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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Biophysical Chemistry 146 (2010) 98–107 Contents lists available at ScienceDirect Biophysical Chemistry j o u r n a l h o m e p a g e : h t t p : / / w w w. e l s ev i e r. c o m / l o c a t e / b i o p h y s c h e m Ligand exchange effects in gold nanoparticle assembly induced by oxidative stress biomarkers: Homocysteine and cysteine Magdalena Stobiecka a, Jeffrey Deeb a, Maria Hepel a,b,⁎ a b Department of Chemistry, State University of New York at Potsdam, Potsdam, NY 13676, USA Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA a r t i c l e i n f o Article history: Received 25 October 2009 Received in revised form 10 November 2009 Accepted 10 November 2009 Available online 14 November 2009 Keywords: RELS spectroscopy Gold nanoparticles Surface plasmon resonance Homocysteine-mediated assembly Oxidative stress biomarkers Elastic light scattering Resonance rayleigh scattering a b s t r a c t The interactions of oxidative stress biomarkers: homocysteine (Hcys) and cysteine (Cys) with the multifunctional gold nanoparticles, important in view of novel biomedical applications in diagnostics and therapy, have been investigated using resonance elastic light scattering (RELS), UV–Vis plasmonic spectroscopy, and highresolution TEM imaging. The Hcys-induced assembly of gold nanoparticles has been observed for non-ionic surfactant-capped gold nanoparticles as well as for negatively-charged citrate-capped gold nanoparticles. We have observed for the first time the de-aggregation of citrate-capped gold nanoparticle ensembles followed by their conversion to citrate-linked Hcys-capped nanoparticle assemblies. The Cys molecules, which are smaller than Hcys by only one CH2 group, show much less activity. The mechanisms leading to this intriguing disparity in the abilities of these two thioaminoacids to ligand exchange with surfactant- or citrate-capping molecules of the gold nanoparticle shells are proposed on the basis of the experimental evidence, molecular dynamics simulations, and quantum mechanical calculations. For citrate-capped gold nanoparticles, we postulate the formation of surface complexes facilitated by electrostatic attractions and formation of double hydrogen bonds for both Hcys and Cys. The conformational differences between these two kinds of complexes result in marked differences in the distance between –SH groups of the biomarkers to the gold surface and different abilities to induce nanoparticle assembly. Analytical implications of these mechanistic differences are discussed. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Biomarkers of oxidative and nitrosative stress have recently been the subject of extensive studies [1,2] as the new evidence demonstrates ever increasing number of related diseases. The oxidative stress has been suggested as the causative factor in aging [3] and many diseases such as cadiovascular, diabetes, cancer, autism spectrum disorders (ASD) [4], and others. Among the biomarkers of oxidative stress are small biomolecules such as: ubiquinol [5] which is very labile in the oxidation of low-density lipoprotein (LDL), glutathione (GSH) which is depleted in the presence of organic radicals and peroxides [6], homocysteine [7,8] which has been found at elevated levels in atherosclerosis [9–14], Alzheimer disease [15,16], dementia [15], and poses an increased risk of birth defects [17]. Some biomarkers of oxidative stress are necessary to maintain healthy homeostasis (e.g. glutathione), while others participate in the development of diseases (e.g. homocysteine). For instance, decreased levels of glutathione and increased levels of oxidized glutathione (GSSG) have been observed in plasma, serum and urine samples from individuals diagnosed with ASD [4,18–20]. Homocysteine (Hcys), which is a sulfur-containing amino acid, is formed during a ⁎ Corresponding author. Department of Chemistry, State University of New York at Potsdam, Potsdam, NY 13676, USA. Tel.: + 1 315 267 2264; fax: + 1 315 267 3170. E-mail address: hepelmr@potsdam.edu (M. Hepel). 0301-4622/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2009.11.001 metabolism of methionine to cysteine but the increased concentration of Hcys in plasma (CHcys N 15 μM) is a risk factor for many disorders, including cardiovascular [9–12], renal [21], Alzheimer's [15,16], and other diseases [22]. Redox-related alterations, measured usually as the change in the concentration ratio of GSH/GSSG which is the main redox level maintaining couple in organisms, may also be heritable. Deviations from healthy biomarker concentration levels may result from deficiency of certain vitamins, e.g. B12 and folic acid (in hyperhomocysteinemia). The investigations of oxidative stress biomarkers are important to understand their behavior and role in organisms and to develop sensors and assays for their rapid detection and diagnosis of stress-related disorders. The reactivities and interactions of the oxidative stress biomarkers have been investigated in conjunction with the development of molecularly-templated polymer films with biorecognition capabilities designed for biomarkers detection [23], fluorimetric assays based on specific reactions [24–26], electrochemical sensors [27,28], colorimetric assays based on nanoparticle assembly [29–32], and the design of immunosensors [33] and other sensors for the analysis of biomarkers or