Role of the Functionalization of the Gold Nanoparticle Surface on the

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Role of the Functionalization of the Gold Nanoparticle Surface on the Formation of Bioconjugates with Human Serum Albumin Fernando Cañaveras, Rafael Madueño, José M. Sevilla, Manuel Blázquez, and Teresa Pineda* Department of Physical Chemistry and Applied Thermodynamics, University of Córdoba, Campus Rabanales, Ed. Marie Curie, E-14014 Córdoba, Spain S Supporting Information *

ABSTRACT: The protein−gold nanoparticle bioconjugates are playing an important role in the studies of biological systems. The nature of the interaction and the magnitude of the binding affinity together with the conformational changes in the protein upon binding are the most addressed topics in relation to the uses of the bioconjugates in different organisms. In this work, we study the human serum albumin (HSA) protein−gold nanoparticle (AuNP) interactions focusing on the nature of the gold nanoparticle surface modification. We have found that the interactions of the HSA with the AuNPs are mainly electrostatic and that the concentration of protein necessary to stabilize the conjugates decreases when the overall negative charge on the nanoparticle surface increases. The changes in the localized surface plasmon resonance (LSPR) signals of the gold nanoparticles (13 nm diameter) are used to determine the number of protein molecules necessary to stabilize the conjugates in a high ionic strength medium. Fluorescence spectroscopy (stationary and time-resolved) is used to characterize the different bioconjugates and determine the binding constants under different experimental conditions. Moreover, the use of an extrinsic fluorescence probe (1-anilino-8-naphthalenesulfonic acid, ANS) gives us some information about the existence of partial unfolding of the protein upon binding to the nanoparticle.

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INTRODUCTION The dynamic layer of proteins formed at the nanoparticle surface when the nanoparticles and proteins are in contact, the protein−nanoparticle corona, determines the interactions with living systems and is responsible for the different cellular responses to the nanoparticles.1,2 Thus, the concept of safe nanoparticles is a result of the precise understanding of nanoparticle−biological system interactions.3 In this sense, a large variety of techniques such as spectroscopy, chromatography, and mass spectrometry, among others, are being used to study the binding affinity, ratio, structure, and mechanism of protein−nanoparticle interactions.4,5 It has been shown that the protein corona is composed of an inner layer of selected proteins with a lifetime of several hours in a slow exchange with the environment (hard corona) and an outer layer of weakly bound proteins which are characterized by a faster exchange rate with the free proteins (the soft corona). It is worth noting that the biological impact of protein-coated NPs is mainly related to the proteins with the highest affinity (i.e., the hard corona) and their specificity and suitable orientation for a particular receptor response, rather than to the effects from low-affinity high-abundance proteins, which might bind initially but are quickly replaced by lower abundance and higher affinity proteins.6−8 The facile bioconjugation of gold nanoparticles (AuNPs) together with the nontoxic nature of gold make them useful platforms to construct protein−nanoparticle assemblies.1,5 The formation of AuNP−protein complexes in which the protein is physically or chemically bound to the nanoparticle surface allows for the stabilization in different experimental conditions © 2012 American Chemical Society

including wide pH and ionic strength ranges where the AuNPs are unstable.9−12 Recently, a systematic and comparative study of the binding association of different human plasma proteins with AuNPs has been reported. It has been found that the AuNPs strongly associate with these essential blood proteins and that the binding constant, the degree of cooperativity, and the thickness of the layer and protein conformational changes depend on particle size and native protein structure. These findings are considered important in understanding the nanoparticle aggregation as well as the interactions in the biological medium.5 Human serum albumin (HSA) is the most abundant protein (up to 60% of the total) in blood plasma.13 It is a globular protein of 66 kDa MW consisting of 585 amino acid residues with only one tryptophan (Trp 214) in its primary sequence. The three-dimensional structure is described in terms of three α-helical homologous domains (I, II, and III) that assemble to form a heart-shaped structure. Each domain is formed by two subdomains (A and B) that possess common structural motifs.14 HSA plays an important role as a shuttle for a broad range of endogenous and exogenous ligands, such as fatty acids, drugs, metabolites, or bile acids. The binding of these ligands results in an increased solubility in plasma, helping their delivery to the target tissues. Although several binding sites have been reported for HSA, it is accepted that there are two Received: March 5, 2012 Revised: April 16, 2012 Published: April 19, 2012 10430

