Gold Nanosphere

Article pubs.acs.org/JPCB

Concentration-Controlled Formation of Myoglobin/Gold Nanosphere Aggregates Paz Sevilla,†,‡ Santiago Sánchez-Cortés,‡ José V. García-Ramos,‡ and Alessandro Feis*,§ †

Departamento de Química Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain Instituto de Estructura de la Materia, IEM-CSIC, C/Serrano 121, 28006 Madrid, Spain § Dipartimento di Chimica “Ugo Schiff”, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy ‡

S Supporting Information *

ABSTRACT: Gold nanoparticles are being increasingly proposed as biotechnological tools for medical diagnosis and therapy purposes. Their safety for human beings and the environment is therefore becoming an emerging issue, which calls for basic research on the interactions between nanostructured gold particles and biological materials, including physicochemical studies of model systems. In this Article, we focus on the “reaction products” of a widely known nanoparticle type, citrate-capped 30 nm gold nanospheres, with a model protein, horse myoglobin. Protein adsorption and partial denaturation were accompanied by the formation of nanoparticle aggregates with strongly distinct optical spectroscopy properties and shapes, as observed by transmission electron microscopy. We singled out the concentration of myoglobin as the determinant of these differences, and verified on this basis that surface-enhanced Raman scattering (SERS) spectra can only be obtained by aggregates with strong interparticle optical coupling, which are obtained at low protein concentration. The results can be useful both in improving the spectroscopy of biomolecules and in understanding the formation of the protein corona in biomedical applications.

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INTRODUCTION Understanding the interactions that take place at the interface between metal nanostructures and biological macromolecules is a currently relevant scientific issue. In fact, metal nanoparticles (NPs) are becoming widespread materials1 and their impact on the biosphere will be more and more likely to occur in the future. Practical applications of noble metal nanostructures, in particular, are being increasingly proposed in medical diagnostics and therapy2−4 because of the rich optical properties deriving from plasmonic excitations.5 It can be therefore envisaged that the complexity of interactions of metals in various NP forms on one hand, and human cellular components on the other one, must be unravelled by first examining the properties of treatable model systems. The experimental results presented in this Article are focused on the mutual changes induced by the formation of complexes between gold nanospheres (AuNS) and myoglobin (Mb). This reaction can be viewed as an extremely simplified example of the events triggered by the encounter of a protein with a nanostructured metal surface. We have soon realized that these events strongly depend on the protein concentration in an unusual fashion. In a way, there is not a univocally defined Mb/ AuNS complex but, rather, AuNS aggregates (where the protein stays adsorbed at the metal surface) with various formation kinetics, shape, and optical properties, all of which depend on the initial protein concentration. © 2014 American Chemical Society

This variability of optical properties has interesting consequences. The excitation of localized surface plasmon resonances (LSPR) in metal NPs results in very strong extinction bands, associated with high enhancements of the local fields at the metal surface. This intensification constitutes the basis for surface enhanced spectroscopies: surface enhanced Raman scattering (SERS), surface enhanced fluorescence (SEF), and surface enhanced infrared absorption (SEIRA). These techniques present high sensitivity for the detection of low concentrations of molecules and are potentially useful for the study of proteins on metal surfaces. SERS displays strong dependence on surface morphology. In NP aggregates, the electromagnetic field is enhanced 5 to 7 orders of magnitude in relation to isolated NPs.6 Moreover, the enhancement depends on the size of the gap between aggregated NPs. Therefore, a deepened knowledge of the Mb/AuNS system would shed light on the controlled formation of aggregates, and consequently on the opportunity of obtaining protein SERS spectra.7 We have verified this aspect by performing SERS on aggregates with different protein concentrations. There are specific reasons to prefer gold to other metals in the field of nanotechnology. AuNS exhibit a particularly high Received: February 26, 2014 Revised: April 28, 2014 Published: April 28, 2014 5082

dx.doi.org/10.1021/jp502008a | J. Phys. Chem. B 2014, 118, 5082−5092

The Journal of Physical Chemistry B

Article

Figure 1. (Left) Extinction spectra (optical path = 1 cm) of AuNS alone (A) and after one-shot addition of Mb, leading to the final concentrations 3.7 × 10−8 M (B), 7.4 × 10−8 M (C), 7.4 × 10−7 M (D), and 7.4 × 10−6 M (E). These spectra were recorded 15 min after mixing the protein with the AuNS. (Right) Selected TEM micrographs of the same samples.

functionality is associated with a secondary and tertiary native structure. For instance, our previous studies have demonstrated that there are substantial changes in the structural properties of bovine serum albumin when this protein is adsorbed on Ag NPs,13 while other authors have found a similar effect for lysozyme adsorbed on AuNS.14 We have selected a very simple and widespread kind of AuNS, i.e., aqueous dispersions of citrate-capped AuNS prepared according to Frens’ method.15 Besides the existing wide characterization of these NPs, an

biocompatibility. They can be used for protein and nucleic acid detection, cancer imaging8 and therapy9 or multistep drugdelivery systems.10 Moreover, they are appreciated in highsensitivity, label-free detection methods. Their use in the presence of proteins,11,12 though, must be carefully evaluated. Whereas small organic molecules usually change their physicochemical properties only to a limited extent when adsorbed on metal surfaces, a different situation arises when the molecules adsorbed on the metal surface are proteins. Their 5083



