Gold nanoparticles capped by peptides

Materials Science and Engineering B 140 (2007) 187–194

Gold nanoparticles capped by peptides ˇ Francesca Porta a,∗ , Giovanna Speranza b , Zeljka Krpeti´c a , Vladimiro Dal Santo c , Pierangelo Francescato b , Giorgio Scar`ı d a

Dipartimento di Chimica Inorganica Metallorganica Analitica, Center of Excellence CIMAINA, INSTM Unit, Via Venezian 21, Milano I-20133, Italy b Dipartimento di Chimica Organica e Industriale, Milano I-20133, Italy c ISTM-CNR, Milano I-20133, Italy d Dipartimento di Biologia, Milano I-20133, Italy

Abstract Two dipeptides (GK and GC) and two 15-aminoacid peptides (GK15 and GC15) were used as capping agents in the preparation of monolayerprotected gold nanoparticles (MPCs). They were characterized by TEM microscopy, UV–vis, NMR and IR spectroscopy. © 2007 Elsevier B.V. All rights reserved. Keywords: Gold; Nanoparticle; Lysine; Glycine; Cysteine

1. Introduction The unique properties of nanoparticles sparked their application in a broad range of fields, including chemistry, physics, biology, materials science, medicine, and catalysis. For example, nanoparticles can be used as labels for optical bio detection, as substrates for multiplexed aqueous bioassays, as probes for cellular imaging or as carriers for therapeutic delivery [1–4]. Metal nanoparticles are attractive because of their easy synthesis and modification as well as their size- and shape-dependent properties [5–7]. In particular gold can be stabilised with a wide variety of molecules such as peptides, proteins, DNA and polymers [8–12]. The interaction between gold and those molecules can be a non-covalent electrostatic interaction, a hydrophobic interaction or a covalent binding [13]. Citrate reduction method represents the first gold hydrosol preparation. The citrate-capped gold nanoparticles [14,15] were further functionalized by other ligands for improving the stability. Bis(␳-sulfonatophenyl)phenylphosphane dihydrate dipotassium salt (BSPP) has been frequently used by Alivisatos’ group [12,16–18] achieving BSPP-gold nanoparticles stable in solution with high ionic strength and usable for further ligandattachment. Mangeney et al. covalently derivatized a hydrophilic polymer monolayer bearing terminal disulfide groups. The

∗

Corresponding author. Tel.: +39 0250314361; fax: +39 025031440. E-mail address: [email protected] (F. Porta).

0921-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2007.03.019

resulting colloids were found extremely stable and ready for biological applications [19]. Chen’s group proposed a two-step approach to attach neutral and positively charged thiols onto the citrate-capped gold particles. They first displaced the citrate and chloride on the particles by thioctic acid (TA), and then exchanged TA by thiols with desired functionality. This method successfully converts negatively charged citrate-capped gold nanoparticles into oppositely charged ones [20]. The procedure developed by Brust and et al. [21] produced monolayer-protected gold nanoparticles of reduced dispersity and controlled size; the resulting organosols exhibit great stability in organic solutions and as dry powders can be redispersed in organic solvents. Subsequently, the Brust route was explored for synthesis of a wide range of monolayer-protected gold clusters (MPCs). The MPCs’ capping layers containing modified thiol ligands can be straight-chain alkanethiolates of different length, glutathione [23], tiopronin [24,25], thiolated poly(ethylene glycol) (PEG) [26–28], p-mercaptophenol [29] and other systems [22]. Although alkanethiol-capped gold nanoparticles are insoluble, many of those modified MPCs are water-soluble [25,24,27,28], thus more compatible with biological applications. Capping agents other than thiolated ligands have also been broadly studied. For example, Heath et al. extended Brust’s protocol to produce amine-capped nanocrystals of 2.5–7.0 nm diameter [30]. Murray and co-workers have developed efficient strategies to functionalize MPCs by “place-exchange” reactions, in which MPCs with alkanethiolate monolayers can

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be replaced by other thiol-ended groups [31,32,22]. Moreover, important studies on peptide-stabilised nanoparticles were succesfully carried out by Rotello et al., that synthetized mixed monolayer-protected gold clusters (MMPCs) [33]. The target of the present research was the preparation of gold nanoparticles with potential biological applications. Thus we prepared nanoparticles stabilized by peptides (GK15 and GC15) bearing several polar functional groups such as NH2 and SH, which could assure a good solubility in water.

to give 610 mg (90% yield) of the title compound; (+) ESI-MS m/z, found 179; calcd. for C5 H10 N2 O3 S 178. 2.3. Synthesis of GK15 and GC15

Sodium tetrachloroaurate(III)dihydrate (99%), sodium borohydride (98.5%) were purchased from Sigma–Aldrich, trisodium citrate dihydrate (99.5%) from Fluka. All reagents for the syntheses of peptides were of commercial quality (Sigma–Aldrich) or purified prior to use by standard methods.