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measurements. For critical flocculation concentration experiments, the complex was prepared in a sodium phosphate buffer 10 mM at pH 7.4, and after a sequential addition of NaCl the LSPR spectra were measured. For the estimation of the protein molecules that strongly interact with the gold nanoparticles (the hard corona) the bioconjugates were prepared in a ratio of protein:AuNP higher than the critical flocculation concentration. After 1 h, the colloidal mixtures were either centrifuged (1 h at 13 600 rpm) or filtrated and the supernatant and/or filtrate measured by using the absorbance at 278 nm (ε HSA = 3.44 × 10 4 M−1 cm−1)24 or the fluorescence intensity by excitation at 295 nm. The choice of absorbance or fluorescence measurements was made on the basis of the absolute amount of protein present in the supernatant. Characterization Techniques. The absorbance spectra of the prepared solutions were recorded using a Jasco V-670 UV− vis−NIR spectrophotometer. The morphology and size distribution of the bioconjugates were analyzed by transmission electron microscopy using a JEOL JEM 2010 instrument (from the Servicio Central de Apoyo a la Investigación (SCAI), Universidad de Córdoba) operating at 80−200 kV and analyzed using Image Pro Plus software. Samples were prepared by casting and evaporating a droplet of the conjugate solution onto Formvar-coated Cu grids (400 mesh, Electron Microscopy Sciences). The negative staining technique was employed using uranyl acetate as the contrast agent. The stationary fluorescence spectra were recorded using a Perkin-Elmer LS50B spectrofluorimeter. The time-correlated single photon counting of samples with ANS was carried out by using a 406.4 nm LED as the excitation source in a FLS920 Edinburg instrument.

major structurally selective binding sites, named site I and site II according to Sudlow’s classifications.15,16 Although many studies on plasma proteins−AuNP bioconjugates have been reported, most of them deal with bovine serum albumin (BSA) and only a few are with the protein from the human source.17−20 In the present work, we have chosen this protein because its tertiary structure has been completely described,14 and it possesses only one tryptophan residue, being the intrinsic fluorescence study more interesting. Then, we present a systematic study of the formation of HSA−gold nanoparticle conjugates by using either citrate protected AuNPs (cit-AuNP) or 6-mercaptopurine protected AuNPs (6MP-AuNP). The study is focused on the influence of the nanoparticle surface functionality on the stability of the conjugates and the type of interaction as well as the occurrence of conformational changes in the protein upon binding.

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EXPERIMENTAL SECTION Human serum albumin (HSA), 6-mercaptopurine (6MP), 1anilino-8-naphthalenesulfonic acid (ANS), and semiconductor grade purity sodium hydroxide were purchased from SigmaAldrich. Hydrogen tetrachloroaurate trihydrate (from 99.999% pure gold) was prepared using a literature procedure21 and stored in a freezer at −20 °C. The rest of the reactants were from Merck analytical grade. All solutions were prepared with deionized water produced by a Millipore system. Synthesis of cit-AuNPs. The synthesis of gold nanoparticles (cit-AuNPs) has been carried out by following the classic method of Turkevic et al.22 which consists of the reduction of HAuCl4 by citrate anions in an aqueous medium. The citrate anions not only serve as a reductor agent but also exert a protection effect against the aggregation of the synthesized particles. Briefly, the gold nanoparticles were prepared as follows. In a 1 L Erlenmeyer flask, 500 mL of 1 mM HAuCl4 was brought to a boil, with vigorous stirring on a magnetic stirring hot-plate. Fifty milliliters of 38.8 mM Na3citrate was added to the solution all at once, with vigorous stirring. The yellow solution turned clear, dark blue, and then a deep red burgundy color within a few minutes. Stirring and boiling was continued for 10−15 min after the burgundy color was observed. The solution was then removed from heat and kept stirring for 15 min. After the Au colloid solution had cooled, the volume was adjusted to 500 mL with H2O. Synthesis of 6MP-AuNPs. The synthesis of 6MP-AuNPs has been carried out by adding a 10-fold excess of 6MP to a citAuNPs alkaline aqueous solution. Under these experimental conditions, only a slight change in the surface plasmon resonance (SPR) band of 3 nm after the modification was observed. The sample of 6MP-AuNPs was dialyzed against an 10 mM NaOH solution to remove the 6MP molecules that had not reacted with the AuNPs. The formation of the 6MP-selfassembled monolayer concomitant to the displacement of citrate ions from the AuNP surface has been demonstrated by FT-IR spectroscopy.23 Flocculation Experiments and Critical Flocculation Concentration. Flocculation experiments were performed to determine the AuNP:protein ratio required to stabilize the gold nanoparticles in 0.1 M sodium phosphate buffer at pH 7.4. In a typical assay an aliquot of gold nanoparticles (17 nM) was added to several vials containing different amounts of HSA with concentrations ranging from 23 to 188 nM in sodium phosphate 0.1 M at pH 7.4. The solutions were kept for 30 min at room temperature before UV−vis spectroscopy