utilizing biomarkers in the sensory film design [34–36]. In particular, in studies of biomolecule-induced gold nanoparticle assembly, the kind of interparticle interactions is the key element of the functionalized nanoparticle self-affinity [37–39]. The interparticle forces include electrostatic [40], zwitterionic [29,40], van der Waals Author's personal copy M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 forces [41], as well as hydrogen bonding forces [41–43]. The investigations of functionalized spherical gold nanoparticles and gold nanorods for application in novel assays for GSH [43], cysteine [32,44–48] and homocysteine [29,30,45,49] have been reported. The gold nanoparticle cores with protective shells of self-assembled monolayers (SAM) of thiolates [50,51], surfactants [47,52,53], citrate ions [48], and others can be utilized in the analysis. A difference in the sensitivity of the gold nanoparticle assembly process to structurally similar cysteine and homocysteine molecules, which differ only by one CH2 group, has been found [32,46,47]. Probing the interactions of biomolecules with gold nanoparticles and their influence on surface plasmon resonance and the elastic light scattering cross-section has potential applications in the development of novel assays for these molecules. The gold nanoparticle assembly process observed upon the addition of biomolecules is believed to be due to the ligand exchange [54] followed by the attractive interparticle interactions [29,48]. According to the thermodynamic stability, the citrate shell is less strongly bound than cysteine shell and the latter is less strongly bound than homocysteine shell. In practice, the kinetic hindrance may slow down considerably the ligand exchange processes. Also, the interparticle molecular-linking may induce an assembly before the completion of a ligand exchange process, as we have recently observed in the case of GSH-induced assembly. The biomolecule-induced gold nanoparticle assembly process can be monitored using surface plasmon absorbance band shifts. The oscillation frequency of the local surface plasmon (SP) is very sensitive to the changes in dielectric environment of nanoparticles and distance between nanoparticles within 5r range (where r is the nanoparticle radius). Theoretical studies of plasmonic oscillations [55–65] and SP absorbance spectra [66–74] have enabled the understanding of mechanisms leading to the absorbance maximum shifts associated with the assembly processes. The hydrodynamic radius of nanoparticle aggregates can be measured using dynamic light scattering although the complex dielectric medium and formation of aggregates of small particles may complicate the analysis. The use of transmission electron microscopy (HR-TEM) has been so far the best in determining the nanoparticle diameters and presenting images of aggregated nanoparticles. In this work, we have applied UV–Vis plasmonic absorbance measurements, HR-TEM, and the resonance elastic light scattering (RELS) spectroscopy [75–87]. The latter provides very sensitive measure of the degree of gold nanoparticle assembly. The gold nanoparticles show enhanced scattering during the assembly process due to the collective oscillation of local surface plasmons in nanoparticles bound in an ensemble. In this work, the assembly of gold nanoparticles induced by oxidative stress biomarkers, homocysteine and cysteine, has been investigated using non-ionic fluorosurfactant-capped gold nanoparticles and negatively-charged citrate-capped gold nanoparticles. The remarkable differences in ligand exchange abilities of the homocysteine and cysteine, have been observed for both the charged and uncharged nanoparticle shells. Different mechanisms leading to these effects for uncharged and charged nanoparticle shells are proposed. The elucidation of these mechanisms is crucial for analytical determination of structurally similar cysteine and homocysteine using rapid and inexpensive measurement techniques important for oxidative stress screening and prevention of environmental pollution effects on human health. 2. Experimental 2.1. Chemicals All chemicals used for investigations were of analytical grade purity. DL-Homocysteine (HS(CH2)3NH2COOH), L-Cysteine (HS (CH2)2NH2COOH), tetrachloroauric(III) acid trihydrate (HAuCl4⋅3H2O) 99 with 99.9+% metals basis, D-Methionine,and L-glutathione (GSH) reduced (minimum 99%), were purchased from Sigma Aldrich Chemical Company (Atlanta, GA, U.S.A.) and used as received. ZONYL FSN-100, a fluorocarbon-ether surfactant (FES), with nominal composition CF3 (CF 2 ) m (C 2 H4 O)n CH 2OH and average molecular mass M = ∼950 g/mol was obtained from Sigma Aldrich. Sodium citrate dihydrate (HOC(COONa)(CH2COONa)2⋅2H2O) was received from J.T. Baker Chemical Co. (Phillipsburg, NJ, U.S.A.). Sodium borohydride (NaBH4) was obtained from Fisher Scientific Company. L(+) Histidine was purchased from Eastman Organic Chemicals (Rochester, NY, U.S. A.). Solutions were prepared using Millipore (Billerica, MA, U.S.A.) Milli-Q deionized water (conductivity σ = 55 nS/cm). They were deoxygenated by bubbling with purified argon. 