Peptide synthesis was carried out by standard Fmoc solidphase protocols with HBTU/HOBt/DIPEA as coupling reagents on a Fmoc-Gly-Wang resin. The functional groups of amino acid side chain were protected as follows: Lys(Boc) and Cys(Trt). Deprotection and cleavage of peptides from the resin were performed with TFA/water (95:5) and TFA/EDT (95:5) mixtures for the lysine- and cysteine-containing peptides, respectively. After purification by semipreparative RP-HPLC, peptides were shown to be >95% homogeneous by analytical RP-HPLC. Their identity and molecular weights were confirmed by mass spectrometry: peptide GK15, (−) ESI-MS, m/z, found 1227.8; calcd. for C50 H92 N20 O16 1228.7; peptide GC15, (+) ESI-MS m/z, found 1104.4; calcd. for C35 H57 N15 O16 S5 1103.3.

2.2. Syntheses of GK and GC

2.4. Citrate-coated gold sol

2.2.1. Abbreviations HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluoro-phosphate; HOBt, N-hydroxybenzotriazole; DIPEA, N,N -diisopropylethylamine; Fmoc, 9-fluorenyl methyloxycarbonyl; Trt, trityl; DMF, dimethylformamide; Boc, tert-butoxycarbonyl; Z, benzyloxy carbonyl; TFA, trifluoroacetic acid; EDT, 1,2-ethanedithiol; TEA, triethylamine; EtOAc, ethyl acetate.

Gold nanoparticles (14.5 nm) were prepared by citrate reduction of HAuCl4 . An aqueous solution of HAuCl4 (200 mL, 2.43 × 10−4 M) was refluxed for approximately 20 min, until the temperature of the initial solution was 63 ◦ C, and aqueous solution of sodium citrate (0.99 mL, 0.295 M) was added quickly. Reflux was continued for another 3 h until a ruby red solution was observed. The solution was filtered through 0.45 ␮m Millipore syringe filters to remove any precipitate.

2. Experimental 2.1. Materials

2.2.2. Gly-Lys (GK) To a cooled solution (0 ◦ C) of benzyloxycarbonylglycine N-hydroxysuccinimide (1.5 g, 5 mmol) in DMF (30 mL), was added TEA (0.7 mL) followed by H-Lys(Z)OH (1.4 g, 5 mmol) and the reaction mixture was stirred at r.t. overnight. The solvent was reduced to approximately a half-volume under reduced pressure, 0.1N HCl (200 mL) was added and the solution extracted with EtOAc (2 × 150 mL). Combined extracts were dried (Na2 SO4 ) and evaporated to give a residue (1.6 g, 75% yield), pure by TLC; which was dissolved in EtOH (100 mL) and stirred under hydrogen (1 atm) in the presence of 5% Pd/C (100 mg) at r.t. for 24 h (TLC control). Filtration of the catalyst through Celite and removal of the solvent gave the desired product as an amorphous solid (640 mg, 90% yield); (+) ESI-MS m/z, found 204; calcd. for C8 H17 N3 O3 203. 2.2.3. Gly-Cys (GC) The same experimental conditions described above were applied for the coupling of t-butyloxycarbonylglycine N-hydroxysuccinimide (1.4 g, 5 mmol) with H-Cys(Trt)-OH (1.8 g, 5 mmol) to afford, after recrystallization from EtOAc/petroleum ether, 1.9 g (78% yield) of protected dipeptide. Cleavage of the protective groups was carried out by treatment with TFA/EDT (95:5) (30 mL) for 2 h at r.t. Removal of the solvent gave a residue which was taken up with diethyl ether, cooled (0 ◦ C) and filtered

