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Preparation of helical peptide monolayer-coated gold nanoparticles
Journal of Colloid and Interface Science 288 (2005) 83–87 www.elsevier.com/locate/jcis
Preparation of helical peptide monolayer-coated gold nanoparticles Nobuyuki Higashi ∗ , Jun Kawahara, Masazo Niwa Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyo-tanabe, Kyoto 610-0321, Japan Received 6 January 2005; accepted 26 February 2005 Available online 9 April 2005
Abstract We describe herein the preparation of polypeptide (poly(γ -benzyl-L-glutamate)) monolayer-covered gold nanoparticles (PBLG(n)SS–Au). Two types of PBLG(n)SS having PBLG segment length n = 20 and 50 were synthesized and successfully attached to the gold nanoparticle surface using the Brust–Schiffern method. The mean sizes of PBLG(n)SS–Au particles and their gold cluster cores in CHCl3 , which were evaluated by means of dynamic light scattering and TEM, respectively, demonstrated that the gold cluster surfaces were covered with PBLG monolayers, and their conformation was found to be mainly in α-helix on the basis of FT-IR spectroscopy. 2005 Elsevier Inc. All rights reserved. Keywords: Gold nanoparticle; Peptide; α-Helix; Monolayer; Gold plasmon
1. Introduction The design, synthesis, and characterization of surfacefunctionalized nanoparticles are of fundamental importance in controlling the mesoscopic properties of new materials and in developing new tools for nanofabrication [1]. Metal colloids functionalized with self-assembled monolayers (SAMs) are inherently nanoscopic entities that provide a building block for microscale constructs. The fabrication of SAM-functionalized gold nanoparticles has been greatly facilitated by the methods developed by Brust et al. [2]. In their approach, chemical reduction of the corresponding metal salt is performed in the presence of capping ligands such as sulfur-containing molecules. In spite of such diversity and versatility of SAM-functionalized metal colloids, ligands are limited to small molecules and the use of macromolecules such as polypeptides is limited [3–5], probably because of difficulties in their synthesis and colloid preparation, including the issues of colloidal stability of nanoscale gold particles. In recent studies, we have reported the controllable orientation of helical peptide rods bound as a SAM at the end of a long-chain alkyl spacer bearing a terminal S–S bond as * Corresponding author. Fax: +81 774 65 6844.
E-mail address: [email protected] (N. Higashi). 0021-9797/$ – see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2005.02.086
an anchor on a planar gold film [6]. In particular, we accomplished efficient immobilization of some functional groups at an appropriate location within such helical SAMs due to specific interhelix interaction [7,8]. Dendrimers have also attracted increasing interest as templates and cores for designing nanoparticles. We have prepared peptide dendrimers, in which polypeptide segments are linked covalently with the peripheries of a polyamidoamine dendrimer [9–11]. Peptide segments of the dendrimers exhibited unique properties in solutions such as enhancement in helicity [9] and enantiomeric binding of amino acids [10] due to assembled structure of helices, different from that in bulk. We report herein the first preparation of polypeptide (poly(γ -benzyl-L-glutamate))-covered gold nanoparticles (PBLG(n)SS–Au). Two types of PBLG(n)SS having an S–S bond as a gold-adsorbable moiety and PBLG segment with length n = 20 or 50 (Fig. 1) have been synthesized, in the same manner as described previously [7]. With the use of the Brust–Schiffern method [2], gold nanoparticles (PBLG(n)SS–Au) were prepared by reduction of metal salts (HAuCl4 ) with NaBH4 in the presence of PBLG(n)SS. In this study, we will describe the characterization of these peptide–gold nanoparticles in view of peptide secondary structures and their dispersion states in solutions and in cast films.
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N. Higashi et al. / Journal of Colloid and Interface Science 288 (2005) 83–87
Fig. 1. Molecular structure of gold-adsorbable peptides.