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RESULTS AND DISCUSSION Stability of HSA−cit-AuNPs Bioconjugates. In the presence of proteins, the AuNP surface is rapidly covered by a corona of proteins that may be loosely divided into a soft component in which rapid dynamical exchange predominates and a hard corona whose constituent protein molecules have a high affinity for the particle surface.6,7 The protein corona stabilizes the nanoparticles under conditions of pH and ionic strength where citrate protected gold nanoparticles (citAuNPs) flocculate. However, the stabilization of bioconjugates protein/cit-AuNP in aqueous solution requires a protein concentration higher than that involved in the direct interaction with the gold core.12 Taking into account, we have carried out experiments to determine the concentration of protein that is necessary for the stabilization of the bioconjugates formed by the HSA protein and cit-AuNPs (HSA/cit-AuNP). To detect the onset of flocculation, the changes in the surface plasmon resonance absorption of the AuNPs are followed. Thus, Figure 1 shows spectra for cit-AuNPs either changing the HSA concentration while keeping the number of nanoparticle constant (Figure 1a) or changing the latter and maintaining the protein concentration (Figure 1b). In order to get the critical conditions for flocculation of the HSA/AuNP system, we have carried out experiments in 0.1 M sodium phosphate buffer at pH 7.4. Under these conditions, the ionic strength is high enough to flocculate the cit-AuNPs and irreversible aggregation is observed after a few seconds of mixing. However, in the presence of protein the cit-AuNPs start to stabilize, and after the addition of more than 35−40 molecules of HSA per nanoparticle, the shape of the spectrum 10431

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The estimation of the number of protein molecules interacting with the AuNPs was made by centrifuging and filtering the complexes formed in an excess of protein. The quantification of the supernatant and/or the filtrate by using fluorescence measurements gave a ratio of 45 ± 5 protein molecules per AuNP, in good agreement with the results described above. In order to check the influence of the ionic strength in the stability, the bioconjugate has been prepared in sodium phosphate buffer 0.01 M at pH 7.4, at a HSA/cit-AuNP ratio of 84/1, 2-fold the ratio necessary for the stabilization at 0.1 M buffer concentration. Under these conditions, the preparation is stable and the ionic strength can be increased by adding sodium chloride to reach the critical flocculation concentration (Figure 3). This parameter is determined by the displacement of the

Figure 1. LSPR spectra of cit-AuNPs in the presence of HSA in 0.1 M sodium phosphate buffer at pH 7.4. (a) The HSA concentration is changed while cit-AuNPs are kept constant. (b) The cit-AuNP concentration is changed while HSA is kept constant. Concentrations are gathered in the figure. The black line corresponds to the cit-AuNPs in citrate buffer 3.5 mM at pH 7.

looks like the cit-AuNP. The comparison of these spectra, however, shows a small difference in the half-width and maximum absorption wavelength to longer values, indicating the adsorption of the protein at the nanoparticle surface (Figure 1a). The localized surface plasmon resonance (LSPR) band for the HSA/cit-AuNP complex shows a ∼ 8 nm bathochromic shift which is consistent with a change in the dielectric function on the nanoparticle surface upon protein adsorption.25−27 It is interesting to note that the introduction of the protein into the nanoparticle suspension did not significantly alter the line shape of the absorption spectrum, indicating that the HSA/cit-AuNP constructs remained as single colloids rather than flock under these conditions. The minimum HSA molecules that stabilize the gold sol is approximately twice the necessary to form a wellpacked HSA monolayer with the proteins adsorbed in an endon orientation.11 This is in agreement with previous work12 that states that protein concentration in excess of those required for monolayer formation affords increased stability as it is expected for a system in equilibrium. TEM images allow visualization of the structures formed under experimental conditions of HSA−cit-AuNPs stability (Figure 2). The nanoparticle constructs remained well separated, indicating the absence of aggregation.