2.5. Preparation of Au–GC and Au–GK sols 2.5.1. Au–GC An aqueous solution of NaAuCl4 (0.4 mL, 2.16 × 10−2 M) was added to 17 mL of mQ water. Under vigorous stirring 0.065 mL of a 2.6% (w/w) aqueous solution of the dipeptide GC was added (Au:GC = 1:0.1, w/w ratio). After 15 min under stirring, NaBH4 was added (0.173 mL, 0.1 M) and bordeaux red sol was immediately formed. The sol was let under stirring for 60 min and then ultracentrifuged for recovering a pellet of peptide-capped gold nanoparticles. The pellet was redispersed in mQ water and the sol ulftracentrifuged for removing the uncoordinated peptide. Then the pellet was redispersed in mQ water and the sol lyophilised. It was stored as lyophilised powder. 2.5.2. Au–GK An aqueous solution of NaAuCl4 (0.3 mL, 4.16 × 10−2 M) was added to 55 mL of mQ water. Under vigorous stirring, 5 mL of a 4.8% (w/w) aqueous solution of dipeptide GK was added (Au:GK = 1:10, w/w ratio). After 15 minutes under stirring, NaBH4 was added (0.173 mL, 0.1 M) and a cherry red sol was immediately formed. The sol was let under stirring for 60 min and purified as in Section 2.5.1.

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2.6. Preparation of Au–GC15 and Au–GK15 sols 2.6.1. Au–GC15 An aqueous solution of NaAuCl4 (0.203 mL, 6.15 × 10−2 M) was added to 25 mL of mQ water. Under vigorous stirring, 0.794 mL of a 1.55% (w/w) aqueous solution of GC15 was added (Au:GC15 = 1:0.5, w/w ratio). After 15 min under stirring NaBH4 was added (0.25 mL, 0.1 M) and a cherry red sol was immediately formed. The sol was let under stirring for 60 min and purified as in Section 2.5.1. 2.6.2. Au–GK15 An aqueous solution of NaAuCl4 (1.319 mL, 8.87 × 10−3 M) was added to 50 mL of mQ water. Under vigorous stirring 5 mL of a 3.53% (w/w) aqueous solution of GK15 was added (Au:GK15 = 1:10, w/w ratio). After 15 min under stirring NaBH4 was added (0.234 mL, 0.1 M) and a dark violet sol was immediately formed. The sol was let under stirring for 60 min and purified as in Section 2.5.1. 2.7. Preparation of Au-citrate–GC15 sol Under vigorous stirring, suitable amounts of the lyophilised GC15 peptide (calculated for having Au/GC15 weight/weight ratios equal to 1:1, 1:5, 1:25, 1:50 and 1:125) were added to 100 mL of a Au-citrate sol (prepared as in Section 2.4). The mixture was let under stirring for 60 min. In all the reactions the colour of the sol changed from ruby red to bordeaux. The sol was purified as in Section 2.5.1. 2.8. Synthesis and characterization techniques 2.8.1. Syntheses of peptides An Applied Biosystems mod 433A synthesizer was used for solid-phase synthesis. Analytical and preparative HPLC was

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performed on Pharmacia AKTA Basic 100 instrument using a Purospher 5 ␮m C18 300A (250 mm × 4 mm) and a preparative Jupiter 15 ␮m C18 300A (250 mm × 21.2 mm). Both analytical and preparative runs used gradient elution from 5% to 10% B in 5 min, to 50% B in 35 min then to 100% B in 40 min at 0.5 mL/min (analytical) and 20 mL/min (preparative), where A was 0.1% TFA in water and B was MeCN/0.1% TFA (8:2). Analytical TLC was performed on silica gel 60 F254 aluminium sheet (Merck) using acetic acid/toluene (1:9) as eluent. Mono- and bidimensional NMR spectra were recorded in H2 O:D2 O = 9:1 volume ratio on a Bruker AVANCE 400 spectrometer. Chemical shifts (δ ppm) were referenced to 3(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (δ Me = 0). Electrospray ionization mass spectra (ESI-MS) were acquired on ThermoFinnigan LCQ Advantage spectrometer. 2.8.2. Characterization of nanoparticles The morphology and size of Au–GC, Au–GK, Au–GC15 and Au–GK15 nanoparticles were investigated by a transmission electron microscopy (EF TEM LEO 912AB) operating at 120 kV potential. Samples for TEM analysis were prepared by placing a drop of peptide sol on carbon-coated copper grid. All the UV–vis spectra were registered by using JASCO V-530 spectrometer employing 1 cm quartz cuvette and diluting the original solution with mQ water (1:10). The powders of lyophilized sols were studied by ATR-FTIR spectrometry. Infrared spectra were recorded with 4 cm−1 resolution and 20 scans using an ATR platform (GOLDEN GATE, Specac) by pressing finely grounded solid sample on the diamond crystal. The ATR was mounted in Biorad FTS-40 spectrophotometer equipped with a KBr beamsplitter and a DTGS detector operating between 400 and 4000 cm−1 and 20 scans. 1 H NMR spectra of samples obtained by dissolving the proper amount of lyophilised sol in H2 O:D2 O (9:1) were recorded.