2.2. Apparatus The imaging analyses of Au nanoparticles were performed using high-resolution transmission electron microscopy (HR-TEM) with Model JEM-2010 (Jeol, West Chester, PA, U.S.A.) HR-TEM instrument (200 kV) and imaging with a Jeol Model JSM-7400F field-emission scanning electron microscope (FE-SEM). The elastic light scattering spectra were recorded using LS55 Spectrometer (Perkin Elmer, Waltham, MA, U.S.A.) equipped with 20 kW Xenon light source operating in 8 μs pulsing mode. Pulse width at half height was less than 10 μs. Separate monochromators for the incident beam and the detector beam enabled to use monochromatic radiation with wavelengths from 200 nm to 800 nm with 1 nm resolution. Additionally, the system was equipped with sharp cut-off filters: 290, 350, 390, 430, 515 nm. The dual detector system consisted of a photomultiplier tube (PMT) and an avalanche photodiode. The RELS spectra were obtained at 90° angle from the incident (excitation) light beam. The excitation beam monochromator was either scanned simultaneously with the detector beam monochromator (Δλ = 0) or set at a constant excitation wavelength. The UV–Vis spectra were recorded using Perkin Elmer Lambda 50 Spectrophotometer in the range 400 to 900 nm or Ocean Optics (Dunedin, FL, U.S.A.) Model R4000 Precision Spectrometer in the range from 340 nm to 900 nm. 2.3. Procedures The Au nanoparticles were synthesized according to the published procedure [88]. Briefly, to obtain 5 nm AuNP, 10 mM HAuCl4 was mixed with 10 mM trisodium citrate solution (ratio 1: 3.75) and poured to distilled water (109 mL). The obtained solution was vigorously stirred and fresh cold NaBH4 solution (5 mM, 8.9 mL) was added dropwise. The solution slowly turned light grey and then ruby red. Stirring was maintained for 30 min. The obtained citratecapped core-shell Au nanoparticles (AuNP) were stored at 4 °C. Their size, determined by HR-TEM imaging and UV–Vis surface plasmon absorption was 5.0 nm. The concentrations of AuNP's are given in moles of particles per 1 L of solution (usually, in the nM range). The RELS and UV–Vis spectra for samples were obtained with 1 min of mixing of AuNP with biomolecule solutions, unless otherwise stated. Quantum mechanical calculations of electronic structures for a model fluorocarbon-ether surfactant, citric acid, cysteine and homocysteine were performed using modified Hartree–Fock methods [89,90] with 6–31G* basis set and pseudopotentials, semi-empirical PM3 method, and density functional theory (DFT) with B3LYP functional. The molecular dynamics simulations and quantum mechanical calculations were carried out using procedures embedded in Wavefunction (Irvine, CA, U.S.A.) Spartan 6. The electron density and local density of states (LDOS) are expressed in atomic units, au− 3, where 1 au = 0.529157 Å and 1 au− 3 = 6.749108 Å− 3. Author's personal copy 100 M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 3. Results and discussion 3.1. Plasmonic spectroscopy of thioaminoacid-induced assembly of gold nanoparticles protected by ZONYL Fluorosurfactants provide similar advantages to other surfactants but, in addition, show high degree of chemical inertness. For these reasons they have recently been applied in chemical analysis [52]. The ZONYL fluorosurfactant is known to form self-assembled monolayers on gold surfaces rendering the surface more hydrophobic and significantly retarding the gold oxide formation processes [91]. In the case of AuNP, it stabilizes gold colloids by forming tight shells around nanoparticle cores with hydrophilic heads oriented toward Au surface and fluorocarbon tails forming hydrophobic non-interacting external surface. Although this surfactant forms water-tight shells, its bonding to a gold surface is not as strong as that of thiolates. Therefore, in their presence, ZONYL is replaced in a ligand exchange process by thiols, including thioaminoacids, homocysteine and cysteine, investigated in this work, provided that sufficiently high concentration of these agents is used and long enough time is allowed. The HR-TEM images of fluorosurfactant-capped AuNP's are presented in Fig. 1 before (a) and after (b–d) homocysteine-induced nanoparticle framework assembly. The ligand exchange process taking place upon addition of homocysteine to ZONYL-capped AuNP can be monitored using SPband absorbance of AuNP, as illustrated in Fig. 2. The UV–Vis spectra 1– 9 were recorded for increasing concentrations of Hcys, from 0 to 22.2 μM and constant concentration of AuNP5 nm (6 nM). It is seen that the SP band shifts toward longer wavelengths and the maximum absorbance increases with increasing CHcys. These observations are consistent with ligand exchange process: AuNP = FESx + yHcys = AuNP = Hcysy + xFES where x ≈y, followed by interparticle molecular linking of AuNP/Hcys through direct Hcys-Hcys interactions. At the pH of these experiments (pH = 6.0), homocysteine exists as a zwitterion with α-amino group − protonated (–NH+ 3 ) and carboxylic group dissociated (COO ). Therefore, the zwitterionic interparticle binding between Hcys-capped AuNP is playing a predominant role as recently discussed by Zhong et al. [29]. Fig. 1. HR-TEM images of ZONYL-capped gold nsanoparticles before (a) and after assembly with 15 μM homocysteine (b–d); CAuNP = 6 nM, CZONYL = 0.22 %, pH = 6; bar size: (a) 50 nm, (b) 50 nm, (c) 10 nm, (d) 5 nm. Author's personal copy M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 101 Extensive studies of the surface plasmon absorbance for various AuNP systems have been carried out by several groups [37,38,42, 50,51,70,86,92–99]. In particular, it follows from studies of the homocysteine-mediated assembly of AuNP that the interparticle zwitterion interaction of the Hcys-Au system is particularly strong [29] and