Figure 3. LSPR spectra of HSA/cit-AuNP at a molar ratio 84/1 in 10 mM sodium phosphate buffer at pH 7.4 in the presence of different concentrations of NaCl (as indicated in the figure). Inset: changes in the maximum wavelength of the SPR band with the addition of NaCl.

maximum absorption wavelength of the SPR band.12 The inset in Figure 3 shows that the displacement of the band from 527 nm takes place at NaCl concentrations higher than 0.3 M and thus, this salt concentration constitutes the critical value above which the bioconjugate is no longer stable and flocculates. Stability of HSA−6MP-AuNPs Bioconjugates. It has been reported that the binding of albumin to citrate-coated gold surfaces proceeds predominantly by an electrostatic mechanism through the surface lysine residues (60) of the protein and the citrate anions, although the presence of hydrophobic interactions and coordination binding cannot been discarded.9 In agreement with this study is the observation that the stability of the bioconjugate persists in the whole pH range, even at the protein isoelectric point. This finding is taken as proof that the steric stabilization should also be considered.10,11 Although there are a considerable number of studies that provide data on the system albumin/AuNP, in the present work we focus on the role of the chemical functionality at the surface of the nanoparticle in its interaction with the protein. Thus, we have studied the interaction of the HSA protein with AuNPs protected by 6-mercaptopurine (6MP-AuNPs). These nanoparticles have been prepared and characterized in aqueous and organic media.23 The 6MP-AuNPs are stable in aqueous solutions in neutral and alkaline media and flocculate at acid pH when the negative charge of the N(9) group of the purine ring is lost (the 6MP molecules are supposed to be

Figure 2. TEM images of the HSA/cit-AuNPs at a molar ratio 40/1. The bar in the bottom right corner corresponds to 200 nm, while that at the bottom of the inset states for 20 nm. 10432

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AuNPs that would involve the interaction of the surface lysine positively charged residues with the citrate anions.10,11 The estimation of the number of protein molecules that remain bound to the 6MP-AuNPs under these conditions has been made by centrifuging the sample and measuring the supernatant by UV−vis spectroscopy. The amount of protein that precipitates with the nanoparticles is higher than that with the HSA−AuNPs bioconjugate (the amount of adsorbed protein is roughly the equivalent to 10 ± 1 monolayers). This finding can be related to the different nature of the interaction of the protein with the nanoparticle surface and the high protein concentration present that favors protein−protein interaction. In order to assess if the interactions are of electrostatic nature, we have carried out experiments at a higher pH where the 6MP-AuNPs are negatively charged (pH = 9). Under these conditions, the results gathered in Figure 5 are obtained.

bound to the gold nanoparticle surface by the SH group exposing the opposites N(3) and N(9)−H groups to the solution) (see Scheme 1 for numbering). However, the Scheme 1. Structure of 6-Mercaptopurine (6MP)

flocculation is reversible, and a stable dispersion is obtained after changing the pH to values higher than the N(9)−H dissociation pKa. The LSPR spectra of the nanoparticles after the addition of protein are shown in Figure 4. At a first glance, it can be said

Figure 5. LSPR spectra of 6MP-AuNPs in the presence of HSA in 0.1 M sodium phosphate buffer at pH 9.0. Concentrations are gathered in the figure.

Figure 4. LSPR spectra of 6MP-AuNPs in the presence of HSA in 0.1 M sodium phosphate buffer at pH 7.4. (a) The HSA concentration is changed while 6MP-AuNPs are kept constant (at 1 nM). (b) The 6MP-AuNP concentration is changed while HSA are kept constant (at 8 μM). Concentrations are gathered in the figure.