Fig. 1. GC, GK, GC15 and GK15 structures.

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3. Results and discussion

3.2. Results

3.1. Design strategy

GK and GC easily stabilized red gold sols that show plasmon resonance peaks at 520 and 515 nm, respectively in the UV–vis spectra, in agreement with crystalline nanoparticles with mean diameters of 7.4 and 7.9 nm, respectively (by TEM). NMR spectra were recorded in H2 O/D2 O (9/1 volume ratio), after dissolution of solid samples obtained by lyophilisation of the original sols. Both dried Au–GC and Au–GK can be stored as powders and were found to be finely re-dispersible in water. This is an important result since few systems can be stored in dry state and then redispersed in water [19]. 1 H NMR spectra highlighted the binding site of peptides. In Au–GC nanoparticles, the binding occurs at the SH group of cysteine. As a matter of fact the CH2 SH signal is strongly downfield shifted, changing multiplicity as well, from a doublet at 3.0 ppm to doublet of doublet at 3.358 ppm [δ = 0.358 ppm; J = 14.8 and 2.8 Hz] passing from GC to Au–GC. Furthermore the signal of amide NH proton (d, 8.652 ppm) shifts upfield to 8.438 ppm [δ = 0.214 ppm], and broadens significantly with respect to the signal of the free peptide, leading to the conclusion that the formation of an Au–GC bond implies the breaking of an hydrogen bond in which the NH group was involved [39a,b]. In Au–GK nanoparticle, the amine group of glycine is the responsible of binding. In fact, 1 H NMR spectrum shows a significant downfield shift (from 3.52 to 3.67 ppm, δ = 0.15 ppm) and broadening of the signal due to CH2 of glycine (Fig. 2), while no shift of the εCH2 group of lysine was observed. The violet Au–GK15 sol was obtained by reduction of gold salt with sodium borohydride in the presence of GK15 as capping agent (Au/GK15 = 1/10, w/w). The reduction was followed by UV–vis spectroscopy observing the decreasing of the AuCl4 − band at 216 nm (until its complete disappearing which indicates the total reduction of gold) and the increasing of the plasmon resonance peak of Au(0) at 528 nm (Fig. 3). Fig. 3 shows also the optical absorption spectra of colloidal gold in 1 h of standing and after purification highlighting the stability of the sol

Two peptides, both composed of 15 amino acidic residues, were prepared using standard Fmoc solid-phase synthesis and purified by preparative HPLC. The amino acid sequences of two peptides are reported in Fig. 1. The peptide GC15 [H2 N–GC(GGC)4 –G–COOH] is composed of 10 glycine and 5 cysteine residues, with a GGC repetitive unit, while GK15 [H2 N–GK(GGK)4 –G–COOH] consists of the same number of glycines and five lysines (GGK is the repetitive unit). GC15 and GK15 were characterized by MS spectrometry, NMR and ATR-FTIR spectroscopies. The sequences of both GC15 and GK15 were planned to allow the peptide, composed by a periodic motif, bind gold nanoparticles along the length of the peptide, as observed for leucine and lysine containing peptide bound to carboxylate-terminated thiolcapped gold nanoparticles [34]. Moreover GC15 bears many potential anchor groups (SH or NH2 ) that can covalently bind gold particle, although it was already stated the superiority of thiol in the covalent bond with gold [35]. GK15 peptide contains only primary amines that potentially can bind in different ways depending on pH of the sol and pI of the peptide [36]. A covalent interaction between GK15 and gold should involve neutral amino groups and Au surface. In addition, an electrostatic binding of GK15 can be assumed, if the NH2 groups should be protonated, taking into account its similarity with poly-lysine [37]. GC15 and GK15 were used as capping agents in two procedures: (a) reduction of aqueous solution of NaAuCl4 by NaBH4 ; (b) addition of peptides to citrate-capped gold sol. Typically 10−4 M solution of AuCl4 − and a Au/reductant weight/weight ratio equal to 1/0.5 (for Au–GC15 sol) and 1/10 (for Au–GK15) were employed. Method (b) was applied only to GC15, varying the w/w Au/GC15 ratio (from 1/1 to 1/125, see Section 2). The most significant results were obtained with 1:1 Au–peptide ratio. With the aim of limiting the presence of zwitterionic forms of peptides, but maintaining amine groups as NH3 + moieties, which favours electrostatic bindings to gold surface [38], all the reactions were carried out in the 2.75–3.00 pH range, adjusting the acidity by adding small amounts of HAuCl4 0.1 M solution. On the contrary, a basic environment was proved to favour self-assembly of gold nanoparticles giving 1D and 2D arrangements, as in the case of Au–lysine sols [36d]. We also prepared two dipeptides, GC and GK, composed of glycine and cysteine and glycine and lysine, respectively and used them as capping ligands in the reduction of NaAuCl4 (Au:GC = 1:0.1 and Au:GK = 1:10). Indeed, we found considerable differences in the chemical behaviour of dipeptides and 15mer-peptides related to the different chain lengths (15 versus 2 aminoacidic residues). In particular we found different binding sites of GK and GK15 to gold surface, i.e. the glycine-NH2 and the side chain lysine-NH2 groups, respectively.

