pubs.acs.org/Langmuir © 2009 American Chemical Society
Novel Synthetic Route to Peptide-Capped Gold Nanoparticles Takeshi Serizawa,*,†,‡ Yu Hirai,†,§ and Mamoru Aizawa§ †
Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, ‡Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, and § Department of Applied Chemistry, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan Received November 21, 2008. Revised Manuscript Received August 20, 2009 A novel synthetic route to peptide-capped gold nanoparticles was demonstrated herein. Tetrachloroaurate ions were reduced with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) under extremely mild conditions (pH 7.2, ambient temperature) in the presence of cysteine-terminal desired peptides, so that peptide-capped spherical nanoparticles were successfully synthesized. Model basic peptides containing the Arg-Pro-Thr-Arg sequence, which is an essential motif that specifically binds to film surfaces composed of isotactic poly(methyl methacrylate), were employed. Particle sizes were approximately 10 nm, and size distributions were narrow. Positive zeta potentials of nanoparticles suggested the presence of the Arg-Pro-Thr-Arg sequence on the outermost surface. Thermogravimetric analysis revealed that peptides were closely packed on the gold’s surface. Parameters affecting reaction rates such as peptide structures and concentrations were investigated. Native peptide functions were conserved on nanoparticles by introducing a certain spacer between cysteine and the Arg-Pro-Thr-Arg sequence, suggesting that designing suitable peptide structures is essential to conserve peptide functions.
Introduction Gold nanoparticles have potential applications in biological and nanotechnological fields due to distinct surface plasmon properties.1 Gold nanoparticles capped with a great number of functional peptides are regarded as protein models demonstrating bioimaging, biosensing, and enzymatic reactions.1d,2 Such nanoparticles are representatively synthesized by adding required peptides chemically modified with N-terminal Cys residues into aqueous nanoparticle solutions, which are conventionally synthesized by reducing tetrachloroaurate ions (AuCl4-) with citric acid, resulting in the exchange of capping reagents from citric acid into peptides.2,3 Exhaustive investigations have revealed that the most excellent peptide sequence with a free C-terminus for stably dispersing gold nanoparticles in aqueous phases is Cys-AlaLeu-Asn-Asn.3 Although the utility of this method has been sufficiently proven with derivatives of the aforementioned peptide,2,4 a systematic study3 clearly demonstrated that peptides abundant with basic amino acids such as Lys and Arg, and particularly predominate at C-termini, except for a certain peptide,4c are not available frequently due to the precipitation of gold nanoparticles during exchange reactions. Therefore, an alternative method to prepare peptide-capped gold nanoparticles is still required. *Telephone/fax: þ81-3-5452-5225. E-mail:
[email protected]. u-tokyo.ac.jp (1) (a) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. (b) Pasquato, L.; Pengob, P.; Scrimin, P. J. Mater. Chem. 2004, 14, 3481–3487. (c) Ghosh, S. K.; Pal, T. Chem. Rev. 2007, 107, 4797–4862. (d) De, M.; Ghosh, P. S.; Rotello, V. M. Adv. Mater. 2008, 20, 4225–4241. (2) Levy, R. ChemBioChem 2006, 7, 1141–1145. (3) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076–10084. (4) (a) Olmedo, I.; Araya, E.; Sanz, F.; Medina, E.; Arbiol, J.; Toledo, P.; Alvarez-Lueje, A.; Giralt, E.; Kogan, M. J. Bioconjugate Chem. 2008, 19, 1154– 1163. (b) Jeong, K. J.; Butterfield, K.; Panitch, A. Langmuir 2008, 24, 8794–8800. (c) Sun, L.; Liu, D.; Wang, Z. Langmuir. 2008, 24, 10293–10297. (d) Nativo, P.; Prior, I. A.; Brust, M. ACS Nano 2008, 2, 1639–1644.