2. Experimental 2.1. Materials Solvents and other chemical reagents were purchased from Wako Pure Chemical Industries Ltd. or Nacalai Tesque Inc. and used without further purification. Aqueous solutions were made with water purified with a Milli-Q purification system (Millipore Co.). PBLG(n)SSs were prepared in the same manner reported previously [7]. The mean degree of polymerization (n) and the molecular weight distribution (Mw /Mn ) for the resulting polypeptides were characterized as n = 20 and 50, and Mw /Mn = 1.06 and 1.02, respectively, by means of size exclusion chromatography (LC5A, Shimadzu Co.), 1 H NMR (JOEL-FX400), and MALDI-TOF mass (Kompact MALDI III, Shimadzu Co.) spectroscopies. 2.2. General procedure for gold nanoparticle preparation According to an established procedure [2], PBLG(50)SS– Au was prepared as follows. A solution of PBLG(50)SS in toluene (80 ml, 1.0 mM) was first prepared; due to the poor solubility of PBLG(50)SS, the mixture was warmed at about 40 ◦ C to completely dissolve it and kept overnight. An aqueous solution of HAuCl4 ·3H2 O (30 ml, 30 mM) was mixed with a solution of tetraoctylammonium bromide in toluene (80 ml, 50 mM). The two-phase mixture was vigorously stirred and the PBLG(50)SS toluene solution was then added. An aqueous solution of NaBH4 (10 ml, 160 mM) was slowly added with vigorous stirring. After further stirring for 40 h the solvents were evaporated. The residue was washed with diethyl ether several times to give a wine-red powder. PBLG(20)SS was also prepared following the same manner. 2.3. Characterization of peptide–gold nanoparticles The optical properties of the peptide–gold nanoparticles and the cast films were characterized by UV–vis spectroscopy (UV-2450, Shimadzu Co.). FTIR spectra (Nexus 470, Thermo Nicolet Co.) were obtained for CHCl3 solutions of peptide–gold nanoparticles. X-ray photoelectron spectroscopy (XPS) was performed on a Shimadzu ESCA1000 system using a MgKα source. The peak locations were corrected based on the C1s line emitted from neutral hydrocarbon. Dynamic light scattering (DLS) measurements
Fig. 2. Absorption spectra of PBLG(50)SS–Au in CHCl3 (6.0 g L−1 , solid line) and the film cast from the CHCl3 solution (dashed line). The inset shows the plot of absorbance at 520 nm as a function of concentration.
were performed in CHCl3 on a DLS 7000 spectrometer (Otsuka Electric Ltd.) equipped with a He–Ne laser (632.8 nm). Samples were filtered through a Fluoropore MillexR-FH filter (with pore size 0.45 µm; Millipore Ltd.) to remove dust particles in solutions prior to measurements. The morphology of gold nanoparticles was observed under a JEOL JEM-2010 transmission electron microscope (TEM) operating at 200 kV. Samples were prepared by slow evaporation of one drop of a CHCl3 solution of nanoparticle on a carboncoated copper mesh grid. The AFM images were collected on a Nanoscope IIIa (Digital Instrument Inc.) operated in a tapping mode using silicon cantilevers (125 µm, tip radius 10 nm).
3. Results and discussion 3.1. PBLG(n)SS–Au nanoparticles First of all, the oxidation state of gold in the PBLG(n)SS– Au nanoparticles was determined by XPS. The appearance of binding energies of the doublet for Au4f7/2 (84 eV) and Au4f5/2 (88 eV) and the disappearance of a band at 85 eV due to AuI indicate that the gold atoms in the clusters must be present as Au0 [12]. Fig. 2 shows the UV–vis spectrum of the prepared PBLG(50)SS–Au in CHCl3 (6.0 g L−1 , solid line). The spectrum indicates the formation of gold nanoparticles with an absorption band at about 520 nm, assigned to a characteristic gold plasmon band. It is found from the inset of the figure that the absorbance increases linearly with increasing concentration, and the absorption maxima (λmax ) do not change with concentration. This spectral feature suggests that PBLG(50)SS–Au is modestly dispersed in CHCl3 without aggregation between particles since the aggregation of gold nanoparticles has been reported to cause a red
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Fig. 3. Size distribution of PBLG(n)SS–Au (a) n = 20 and (b) n = 50 in CHCl3 , measured by dynamic light scattering.