Fig. 2. 1 H NMR spectra in 1–5 ppm region of GK and Au–GK in H2 O/D2 O (9:1).

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Fig. 3. UV–vis spectra recorded during the reduction of NaAuCl4 and formation of plasmon band of Au–GK15 sol. In the inset, plasmon resonance peaks after 2, 30, and 60 min and after purification.

with respect to time. The surface plasmon bands exhibit a light red shift after purification. TEM analyses revealed nanoparticle sizes with mean diameters in the 7–9 nm interval and crystalline phase (Fig. 4). ATR-FTIR spectra of the lyophilised sol are presented in Fig. 5 a and b. Pure GK15 peptide (Fig. 5a, spectrum A) shows two bands in the amide A region respectively located at 3294 and 3081 cm−1 . In the same region IR spectra of Au–GK15 (Fig. 5a, spectrum B) show, besides the above-mentioned bands, a new band located at 3205 cm−1 grew up. The band is similar to the one found in Au–GC15 spectra (vide infra). In amide I and II region the differences are smaller since pure peptide (Fig. 5b, spectrum A) shows a band located at 1647 and a broad band centred at 1542 cm−1 . In Au–GK15 sample (Fig. 5b, spectrum B) only two broad bands are present, at 1647 and 1526 cm−1 . Deconvolution of amide I band reveals at least 3 components:

Fig. 4. TEM micrograph and electronic diffraction pattern of Au–GK15 nanoparticles. Bar chart 100 nm.

Fig. 5. ATR-FTIR spectra of GK15 (A) and Au–GK15 (B) in 3500–2500 cm−1 region (a) and 1800–1350 cm−1 region (b).

1739 (weak), 1675 (medium) and 1643 (strong). Summarizing, we can affirm that the shifts observed in amide A and amide I and II regions are clearly indicative of GK15-capped gold nanoparticles. The band close to 3200 cm−1 found in Au–GK15 (and in Au–GC15, see later) falls between amide A and B typical regions. It could be accounted for a very strong H-bond

Fig. 6. 1 H NMR spectra (1–4.6 ppm region) of Au–GK15 and GK15 in H2 O/D2 O (9:1).

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interaction involving amide groups due to intra-monolayer interaction arising in the formation of MPCs [39] and/or the presence of NH3 + cation(s). The 1 H NMR spectra of the re-dispersed lyophilized sols (Figs. 6 and 7) did not show peculiar differences in the chemical shifts of protons passing from of GK15 to Au–GK15. A broadening of the signal of ␧-CH2 group (3.0 ppm) of the side chain of lysine strongly suggests the interaction of amine group of lysine(s) with gold surface. Moreover a signal due to lysine and glycine NH2 groups, which is not observable in the 1 H NMR spectrum of GK15, is present as a broad singlet at 7.52 ppm in Au–GK15, indicative of NH3 + moieties. Pure GK15 exhibits ␣-protons of glycine as a broad signal at 3.99 ppm plus a doublet of doublet at 3.78 ppm (AB system) correlated to the highest NH signal at 8.09 (t, J = 6.2 Hz). When the peptide is capped to gold, these glycine signals remain practically unchanged (CH2 3.80 ppm, NH2 8.168 ppm, δ = 0.079). However, it is interesting to note a large downfield shift of an amide NH signal of a lysine from 8.25 to 8.70 ppm (δ = 0.45 ppm), indicative of the presence of hydrogen bonds. The combination of IR and NMR information strongly support the presence of electrostatic bindings between the protonated NH2 group(s) of lysine and gold surface. It is known that borohydride-reduced gold nanoparticles are negatively charged for the presence on the surface of bound AuCl4 − and AuCl2 − anions [40,9,38b]. In this hypothesis GK15 could lay parallel to gold surface with some of the NH3 + groups sticking out in the solvent H2 O, assuring its solubility and re-dispersibility. The hydrogen bonds could play also a role in gold Au–GK15 nanoparticle stabilization, involving amide NH group of the backbone (downfield shift of NH from 8.25 to 8.70 ppm) [39]. A cherry red monodispersed sol (Au–GC15) was obtained by reduction of Au(III) salt in the presence of GC15 (Au/GC15 = 1/0.5, w/w). UV–vis spectra shows the plasmon peak at 514 nm related to mean diameters of particles (by TEM) variable in 7.0–7.5 nm interval. IR spectra of lyophilised Au–GC15 display some differences respect to pure GC15. Pure peptide shows 3291 cm−1 band in amide A region typical of strongly H–bonded NH groups, upon interaction with gold par-