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× 10−6 M. In all the measurements we present, small volumes of concentrated Mb solutions in pure water were added to the unbuffered, undiluted AuNS aqueous dispersions (pH 4.0− 4.5). The first series of measurements is shown on the left side of Figure 1. The extinction spectrum of AuNS alone displayed a maximum at 525 nm in the LSPR region. The position of the maximum is that expected for a 30 nm diameter AuNS on the basis of Mie theory (see Supporting Information Figure 1). After the addition of concentrated Mb solution to a final concentration 3.7 × 10−8 M Mb, the LSPR band decreased and shifted to 529 nm, while a very broad band, peaking at 700 nm, appeared in the long wavelength region. These changes occurred on a time scale of minutes, the detailed kinetics for this process being presented below. The spectra in Figure 1 were recorded when an equilibrium was reached, that is, when the LSPR band position did not appreciably change in time. This occurred within ∼15 min. A slow sample precipitation started at longer times, and could be detected by visual inspection. The precipitation caused an overall slight decrease of the extinction spectrum, without changes in the relative band intensities. Further increases of Mb concentration, after the equilibrium was reached, did not lead to substantial extinction changes (Supporting Information Figure 2). At variance, when Mb was added to the AuNS sample, reaching a 7.4 × 10−8 M protein concentration in one shot, the longer-wavelength band was less shifted, its band maximum lying at 640 nm. We obtained largely different results at higher Mb concentrations, provided the protein was added in one shot. At 7.4 × 10−7 M Mb, a single LSPR band was observed, peaking at 529 nm with a slightly increased intensity. The same was obtained at 7.4 × 10−6 M Mb, a single LSPR band being present at 529 nm. The additional band at 408 nm is an absorption band of the protein, namely, the one due to the allowed π−π* transition of the heme chromophore (Fe-protoporphyrin IX). The right-hand panel of Figure 1 shows a selection of TEM micrographs of the same samples. Protein-free AuNS displayed an approximately spherical shape, with 30 nm diameter in most cases. We only encountered isolated particles in the TEM sample. In contrast, all the protein/AuNS samples appeared to be formed by NP aggregates. Most of the aggregates (within the limits of the low statistics allowed by TEM images) consisted of rows, or parallel strands, at 3.7 × 10−8 M protein concentration, whereas more extended aggregates were present at 7.4× 10−8 M Mb (see Supporting Information Figure 3). A thin protein layer, highlighted by the uranyl acetate treatment, surrounded most AuNS in the latter sample. In the presence of higher protein concentrations, namely, 7.4 × 10−7 and 7.4 × 10−6 M, the protein layers were more evident and possibly thicker. The aggregation kinetics was studied by following the decrease of the extinction at 525 nm. In fact, the extinction spectra did not display any neat isosbestic point as time proceeded (see Supporting Information Figure 4). On the other hand, the 525 nm extinction change represents the disappearance of the isolated, protein-free AuNS and therefore this variation can be considered as an effective indicator of the aggregation process. Figure 2 shows the aggregation kinetics at the four different final Mb concentrations we examined. A clear effect of Mb concentration can be detected. The aggregation at the lowest (3.7 × 10−8 M) concentration can be fit by a double exponential decay: a prompt one with a characteristic time 7 ± 1 s, actually falling below our time resolution (10 s), and a

advantage of this system is the minimal presence of chemicals which can interfere in the protein/metal interaction, e.g., surfactants or polymers. The choice of the protein is another determinant. Mb (from horse muscle) is a relatively small, globular protein consisting of 153 amino acids.16 It lacks cysteine residues, which rules out the formation of covalent bonds with the Au surface. We can therefore foresee that the formation of Mb/AuNS complexes is driven by electrostatic and hydrophobic interactions, making the process a representative example of protein/NPs conjugation in the absence of specifically designed binding sites. Moreover, the number and diversity of studies about Mb is outstanding and ranges from crystallography17 to spectroscopy on Mb in different forms (i.e., free and ligated) and chemical environments.18−22 For these reasons, Mb can be considered as a simple yet versatile model protein.

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MATERIALS AND METHODS All solutions were prepared with water from a Milli-Q Integral A10 system from Millipore, yielding water with resistivity in the range 10−15 MΩ × cm. Mb (from horse skeletal muscle) was purchased from Biozyme. All other reagents were purchased from Sigma-Aldrich. AuNS were obtained by citrate reduction of HAuCl4 according to Frens’ method.15 Transmission electron microscopy (TEM) images were obtained on a JEOL 1010 microscope operating at 80 kV from Japan Electron Optics Laboratory Co. Ltd., installed in the Centre for Electron Microscopy at the University Complutense of Madrid. UV−visible absorption spectra were recorded on a Shimadzu UV-3600 spectrophotometer using quartz cells with 1 cm of path length. The kinetics of AuNS aggregation has been monitored by the change in extinction at 525 nm after one-shot addition of concentrated Mb solution, leading to the final desired concentration. Circular dichroism (CD) experiments were performed on a JASCO 710 instrument between 190 and 250 nm. The cell length was 1 cm. The slit width was 1500 μm, yielding a resolution of 0.5 nm. The spectra were recorded at 50 nm/min speed with a response time constant of 4 s. CD measurements gave directly the ellipticity, θ (mdeg), and the mean residue ellipticity (MRE) was calculated using the following expression: MRE = θ /(CPNr)