It can be observed that at pH 9 the presence of a 10-fold excess of protein molecules over nanoparticles avoids the flocculation of the 6MP-AuNPs and that a little increase of the concentration of protein produces a high stabilization of the bioconjugate. As expected, the uniform negative charge of the 6MP molecules attached to the nanoparticle surface is responsible for the interaction of the protein. This stronger interaction together with the overall negative charge of the protein (the isoelectric point of HSA is 4.7) makes the complex more stable even at a very low protein concentration. Moreover, the complexes remain stable in the presence of NaCl concentration higher than 1 M. As in the case of the HSA−AuNPs conjugate at pH 7.4, the number of HSA molecules that precipitate with the 6MP-AuNPs under these conditions (pH 9.0) is approximately the equivalent to two protein monolayers. Fluorescence Studies. Intrinsic Fluorescence. The presence of a single Trp residue (Trp 214) in the primary structure of the HSA protein makes the fluorescence study of the HSA−gold nanoparticle bioconjugate very interesting. Thus, the changes in the intrinsic fluorescence properties of the bioconjugate can be interpreted as changes in the local environment of the fluorofore. Figure 6a shows the fluorescence spectra of HSA protein by excitation at 295 nm (the choice of this excitation wavelength

that the amount of protein necessary to stabilize the 6MPAuNPs in 0.1 M sodium phosphate buffer at pH 7.4 is much higher than that for the cit-AuNPs. In Figure 4a, the spectra of a 1 nM solution of 6MP-AuNPs in the presence of different concentrations of HSA are shown. As can be observed, only after the addition of 16 800 protein molecules over nanoparticle the dispersion becomes stable. The corresponding spectra show two bands at 278 and 528 nm corresponding to HSA and nanoparticle absorption signals, respectively. The displacement of the LSPR band upon protein interaction is similar to that found for cit-AuNPs. The displacement of the absorption band to 645 nm, when the protein concentration decreases, is in agreement with the lower stabilization ability of the protein for the modified nanoparticles under these experimental conditions. Figure 4b shows the titration of a solution of HSA (8 μM) with 6MP-AuNPs, and only at the lower nanoparticle concentration the shape of the spectrum is that of the stable bioconjugate. The inability of the protein to stabilize the 6MP-AuNPs in a medium of high ionic strength should be related to the low fraction of negative charge of the particles at pH 7.4. This finding is in agreement with the conclusions for the HSA/cit10433

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described in the precedent section, where a much higher concentration of protein was necessary for the stabilization of the nanoparticles in a high ionic strength medium. The layer structure of the 6MP-AuNP has been characterized by our group in previous works.23,29,30 Regarding the present experiments, it is worth noting that the apparent surface pKa for the N(9)H group of the molecule is close to pH 7. Thus, under the conditions of this experiment, the 6MP-AuNPs can exhibit a certain fraction of negative charge (that necessary to keep the dispersion stability), but it should be in a lower degree than that in the cit-AuNPs. In order to demonstrate the role of the charge in the binding affinity of HSA for the gold nanoparticles, a quenching titration at higher pH (pH 9) has been carried out. Under these conditions, it has been found that the quenching efficiency is higher than in the cases described above (Figure S2, Supporting Information). A binding constant of 3.44 × 109 M−1 has been obtained, which supposes an order of magnitude higher than at pH 7. The possibility of the influence of conformational changes with pH in the affinity constant is ruled out on the basis of previous work9 that find a similar binding behavior of the BSA protein at pH 3.8, 7, and 9. Thus, the fact that the 6MP-AuNPs exhibit a completely dissociated monolayer should be responsible for the higher binding affinity of the protein for the 6MP-AuNP surface. Extrinsic Fluorescence. One of the most used organic probes in fluorescence studies of proteins is 1-anilinonaphthalene-8-sulfonate (ANS) (Scheme 2). This molecule is

Figure 6. (a) Fluorescence emission spectra of 7.6 μM HSA in the absence and presence of AuNPs with the concentration in the range of 0.12−1.0 nM upon excitation at 295 nm in 0.01 M sodium phosphate buffer at pH 7.4. (b, c) Fractional fluorescence and logarithmic plots of HSA with the concentration of AuNPs.