Fig. 7. 1 H NMR spectra (7–9 ppm region) of Au–GK15 and GK15 in H2 O/D2 O (9:1).

Fig. 8. ATR–FTIR spectra of GC15 (red line) and Au–GC15 (black line) in 3700–2500 cm−1 region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

ticles a new band arises at 3217 cm−1 (Fig. 8). This band falls outside typical amide A region (3450–3270 cm−1 ) and could be accounted for a NH stretching of a very strongly H-bonded amide group. There is also a variation of intensity for 1664 and 1627 cm−1 amide I band components, being the latter less intense in Au–GC15. Unlikely, the lyophilized Au–GC15 sol was not water-dispersible, thus NMR spectra is lacking. However further studies in solvents like DMSO-d6 and CD2 Cl2 are in progress. A possible reason of the low re-dispersibility of Au–GC15 in water can be the aggregation of the particles, caused by multiple interactions of the peptide with different gold particles during the lyophilisation step. However stability and re-dispersibility of both Au–GC15 and Au–GK15 were qualitatively investigated only by spectroscopic evidences (UV–vis and NMR spectra). Thus we decided to investigate also another preparative procedure to better investigate the interaction between gold and GC15. By adding suitable amounts of the lyophilised GC15 to a citrate-capped gold sol (Au/GC15 = 1:1, w/w) we obtained a bordeaux Au-citrate–GC15 colloid, which presents a characteristic plasmon peak at 524 nm in the UV–vis spectrum. TEM images of the constituting MPCs shown spherical nanoparticles similar to those of citrate-capped particles, varying the mean diameter in 15–17 nm range (Fig. 9). In addition, the formation of nanowire and pearl-necklace structures was observed highlighting mono and bidimensional aggregation [36d]. ATR spectra revealed the presence of both citrate and GC15 on the gold surface. Trisodium citrate shows a sharp strong peak located at 1582 cm−1 ascribable to C O stretching of carboxylate anions that upon interaction with gold particles undergoes to a red shift down to 1557 cm−1 . Upon addition of increasing amounts of GC15 to gold citrate sol, IR spectra show the development of features typical of CG15 and the bands located at 1644 and 1514 cm−1 increased. The exhibited bands of Au-citrate–GC15 are very similar to pure GC15 ones and are indicative of a weak interaction of GC15 with gold surface. 1 H NMR spectra confirmed the presence of both cit-

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state and only Au–GK15 was studied in aqueous solution by NMR spectroscopy, being the only one water-re-dispersible. The results suggest electrostatic multiple interactions of protonated NH2 groups of GK15 with anions present on negative gold surface, observing breaking and formation of H-bondings. The reaction of citrate-capped gold sol with GC15 produced the red sol Au-citrate–GC15. NMR and IR studies confirmed the presence of both capping agents in bioconjugate but did not indicate a definite interaction of GC15 and gold. Certainly we consider the present results as a preliminary investigation and the matter worthy of an in-depth further study. Moreover a further study will be dedicated to the admission of these peptide monolayer-protected gold clusters into cancer cells, as in the case of Au-aminovaleric acid nanoparticles [41]. Acknowledgments

Fig. 9. TEM micrograph of Au-citrate–GC15 nanoparticles. Bar chart 50 nm.