shift of the peak wavelength (λmax ) and broadening of the spectrum [3,13]. Presumably, the helical PBLGs must exist densely on colloidal gold surface and as a result it becomes difficult to aggregate through interhelix interaction. A similar spectral feature was observed for PBLG(20)SS–Au; the particles were stably present in CHCl3 without any aggregation although the particle size seems to depend on the peptide segment length (n) as described later. The UV–vis spectrum of the cast film prepared from the CHCl3 solution of PBLG(50)SS–Au, a typical sample, was observed to have an absorbance at 520 nm (Fig. 2, dashed line) that is consistent with that in solution, meaning that interaction between particles would not be so important even in the cast film. To elucidate the size of these particles in solutions, we performed dynamic light scattering (DLS). Fig. 3 displays histograms of size distributions for PBLG(20)SS–Au and PBLG(50)SS–Au particles. They exhibit unimodal and narrow distributions. The diameters are evaluated to be about 8 and 16 nm for PBLG(20)SS–Au and PBLG(50)SS–Au, respectively. It needs the core size of gold cluster to estimate the real thickness of peptide layers. TEM observation was thus employed (Fig. 4). The CHCl3 solutions of these PBLG(n)SS–Au were dropcast onto TEM grids. The images revealed the formation of gold nanospheres, which were homogeneously distributed. The particles were found to be spherical although the mean diameters of gold cores were different between them: about 4 nm for PBLG(20)SS– Au and 10 nm for PBLG(50)SS–Au. It has been reported that manipulation of the preparative reaction conditions such
Fig. 4. TEM images of (a) PBLG(20)SS–Au and (b) PBLG(50)SS–Au cast on copper TEM grids from CHCl3 solutions.
as ligand-to-gold ratio can affect changes in cluster dimensions [14]. The molecular size, including polymer molecular weight, of ligands has also affected the size of gold nanoparticles [4]. The observed discrepancy in core size must be derived from the latter effect of polymer molecular weight since the preparative conditions were the same. From the results of both TEM and DLS observations, one can roughly estimate the mean thickness of the coated PBLG layer to be 4 and 6 nm for PBLG(20)SS–Au and PBLG(50)SS–Au, respectively. By assuming a complete α-helix conformation, the helix length of PBLG can be computed to be 5 and 10 nm for n = 20 and n = 50, respectively, using the occupied length (0.15 nm) of one α-amino acid residue along the helix axis. These values are comparable with those evaluated by TEM and DLS. 3.2. Secondary structures of PBLG segments We have previously demonstrated that the two- or threedimensional assembly of peptides at some interfaces induces secondary structural changes and consequently provides more highly ordered peptide organizations [15]. It is thus important to reveal the secondary structure of PBLG segments on gold clusters. FTIR and 1 H NMR spectroscopy are employed. The spectra of gold-free PBLG(n)SS themselves were also measured for comparison. Fig. 5 displays FTIR
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Table 1 Helix content of PBLG(n)SS and PBLG(n)SS–Au particles in CHCl3 based on 1 H NMR spectroscopy n 20 50
Helix contents (%) PBLG(n)SS (FTIRa )
PBLG(n)SS–Au (FTIRa )
69 (59) 86 (83)
87 (85) 92 (94)
a Estimated by the peak deconvolution method applied to the amide II band region in FTIR spectra.
Fig. 5. FTIR spectra of (a) PBLG(20)SS–Au and (b) PBLG(20)SS in CHCl3 at 20 ◦ C.
ble 1, together with data on PBLG(50)SS and PBLG(50)SS– Au and from FTIR analyses. The immobilization of PBLG segments onto gold cluster is found to bring a marked increment of α-helix content, compared with the gold-free PBLG(n)SS; in particular, in the case of PBLG(20)SS–Au having the shorter PBLG segment, the increment of helicity becomes more drastic. A similar enhancement in helicity has been found for the PBLG-attached dendritic nanoparticles [9]. The observed helix enhancement is probably due to PBLG side chain interactions such as π–π stacking of benzyl groups, resulting from characteristic alignment of PBLG segments on gold clusters, at which they are forced to assemble densely.