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Recently, it was demonstrated that 2-[4-(2-hydroxyethyl)1-piperazinyl]ethanesulfonic acid (HEPES), which is frequently utilized as a Good buffer, reduces AuCl4- in aqueous phases through the oxidation of its N-substituted piperazine ring to an N-centered cationic free radical, producing gold nanoparticles under mild conditions (ambient temperature and neutral pH).5 Subsequent investigations led to preparations with branched6 and flowered7 gold nanoparticles, demonstrating the possibility to control nanoparticle shape. Branched nanoparticles exhibited strong surface-enhanced effects which were utilized in the design of Raman-active tags.7b HEPES also reduces silver ions (Agþ) to produce silver nanoparticles, suggesting further utility for reducing various metal ions.8 Other Good buffers with piperazine and morpholine rings are also available to reduce AuCl4-.5 In all cases, excess amounts of Good buffers are utilized as stabilizers for metal nanoparticles. Furthermore, HEPES reduction in the presence of gold-binding peptides results in the preparation of spherical9 and superstructured10 gold nanoparticles. Therefore, when other reagents such as thiol compounds, which coordinate more strongly to the gold’s surface than Good buffers, coexist in reaction media, reagents could cap gold nanoparticles instead of Good buffers and modulate nanoparticle formation. The aforementioned concept demonstrates a general method to produce gold nanoparticles capped with desired molecules. This paper focuses on the synthesis and characterization of gold nanoparticles capped with Cys-terminal peptides. AuCl4(5) Habib, A.; Tabata, M.; Wu, Y. G. Bull. Chem. Soc. Jpn. 2005, 78, 262–269. (6) Xie, J.; Lee, J. Y.; Wang, D. I. C. Chem. Mater. 2007, 19, 2823–2830. (7) (a) Jena, B. K.; Raj, C. R. Langmuir 2007, 23, 4064–4070. (b) Xie, J.; Zhang, Q.; Lee, J. Y.; Wang, D. I. C. ACS Nano 2008, 2, 2473. (8) (a) Sun, R. W.-Y.; Chen, R.; Chung, N. P.-Y.; Ho, C.-M.; Lin, C.-L. S.; Che, C.-M. Chem. Commun. 2005, 5059–5061. (b) Tan, S.; Erol, M.; Attygalle, A.; Du, H.; Sukhishvili, S. Langmuir 2007, 23, 9836–9843. (9) (a) Slocik, J. M.; Stone, M. O.; Naik, R. R. Small 2005, 1, 1048–1052. (b) Diamanti, S.; Elsen, A.; Naik, R.; Vaia, R. J. Phys. Chem. C 2009, 113, 9993–9997. (10) Chen, C. L.; Zhang, P.; Rosi, N. L. J. Am. Chem. Soc. 2008, 130, 13555– 13557.
Published on Web 09/21/2009
DOI: 10.1021/la9021799
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Figure 1. Schematic representation of this study.
was reduced in HEPES buffer in the presence of simply designed peptides (Figure 1). As a model basic peptide, Arg-Pro-Thr-Arg (RPTR), which is an essential motif that specifically binds to film surfaces composed of isotactic (it) poly(methyl methacrylate) (PMMA),11 was employed. The 7-mer peptide Glu-Leu-TrpArg-Pro-Thr-Arg was originally identified by affinity selection for the it-PMMA film surface from genetically engineered phage libraries displaying random peptides.11a Subsequently, the essential motif was determined by detailed binding analysis of derivative peptides based on surface plasmon resonance measurements.11b Considering the directional affinity of the 7-mer peptide for it-PMMA from its C-terminus,12 the N-terminus was modified with Cys (C), to prepare CRPTR with an amidated C-terminus. Gold nanoparticles capped with C(EG)4RPTR, which further introduced a tetraethyleneglycol, (EG)4, spacer between Cys and RPTR, were similarly synthesized. To demonstrate whether peptide functions were conserved on gold nanoparticles, gold nanoparticles adsorbed onto films composed of target it-PMMA and reference syndiotactic (st) PMMA were analyzed quantitatively by quartz crystal microbalance (QCM) measurements. Herein, a novel synthetic route to gold nanoparticles capped with diverse peptides under extremely mild conditions, which is suited for handling biomolecules, was substantiated successfully.