rate group (AB system at 2.63 ppm) and GC15 capped on gold, even if Au-citrate–GC15 lyophilised sol was not easily waterredispersible. The partial displacement of citrate anions is well known in the preparation of mixed-ligand-capped nanoparticle [20,33]. We believe that in our system only small amounts of citrate have been exchanged by GC15 peptide, as we experienced that w/w Au/peptide ratios larger than 1/5 produced a remarkable aggregation of the particles. Moreover NMR spectra revealed a peculiar aspect of Au-citrate–CG15: light differences in the chemical shifts of methylene protons of CH2 SH group prior and after interaction with gold (from 2.94 to 2.99 ppm after coordination). This confirms the feeble interaction (or at least not clear in this experimental condition) between SH group and gold surface in agreement with IR data. Further studies and NMR experiments in various solvents are in progress to clarify this particular aspect. However changes of glycine signals were observed passing from pure GC15 to Au-citrate–GC15, indicative of an additional interaction of the gold surface with the terminal NH2 group of glycine. 4. Conclusions Two red sols, Au–GK and Au–GC, were obtained by direct reduction of NaAuCl4 by NaBH4 in the presence of the investigated dipeptides. The sols were lyophilized and stored as dry powders without observable aggregation. The complete water redispersibility, without changing of their characteristic (checked by UV–vis spectroscopy) allowed us to record NMR spectra that indicated the binding sites of GK and GC dipeptides to gold surface. They are: SH cysteine group in Au–GC and NH2 terminal glycine group in Au–GK. Two violet/red Au–GK15 and Au–GC15 sols were obtained by direct reduction of auric salt by sodium borohydride. Both colloids were studied by ATR-FTIR spectroscopy in the solid

We gratefully acknowledge the excellent assistance of Dr. Teresa Recca for NMR analyses and Dr. Nadia Santo for TEM micrographs. References [1] S. Schultz, Proc. Natl. Acad. Sci. USA 97 (2000) 996–1001. [2] A.G. Tkachenko, H. Xie, D. Coleman, W. Glomm, J. Ryan, M.F. Anderson, S. Franzen, D.L. Feldheim, J. Am. Chem. Soc. 125 (2003) 4700–4701. [3] Y.W. Cao, R. Jin, C.A. Mirkin, J. Am. Chem. Soc. 123 (2001) 7961–7962. [4] C.M. Feldherr, R.E. Lanford, D. Akin, Proc. Natl. Acad. Sci. USA 89 (1992) 11002–11005. [5] D.A. Handley, in: M.A. Hayat (Ed.), Colloidal Gold: Principles, Methods and Applications, Academic Press, San Diego, 1989, pp. 13–32. [6] D.L. Feldheim, C.A. Foss, Metal Nanoparticles: Synthesis, Characterization, and Application, Marcel Dekker, New York, 2002. [7] M.C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293–346. [8] M.A. Hayat (Ed.), Colloidal Gold: Principles, Methods, and Applications, Academic Press, San Diego, 1989. [9] C.D. Keating, J. Chem. Ed. 76 (1999) 949–955. [10] A.P. Alivisatos, K.P. Johnsson, X. Peng, T.E. Wilson, C.J. Loweth, M.P. Bruchez Jr., P.G. Schultz, Nature (London) 382 (1996) 609–611. [11] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, Nature 382 (1996) 607–609. [12] C.J. Loweth, W.B. Caldwell, X. Peng, A.P. Alivisatos, P.G. Schultz, Angew. Chem. Int. Ed. 38 (1999) 1808–1812. [13] C.M. Niemeyer, Angew. Chem. Int. Ed. 40 (2001) 4128–4158. [14] J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. (1951) 55–56. [15] G. Frens, Nat.- Phys. Sci. 241 (1973) 20–22. [16] W.J. Parak, T. Pellegrino, C.M. Micheel, D. Gerion, S.C. Williams, A.P. Alivisatos, Nano Lett. 3 (2003) 33–36. [17] D. Zanchet, C.M. Micheel, W.J. Parak, D. Gerion, S.C. Williams, A.P. Alivisatos, J. Phys. Chem. B 106 (2002) 11758–11763. [18] D. Zanchet, C.M. Micheel, W.J. Parak, D. Gerion, A.P. Alivisatos, Nano Lett. 1 (2001) 32–35. [19] C. Mangeney, F. Ferrage, I. Aujard, V. Marchi-Artzner, L. Jullien, O. Ouari, E.-D. Rekai, A. Laschewsky, I. Vikholm, J.W. Sadowski, J. Am. Chem. Soc. 124 (2002) 5811–5821. [20] S.-Y. Lin, Y.-T. Tsai, C.-C. Chen, C.-M. Lin, C.-H. Chen, J. Phys. Chem. B 108 (2004) 2134–2139. [21] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, J. Chem. Soc. Chem. Commun. (1994) 801–802. [22] A.C. Templeton, M.P. Wuelfing, R.W. Murray, Acc. Chem. Res. 33 (2000) 27–36. [23] T.G. Schaaff, G. Knight, M.N. Shafigullin, R.F. Borkman, R.L. Whetten, J. Phys. Chem. B 102 (1998) 10643–10646.