was to avoid the contribution from the tyrosine residues) of free native HSA and HSA−cit-AuNPs at different concentrations of cit-AuNPs. It can be observed that fluorescence is quenched upon the addition of cit-AuNPs. This gradual decrease in fluorescence intensity takes place without a noticeable change in the emission wavelength. The quenching of the protein fluorescence has been generally analyzed by a mechanism of static quenching by using the Stern−Volmer equation:28 F0 = 1 + kqτ0 = 1 + KSV[Q] (1) F where F0 and F are the maximum fluorescence intensities in the absence and presence of quencher, respectively, kq is the bimolecular quenching constant, which is a measurement of the efficiency of quenching, and [Q] is the quencher concentration. From Figure 6b which shows the Stern−Volmer plot, F0/F versus [AuNPs], according to eq 1, a KSV value of 6.83 × 108 M−1 is obtained. If a value of τ0 ≈ 5 × 10−9 s28 for the Trp residue is used, kq = 1.36 × 1017 M−1 s−1 which is much higher than the values for diffusion-controlled quenching processes. Therefore, the process that gives place to the decrease in fluorescence must have a different origin than the collisional quenching. In fact, the observation that the data obey the Stern−Volmer equation is not a proof of the existence of a dynamic quenching process. Similar behaviors have been treated by using the static quenching approach and have been analyzed as being due to the formation of nonfluorescent protein−NP complexes. For these systems, eq 2 can be used to obtain the binding constant (K) and the number of binding sites (n): ⎛F − F⎞ ⎟ = log K + n log[Q] log⎜ 0 ⎝ F ⎠

Scheme 2. 1-Anilino-8-naphthalenesulfonic Acid (ANS)

highly fluorescent when bound to hydrophobic sites of the protein, but its emission fall dramatically in polar environments. Thus, the changes in the fluorescence of this probe upon the formation of the bioconjugates can also be used to monitor the changes in the tertiary structure of the protein. The fluorescence properties of ANS can be summarized as a blue shift of the maximum of the emission spectrum and the increase of quantum yield and lifetime when transferred from water to proteins. Given the structure of the ANS molecule, the interaction with protein sites has been shown to take place through ion pair formation between the sulfonate group and proximate positively charged side chains and, on the other hand, through hydrophobic interactions in sites already present or induced in the protein by accommodating the naphthalene and aniline moieties.31 Binding of ANS to albumin has been extensively studied but only recently an integrated thermodynamic and kinetic analyses have provided a clear description of the binding process. Thus, three ANS molecules could bind at hydrophobic cavities that can be grouped in two classes with slight differences between their binding constant. The sites are located at subdomains IIIA and IIA, one with higher affinity (class H; kH = 8 × 106 M−1) and two with lower values (class L; kL = 4.5 × 105 M−1).31 Docking of ANS at subdomain IIIA shows that the molecule is clustered in a binding pocket oriented with its sulfonate group toward a polar patch formed by residues R410, K414, and S489. A salt bridge between the

(2)

Figure 6c shows the plot of log[(F0 − F)/F] versus log[Q]. A straight line where the slope and the intercept allow us to determine n = 1.00 and K = 6.65 × 108 M−1 can be obtained. The value for the binding constant is in agreement with the existence of strong interactions between HSA and the gold nanoparticle surface. A similar behavior of this found in the interaction of HSA with cit-AuNPs has been obtained in the bioconjugate HSA/ 6MP-AuNPs, under similar experimental conditions (Figure S1, Supporting Information). A binding constant of 8.7 × 108 M−1 that is of the same order of the HSA−cit-AuNPs conjugate is obtained. This finding contrasts with the results of flocculation 10434

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sulfonate group and Y411 is observed.32 The naphthalene and aniline rings of this potentially fluorescent ANS molecule are mainly surrounded by nonpolar residues.31,32 In this work we have used a HSA−ANS complex formed by adding ANS (3 μM) to a protein solution 10 μM. Under these experimental conditions, we can assume that the ANS molecules will be mainly bound to the site of high affinity (at the subdomain IIIA), and therefore, the fluorescence signal observed for ANS should be due to the environment close to this binding site.33 Figure 7 shows the fluorescence spectra of

that, under these experimental conditions, the local environments of Trp214 (subdomain IIA) and ANS binding site (subdomain IIIA) do not suffer any noticeable structural changes upon interaction with AuNPs. The decay times and relative contributions of decay components are sometimes related to ANS binding sites with different degrees of exposure to aqueous environment, pointing to the heterogeneous nature of this probe binding to proteins.35 Figure 8 shows the decay times HSA−ANS and HSA−ANS