Experimental Section Materials. HAuCl4 3 3H2O (Aldrich) and HEPES (nacalai tesque) were used without further purification. Peptides were synthesized by conventional solid-phase syntheses, purified by high performance liquid chromatography, and characterized by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS). 15-Amino-N-(9-fluorenylmethoxycabonyl)4,7,10,13-tetraoxapentadecanoic acid (Quanta BioDesign) was utilized for a tetraethyleneglycol spacer. HEPES buffer (100 mM, pH 7.2) was prepared by adding sodium hydroxide. It- and stPMMAs with number-average molecular weights of 32 900 (PDI = 1.3, 99% isotactic content) and 30 000 (PDI = 1.2, >85% syndiotactic content) were purchased from Polymer Source and were used as received. Ultrapure water was provided by a Milli-Q system. Nanoparticle Preparation and Characterization. Peptides were dissolved in HEPES buffer, and AuCl4- was added into solutions at 25 °C. Ultraviolet-visible (UV-vis) spectra were recorded by using a spectrometer model V-670 (Jasco) at 25 °C. Transmission electron microscopic (TEM) images were obtained by using a JEM 2100F (Jeol) instrument at an accelerated voltage of 200 keV using carbon-coated copper grids (300 mesh, Agar Scientific). Before casting on TEM grids, gold nanoparticles were centrifuged at 15 000 rpm for 1 h and redispersed in water. Dynamic light scattering (DLS) of gold nanoparticles in HEPES buffer was measured by using a Zetasizer Nano ZS (11) (a) Serizawa, T.; Sawada, T.; Matsuno, H.; Matsubara, T.; Sato, T. J. Am. Chem. Soc. 2005, 127, 13780–13781. (b) Serizawa, T.; Sawada, T.; Matsuno, H. Langmuir 2007, 23, 11127–11133. (12) Date, T.; Tanaka, K.; Nagamura, T.; Serizawa, T. Chem. Mater. 2008, 20, 4536–4538.
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(Malvern Instruments) instrument equipped with a He-Ne laser (output power = 4 mW at λ0 = 632 nm) at ambient temperature, assuming a solution density and a refractive index to be 0.95 mPa S and 1.3345, respectively. Surface zeta potentials of gold nanoparticles were obtained with a Zetasizer Nano ZS (Malvern Instruments) instrument using the Smolouchowski approximation in water at ambient temperature after gold nanoparticles were centrifuged and redispersed in water. Thermogravimetric analysis (TGA) was demonstrated by using a TGA-50 (Shimadzu) instrument with a platinum cell in nitrogen gas atmosphere at a heating rate of 10 °C min-1. X-ray photoelectron spectra (XPS) were obtained by using a Kratos AXIS-NOVA (Shimadzu) instrument employing Al KR radiation. Peaks were referenced to carbon at 285.0 eV to account for the sample charging. For both TGA and XPS measurements, gold nanoparticles capped with CRPTR and C(EG)4RPTR were centrifuged and freeze-dried. Nanoparticle Adsorption. The particle concentration was determined by using an extinction coefficient for gold nanoparticles with 10 nm diameters, as per previous reports.13 PMMA films with 45 nm thickness were spin-coated on 9 MHz QCM substrates (USI System). Tapping-mode atomic force microscopic (SPM-9600, Shimadzu) observations estimated the mean surface roughness of it- and st-PMMA films on the QCM substrates to be 1.8 nm. After mounting nanoparticle solutions onto substrates at 4 °C, substrates were gently rinsed with pure water, and frequency decreases were measured in air. Frequency decreases were converted to mass of adsorbed nanoparticles using Sauerbrey’s equation.14 Experiments were repeated four times.