194

F. Porta et al. / Materials Science and Engineering B 140 (2007) 187–194

[24] A.C. Templeton, S. Chen, S.M. Gross, R.W. Murray, Langmuir 15 (1999) 66–76. [25] O. Kohlmann, W.E. Steinmetz, X.-A. Mao, W.P. Wuelfing, A.C. Templeton, R.W. Murray, C.S. Johnson Jr., J. Phys. Chem. B 105 (2001) 8801–8809. [26] W.P. Wuelfing, S.M. Gross, D.T. Miles, R.W. Murray, J. Am. Chem. Soc. 120 (1998) 12696–12697. [27] A.G. Kanaras, F.S. Kamounah, K. Schaumburg, C.J. Kiely, M. Brust, Chem. Commun. (2002) 2294–2295. [28] M. Bartz, J. Kuther, G. Nelles, N. Weber, R. Seshadri, W. Tremel, J. Mater. Chem. 9 (1999) 1121–1125. [29] M. Brust, J. Fink, D. Bethell, D.J. Schiffrin, C. Kiely, J. Chem. Soc. Chem. Commun. 16 (1995) 1655–1656. [30] D.V. Leff, L. Brandt, J.R. Heath, Langmuir 12 (1996) 4723–4730. [31] A.C. Templeton, M.J. Hostetler, C.T. Kraft, R.W. Murray, J. Am. Chem. Soc. 120 (1998) 1906–1911. [32] M.J. Hostetler, A.C. Templeton, R.W. Murray, Langmuir 15 (1999) 3782–3789. [33] C.M. McIntosh, E.A. Esposito III, A.K. Boal, J.M. Simard, C.T. Martin, V.M. Rotello, J. Am. Chem. Soc. 124 (2002) 5796–5810. [34] P.V. Bower, E.A. Louie, J.R. Long, P.S. Stayton, G.P. Drobny, Langmuir 21 (2005) 3002–3007. [35] (a) R. Levy, N.T.K. Thanh, R.C. Doty, I. Hussain, R.J. Nichols, D.J. Schiffrin, M. Brust, D.G. Fernig, J. Am. Chem. Soc. 126 (2004) 10076–10084;

[36]

[37] [38]

[39]

[40] [41]

(b) J. Zhang, Q. Chi, J.U. Nielsen, E.P. Friis, J.E.T. Andersen, J. Ulstrup, Langmuir 16 (2000) 729–7237. (a) Z. Zhong, A.S. Subramanian, J. Highfield, K. Carpenter, A. Gedanken, Chem. Eur. J. 11 (2005) 1473–1478; (b) Z. Zhong, S. Patskovskyy, P. Bouvrette, J.H.T. Luong, A. Gedanken, J. Phys. Chem. B 108 (2004) 4046–4052; (c) M. Larsson, J. Lindgren, J. Raman Spectrosc. 36 (2005) 394– 399; (d) Z. Zhong, J. Luo, T.P. Ang, J. Highfield, J. Lin, A. Gedanken, J. Phys. Chem. B 108 (2004) 18119–18123. C.E. Jordan, B.L. Frey, Kornguth, R.C. Corn, Langmuir 10 (1994) 3642–3648. (a) M.G. Bellino, E.J. Calvo, G. Gordillo, Phys. Chem. Chem. Phys. 6 (2004) 424–428; (b) H. Joshi, P.S. Shirude, V. Bansal, K.N. Ganesh, M. Sastry, J. Phys. Chem. B 108 (2004) 11535–11540. (a) A.K. Boal, V.M. Rotello, Langmuir 16 (2000) 9527–9532; (b) L. Fabris, S. Antonello, L. Armelao, R.L. Donkers, F. Polo, C. Toniolo, F. Maran, J. Am. Chem. Soc. 128 (2006) 326–336. A. Kumar, S. Mandal, P.R. Selvakannan, R. Parischa, L.E. Mandale, M. Sastry, Langmuir 19 (2003) 6277–6282. Z. Krpeti´c, F. Porta, G. Scar`ı, Gold Bull. (London, United Kingdom) 39 (2006) 66–68.