(1)

where C is the molar concentration (dmol/L), P is the path length of the cell (cm), and Nr is the number of protein residues, which is 153 for horse Mb. The SERS spectra were obtained with a confocal Raman microscope Renishaw Invia equipped with a Leica microscope and an electrically refrigerated CCD camera. Laser excitation lines were provided by a diode laser (785 nm) and a Renishaw Nd;YAG laser (532 nm). The measurements were carried out using a macro configuration. The laser beam (0.2 mW power at 785 nm, 2 mW at 532 nm) was focused inside a quartz tube containing the sample. The frequencies were calibrated with silicon. Mie calculations were performed with the program MiePlot (2011) by Philip Laven.

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RESULTS We have characterized the interactions of AuNS with Mb in a wide protein concentration range, i.e., from 3.7 × 10−8 M to 7.4 5084



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Figure 4. Kinetics of secondary structure changes after mixing AuNS with Mb at 7.4 × 10−8, 7.4 × 10−7, and 7.4 × 10−6 M concentration. For the most diluted sample, the average MRE in the 218−226 nm range is plotted, whereas the MRE at 222 nm is plotted for the other two samples. Exponential fitting plots are also shown for the highest and lowest concentrations.

Figure 2. Kinetics of AuNS aggregation following the addition of 3.7 × 10−8, 7.4 × 10−8, 7.4 × 10−7, and 7.4 × 10−6 M Mb. The extinction change at 525 nm is plotted as a function of time. Double exponential fitting plots are also shown for the lower concentrations.

slower one, with a characteristic time 220 ± 30 s. At 7.4 × 10−8 M Mb the decay stays biexponential, the slower time becoming 280 ± 80 s. In contrast, at higher Mb concentrations, only a prompt change can be observed. The extinction change is reduced, as the spectral variations are small (Figure 1D, E). We successively investigated the variations with time induced by Mb/AuNS interactions on the protein secondary structure by ultraviolet CD spectroscopy. Figure 3 compares the CD spectra−in the 190−250 nm range−of Mb in the absence of AuNS with those of Mb 15 min after addition to Au colloid, at increasing final protein concentration. Below 7.4 × 10−8 M protein, the signal-to-noise ratio precludes the measurement. The interaction with AuNS leads to an intensity decrease of the negative bands at 209 and 222 nm, as well as of the positive band at 192 nm. This is especially apparent at the lowest Mb concentration, that is, 7.4 × 10−8 M. The interpretation of these changes is well established in the literature, being related to a decrease of the α-helix content of the protein.23,24 Figure 4 displays the kinetics for the CD changes. The disappearance of the 222 nm band is plotted for 7.4 × 10−7 and 7.4 × 10−6 M protein concentrations. For 7.4 × 10−8 M concentrations, owing to the lower signal-to-noise ratio, the average CD in the 218−226 nm range was plotted as a function of time. The data could be fit by single exponentials with 170 ± 30 and 139 ± 4 s decay times for 7.4 × 10−8 and 7.4 × 10−6 M

Mb, respectively. For the intermediate concentration, exponential fitting is not adequate. It can be nevertheless observed that the CD variations are in-between those measured at lower and higher concentration. The concentration dependence of Mb/AuNS interaction has a strong influence on the possibility to obtain SERS spectra, as indicated by the following results. Figure 5A compares the Raman spectra of Mb/AuNS at 3.7 × 10−8 and 7.4 × 10−7 M Mb concentration with 785 nm excitation wavelength. The latter sample only displayed broad background bands due to water and to the quartz sample holder. In contrast, the sample at lower Mb concentration yielded an intense SERS spectrum. This difference is clearly related to the different plasmon resonance conditions: it can be seen from Figure 1B that the 785 nm excitation wavelength matches the LSPR band of the 3.7 × 10−8 M Mb sample, whereas it is largely detuned from the LSPR of the 7.4 × 10−7 M Mb sample. The spectrum in Figure 5B yields further evidence that the SERS enhancement mechanism is related to the plasmonic properties of the different kinds of Mb/AuNS aggregates. In this case, the protein concentration was 3.7 × 10−8 M and the excitation wavelength was 532 nm, matching the LSPR band at shorter wavelength. The spectrum only displays the broad background features without any SERS bands. A “blank” SERS spectrum,

Figure 3. CD spectra of Mb aqueous solutions at 7.4 × 10−8, 7.4 × 10−7, and 7.4 × 10−6 M concentration in the absence (black line) and in the presence (red line) of AuNS. The latter spectra were measured 15 min after mixing the protein with the AuNS. 5085