that the Hcys-mediated assembly of AuNP can be accelerated by an increased temperature and ionic strength of the solution thus reducing the barrier for Hcys attachment to gold nanoparticle surface [29]. Also, the assembly can be reversed by the pH change [29,30]. Similar experiments performed with cysteine indicate that at higher concentrations (C N 15 μM) the kinetics of ligand exchange for both Hcys and Cys is very fast and the exchange is completed within 1 min of mixing AuNP solution with the thioaminoacids. However, at lower concentrations, the ligand exchange is considerably faster for Hcys than for Cys. 3.2. Resonance scattering of the thioaminoacid-mediated ZONYL-capped gold nanoparticle assembly process Typical light scattering spectrum for a ZONYL FSN surfactant-capped 5 nm diameter Au nanoparticles (AuNP5 nm) in solution is presented in Fig. 3, curve 1, for AuNP5 nm concentration of 6 nM and a constant excitation wavelength λex = 550 nm (1.94 eV). The strong resonant Rayleigh scattering from AuNP5 nm nanoparticles in solution results from the absorption of photons at 550 nm followed by secondary emission without any energy loss. Thus, the coherent elastic Rayleigh scattering with Gaussian peak shape centered at λem = λex = 550 nm is observed. The narrow linewidth of Δλ = 15 nm confirms that the effects due to radiation broadening, density fluctuation, fluorescence, and inelastic Raman scattering are negligible. Note that the background intensity is very low (virtually zero), which is leading to the well defined RELS peaks. The addition of homocysteine to the ZONYL-capped AuNP5 nm nanoparticles results in strong enhancement of Rayleigh scattering, as indicated in Fig. 3, curves 2–9, obtained for 6 nM AuNP5 nm + x μM Hcys, where x = 0 ... 22.2 μM. Upon addition of Hcys, the solution pH was maintained at pH = 6.0. This pH value is within the range of predominantly neutral (zwitterionic) form of homocysteine (pH= 2.22 to 8.87; pKa,1 = 2.22 (COOH), pKa,2 = 8.87 (NH2), pKa,3 = 10.86 (SH)). The strong enhancement of RELS from AuNP5 nm by Hcys molecules is expected since any size increase of AuNP due to the aggregate formation associated with interparticle interactions with zwitterionic Hcy-Hcys cross-linking should result in stronger scattering. The strong sixth-power dependence of elastic scattering intensity Isc on the Fig. 2. (a) Absorbance spectra for ZONYL-capped AuNP for different concentrations of homocysteine, CHcys [μM]: (1) 0, (2) 2.22, (3) 3, (4) 4.44, (5) 5.56, (6) 11.11, (7) 16, (8) 18, (9) 22.22. CAuNP = 6 nM, CZONYL = 0.22 %, pH = 6; (b–c) dependence of (b) λmax and (c) Amax vs. CHcys. The bathochromic shift of the surface plasmon peak (Δλmax = 36 nm, for 16 μM Hcys) corresponds to the formation of small Hcys-linked AuNP ensembles. The increase of SP absorbance by 21% (from 0.253 to 0.305, Fig. 1b) indicates on the collective oscillations of local surface plasmons in AuNP that form these ensembles. The collective oscillation of local surface plasmons is excited when the distance d between AuNP is: d b 5r, where r is the AuNP radius. The absorbance maximum increases with CHcys and reaches the saturation value at CHcys N 7 μM, with the half-absorbance change appearing at CHcys = 3.38 μM. The value of λmax also reaches saturation at CHcys N 7 μM (Fig. 1c). Therefore, we can assume that above 7 μM Hcys concentration the ligand exchange process has completed and nanoparticle shells are saturated with Hcys. Fig. 3. Resonance elastic light scattering spectra for ZONYL-capped AuNP5 nm for different concentrations of homocysteine, CHcys [μM]: (1) 0, (2) 2.22, (3) 3, (4) 4.44, (5) 5.56, (6) 14, (7) 16, (8) 18, (9) 22.22. CAuNP = 6 nM, CZONYL = 0.22 %, pH= 6, λex = 550 nm. Author's personal copy 102 M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 nanoparticle diameter a follows from the Rayleigh equation for light scattering from small particles:

 1 + cos2 θ 2π4 ½n2 −n1 Š2 −1
a6   Isc = I0 N λ 2R2 ½n2 −n1 Š2 + 2 2 ð1Þ where n1 and n2 are the refractive indices for the solution and particles, respectively, λ is the wavelength of incident light beam, θ is the scattering angle, N is the number of particles, and I0 is the constant. For λ = const and other experimental conditions (θ, R, I0) unchanged, one obtains: Isc;2 N a6 6 = 2 62 = crel arel Isc;1 N1 a1 3.3. Ligand exchange processes for ZONYL-capped gold nanoparticles ð2Þ where indices 1,2 stand for the particles before and after Hcys addition, respectively, crel = N2/N1 is the relative concentration of particles after addition of Hcys, and arel = a2/a1 is the relative diameter of particles after addition of Hcys. Therefore, the increase in the particle diameter can be estimated as follows: arel " Isc;2 = crel Isc;1 #1 = 6 ð3Þ Furthermore, the relative concentration crel, which is equal to 1 for a no-aggregation condition and less than 1 for aggregation, can be expressed by: crel = N2 V = 1 N1 V2 −3 Isc = A2 + ðA1 –A2 Þ = ð1 + exp½ðC−C1 = 2 Þ = sŠÞ ð5Þ and: Isc;2 3 = arel Isc;1 ð6Þ Therefore, the increase of the particle diameter can be estimated as follows: arel sffiffiffiffiffiffiffiffi 3 Isc;2 = Isc;1 It is interesting to compare the ligand exchange processes for different aminoacid ligands and ZONYL-capped AuNP. As reported earlier [24,52], these processes differ considerably between aminoacids and these differences are due to highly selective ZONYLreplacement abilities of the particular aminoacids. The plots of RELS