fitting algorithm was based on Monte Carlo enumeration of percentages of aggregates of all studied geometries and formation of a summed absorption spectrum, which was fitted to the experimental data by means of a least-squares technique. Fitting results for the case of AuNP solutions after the addition of thiourea up to 3 μM are depicted in Figure 9b. After enumeration of one billion aggregate percentage sets, a rootmean-square deviation of 7% was achieved. As can be seen from the histogram (Figure 9b, inset), the optical response is formed mainly by linear nanoparticle aggregates consisting of 2 (20.7%), 3 (59.6%), and 6 (14.1%) nanoparticles and spherically symmetric nanoparticle aggregates consisting of 13 (4.1%) nanoparticles, while the contributions of other geometries are lower than 1% for each. This proves our assumption about the origin of optical response, which is due to contributions from both chainlike and spherically shaped aggregates.

within NaCl. This is similar to the Hofmeister series, where NH4+ is stronger than Na+. However, some features of the molecular structure, e.g., presence of three hydroxyl groups per molecule of TRIS, can provide some stability to AuNPs aggregates in colloidal solution. Two groups of the compounds (those that modify but do not cause aggregation of nanoparticles and those that caused their aggregation and precipitation, at the same time causing some shifts to the initial absorption spectra of AuNPs) can be used for development of a LSPR chemical and biomolecular sensing platform because the interaction of citrate-stabilized AuNPs with the aforementioned compounds is a very sensitive easy-to-visualize process. However, interpretation of experimental results requires the clear understanding of physicochemical processes that take place upon the interactions of AuNPs with the analyte, and it requires further investigations. A theoretical model based on the FDTD method was proposed for consideration of evolution of the AuNPs system light extinction properties during the aggregation process. It was shown that the optical response of an aggregated Au colloid should be formed both by close to spherical and chain-like nanoparticle aggregates present in solution. The theoretical approach applied was demonstrated to be useful for estimation of the distribution of aggregate shapes corresponding to the experimental absorption spectrum. The knowledge obtained will be of great use in various applications, especially for sensing. A deep understanding of aggregation of AuNPs in the presence of organic modifiers is indispensable in designing sensing systems using AuNPs hybridized with organic recognition functionalities. There exist possibilities for the introduction of various organic-functionalized recognition systems, which have been reported for the detection of amino acids,46,47 nucleic acid base families,48,49 and chiral substances50,51 to nanoparticle-based advanced sensing systems.

4. CONCLUSIONS The experiments presented here illustrate that at least 3 features of potentially modifying compounds could influence citrate-stabilized AuNPs: (1) the presence of sulfur atoms, which can form covalent bonds with gold atoms; (2) the presence of ionizable functional groups; and (3) the sign (+ or
) of ionizable functional groups. The presence of sulfur atoms within the thiol or thiocarbonyl groups (e.g., in Cys, MCH, GSH, thiourea) allow these compounds to covalently bind to gold atoms at the surface of AuNPs and to efficiently replace citrate ions from the surface. The fate of the modified AuNPs depends on what charge is carried by the modifying compounds: negatively charged compounds increase the stability of nanoparticle dispersion, positively charged compounds weaken negative zeta potential and cause fast aggregation and precipitation of AuNPs, and compounds with neutral (nonionizable) functionalities lead to quite limited stability with a high probability of aggregation. Of the compounds studied, which cannot create S
Au bonds, only positively charged ones (primarily those carrying ionizable amine groups) influenced the stability of citrate-stabilized AuNPs by the formation of a diffuse layer near the AuNP surface, partially neutralizing their negative charge. The importance of this probably depends on several factors, which can be ranked in a similar manner to the Hofmeister series for salting-out proteins. The charged amine group within TRIS has been shown here to influence the properties of AuNPs much stronger than Na+

’ AUTHOR INFORMATION Corresponding Author

*(V.C.) Fax: +38-44-525-18-27. E-mail: [email protected]. (S.I.) Fax: +81-29-860-4832. E-mail: ISHIHARA.Shinsuke@ nims.go.jp.

’ ACKNOWLEDGMENT This work was partly supported by World Premier International Research Center Initiative (WPI Initiative); MEXT; Japan and Core Research for Evolutional Science and Technology (CREST) program of Japan Science and Technology Agency (JST), Japan; National Academy of Science of Ukraine in the frame of national program “Nanotechnology and Nanomaterials”; and complex program of National Academy of Sciences of Ukraine “Sensor systems for medical, ecological and industrial purposes”. ’ REFERENCES (1) Thaxton, C. S.; Georganopoulou, D. G.; Mirkin, C. A. Clin. Chim. Acta 2006, 363, 120. (2) Caruthers, S. D.; Wickline, S. A.; Lanza, G. M. Curr. Opin. Biotechnol. 2007, 18, 26. (3) Jain, K. K. Clin. Chem. 2007, 53, 2002. (4) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Elst, L. V.; Muller, R. N. Chem. Rev. 2008, 108, 2064. 2689

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