4. Conclusions
Fig. 6. 1 H NMR spectra of (a) PBLG(20)SS–Au and (b) PBLG(20)SS in CDCl3 at 20 ◦ C.
spectra of the C=O stretching band region for PBLG(20)SS and PBLG(20)SS–Au nanoparticles in CHCl3 at a concentration of 5.0 g L−1 . The peaks appearing at 1732 and 1655 cm−1 can be assigned to the C=O stretching band of the ester group in the side chain and the amide I band in the main chain, respectively. In the amide II band region, there is a sharp peak at 1547 cm−1 that is based on an α-helix structure and a broad shoulder around 1535 cm−1 that is derived from a random coil conformation [16]. Focusing on the amide II band region, it can be clearly seen that the attachment of PBLG segment onto gold cluster significantly enhances the helix content in comparing PBLG(20)SS and PBLG(20)SS–Au nanoparticles, although a minor existence of random coil conformation may not be excluded. To obtain more quantitative information on the secondary structure, 1 H NMR spectra were measured in CDCl under the same 3 conditions (Fig. 6). The α-CH resonance signal of the PBLG main chain was known to give a peak at 3.95 ppm ascribed to the α-helix conformation, while giving an apparent lowfield shift on going from the helix to the random coil form [17]. From signal areas based on α-helix and random coil forms, the helix contents were evaluated and summarized in Ta-
This study demonstrates that novel polypeptide monolayer-coated gold nanoparticles are successfully prepared by in situ reduction of HAuCl4 in the presence of PBLG(n)SS (n = 20, 50). The particles were present stably in CHCl3 without any aggregation, although their sizes (8–16 nm) were found to depend upon the peptide segment length (n). The PBLG segments attached on gold cluster took α-helix conformation with nearly 100% content, resulting from enhancement in helicity due to assembled structure. It is expected that these helical peptide-coated gold nanoparticles would cause specific aggregation between particles and guest peptide molecules through interhelix interaction if the surface density of helices could be adjusted.
Acknowledgments This material is based upon work supported in part by the project “Hybrid Nanostructured Materials and Its Application” of the High Technology Center at RCAST of Doshisha University. We thank Dr. M. Morikawa at Professor Kimizuka’s lab (Kyusyu University) for TEM observation.
References [1] For a recent review on surface-functionalized nanoparticles, see R. Shenhar, V.M. Rotello, Acc. Chem. Res. 36 (2003) 549–561, and references cited therein.
N. Higashi et al. / Journal of Colloid and Interface Science 288 (2005) 83–87
[2] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, J. Chem. Soc. Chem. Commun. (1994) 801–802. [3] H. Otsuka, Y. Akiyama, Y. Nagasaki, K. Kataoka, J. Am. Chem. Soc. 123 (2001) 8226–8230. [4] R.G. Shimmin, A.B. Schoch, P.V. Braun, Langmuir 20 (2004) 5613– 5620. [5] L. Maya, C. Muralidharan, T.G. Thundat, E.A. Kenik, Langmuir 16 (2000) 9151–9154. [6] M. Niwa, M.-a. Morikawa, N. Higashi, Angew. Chem. Int. Ed. 39 (2000) 960–963. [7] M. Niwa, M.-a. Morikawa, N. Higashi, Langmuir 15 (1999) 5088– 5092. [8] M. Niwa, M.-a. Morikawa, T. Nabeta, N. Higashi, Macromolecules 35 (2002) 2769–2775. [9] N. Higashi, T. Koga, M. Niwa, Chem. Commun. (2000) 361– 362.
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[10] N. Higashi, T. Koga, M. Niwa, ChemBioChem (2002) 448–454. [11] N. Higashi, T. Koga, M. Niwa, J. Nanosci. Nanotechnol. (2001) 309– 315. [12] A. McNeillie, D.H. Brown, W.E. Smith, M. Gibson, L. Watson, J. Chem. Soc. Dalton Trans. (1989) 767–770. [13] N. Nath, A. Chilkoti, J. Am. Chem. Soc. 123 (2001) 8197–8202. [14] M.J. Hostetler, J.E. Wingate, C.-J. Zhong, J.E. Harris, R.W. Vachet, M.R. Clark, J.D. Londono, S.J. Green, J.J. Stokes, G.D. Wignall, G.L. Glish, M.D. Poster, N.D. Evans, R.W. Murray, Langmuir 14 (1998) 17–30. [15] N. Higashi, T. Koga, M.-a. Morikawa, M. Niwa, in: H.S. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 5, Academic Press, San Diego, 2001, pp. 167–205, and references cited therein. [16] T. Miyazawa, E.R. Blout, J. Chem. Phys. 83 (1961) 712–719. [17] E.M. Bradburg, C. Crane-Robinson, H. Goldman, H.W.E. Rattle, Nature 217 (1968) 812–816.