intensity vs. aminoacid concentration measured at λex = 550 nm for Hcys, methionine, alanine, histidine, and glutathione, are presented in Fig. 4. They show a strong increase of Isc with C for homocysteine and apparent no response for other aminoacids and glutathione. The Isc vs. CHcys dependence is sigmoidal with an inflection point at low Hcys concentration indicating a high affinity of Hcys for Au surface, higher than that of ZONYL. From a Boltzmann function fitted to the experimental data for Hcys and ZONYL, we obtain: ð9Þ ð4Þ where Vi is the effective volume of a single aggregate i. Substituting Vi = (4/3)π(ai/2)3, one obtains: crel = arel clude that Hcys-mediated assembly of AuNP's occurs upon addition of Hcys to the ZONYL-capped AuNP solution and the effective diameter of assemblies is: a = 1.99 a0 (where a0 is the diameter of Hcys-capped AuNP). This assembly results in a large increase in Isc in accord with the data of Fig. 3. Because there are only very weak interactions between the Hcys molecules and hydrophobic tail of ZONYL, any Hcys-mediated bridging of ZONYL-capped AuNP's, such as that observed upon addition of GSH to citrate-capped AuNP's, cannot take place. Hence, the ligand exchange is the first stage of the interactions between Hcys and ZONYL-capped AuNP and it is followed by HcysHcys interparticle interactions leading to AuNP assembly. ð7Þ where A1, A2 — are the lower and higher Isc plateaus, C1/2 is the concentration at the inflection point, and s is the slope parameter. The ⁎ describing value of C1/2 = 3 μM and the characteristic constant K1/2 the “half-reaction” state of the ligand exchange in the ZONYL ⁎ = 3.3 × 105 M− 1 (note that the value replacement by Hcys is: K1/2 and units of this phenomenological half-reaction-state equilibrium constant are typically different than those for a thermodynamic equilibrium constant for higher order reactions involving more than ⁎ confirms a high affinity of single molecules). The high value of K1/2 Hcys to the gold surface in comparison to that of the ZONYL surfactant. The longer elution time for Hcys than for Cys observed in C18 column chromatography experiments [24,52] is consistent with higher affinity of Hcys than Cys to hydrophobic chains. In the setting of a ZONYL-capped AuNP, this would translate to a slower transfer of Hcys through a ZONYL shell and a slower kinetics of the ligand From the data of Fig. 3, the scattering intensity increase is: Isc,2/ Isc,1 = 80.44/10.26 = 7.84 and, hence, arel = 1:99 ð8Þ This means that most likely small aggregates composed of only few nanoparticles (e.g. 2–6) are formed. Since a small contribution to the change in particle diameter is also due to the ligand exchange, we have to estimate this contribution. The thickness of the ZONYL shell around AuNP is 1.1 nm (vertical, fully extended orientation, ZONYL FSN-100, with formula CF3(CF2) m(C2H4O) nCH2OH and average m = 12, n = 6 assumed on the basis of molmass M = 950 g/mol) and the height of Hcys molecule adsorbed on Au is on the order of 0.5 nm based on quantum mechanical evaluation for Hcys adsorbed on a solid Au surface. The structure and dimensions of ZONYL and Hcys molecules are shown later on (Figs. 8–11). Hence, the diameter of a single AuNP, with core of 5 nm diameter would decrease from ca. 7.2 nm to 6.0 nm. Obviously, the diameter decrease cannot explain the observed ∼ 8-fold scattering intensity increase. Therefore, we can con- Fig. 4. Dependence of elastic light scattering intensity maximum Isc,max for ZONYL-capped AuNP on concentration of analytes: (1) homocysteine, (2) methionine, (3) alanine, (4) histidine, (5) glutathione, CAuNP = 6 nM, CZONYL = 0.22%, pH = 6, λex = 550 nm. Author's personal copy M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 103 exchange process. Since the opposite is observed, this means that other factors play a role in the ligand exchange mechanism. The interactions of Hcys and Cys with ZONYL molecules are further discussed later on by employing molecular dynamic simulations of a model ZONYL and biomarker molecules. 3.4. Interactions of thioaminoacids with citrate ligands of core-shell gold nanoparticles Upon addition of homocysteine to citrate-capped AuNP, an increase in resonance elastic light scattering, similar to the one described for ZONYL-capped AuNP, is also observed (Fig. 5), provided that the solution pH is carefully controlled. The RELS spectra in Fig. 5 were obtained at pH = 5.0 for λex = 560 nm, for increasing concentrations of Hcys from CHcys = 0 to 15 μM. The increase in scattering intensity upon addition of 15 μM Hcys is Isc,2/Isc,1 = 36.2/1.91 = 19.0 (mean of 5 measurements). The 19-fold increase in scattering intensity clearly indicates on the homocysteine-induced assembly of AuNP. Utilizing again Eq. (7), we obtain for the increase of particle diameter: arel = 2.7. Similar RELS experiments carried out for other aminoacid ligands and glutathione, presented in Fig. 6, show that the RELS response is highly selective to Hcys, consistent with recent findings [29,43,46] showing that thiol-containing aminoacids adsorb preferentially on a gold surface while glutathione (at neutral pH) is repelled from the citrate shell of nanoparticles. The mechanisms leading to this high selectivity are not well understood, though the importance of this selectivity for analytical determinations of homocysteine in a matrix of aminoacids and glutathione is high. In order to explore the effects of protonation