Figure 7. Influence of the cit-AuNP concentration on the fluorescence emission properties of the complex HSA−ANS. (a) Fluorescence spectra of 10 μM HSA in the absence (black) and presence of 3.2 μM ANS (red) and cit-AuNPs with concentration in the range of 0.1−0.7 nM upon excitation at 295 nm in 0.01 M sodium phosphate buffer at pH 7.4. The spectrum (···) has been recorded by excitation at 350 nm. (b) Fluorescence spectra of the complex HSA−ANS in the presence of cit-AuNPs with concentration in the range of 0.1−0.7 nM upon excitation at 350 nm in 0.01 M sodium phosphate buffer at pH 7.4. Figure 8. Fluorescence decays of the complex HSA−ANS (concentrations of HSA and ANS are 10 and 3 μM, respectively; concentration of cit-AuNPs is 0.7 nM) excited at 406.4 nm and probed at 480 nm in the absence (red) and presence of cit-AuNPs (blue). The decay curves were analyzed by using a biexponential function giving average lifetimes of 14 ns.

the free HSA and HSA−ANS and the complex in the presence of varying amounts of AuNPs. The HSA protein shows an emission spectrum centered at 345 nm by excitation at 295 nm. In the presence of ANS, the emission wavelength is maintained at 345 nm, but the intensity decreases by 30% of its original value. The decrease in emission intensity together with the apparition of a second emission band at 470 nm can be explained by the occurrence of a process of radiationless energy transfer (FRET) where the Trp214 acts as a donor and the ANS bound to subdomain IIIA as an acceptor.31,34 The addition of cit-AuNPs to the solution containing the HSA−ANS complex brings about a parallel decrease of the fluorescent intensity at both the 345 and 470 nm bands. These changes in intensity are ascribed to a static quenching mechanism, as in the case of uncomplexed HSA. If the fluorescence intensities in the absence of quencher are taken as those at 345 and 470 nm in the presence of ANS, we can obtain K = (1.50 ± 0.04) × 109 M−1 (Figure S3, Supporting Information). Moreover, by using the data obtained by excitation at 350 nm (that is, the spectra corresponding to ANS bound to HSA), a similar K value is obtained. Therefore, it can be said that the cit-AuNPs quench the ANS emission in the same way as the intrinsic protein fluorescence. A logarithmic plot (eq 2) gives a K = 1.05 × 1010 M−1 and a value of n = 1.09. This value is 15-fold higher than that for the binding constant in the absence of ANS, indicating that the presence of a molecule bound to the site IIIA produces a stronger complex with the cit-AuNPs. The interaction of the sulfonic acid with Lys414 through a salt bridge should increase the overall negative charge of the conjugate and make the binding affinity stronger. These results allows us to conclude

conjugated with cit-AuNPs. It can be observed that the lifetime decays are identical, indicating that the overall lifetime does not change upon conjugation with the gold nanoparticle. This feature is a strong indication that the mechanism of quenching is static and that the changes observed in fluorescence spectra can be analyzed by using eq 2. Recent studies have dealt with the fluorescent decay kinetics of the ANS probe bound to albumins and the changes observed in these parameters when the ANS−protein complex is bound to gold nanoparticle of different sizes.20,36 The results are also very dependent on the type of construct. In this sense Patra and co-workers20 prepared AuNPs in the presence of HSA protein in a way that the ratio HSA:AuNP obtained is 1:1. The addition of ANS to these conjugate brings about a sequential decrease of the decay times upon increasing nanoparticle size. They analyzed these findings as a surface energy transfer process where ANS is located in binding site IIIA and the gold nanoparticle in subdomain IA linked to Cys53-Cys62 of the primary structure of the HSA protein. On the other hand, a study by Glomm and co-workers36 used bioconjugates prepared in the presence of high ratio of protein to AuNP (2000:1), and the addition of ANS was made after the construct preparation. Thus, the decrease in ANS fluorescence in the presence of the nanoparticle is interpreted as being due to the partial unfolding of the protein and higher exposure of the probe to water. 10435

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of HSA 0.1 μM) can be explained by the release of some ANS molecules from the binding sites of the protein upon partial unfolding. This effect has been recently described to take place in the first stage of albumin unfolding at a low denaturant concentration33 and is explained either by the release of the ligands from its binding site or by the increase of the distance between the Trp214 and the ANS bound molecules upon partial unfolding. Then, the occurrence of partial structural change in HSA upon HSA:AuNP bioconjugates formation cannot be discarded.