Preparation of helical peptide monolayer-coated gold nanoparticles Nobuyuki Higashi ∗ , Jun Kawahara, Masazo Niwa Department of Molecular Science & Technology, Faculty of Engineering, Doshisha University, Kyo-tanabe, Kyoto 610-0321, Japan Received 6 January 2005; accepted 26 February 2005 Available online 9 April 2005
Abstract We describe herein the preparation of polypeptide (poly(γ -benzyl-L-glutamate)) monolayer-covered gold nanoparticles (PBLG(n)SS–Au). Two types of PBLG(n)SS having PBLG segment length n = 20 and 50 were synthesized and successfully attached to the gold nanoparticle surface using the Brust–Schiffern method. The mean sizes of PBLG(n)SS–Au particles and their gold cluster cores in CHCl3 , which were evaluated by means of dynamic light scattering and TEM, respectively, demonstrated that the gold cluster surfaces were covered with PBLG monolayers, and their conformation was found to be mainly in α-helix on the basis of FT-IR spectroscopy. 2005 Elsevier Inc. All rights reserved. Keywords: Gold nanoparticle; Peptide; α-Helix; Monolayer; Gold plasmon
1. Introduction The design, synthesis, and characterization of surfacefunctionalized nanoparticles are of fundamental importance in controlling the mesoscopic properties of new materials and in developing new tools for nanofabrication [1]. Metal colloids functionalized with self-assembled monolayers (SAMs) are inherently nanoscopic entities that provide a building block for microscale constructs. The fabrication of SAM-functionalized gold nanoparticles has been greatly facilitated by the methods developed by Brust et al. [2]. In their approach, chemical reduction of the corresponding metal salt is performed in the presence of capping ligands such as sulfur-containing molecules. In spite of such diversity and versatility of SAM-functionalized metal colloids, ligands are limited to small molecules and the use of macromolecules such as polypeptides is limited [3–5], probably because of difficulties in their synthesis and colloid preparation, including the issues of colloidal stability of nanoscale gold particles. In recent studies, we have reported the controllable orientation of helical peptide rods bound as a SAM at the end of a long-chain alkyl spacer bearing a terminal S–S bond as * Corresponding author. Fax: +81 774 65 6844.
E-mail address: [email protected] (N. Higashi). 0021-9797/$ – see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2005.02.086
an anchor on a planar gold film [6]. In particular, we accomplished efficient immobilization of some functional groups at an appropriate location within such helical SAMs due to specific interhelix interaction [7,8]. Dendrimers have also attracted increasing interest as templates and cores for designing nanoparticles. We have prepared peptide dendrimers, in which polypeptide segments are linked covalently with the peripheries of a polyamidoamine dendrimer [9–11]. Peptide segments of the dendrimers exhibited unique properties in solutions such as enhancement in helicity [9] and enantiomeric binding of amino acids [10] due to assembled structure of helices, different from that in bulk. We report herein the first preparation of polypeptide (poly(γ -benzyl-L-glutamate))-covered gold nanoparticles (PBLG(n)SS–Au). Two types of PBLG(n)SS having an S–S bond as a gold-adsorbable moiety and PBLG segment with length n = 20 or 50 (Fig. 1) have been synthesized, in the same manner as described previously [7]. With the use of the Brust–Schiffern method [2], gold nanoparticles (PBLG(n)SS–Au) were prepared by reduction of metal salts (HAuCl4 ) with NaBH4 in the presence of PBLG(n)SS. In this study, we will describe the characterization of these peptide–gold nanoparticles in view of peptide secondary structures and their dispersion states in solutions and in cast films.
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N. Higashi et al. / Journal of Colloid and Interface Science 288 (2005) 83–87
Fig. 1. Molecular structure of gold-adsorbable peptides.