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the assignment can only be correlative. We can expect three main groups of vibrational bands to be present in the SERS spectrum: heme group bands, amino acid side chain bands, and peptide backbone bands. It is noteworthy that the bands of the heme group, because of the resonance conditions, are weak or absent in the SERS spectrum. Possibly, only the bands at 753 and 1123 cm−1 can be assigned to heme modes, namely, to the υ15 and υ5 porphyrin modes, respectively.25 Concerning the side chains, the aromatic amino acid groups should display the most intense bands. Horse Mb owns two tyrosine residues, two tryptophans, and six phenylalanines. Tryptophan bands are normally outstanding, but seem to be silent in our spectrum. Conversely, a large number of bands are consistent with the reported Raman and SERS spectra of tyrosine (see Table 1). Finally, the phenyalanine spectrum, which is characterized by an intense and narrow band at 1003 cm−1, does not contribute to our SERS spectrum of Mb. As it concerns the rest of the amino acids, the spectrum is dominated by the vibrational bands of lysine−possibly both in the charged and neutral states−and by the carboxylate groups of glutamate, aspartate, and/or of the coadsorbed citrate molecules (see Table 1). The assignments reported in the table are based on correlations with reported SERS spectra of single amino acids and peptides. Specific references are cited in Table 1 itself. Moreover, we identify the peptide backbone bands, amide I and III, as the bands at 1258 and 1666 cm−1, and discuss them below.

Figure 5. (A) Raman spectra of Mb/AuNS complexes at 3.7 × 10−8 and 7.4 × 10−6 M protein concentration (corresponding to the samples in Figure 1 B and E, respectively) with 785 nm excitation wavelength. (B) Raman spectrum of the Mb/AuNS complex at 3.7 × 10−8 M protein concentration with 532 nm excitation.

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that is, one obtained from the same colloid, aggregated with potassium nitrate in the absence of Mb, is displayed in Supporting Information Figure 6. A tentative assignment of the SERS bands in Figure 5A is reported in Table 1. Owing to the complexity of the protein,

DISCUSSION Concentration Dependence. The interactions between metal NPs and proteins display several levels of complexity,11 implying that the experimental conditions must be very carefully controlled in the study of these interactions. First of all, general rules for the reactivity of proteins in the presence of metal NPs are not yet established. In fact, it is possible that they will not be found because of the heterogeneity of protein chemical structure on one side, and of the multiplicity of NPs structure on the other one. Moreover, even when an experimental study is focused on the interactions between a single protein and a simple nanostructure, as in our case, the effects of the variations of several physical and chemical parameters must be considered. We have focused our investigation on the effects due to the concentration of the protein itself, and the results are synthetically presented in Table 2. Apparently, there is not a

Table 1. Proposed Assignment of the SERS Spectrum of Mb/AuNS wavenumber/cm−1 245 325 409 492 523 584 644 675 707 725 753 831 876 938 996 1123 1168 1201 1258 1307 1371 1447 1497 1535 1597 1666 1681

assignment ν (Au−Cl)26 δ (Au-CN) CN− impurity δ (COO−)27 Tyr28 δ (COO−)27

Table 2. Synopsis of the Results

heme ν1525 Tyr28,29

myoglobin concentration (M) 3.7 7.4 7.4 7.4

ν (C−COO−)27,29 ν (C−C)30 heme ν525 Tyr,28 Lys ω (NH3+)30,27 Tyr28 amide III, disordered31−34 Lys δ (NH2)/δ (CH2), twist (CH2)27 νs (COO−),27 ν (C-NH3+)30 CH deformation Lys δ (NH3+),27 Tyr35

× × × ×

10−8 10−8 10−7 10−6

LSPR extinction maximum (nm)

CD reduction

700 640 529 529

∼100% 42% 31%

SERS spectrum with 785 nm excitation yes

no

progressive variation of the Mb/AuNS properties when the protein concentration is increased: we rather observe that the effects are leveled at high protein concentration. Considering the extinction changes first, a double LSPR band is present in the spectra below 10−7 M Mb (Figure 1B, C), which suggests the formation of Mb/AuNS aggregates. Above this concentration, there is a single LSPR band which is slightly displaced in comparison with the one of AuNS without protein, its maximum being independent of the concentration (Figure 1D,