Although the average decay times are roughly the same in the absence and the presence of AuNPs of different sizes, the changes in the individual contributions are also correlated with the above-mentioned conformational changes. In the present work, however, we have prepared the bioconjugates with the protein already modified with ANS in an extent that only one ANS molecule should be bound per HSA protein. Thus, the absence of any change in ANS lifetime before and after conjugation allows us to explain the decrease in fluorescence by the formation of a nonfluorescent complex protein−nanoparticle, and neither conformational changes nor energy transfer mechanism can be concluded under these experimental conditions. In fact, the identical extents of quenching measured by excitation at 295 nm (Trp) and 350 nm (ANS) are in agreement with this finding. The structure of the protein corona, however, must be strongly influenced by the deposition kinetics, being the adsorption/spreading kinetics and intermolecular interactions very important aspects to be taken into account.37 In this sense, the protein concentration is an important factor in the interplay between the time required for the transport of the molecule to the surface and that for reorientation/spreading on the surface. Thus, a high protein concentration should be appropriate to reach high surface coverage and avoid the protein to spread and consequently unfold before it is surrounded by other molecules. In order to rule out the denaturation of the HSA proteins upon binding to the nanoparticle surface, we have carried out AuNP titrations and monitored the changes in the ANS fluorescence under different HSA:ANS concentration ratios. Figure 9 shows the normalized fluorescence decreases as a function of AuNP concentrations.

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CONCLUSIONS The formation of bioconjugates HSA−cit-AuNPs and HSA− 6MP-AuNPs takes place mainly through electrostatic interactions between the negative charges on the nanoparticle surface and the positive charged lysine residues in the protein. This is evidenced by the higher stability of the bioconjugates upon increasing the fraction of negative charges at the nanoparticle surface, being the number of protein molecules necessary for stabilization lower upon increasing this parameter. In all the cases studied the critical flocculation concentration is higher than the average ionic strength existent under physiological conditions. The decreases in emission intensity observed in all the cases studied are interpreted as due to a static quenching mechanism upon the formation of nonfluorescent complexes. An interesting finding in this work is the parallel quenching observed in the fluorescence of two probes, the Trp214 (localized in domain IIA) and the ANS molecule bound to a site in the domain IIIA of the protein. The fact that very similar association constants are obtained from the different data points to the integrity of the tertiary structure of the protein upon binding. However, partial unfolding of the protein can take place when the bioconjugates are formed in the presence of low protein concentration. Finally, the dependence of the affinity of the protein albumin on the surface charged state of the gold modified surface can be used to modulate the binding of this protein in the presence of others more or less abundant or with different binding affinity constants.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1 and S2: fluorescence spectra and fractional fluorescence and logarithmic plots of HSA/6MP-AuNPs at pH 7.4 and 9.0; Figure S3: fractional fluorescence plots of HSA−ANS complex with the concentration of AuNPs obtained from data of fluorescence emission intensities measured by excitation at 295 and 350 nm. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 9. Titration curves of the HSA/ANS complexes with AuNPs measured by fluorescence spectroscopy (λexc = 350 nm). Sodium phosphate 0.010 M, pH 7.4.

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Two different trends can be observed. On one hand, there is no noticeable influence of the ANS concentration at constant HSA values and, on the other, the extent of quenching depends on the protein concentration at least up to protein/AuNP surface site ratio of around 300/1. It is interesting to note that under these experimental conditions the quenching observed in the intrinsic fluorescence (data not shown) is independent of both the protein concentration and the HSA/ANS ratio and amounts for an extent similar to that observed in ANS at the higher HSA concentrations (around 40%). If we assume that the 40% decrease is solely due to the complex formation, then the additional decrease obtained (up to 60% at a concentration

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Ph +34-957-218646; Fax +34-957218618. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministerio de Ciencia e Innovación (MICINN) (Project CTQ2010-16137) Junta de Andaluciá and University of Córdoba for financial support of this work. 10436

dx.doi.org/10.1021/jp3021497 | J. Phys. Chem. C 2012, 116, 10430−10437

The Journal of Physical Chemistry C

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Article

(35) Togashi, D. M.; Ryder, A. G. J. Fluoresc. 2006, 16, 153−160. (36) Lystvet, S. M.; Volden, S.; Yasuda, M.; Halskau, O.; Glomm, W. R. Nanoscale 2011, 3, 1788−1797. (37) Glomm, W. R.; Halskau, O., Jr.; Hanneseth, A.-M. D.; Volden, S. J. Phys. Chem. B 2007, 111, 14329−14345.

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