2. Experimental 2.1. Materials Solvents and other chemical reagents were purchased from Wako Pure Chemical Industries Ltd. or Nacalai Tesque Inc. and used without further purification. Aqueous solutions were made with water purified with a Milli-Q purification system (Millipore Co.). PBLG(n)SSs were prepared in the same manner reported previously [7]. The mean degree of polymerization (n) and the molecular weight distribution (Mw /Mn ) for the resulting polypeptides were characterized as n = 20 and 50, and Mw /Mn = 1.06 and 1.02, respectively, by means of size exclusion chromatography (LC5A, Shimadzu Co.), 1 H NMR (JOEL-FX400), and MALDI-TOF mass (Kompact MALDI III, Shimadzu Co.) spectroscopies. 2.2. General procedure for gold nanoparticle preparation According to an established procedure [2], PBLG(50)SS– Au was prepared as follows. A solution of PBLG(50)SS in toluene (80 ml, 1.0 mM) was first prepared; due to the poor solubility of PBLG(50)SS, the mixture was warmed at about 40 ◦ C to completely dissolve it and kept overnight. An aqueous solution of HAuCl4 ·3H2 O (30 ml, 30 mM) was mixed with a solution of tetraoctylammonium bromide in toluene (80 ml, 50 mM). The two-phase mixture was vigorously stirred and the PBLG(50)SS toluene solution was then added. An aqueous solution of NaBH4 (10 ml, 160 mM) was slowly added with vigorous stirring. After further stirring for 40 h the solvents were evaporated. The residue was washed with diethyl ether several times to give a wine-red powder. PBLG(20)SS was also prepared following the same manner. 2.3. Characterization of peptide–gold nanoparticles The optical properties of the peptide–gold nanoparticles and the cast films were characterized by UV–vis spectroscopy (UV-2450, Shimadzu Co.). FTIR spectra (Nexus 470, Thermo Nicolet Co.) were obtained for CHCl3 solutions of peptide–gold nanoparticles. X-ray photoelectron spectroscopy (XPS) was performed on a Shimadzu ESCA1000 system using a MgKα source. The peak locations were corrected based on the C1s line emitted from neutral hydrocarbon. Dynamic light scattering (DLS) measurements
Fig. 2. Absorption spectra of PBLG(50)SS–Au in CHCl3 (6.0 g L−1 , solid line) and the film cast from the CHCl3 solution (dashed line). The inset shows the plot of absorbance at 520 nm as a function of concentration.
were performed in CHCl3 on a DLS 7000 spectrometer (Otsuka Electric Ltd.) equipped with a He–Ne laser (632.8 nm). Samples were filtered through a Fluoropore MillexR-FH filter (with pore size 0.45 µm; Millipore Ltd.) to remove dust particles in solutions prior to measurements. The morphology of gold nanoparticles was observed under a JEOL JEM-2010 transmission electron microscope (TEM) operating at 200 kV. Samples were prepared by slow evaporation of one drop of a CHCl3 solution of nanoparticle on a carboncoated copper mesh grid. The AFM images were collected on a Nanoscope IIIa (Digital Instrument Inc.) operated in a tapping mode using silicon cantilevers (125 µm, tip radius 10 nm).
3. Results and discussion 3.1. PBLG(n)SS–Au nanoparticles First of all, the oxidation state of gold in the PBLG(n)SS– Au nanoparticles was determined by XPS. The appearance of binding energies of the doublet for Au4f7/2 (84 eV) and Au4f5/2 (88 eV) and the disappearance of a band at 85 eV due to AuI indicate that the gold atoms in the clusters must be present as Au0 [12]. Fig. 2 shows the UV–vis spectrum of the prepared PBLG(50)SS–Au in CHCl3 (6.0 g L−1 , solid line). The spectrum indicates the formation of gold nanoparticles with an absorption band at about 520 nm, assigned to a characteristic gold plasmon band. It is found from the inset of the figure that the absorbance increases linearly with increasing concentration, and the absorption maxima (λmax ) do not change with concentration. This spectral feature suggests that PBLG(50)SS–Au is modestly dispersed in CHCl3 without aggregation between particles since the aggregation of gold nanoparticles has been reported to cause a red
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85
Fig. 3. Size distribution of PBLG(n)SS–Au (a) n = 20 and (b) n = 50 in CHCl3 , measured by dynamic light scattering.