equilibria for species in solution and in the protective SAM environment of gold nanoparticle shells, we have performed RELS measurements for Hcys and citratecapped AuNP at three different solution pH: 2.0, 5.0, and 9.0. The plot of scattering intensity Isc vs. CHcys for these three media is presented in Fig. 7. The three dependencies of Isc vs. CHcys for different pH values show completely different behaviors. The curve 1 for pH = 2.0 shows a scattering intensity decrease with increasing CHcys and establishment of a plateau for CHcys N 4 μM. Curve 2 shows a sigmoidal shape with the onset of scattering at CHcys = 5 μM and establishment of a new level of scattering intensity plateau for CHcys N 7 μM. In the case of the third curve, for pH = 9.0, there is virtually no scattering change seen for the entire concentration range of Hcys examined and the level of scattering is very low (Isc ≈ 8, for 20 μM Hcys). Note that the scattering intensity levels established for pH = 2.0 and pH = 5.0 at higher concentrations of Hcys, are different. Fig. 5. Resonance elastic light scattering spectra for citrate-capped AuNP5 nm for different concentrations of homocysteine, CHcys [μM]: (1) 0, (2) 5, (3) 5.5, (4) 5.75, (5) 6.75, (6) 15, CAuNP = 3.8 nM, pH= 5, λex = 560 nm. Fig. 6. Dependence of elastic light scattering intensity maximum Isc,max for citrate-capped AuNP5 nm on concentration of analytes: (1) homocysteine, (2) methionine, (3) alanine, (4) histidine, (5) glutathione, (6) cysteine, CAuNP = 3.8 nM, pH = 5, λex = 560 nm. The elucidation of the mechanism of processes leading to the complex behavior of the citrate-capped AuNP — homocysteine system is a key element to understanding the reactivity and assembling properties of functionalized AuNP and their interactions with small biomolecules. The three situations represented by the data of Fig. 7 can be analyzed as follows: (i) The low elastic scattering intensity observed at pH = 9 (curve 3) for all Hcys concentrations examined is certainly due to the high gold colloid stability which is associated with strong electrostatic interparticle repulsions between deprotonated carboxyl groups that exist in the citrate shell before and in the Hcys-shell after the ligand exchange has taken place. (ii) The situation changes at pH=5 (curve 2) where citrates are still predominantly deprotonated (pK a,1 = 3.09, pK a,2 = 4.75, pKa,3 = 5.41) but homocysteine exists as a zwitterion with − group (pKa,1 = protonated –NH+ 3 group and dissociated COO 2.22 (COOH), pKa,2 =8.87 (NH2)). Thus, at low Hcys concentrations (CHcys b 5 μM), scattering is low since it is dominated by interparticle repulsions of negatively-charged citrate shells. As the ligand exchange process progresses, the citrate ions are being replaced by the neutral Hcys molecules. The progression is accelerated at higher Hcys concentrations. The switch from low elastic light scattering intensity to high intensity is observed in the concentration range: 5 μMbCHcys b 7 μM. At CHcys =7 μM, the Fig. 7. Dependence of elastic light scattering intensity maximum Isc,max on concentration of homocysteine CHcys for citrate-capped AuNP5 nm for different solution pH: (1) pH = 5 and (2) pH = 2. Author's personal copy 104 M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 saturation level is attained. This level can be ascribed to small ensembles of Hcys-linked AuNP where the interparticle attractions are attributed to strong Hcys-Hcys zwitterionic interactions. (iii) In an acidic solution at low pH (curve 1, pH = 2) and in the absence of homocysteine, a strong scattering intensity is observed which is due to the extensive interparticle hydrogen bonding. This occurs because at this pH citrates are predominantly undissociated (pKa,1 = 3.09, pKa,2 = 4.75, pKa,3 = 5.41, for citric acid) rendering the gold colloid unstable. The hydrogen bonding is responsible for the formation of gold nanoparticle networks and since the scattering intensity strongly increases with the aggregate size, a high scattering intensity is observed. Upon the addition of homocysteine, the light scattering intensity unexpectedly decreases to a new level, approximately at 50% of the initial scattering intensity value. This can be rationalized by assuming the dismantling of the initial citrate-linked gold nanoparticle ensembles and replenishing the nanoparticle shells with homocysteine in a ligand exchange process. While the newly formed shells are more strongly bound to the gold cores than citric acid based shells do, the Hcys molecules at pH = 2 are partially positively charged and cannot form as large the nanoparticle aggregates as citrate-capped AuNP do. In fact, one should expect interparticle repulsions of Hcys-capped AuNP at pH = 2 since pKa,1 = 2.22 (COOH), pKa,2 = 8.87 (NH2) for homocysteine. There are two plausible explanations of this behavior. On one hand, the reported value of pKa,1 for Hcys, which has been determined for the solution phase, is not relevant to Hcys molecules adsorbed on gold. A shift of the value of pKa,1 to somewhat lower values, would make the Hcys molecules still zwitterionic at pH = 2. However, to evaluate this possibility, the pKa values for surface bound homocysteine should be determined. On the other hand, some of the partially dissociated citrate molecules may participate in the neutralization and cross-linking of Hcys-capped AuNP. The level of light scattering intensity indicates that the nanoparticle ensembles