Results and Discussion When CRPTR was simply added into aqueous solutions of gold nanoparticles prepared by citric acid reduction, followed by conventional exchange reactions,2-4 red solutions immediately turned blue, suggesting nanoparticle aggregation, and precipitates were observed gradually (Figure S1 in the Supporting Information). When citric acid on nanoparticles was partially exchanged by CRPTR, the cationic CRPTR on nanoparticles might have electrostatically interacted with citric acid on other nanoparticles, thus resuting in nanoparticle aggregation. Otherwise, electrostatic adsorptions of CRPTR onto nanoparticles capped with citric acid might result in nanoparticle aggregation due to charge shielding.15 Accordingly, the importance of developing the present method was confirmed. Figure 2a shows an UV-vis absorption spectrum of aqueous solutions containing AuCl4- (0.5 mM), CRPTR (0.25 mM), and HEPES (100 mM, pH 7.2) after reactions for 48 h at 25 °C. Light yellow AuCl4- solutions, which are derived from broad absorption bands of AuCl4- at around 300-350 nm, turned clear within 5 miniutes, suggesting that AuCl4- was rapidly reduced by excess amounts of HEPES, in which the N-substituted piperazine ring of HEPES was oxidized to an N-centered cationic free radical.5 The reducing activity of HEPES is thought to be similar to that of triethylamine.16 Then solutions gradually turned red (the inset of Figure 2a), suggesting that resulting Au(0) atoms formed nuclei and subsequently grew into nanoparticles based on diffusion limited processes, as shown in a previous study.9b λmax at 527 nm was representative of the formation of spherical gold nanoparticles with approximately 10 nm.1-3 Reactions monitored at λmax were almost saturated after 20 h (Figure 2b). (13) Liu, X.; Atwater, M.; Wang, J.; Huo, Q. Colloids Surf., B 2007, 58, 3–7. (14) Sauerbrey, G. Z. Phys 1959, 155, 206–222. (15) (a) Oishi, J.; Asami, Y.; Mori, T.; Kang, J.-H.; Tanabe, M.; Niidome, T.; Katayama, Y. ChemBioChem 2007, 8, 875–879. (b) Oishi, J.; Asami, Y.; Mori, T.; Kang, J.-H.; Niidome, T.; Katayama, Y. Biomacromolecules 2008, 9, 2301–2308. (16) Newman, J. D. S.; Blanchard, G. J. Langmuir 2006, 22, 5882–5887.
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Figure 2. (a) UV-vis absorption spectra of CRPTR-capped gold nanoparticles and (b) time-dependence of absorbance at 527 nm. The inset of (a) shows a picture of the nanoparticle solution.
The slow growth process of gold nanoparticles possibly led to the narrow size distributions (see TEM and DLS analyses). It is noted that the pH of the solution remained constant throughout reactions. In the absence of peptides, spectra of solutions showed λmax at 680 nm (Figures S2 and S3a in the Supporting Information), similar to a previous report.6 In the presence of RPTR (without Cys), gold solids were precipitated after approximately 30 min (Figure S3b in the Supporting Information), suggesting that HEPES-capped nanoparticles were cross-linked by RPTR with multiple amino groups through electrostatic interactions, by charge shielding15 after electrostatic adsorptions of RPTR onto HEPES-capped nanoparticles, or by both. These observations all suggest that, during reactions of AuCl4- in HEPES buffer,5-7 CRPTR was preferentially introduced onto gold nanoparticle surfaces instead of excess amounts of HEPES through strong gold-thiol chemical bonds. It is noted that HEPES seems to hardly reduce thiolate groups on the nanoparticle surface to release CRPTR, since resulting nanoparticles are extremely stable in HEPES buffer (see below). Figure 3a and b shows TEM images of gold nanoparticles prepared in the presence and absence of CRPTR, respectively. Morphologies were clearly different. The former nanoparticles were spherical, similar to previously prepared nanoparticles capped with gold-binding peptides.9,10 On the other hand, the latter were branched (and partly flower-shaped), similar to previous reports.6,7 These observations also suggested that the former gold nanoparticles were preferentially capped with CRPTR rather than HEPES. Diameters of CRPTR-capped nanoparticles, which were estimated by an average of major and minor axes for 200 nanoparticles, as well as hydrodynamic diameters analyzed by DLS measurements are shown in Figure 3c. These analyses showed almost the same numberaverage diameter of approximately 10 nm with narrow size distributions, although DLS measurements detected slightly larger sizes than TEM analysis. Accordingly, it was found that spherical gold nanoparticles capped with basic peptides, which were monodispersed in water, were successfully synthesized by reducing AuCl4- with HEPES in the presence of Cys-terminal peptides. CRPTR-capped gold nanoparticles stably dispersed in HEPES buffer for at least 6 months at ambient temperature. Even when Langmuir 2009, 25(20), 12229–12234
Figure 3. TEM images of (a) CRPTR-capped and (b) HEPEScapped gold nanoparticles, and (c) histograms of particle sizes estimated from TEM images of (a). The solid line in (c) is DLS data.