Tyr,36,29,35 Lys δ (NH2),37 νas (COO−)27,30 amide I, disordered31−34 Tyr36 5086



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correspondence between the extinction spectra and the structure of Mb/AuNS cannot be found at the present stage. AuNS in the aggregates formed at Mb concentrations above 7.4 × 10−7 M are possibly separated by several protein layers, so that their LSPR bands are much less displaced than they are at lower protein coverage because the interparticle coupling of the surface plasmonic excitations is minimized. As it concerns the maximum position, that is, 529 nm, and the slight (10%) extinction increase of the LSPR band (compared to the LSPR of AuNS without Mb), we note that both features can be explained by a change of the dielectric constant of the medium surrounding the AuNS. The adsorption of Mb on the metal surface should lead to a considerable displacement of water and citrate from the surface. This can increase the dielectric constant and, in fact, a Mie theory calculation shows that the above spectral changes can be explained by a variation of the refractive index of the medium surrounding the AuNS from 1.33 to 1.41 (Supporting Information Figure 5). This interpretation is confirmed by the similarity between our spectra and the reported spectra of peptide-capped AuNS (see below).42 SERS Spectroscopy. Three principal factors are determinant in obtaining good SERS experiments on metal colloids: (i) adsorption of the molecule on the metal surface, (ii) ability of the nanostructures to aggregate and create “hot spots” (consisting mostly of nanogaps and nanotips43,44), and, as a consequence, (iii) matching the excitation laser wavelength with the LSPR. The first one depends on the electronic composition of the molecule and on the metal surface properties. If adsorption is not effective to obtain SERS, it can be increased by adding an extra molecule to the metal NPs, acting as a linker,45 or adding a reactive functional group to the analyte, typically, the thiol group of cysteine in the case of proteins.46 The second factor depends on the fabrication of the substrate; the simplest are suspensions of metallic colloids with spherical shape, aggregated by the addition of salts. Nowadays literature is plenty of recipes to obtain colloids of nanostructures with different size and forms.47 Control of the formation of hot spots is not easy, and it is necessary to find specific conditions for every molecule. The third factor is often made unpredictable by the presence of the hot spots. In fact, the optimum excitation conditions cannot be decided on the basis of the LSPR band position alone, as this represents the extinction of all the NPs. Sometimes the few aggregates in the ensemble contribute to noticeable SERS enhancements, although their extinction is hardly detectable.48 In our case, we have already pointed out that Mb concentration determines the plasmonic properties of the aggregates. These properties, in turn, make the observation of SERS feasible, as it occurs at 3.7 × 10−8 M Mb when the excitation is tuned to 785 nm, or unfeasible, as it occurs at higher Mb concentration owing to low AuNS aggregation. Most of the previously published SERS spectra of Mb were dominated by the vibrational bands of the heme group, because the excitation wavelength was resonant with its strongly allowed electronic transitions.49,50 Our SERS spectra can be rather contrasted with those excited at 830 nm,51,52 which display vibrational bands of the protein matrix, and in particular, of the aromatic amino acid residues. The fact that we observe a selective enhancement of tyrosine (see below), without contributions from tryptophan or phenylalanine, suggests a different interaction with the gold substrate due to

E). This clear-cut difference can be correlated with the maximum surface coverage of the NS. Following Calzolai’s work,38 we estimate the maximum number of Mb molecules on the NS surface, Nmax, according to the following equation: Nmax = 0.65[(RAuNS + 2RMb)3 − RAuNS3]/RMb3

(2)

where RAuNS and RMb are the radii of AuNS and Mb, respectively, yielding Nmax = 192. This is a very rough calculation, modeling Mb as a hard sphere with 2.5 nm radius. The AuNS concentration can be evaluated (on the basis of gold density and AuNS size, assuming a total reduction of the initial HAuCl4 salt) as 3.5 × 10−10 M. Therefore, AuNS should be completely covered at 7 × 10−8 M Mb. Above this concentration, the protein molecules should either form additional layers or stay in solution. In fact, this interpretation explains why the maximum of the LSPR band in the spectra at 7 × 10−7 and 7 × 10−6 M Mb (Figure 1D, E) is identical: the increased protein concentration only increases the number of Mb molecules in the aqueous phase, or in the outer layers, without changes at the metal surface. At the end of the Discussion, we will relate this observation to the fundamental issue of the protein corona formation around NPs in biological environments. The line of reasoning described above may be applied to the concentration dependence of the CD spectra, which are displayed in Figure 3. At all concentrations studied, the interaction of the protein with the AuNS determines a secondary structure change, and in particular, a loss of the αhelix structure. The effect, on the other hand, is most apparent at the lowest final Mb concentration (7.4 × 10−8 M). As CD spectroscopy probes all the protein molecules−both those in the solvent/outer layers and those lying immediately on the AuNS surface−and as the number of interacting protein molecules is limited by the surface coverage, the ellipticity variations at higher Mb molarity will be “masked” by the CD of unaltered protein molecules. Plasmonic Properties of Mb/AuNS Complexes. The concentration-dependent extinction changes can be understood on the basis of the effect of AuNS aggregation on the LSPR. The extinction properties of AuNS aggregates, ranging from the simplest case, a dimer of identical AuNS, to extended linear or bidimensional arrays, have recently been investigated both theoretically and experimentally.39 Briefly, the near-field interparticle interactions give rise to plasmonic modes which are a combination of those of the individual AuNS and depend strongly both on the number of particles in the aggregate and on the interparticle distance. For example, the LSPR band maximum of a linear aggregate of 40 nm AuNS separated by 1 nm will change from ∼680 nm (in a dimer) to ∼730 nm (in a linear trimer) up to 860−900 nm (for aggregates of 10−15 NS). The structure of Mb/AuNS complexes is predictably heterogeneous, and this is confirmed by the micrographs in Figure 1. In spite of structural heterogeneity, the corresponding extinction spectra suggest that some structures prevail. Few NPs aggregates appear to be the prevalent form at 3.7 × 10−8 and 7.4 × 10−8 M Mb, yielding LSPR bands with maxima at 700 and 640 nm, respectively. Additional effects should be considered. For instance, calculations have demonstrated that a variation of the interparticle distance40 can shift the LSPR position of a AuNS dimer by several tens of nanometers. Moreover, experiments on isolated aggregates have shown that AuNS trimers of linear and triangular shape41 display very different LSPR extinction bands. Therefore, a more detailed 5087