formed are larger than those formed at pH = 5 where pure zwitterionic interactions have been found. Therefore, participation of citrate ligands in the gold nanoparticle cross-linking is likely to occur. Fig. 8. Model ZONYL-capped gold nanoparticle; electron density surfaces for d=0.08 au− 3, calculated for a fluorosurfactant molecule with formula CF3(CF2)m(C2H4O)nH with m=6, n =4, with electrostatic potential map (color coded from negative — red, to positive — blue). electrode surfaces [91]. In order to understand the behavior of cysteine and homocysteine in the surroundings of a fluorosurfactant shell, we have performed molecular dynamics simulations of the interactions of a biomarker with different parts of the fluorosurfactant molecule: (a) the top –CF3 group of the molecule, (b) the side of the –(CF2)m– tail, and (c) the side of the ethoxy chain. In Fig. 9, shown are cysteine molecules interacting with a fluorosurfactant molecule at these three positions. While there is virtually no effect of cysteine on the conformation of the fluorosurfactant molecule when cysteine interacts at the top or at the side of the hydrophobic tail, there is a change of the conformation observed when cysteine interacts with the ethoxylated part of the fluorosurfactant. A tendency of the ethoxy chain toward surrounding the cysteine molecule is observed in later stages of the simulation. Similar molecular dynamics simulations were performed for homocysteine. Fig. 10 illustrates the interactions of homocysteine with the top of the surfactant molecule, the side of the hydrophobic tail and the side of the ethoxylated chain. Again, there are no conformational changes in the surfactant molecule when homocysteine interacts with the hydrophobic tail. There are some In summary, we have observed for the first time the scattering spectra for the de-aggregation of citrate-capped gold nanoparticle ensembles followed by their conversion to citrate-linked Hcys-capped nanoparticle assemblies. 3.5. Molecular dynamics and quantum mechanical analysis of ligand exchange processes for core-shell gold nanoparticles The two main monolayer-protective types of shells for AuNP examined in this work differ considerably in their composition and properties, yet they both provide selectivity toward homocysteine versus cysteine in the nanoparticle assembly process. In order to elucidate the intriguing difference one methylene group makes in the behavior of cysteine (HS–(CH2)2–NH2–COOH) and homocysteine (HS– (CH2)3–NH2–COOH), we have performed molecular dynamics and quantum mechanical calculations to characterize the kind of intermediate structures that form on approach of Cys and Hcys molecules to a charged citrate-capped gold nanoparticle. Molecular dynamics simulations have also been carried out to evaluate the interactions of Hcys and Cys with a non-ionic fluorosurfactant-capped gold nanoparticle. A model gold nanoparticle coated with a monolayer of a fluorocarbon-ether surfactant is presented in Fig. 8. The fluorosurfactant used for model calculations has a composition CF3(CF2)m (C2H4O)nH and consists of a hydrophobic fluorocarbon tail and an ethoxylated chain –(C2H4O)n–, with assumed chain lengths: m = 6 and n = 4. The formation of a tight hydrophobic shell is consistent with the ZONYL-AuNP core-shell structure following studies on Au solid Fig. 9. Molecular dynamics simulations of interactions of cysteine with a model fluorosurfactant molecule with formula CF3(CF2)m(C2H4O)nH with m = 6, n = 4; positions of cysteine: (a) at the top of the surfactant molecule, (b) at the side of the hydrophobic –(CF2)m– chain, and (c) at the side of ethoxy chain. Author's personal copy M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 105 Fig. 10. Molecular dynamics simulations of interactions of homocysteine with a model fluorosurfactant molecule with formula CF3(CF2)m(C2H4O)nH with m = 6, n = 4; positions of homocysteine: (a) at the top of the surfactant molecule, (b) at the side of the hydrophobic –(CF2)m– chain, and (c) at the side of ethoxy chain. conformational changes in the surfactant molecule when homocysteine interacts with ethoxyleted chain, but these changes are much smaller than in the case of cysteine. This may be due to higher polarization of cysteine than homocysteine. The ethoxylated chain attempts to surround the smaller cysteine molecule while lowering the system energy. Therefore, it seems that the stronger interaction of cysteine with the fluorosurfactant may slow down considerably the adsorption competition between cysteine and fluorosurfactant at the surface of an Au substrate and hinder the ligand exchange process. It has been suggested earlier that the stronger affinity of homocysteine to fluorocarbon tail facilitates faster transport of homocysteine than cysteine, which is however, contradicted by the results of C18 column chromatography experiments [52] showing clearly faster elution of Cys than Hcys, consistent with stronger interactions of Hcys with a hydrophobic chain [24]. In addition to that, the shorter cysteine forms less strongly bound film of SAM on gold than longer homocysteine so in the adsorption competition, cysteine is a weaker competitor to the fluorosurfactant than homocysteine. In summary, there seem to be both thermodynamic as well as kinetic aspects of the ligand exchange between the thioaminoacids and the fluorosurfactant that lead under carefully selected conditions to a much higher effectiveness of