gold nanoparticles were collected by centrifugation at 15 000 rpm for 1 h, precipitates could be readily redispersed in water with the same λmax (Figure S4 in the Supporting Information). In addition, freeze-dried nanoparticles were similarly redispersed in water (Figure S5 in the Supporting Information). Furthermore, UV-vis absorption spectra hardly changed even after heating at 80 °C (Figure S6 in the Supporting Information), although λmax slightly decreased, similarly to a previous report.6 These observations suggest that resulting nanoparticles are significantly stable possibly due to stably and densely capped peptides on nanoparticles. The concentration of CRPTR was an essential factor to satisfactorily synthesize gold nanoparticles. When the peptide concentration was changed from 0.025 to 0.25 mM at constant concentrations of AuCl4- (0.5 mM) and HEPES (100 mM, pH 7.2), stably dispersed nanoparticles were obtained at more than 0.20 mM (Figure S7 in the Supporting Information). This observation is reasonable when considering that greater amounts of capping reagents are present in reaction media. In other words, nanoparticles partially capped with HEPES at lower CRPTR concentrations seemed to aggregate with one another based on a similar mechanism for aggregation of citric-acid-capped nanoparticles in the presence of CRPTR, as mentioned in Figure S1b in the Supporting Information. Reaction rates decreased with increasing peptide concentrations (Figure 4a and b), similar to a previous report.9b Since CRPTR should form complexes with Au(0) atoms through electrostatic and/or coordinate interactions, an increase in peptide concentrations seemed to delay nucleus formation and nanoparticle growth due to electrostatic and/or steric repulsion derived from interacted peptides. In fact, gold nanoparticles capped with an amino acid Cys were more rapidly produced than those capped with CRPTR (Figure 4c and Figure S8 in the Supporting Information), thus indicating the strong affect of the RPTR sequence on reaction rates. Accordingly, reaction rates and essential concentrations of peptides were changed by peptide species (also see the next paragraph), suggesting different levels DOI: 10.1021/la9021799
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Figure 4. Time-dependences of absorbance at λmax at the CRPTR concentrations of (a) 0.10 mM and (b) 0.25 mM, and (c) at a Cys concentration of 0.10 mM.
Figure 6. (a) TEM image of C(EG)4RPTR-capped gold nanoparticles and (b) histogram of particle sizes estimated from TEM images. The solid line in (b) is DLS data.
Figure 5. (a) UV-vis absorption spectra of C(EG)4RPTRcapped gold nanoparticles and (b) time-dependence of absorbance at 527 nm. The inset of (a) shows a picture of the nanoparticle solution.
of peptide interactions with Au(0) atoms. In other words, reaction conditions are necessary to be optimized, depending on capping species. Other peptides such as C(EG)4RPTR with a tetraethyleneglycol spacer could be applied to the present synthesis. When reacted with aqueous solutions containing AuCl4- (0.5 mM), C(EG)4RPTR (0.30 mM), and HEPES (100 mM, pH 7.2), λmax at 527 nm almost saturated after 50 h (Figure 5), and TEM observations revealed spherical gold nanoparticles with a mean size of approximately 10 nm (Figure 6). It is interesting that DLS measurements exhibited a greater size of nanoparticles (Figure 6b) compared with that estimated from TEM images, thereby suggesting that water-swollen peptides stretch out to aqueous phases, although a similar tendency was slightly observed for CRPTR-capped nanoparticles (Figure 3c). Reactions in the presence of C(EG)4RPTR were obviously slower than those in the presence of CRPTR (Figure 4a and b). In addition, the essential concentration (approximately 0.30 mM) of C(EG)4RPTR required to obtain stable gold nanoparticles was slightly greater than that of CRPTR. C(EG)4RPTR seemed to be difficult to cap Au(0) 12232 DOI: 10.1021/la9021799