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molecules lying immediately on the surface must be selectively probed. Therefore, the opportunity to compare CD69,70 and SERS spectra obtained from the same sample appears to be a very promising one. The CD spectra in Figure 3 clearly point to an extensive destruction of the secondary structure of Mb, which consists of eight α-helices. This change cannot be ascribed to the low pH of the AuNS dispersions, as the acidic denaturation of horse Mb requires a pH < 4;31 moreover, it would be evidenced by a shift of the heme absorption band from 408 to 370 nm.71 Therefore, the α-helix decrease should be rather considered as an effect induced by the adsorption to the metal surface. Interestingly, the CD spectra in the presence of AuNS are different from those of Mb complexed with ultrafine polystyrene particles,72 but rather tend to resemble the spectra obtained at a water-in-oil emulsion interface,73 or upon adsorption onto zirconia NPs grafted with phosphoric acid,74 or else upon thermal unfolding.75 The latter spectra display three features in common with those shown in Figure 3: (i) intensity loss of the positive band at 192 nm, (ii) intensity decrease of both negative bands, with a more pronounced decrease of the 222 nm band, and (iii) a blue shift of the band at 209 nm. This thermal unfolding is reportedly reversible upon lowering the temperature. It would be interesting to verify whether the AuNS-induced unfolding is also a reversible process: attempting to revert the adsorption, though, seems to be a difficult experimental task. The SERS spectroscopy results are equally consistent with AuNS-induced denaturation. Although the assignment of the SERS spectra of such a complex system must be necessarily considered as a tentative one, the presence of some spectral features gives a strong indication of non-native protein structure. Amide I bands are expected to be encountered between 1640 and 1680 cm−1.76 They have a special diagnostic importance, as they are sensitive to the protein secondary structure and they lie in a spectral region which is normally free from other bands. Thus, the band at 1666 cm−1 can be assigned as the amide I band of an unordered peptide backbone.31 Consistently, the intense broad band at 1258 cm−1 is a typical amide III band of denatured peptides and proteins, according both to modeled spectra31 and experimental findings32−34 based on UV resonance Raman spectroscopy. What is the origin of AuNS-induced conformational changes? Possibly, the first step in Mb/AuNS complex formation is an electrostatic interaction between the protein surface and the NS surface. We performed our characterization at pH 4 where Mb has a net positive charge, mainly due to the large amount of surface lysine residues. Thus, it is possible that ion pairs are formed between these residues and the metal-adsorbed anions (i.e., citrate and chloride from the reduction reaction). In spite of the extensive loss in secondary structure, heme loss, which accompanies chemical denaturation,77 is not so apparent as it was in previous SERS studies showing that Mb adsorption on Ag hydrosols rapidly leads to the displacement of the heme group onto the metal surface.49,50 This different behavior of Ag and Au NS is possibly explained by the generally higher reactivity of AgNS surfaces. Moreover, the enhancement of tyrosine bands in the SERS spectrum (Figure 5 and Table 1) indicates that Mb prevalently interacts with AuNS through the C-terminal part of its sequence, corresponding to the G- and H-helices in the native structure,78 where the only two tyrosine residues of horse Mb (residues 103 and 146) are situated. Consistently with this