shift of the peak wavelength (λmax ) and broadening of the spectrum [3,13]. Presumably, the helical PBLGs must exist densely on colloidal gold surface and as a result it becomes difficult to aggregate through interhelix interaction. A similar spectral feature was observed for PBLG(20)SS–Au; the particles were stably present in CHCl3 without any aggregation although the particle size seems to depend on the peptide segment length (n) as described later. The UV–vis spectrum of the cast film prepared from the CHCl3 solution of PBLG(50)SS–Au, a typical sample, was observed to have an absorbance at 520 nm (Fig. 2, dashed line) that is consistent with that in solution, meaning that interaction between particles would not be so important even in the cast film. To elucidate the size of these particles in solutions, we performed dynamic light scattering (DLS). Fig. 3 displays histograms of size distributions for PBLG(20)SS–Au and PBLG(50)SS–Au particles. They exhibit unimodal and narrow distributions. The diameters are evaluated to be about 8 and 16 nm for PBLG(20)SS–Au and PBLG(50)SS–Au, respectively. It needs the core size of gold cluster to estimate the real thickness of peptide layers. TEM observation was thus employed (Fig. 4). The CHCl3 solutions of these PBLG(n)SS–Au were dropcast onto TEM grids. The images revealed the formation of gold nanospheres, which were homogeneously distributed. The particles were found to be spherical although the mean diameters of gold cores were different between them: about 4 nm for PBLG(20)SS– Au and 10 nm for PBLG(50)SS–Au. It has been reported that manipulation of the preparative reaction conditions such
Fig. 4. TEM images of (a) PBLG(20)SS–Au and (b) PBLG(50)SS–Au cast on copper TEM grids from CHCl3 solutions.
as ligand-to-gold ratio can affect changes in cluster dimensions [14]. The molecular size, including polymer molecular weight, of ligands has also affected the size of gold nanoparticles [4]. The observed discrepancy in core size must be derived from the latter effect of polymer molecular weight since the preparative conditions were the same. From the results of both TEM and DLS observations, one can roughly estimate the mean thickness of the coated PBLG layer to be 4 and 6 nm for PBLG(20)SS–Au and PBLG(50)SS–Au, respectively. By assuming a complete α-helix conformation, the helix length of PBLG can be computed to be 5 and 10 nm for n = 20 and n = 50, respectively, using the occupied length (0.15 nm) of one α-amino acid residue along the helix axis. These values are comparable with those evaluated by TEM and DLS. 3.2. Secondary structures of PBLG segments We have previously demonstrated that the two- or threedimensional assembly of peptides at some interfaces induces secondary structural changes and consequently provides more highly ordered peptide organizations [15]. It is thus important to reveal the secondary structure of PBLG segments on gold clusters. FTIR and 1 H NMR spectroscopy are employed. The spectra of gold-free PBLG(n)SS themselves were also measured for comparison. Fig. 5 displays FTIR
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Table 1 Helix content of PBLG(n)SS and PBLG(n)SS–Au particles in CHCl3 based on 1 H NMR spectroscopy n 20 50
Helix contents (%) PBLG(n)SS (FTIRa )
PBLG(n)SS–Au (FTIRa )
69 (59) 86 (83)
87 (85) 92 (94)
a Estimated by the peak deconvolution method applied to the amide II band region in FTIR spectra.
Fig. 5. FTIR spectra of (a) PBLG(20)SS–Au and (b) PBLG(20)SS in CHCl3 at 20 ◦ C.
ble 1, together with data on PBLG(50)SS and PBLG(50)SS– Au and from FTIR analyses. The immobilization of PBLG segments onto gold cluster is found to bring a marked increment of α-helix content, compared with the gold-free PBLG(n)SS; in particular, in the case of PBLG(20)SS–Au having the shorter PBLG segment, the increment of helicity becomes more drastic. A similar enhancement in helicity has been found for the PBLG-attached dendritic nanoparticles [9]. The observed helix enhancement is probably due to PBLG side chain interactions such as π–π stacking of benzyl groups, resulting from characteristic alignment of PBLG segments on gold clusters, at which they are forced to assemble densely.