homocysteine, in relation to that of cysteine, in replacing ZONYL from the gold nanoparticle protective shell. The interactions of cysteine and homocysteine with citratecapping film have also been considered. At the pH of measurements (pH 5–6), the citrate shell is charged negatively providing a long-term stability for the gold colloid, whereas both cysteine and homocysteine are in the form of zwitterions with protonated –NH+ 3 group and dissociated –COO− group. The main interaction of the electrostatic nature between –COO− group of the nanoparticle shell and –NH+ 3 group of the approaching thioaminoacid is expected with strong repulsions between dissociated carboxylate groups of the citrate and Cys or Hcys molecules. The results of molecular dynamics simulations and quantum mechanical calculations obtained are presented below. In Fig. 11, the interactions of cysteine and homocysteine with citrate ions in a ligand exchange process are analyzed. It is seen that both Cys and Hcys form intermediate surface complexes on approaching to a citrate-capped gold nanoparticle. Within the framework of electrostatic Fig. 11. Interactions of cysteine and homocysteine with citrate ions in a ligand exchange process: (a, b) surface complex formation through hydrogen bonding calculated for (a) CitCys and (b) Cit-Hcys using molecular dynamics, and (c, d) electron density surfaces for d= 0.08 au− 3, with electrostatic potential map for (c) Cit-Cys and (d) Cit-Hcys; electrostatic potential: color coded from negative – red to positive – blue. attractions between COO− group of the nanoparticle shell and NH+ 3 group of the thioaminoacid, a double hydrogen bond is formed for both the Cit-Cys and Cit-Hcys complexes. Immediately seen is, however, a completely different configuration of the thioaminoacid in the surface complex formed. Whereas a cysteine molecule forms a kind of axial (linear) configuration extending out of the citrate protective SAM, the homocysteine tends to bend out of the axial conformation and toward the citrate side-chain and the electrode surface. The lack of flexibility of the cysteine molecule has already been pointed out when comparing ring-forming abilities of these two molecules [32]. Here, the bending toward the citrates side chain results in the substantial difference in the distance of the thiol group to the gold surface. This distance is 0.75 nm for Cit-Cys surface complex and only 0.38 nm for Cit-Hcys complex. This difference can be translated to classifying the thioaminoacid position as being outside of the shell (in the case of cysteine) or inside the shell (in the case of homocysteine). The large difference in the observed light scattering intensity between Cys and Hcys can be explained by easier and faster penetration of Hcys into the citrate-dominated gold nanoparticle shell followed by citrate ligand replacement. After the ligand exchange has been completed, the zwitterion-type interactions begin to operate leading to the nanoparticle assembly and manifested by the sharp increase in the resonance elastic light scattering, as observed experimentally. On the other hand, in the case of cysteine, the ligand exchange process is strongly hindered by cysteine inability to enter the citrate protective shell due to the axial conformation of the surface complex Cit-Cys. 4. Conclusion The results demonstrate clearly the differences between cysteine and homocysteine in their ability to ligand exchange with non-ionic Author's personal copy 106 M. Stobiecka et al. / Biophysical Chemistry 146 (2010) 98–107 fluorosurfactant-capped AuNP, as well as with negatively charged citrate-capped AuNP. These intriguing differences appear as an amplification of a small structural difference in the molecular buildup (one methylene group), which progresses through several stages leading to the final sensitive detection in the gold nanoparticle assembly process. The selective stages include kinetic retardation due to interactions of the thioaminoacids with the fluorosurfactant chain or formation of charge-induced H-bonded complexes, as in the case of citrate-capped AuNP. Conformational differences in these surface complexes on one hand prevent cysteine from entering the citrate shell and on the other hand pull the homocysteine into the citrate film, thus shortening the distance between the thiol part of the biomarker and the Au surface and making it easier to form Au thiolate bond. We have observed for the first time the RELS characteristics for de-aggregation of citrate-capped gold nanoparticle ensembles followed by their conversion to citrate-linked Hcys-capped nanoparticle assemblies. The ligand exchange effects and gold nanoparticle assembly induced by thioaminoacid zwitterionic interparticle interactions are important in understanding physicochemical aspects of small biomolecule interactions with metal nanoparticles as the use of the latter is widely being explored for new nanomedical applications. The observed differences in the behavior of structurally similar cysteine and homocysteine have profound implications in their analytical determinations using rapid and inexpensive measurement techniques important for the oxidative stress screening and prevention of environmental pollution effects on human health. Acknowledgements This work was supported by the U.S. DoD Research Program “Idea”, Grant No. AS-073218. References [1] M. Noble, M. Mayer-Proschel, C. 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