the different way of producing the sample, which, in the former case, was obtained by protein drop casting on Au nanograins. Comparison with Other Peptide/AuNS and Protein/ AuNS Complexes. The formation of protein/AuNS complexes can involve, in principle, several kinds of interactions, that is, covalent bonds with the metal surface, electrostatic interactions with the metal-adsorbed citrate anions, and hydrophobic interactions.53,54 Therefore, it is interesting to compare Mb/AuNS complexes with other peptide/AuNS and protein/AuNS complexes in order to trace common features and contrasting aspects. It has been reported42 that the phosphate-induced aggregation of (12.3 nm diameter) AuNS can be prevented by functionalization of the AuNS surface with a short peptide (Cys-Ala-Leu-Asn-Asn). Interestingly, the extinction spectrum of the phosphate aggregates resembles that of Mb/AuNS at low Mb molarity, whereas the spectrum of the peptide-functionalized AuNS displays the same features (slight extinction increase and red shift) as that of Mb/AuNS at high Mb concentration (Figure 1). In a very simplified picture, these findings indicate that Mb at low concentration acts as a salt, that is, electrostatically, whereas Mb at higher concentrations gives rise to a real aggregate with one or more layers packed around the surface of each AuNS, thus generating a stable colloid of quasi-isolated particles.55−57 A previous study14 showed that the extinction properties of lysozyme/AuNS complexes was strongly dependent on protein concentration (in the 10−9−10−5 M range for 10−12 M AuNS concentration, diameter = 90 nm). The LSPR band, though, changed in a completely different way: there was a sort of threshold at 10−8 M, where the lysozyme/AuNS complexes displayed an additional broad LSPR band at about 900 nm, whereas an overall extinction decrease was observed between 10−7 and 10−5 M. It appears from this study that complexes of the kind obtained at high Mb concentrations, that is, fully covered and relatively isolated AuNS, can never be reached when the interacting protein is lysozyme. The formation of bovine serum albumin/AuNS complexes reportedly led to a moderate red shift and a slight extinction increase of the LSPR band, similar to that of Mb/AuNS at high concentrations.58−60 At variance, in another case the addition of bovine serum albumin to AuNS gave rise to extensive aggregation.61 The interactions between human serum albumin and AuNS62 led to stable complexes after the addition of twice the number of protein molecules necessary to form a packed monolayer adsorbed on the metal surface. The interaction between AuNS and bovine hemoglobin gave rise to aggregates which could be detected by TEM.63 Concentration dependence was not assessed in that case. Moreover, there are previous examples of protein/AuNS complexes formed by the encounter with physiological fluids. The extinction spectrum of AuNS, used as photoacoustic tracers in mesenchymal stem cells,64 closely resembles our spectrum of Figure 1B. In addition, 10 nm AuNS in the presence of serum with and without serum proteins, display extinction spectra with the features of those we obtained above and below 10−7 M Mb, respectively.65 Conformational Changes. The fate of proteins interacting with metallic, and nonmetallic, NS is a very debated topic.12,66−68 Possibly, the formation of complexes between proteins and NS is always accompanied by changes in the secondary and tertiary structure, with a very high degree of variability from small rearrangements to true denaturation, depending on the protein stability and the interaction strength. Detecting these changes can be an elusive task, as the protein 5088



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indication that also the formation of external protein layers occurs faster than the NP aggregation. On the other hand, CD changes are due to the loss of αhelical structure. The time dependence of this unfolding process needs not, in principle, be correlated with the aggregation of the NS, as it should originate from an interaction between protein and metal surface, independently of the interNS interactions. In fact, at the protein final concentration 7.4 × 10−8 M where the extinction changes can be directly compared with the CD changes, the latter are the faster ones. In agreement with the extinction kinetics results, we can infer that the dynamics of the protein/AuNS interactions, including the adsorption and unfolding processes, which we cannot distinguish by CD, is faster than the NS aggregation. The rate of protein unfolding at interfaces is a fundamental question in itself.85 Most experimental studies have been focused on unfolding dynamics on fast time scales. Slow unfolding, though, is not unprecedented. The adsorption of lysozyme on the surface of various nanoporous substrates induced protein unfolding on the time scale of 1−1000 s.86 Relevance of the Results in Understanding the Protein Corona Formation. The formation of Mb/AuNS complexes is a very simple example of protein adsorption at a solid/liquid interface, a topic which has been especially discussed in the frame of colloidal chemistry.87 A central issue in the related literature is whether the adsorption is a reversible or irreversible process. In fact, irreversibility has been tracked in some instances88−90 and is a consequence of (i) the diverse chemical composition of protein surfaces, which allows for a manifold of interactions with solid surfaces, and (ii) the intrinsic plasticity of protein structure, which will react with (often irreversible) changes after the binding at the interface is established. Therefore, adsorption can induce proteins to reach a non-native structure, which can make the mirror process of desorption difficult, if not impossible, which will appear as irreversibility. In our case, it has to be considered that additional complexity is given by the irreversibility of the aggregation of the metal NPs themselves. The most apparent consequence is that different Mb/AuNS complexes are obtained for the same protein concentration, depending on the preparation path (see Supporting Information Figure 2). The (largely) irreversible interactions occurring at the interface between NPs and solvent are the basis for the formation of the “hard corona”, which consists of a tightly bound layer of biomolecules, mainly proteins, coating the surface of NPs when they are exposed to physiological fluids.91 The importance of this process is extreme, as the biological effects of nanosized materials will be determined by the metamorphoses of the NPs in living organisms.92 In addition to the hard corona, more layers can be attracted onto the NPs through intermolecular interactions with the adsorbed molecules of the hard corona, leading to the formation of a “soft corona”. Owing to the weaker binding, exchange with the solvent is faster for the molecules of the soft corona.88 In our study, we have found a concentration threshold (on the order of 10−7 M myoglobin) above which the properties of the Mb/AuNS complexes are changed, and we have correlated this threshold with the complete NS surface coverage by the protein, as outlined at the beginning of the Discussion section. It is tempting, then, to identify the Mb/AuNS complexes at low protein concentrations as a model for the hard corona, whereas the complexes at Mb concentrations above the threshold would be a model for the soft corona. We try now to summarize

picture, the bands of the tryptophan residues, both lying close to the N-terminal, are silent in our SERS spectrum. A pictorial representation of the orientation of the Mb molecules adsorbed on AuNS is given in Figure 6.

Figure 6. Three-dimensional structure of horse Mb, as determined by X-ray crystallography,78 PBD code 1WLA. The black ellipse highlights the protein moiety which lies close to the metal surface according to the SERS results, and in particular, both tyrosine residues (red). Some nearby lysine residues are shown in violet, and both tryptophan residues, which cannot be observed in our SERS spectra, in green.