atoms or nuclei compared with CRPTR due to steric repulsion between capped peptides. Therefore, greater C(EG)4RPTR concentrations were necessary to effectively introduce C(EG)4RPTR instead of HEPES. It is noted that once C(EG)4RPTR was introduced at adequate peptide concentrations, the growth to nanoparticles was slow (Figure 4), and was somewhat suppressed (see the next paragraph), compared with the case of CRPTRcapped gold nanoparticles. When almost saturated absorbance was compared between CRPTR-capped (Figure 2b) and C(EG)4RPTR-capped (Figure 5b) nanoparticles, the latter was smaller than the former, even though λmax values were the same. This observation suggests that the latter conversion to nanoparticles is smaller than the former. Greater amounts of Au(0) atoms or clusters that interacted with C(EG)4RPTR might still remain in reaction solutions compared with those that interacted with CRPTR, due to suppresion of nucleus formation and growth by greater steric repulsion of C(EG)4RPTR. In fact, MALDI-TOF MS measurements of supernatants after reactions detected notable peaks (Figure S9 in the Supporting Information), which are assignable to complexes between thepeptide and Au(0), although the peaks could not be discussed quantitatively. It is known that reactions for the preparation of gold nanoparticles capped with goldbinding peptides in HEPES buffer are extremely slow (>100 h) at high peptide concentrations due to the formation of peptideAu(0) complexes.9b It is noted that total charges of peptides were not necessarily basic. Gold nanoparticles capped with acidic or zwitterionic peptides could also be obtained (Cys-capped nanoparticles could be prepared, as discussed in Figure 4c and Figure S8 in the Supporting Information). Peptides with C-terminal Cys residues, which were not suitable for conventional exchange reactions,2-4 were also available (data not shown), thereby suggesting potential utilities of the present method. Synthetic details will be reported elsewhere. Resulting gold nanoparticles were further characterized. Zeta potentials of CRPTR-capped (Figure 2 sample) and C(EG)4RPTR-capped (Figure 5 sample) nanoparticles were estimated Langmuir 2009, 25(20), 12229–12234
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Figure 8. QCM analysis of adsorptions of (a) CRPTR-capped and (b) C(EG)4RPTR-capped gold nanoparticles onto film surfaces of it- and st-PMMAs. Nanoparticles prepared under conditions in Figures 2 and 5 were analyzed here, respectively.
Figure 7. TGA charts for (a) CRPTR-capped and (b) C(EG)4RPTR-capped gold nanoparticles. Nanoparticles prepared under conditions in Figures 2 and 5 were analyzed here, respectively.
to be þ29.5 and þ45.1 mV, respectively. These observations substatiate that cationic peptides are presented on the nanoparticle surface and that RPTR units are more exposed on the latter nanoparticles. On the other hand, the potential of HEPES-capped nanoparticles (Figure 3b sample) was estimated to be -35.8 mV, indicating the presence of HEPES on the nanoparticle surface, similarly to a previous report.6 TGA for CRPTR-capped and C(EG)4RPTR-capped nanoparticles revealed 13.4% and 16.4% mass decreases between 200 and 500 °C, respectively (Figure 7). Estimating the average number of gold atoms per nanoparticle from number-average diameters,13 peptide densities on gold nanoparticles were determined to be 2.46 and 2.52 peptides/nm2, respectively (for calculations, see following XPS discussion). Therefore, peptide densities were similar between the nanoparticles even though molecular sizes were different. Compared with other peptide densities for gold nanoparticles (1.67-1.93 peptides/nm2),3 the present densities were slightly large, and peptides seemed to be densely packed on the nanoparticle surface. XPS for CRPTR-capped and C(EG)4RPTR-capped nanoparticles (Figure S10 in the Supporting Information) showed C1s shoulder peaks at 288 eV, which are representative of carbonyl groups of peptide linkages, suggesting the presence of peptides on the nanoparticle surfaces. S2p peaks were predominantly observed at 168 eV, possibly suggesting that HEPES containing SO3- is present as counterions of two Arg residues and the peptide N-terminus (it is noted that S2p peaks for sodium HEPES were observed at 166 eV). Therefore, the aforementioned peptide densities based on TGA were estimated by assuming that CRPTR and C(EG)4RPTR have three HEPES counterions to two Arg and an N-terminus. Unexpectedly, S2p peaks assignable to sulfur coordinating to the gold’s surface17 were not observed at around 162 eV (trace level). Since TGA indicated that peptides are closely (17) Lu, H. B.; Campbell, C. T.; Castner, D. G. Langmuir 2000, 16, 1711–1718.
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packed on the nanoparticle surface, the sulfur might not be detected by XPS due to limitations of measurement depth (