4. Conclusions
Fig. 6. 1 H NMR spectra of (a) PBLG(20)SS–Au and (b) PBLG(20)SS in CDCl3 at 20 ◦ C.
spectra of the C=O stretching band region for PBLG(20)SS and PBLG(20)SS–Au nanoparticles in CHCl3 at a concentration of 5.0 g L−1 . The peaks appearing at 1732 and 1655 cm−1 can be assigned to the C=O stretching band of the ester group in the side chain and the amide I band in the main chain, respectively. In the amide II band region, there is a sharp peak at 1547 cm−1 that is based on an α-helix structure and a broad shoulder around 1535 cm−1 that is derived from a random coil conformation [16]. Focusing on the amide II band region, it can be clearly seen that the attachment of PBLG segment onto gold cluster significantly enhances the helix content in comparing PBLG(20)SS and PBLG(20)SS–Au nanoparticles, although a minor existence of random coil conformation may not be excluded. To obtain more quantitative information on the secondary structure, 1 H NMR spectra were measured in CDCl under the same 3 conditions (Fig. 6). The α-CH resonance signal of the PBLG main chain was known to give a peak at 3.95 ppm ascribed to the α-helix conformation, while giving an apparent lowfield shift on going from the helix to the random coil form [17]. From signal areas based on α-helix and random coil forms, the helix contents were evaluated and summarized in Ta-
This study demonstrates that novel polypeptide monolayer-coated gold nanoparticles are successfully prepared by in situ reduction of HAuCl4 in the presence of PBLG(n)SS (n = 20, 50). The particles were present stably in CHCl3 without any aggregation, although their sizes (8–16 nm) were found to depend upon the peptide segment length (n). The PBLG segments attached on gold cluster took α-helix conformation with nearly 100% content, resulting from enhancement in helicity due to assembled structure. It is expected that these helical peptide-coated gold nanoparticles would cause specific aggregation between particles and guest peptide molecules through interhelix interaction if the surface density of helices could be adjusted.
Acknowledgments This material is based upon work supported in part by the project “Hybrid Nanostructured Materials and Its Application” of the High Technology Center at RCAST of Doshisha University. We thank Dr. M. Morikawa at Professor Kimizuka’s lab (Kyusyu University) for TEM observation.
References [1] For a recent review on surface-functionalized nanoparticles, see R. Shenhar, V.M. Rotello, Acc. Chem. Res. 36 (2003) 549–561, and references cited therein.
N. Higashi et al. / Journal of Colloid and Interface Science 288 (2005) 83–87
[2] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, J. Chem. Soc. Chem. Commun. (1994) 801–802. [3] H. Otsuka, Y. Akiyama, Y. Nagasaki, K. Kataoka, J. Am. Chem. Soc. 123 (2001) 8226–8230. [4] R.G. Shimmin, A.B. Schoch, P.V. Braun, Langmuir 20 (2004) 5613– 5620. [5] L. Maya, C. Muralidharan, T.G. Thundat, E.A. Kenik, Langmuir 16 (2000) 9151–9154. [6] M. Niwa, M.-a. Morikawa, N. Higashi, Angew. Chem. Int. Ed. 39 (2000) 960–963. [7] M. Niwa, M.-a. Morikawa, N. Higashi, Langmuir 15 (1999) 5088– 5092. [8] M. Niwa, M.-a. Morikawa, T. Nabeta, N. Higashi, Macromolecules 35 (2002) 2769–2775. [9] N. Higashi, T. Koga, M. Niwa, Chem. Commun. (2000) 361– 362.
87
[10] N. Higashi, T. Koga, M. Niwa, ChemBioChem (2002) 448–454. [11] N. Higashi, T. Koga, M. Niwa, J. Nanosci. Nanotechnol. (2001) 309– 315. [12] A. McNeillie, D.H. Brown, W.E. Smith, M. Gibson, L. Watson, J. Chem. Soc. Dalton Trans. (1989) 767–770. [13] N. Nath, A. Chilkoti, J. Am. Chem. Soc. 123 (2001) 8197–8202. [14] M.J. Hostetler, J.E. Wingate, C.-J. Zhong, J.E. Harris, R.W. Vachet, M.R. Clark, J.D. Londono, S.J. Green, J.J. Stokes, G.D. Wignall, G.L. Glish, M.D. Poster, N.D. Evans, R.W. Murray, Langmuir 14 (1998) 17–30. [15] N. Higashi, T. Koga, M.-a. Morikawa, M. Niwa, in: H.S. Nalwa (Ed.), Handbook of Surfaces and Interfaces of Materials, vol. 5, Academic Press, San Diego, 2001, pp. 167–205, and references cited therein. [16] T. Miyazawa, E.R. Blout, J. Chem. Phys. 83 (1961) 712–719. [17] E.M. Bradburg, C. Crane-Robinson, H. Goldman, H.W.E. Rattle, Nature 217 (1968) 812–816.