Kinetic Measurements. The time-dependent changes we observed by extinction and CD spectroscopy originate from subtly interrelated, yet different processes. Extinction changes stem from the formation of AuNS aggregates. As such, they should be compared with the time-dependent aggregation induced by, for example, inorganic salts, small organic molecules, or polypeptides. The coagulation of gold colloids induced by inorganic electrolytes is a classical experiment,79 which can be rationalized on the basis of various theoretical descriptions.80 A characteristic feature of this kind of aggregation is a marked dependence of the aggregation rate on the electrolyte concentration, including a critical concentration. The interaction with small organic molecules81−83 leads to extremely variable kinetics, which can be related to the chemical properties of the interacting molecules, and specifically, to the formation of chemical bonds or to electrostatic interactions. Our results rather resemble the kinetics of AuNS heteroassociation through specially designed polypeptides.84 In that case, the time scale for the observed extinction changes was very similar to ours (2−30 min). Moreover, the spectral variations depended on the polypeptide concentration in a complicated way, the highest polypeptide concentrations inhibiting the aggregation to some extent. The latter observation is in agreement with the most noticeable observation we have made by kinetics measurements: Mb at increasing concentrations slows down the aggregation of AuNS. This delaying effect occurs, in particular, when changing from 3.7 × 10−8 to 7.4 × 10−8 M Mb (Figure 2), and indicates that protein adsorption to the metal is faster than the intermolecular interactions eventually leading to AuNS aggregation. Beyond 7.4 × 10−8 M Mb, this process is completely prevented, therefore a real comparison with the kinetics at lower concentrations is not possible. Nevertheless, the absence of AuNS aggregation at high Mb concentration gives the 5089



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common aspects of the results we have obtained, by means of our four experimental techniques, below and above the protein concentration threshold. Below threshold, extensive proteininduced AuNS aggregation (observed in TEM images and consistent with broad and red-shifted plasmonic bands) is accompanied by Mb helical structure loss and SERS spectra showing denaturation features. This picture indicates strong Mb/AuNS interactions, leading to a tightly bound Mb layer at the NS surface: something we can identify as a Mb hard corona. Above threshold, we observe a full protein coating in TEM images, and decoupled plasmonic bands in the extinction spectra as a consequence of effective interparticle separation. Moreover, CD changes become relatively less apparent at equilibrium, their kinetics being also slowed down. It is possible, then, that the variations we measure are due to additional layers around the first one, that is, that a kind of Mb soft corona is formed. This issue could be better defined by additional studies, employing size-related optical techniques, for example, dynamic light scattering and fluorescence correlation spectroscopy, and/or binding of labeled Mb.

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS MINECO Project POEMS (FIS2010-15405) and Comunidad de Madrid Project MICROSERES II (S2009TIC-1476).

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ABBREVIATIONS NPs, nanoparticles; AuNS, gold nanospheres; Mb, myoglobin; LSPR, localized surface plasmon resonance; SERS, surface enhanced Raman scattering; SEF, surface enhanced fluorescence; SEIRA, surface enhanced infrared absorption; TEM, transmission electron microscopy; CD, circular dichroism; MRE, average molar residue ellipticity

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REFERENCES

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CONCLUSIONS We have highlighted how protein concentration is a determinant in the formation of very different Mb/AuNS aggregates, by keeping the NS concentration constant and adding protein amounts varying over 3 orders of magnitude. Dramatic changes are observed in the extinction spectra at the lowest Mb concentrations employed, pointing to the formation of optically coupled NS dimers, trimers, chains, arrays. These structures are, in fact, revealed by electron microscopy. In contrast, only slight extinction changes occur at protein concentrations exceeding the nanoparticle surface coverage. It appears, in the latter case, that protein adsorption to the metal surface prevails on−or, it is faster than−protein-induced AuNS aggregation. Protein adsorption leads to the formation of protein layers, maybe facilitated by lateral interprotein interactions. This protein coating effectively prevents the AuNS from aggregating and/or optically coupling. As a consequence, SERS spectra cannot be observed at relatively high Mb concentrations. Moreover, the interaction at the metal surface appears to be strong enough to induce slow unfolding, namely, α-helical loss, which can be detected by a careful examination of CD changes. We have focused our study on a single model protein interacting with simple model NS. Therefore, we expect this behavior to be observable in many common cases of protein/NS interactions, if not in general. For example, suggestive comparisons can be made between the properties of Mb/AuNS complexes and those of the protein corona which surrounds NPs when they are exposed to biological environments.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information Figure 1: Calculated LSPR for 30 nm AuNS. Supporting Information Figure 2: Extinction spectra of AuNS samples with 7.4 × 10−7 M Mb, obtained through different pathways. Supporting Information Figure 3: Supplementary TEM micrographs. Supporting Information Figure 4: Time evolution of the extinction spectra at 3.7 × 10−8 M Mb. Supporting Information Figure 5: Mie theory simulations as a function of the refraction index. Supporting Information Figure 6: SERS spectrum of the blank This material is available free of charge via the Internet at http://pubs.acs.org. 5090



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