NANO LETTERS
Organization of Metallic Nanoparticles Using Tobacco Mosaic Virus Templates
2003 Vol. 3, No. 3 413-417
Erik Dujardin,†,| Charlie Peet,† Gerald Stubbs,‡ James N. Culver,§ and Stephen Mann*,† School of Chemistry, UniVersity of Bristol, Cantock’s Close, Bristol BS8 1TS, UK, Department of Biological Sciences, Vanderbuilt UniVersity, NashVille, Tennessee 37235, and Center for Biosystems Research, UniVersity of Maryland Biotechnology Institute, College Park, Maryland 20742 Received January 6, 2003; Revised Manuscript Received January 31, 2003
ABSTRACT Self-assembled cylindrical particles of wild type and recombinant tobacco mosaic virus (TMV) were used as organic templates for the controlled deposition and organization of Pt, Au, or Ag nanoparticles. Chemical reduction of [PtCl6]2- or [AuCl4]- complexes at acidic pH gave rise to the specific decoration of the external surface of wild-type TMV rods with metallic nanoparticles less than 10 nm in size. In contrast, photochemical reduction of Ag(I) salts at pH 7 resulted in nucleation and constrained growth of discrete Ag nanoparticles aligned within the 4 nm-wide internal channel. The number of encapsulated nanoparticles increased when Ag benzoate rather than Ag nitrate was used due to reduced supersaturation associated with the lower Ag/benzoate redox couple, which enhanced the surface-templating effect of the channel wall carboxylates compared with nucleation in solution. Similar experiments using a mutant TMV with reduced negative charge along the central cavity confirmed that glutamic and aspartate acid groups were involved in site-specific deposition. Our results suggest that it should be possible to prepare 1-D arrays for a wide range of inorganic quantum dots by molecular engineering of the internal and external surfaces of self-assembled TMV tubules.
1. Introduction. Reflecting the growing interest in biomimetic chemistry as a powerful approach to combine complexity and functionality in new materials,1,2 the design of versatile nanoparticle-based superstructures has recently evolved into a dynamic discipline where biological concepts and entities are playing a crucial role.3,4 For example, welldefined nanoparticles have been prepared by the biomineralization of cage-like proteins5,6 and spherical viruses.7 Biomolecules, such as oligonucleotides,8,9 antibodies,10 and biotinylated derivatives,11 have been used to interconnect metallic or semiconductor nanoparticles in solution and preimmobilized proteins,12,13 viruses,14 or DNA strands15 have been employed to position nanoparticles on solid surfaces. Motivated by the need for 1D-nanometric objects for applications in nanoelectronics and nanobiotechnology, methods for the anisotropic self-assembly of nanoparticles using biological interactions, such as duplex formation between DNA-functionalized gold nanorods16 or metallization of self-assembled lipid microtubules,17-19 have been recently investigated. Here, we show that cylindrical particles of tobacco mosaic virus (TMV) can be used as a template * Corresponding author. E-mail:
[email protected]. † University of Bristol. ‡ Vanderbuilt University. § University of Maryland Biotechnology Institute. | Present address: Groupe Nanosciences, CEMES/CNRS UPR 8011, 29 rue J. Marvig, BP 4347, 31055 Toulouse Cedex, France. 10.1021/nl034004o CCC: $25.00 Published on Web 02/12/2003
© 2003 American Chemical Society
for in situ formation of anisotropic assemblies of spherical metallic nanoparticles. TMV particles are composed of about 2130 identical coat protein molecules following the right-handed helix of an associated RNA strand to produce a 300 nm-long hollow cylinder with an outer diameter of 18 nm and a 4 nm-wide inner cavity.20 The TMV cylinders are robust toward thermal and chemical treatments, and consist of chemically functionalized surfaces associated with the repetition of specific amino acid side chains. Together, these properties have been used in template-directed mineralization reactions to prepare high aspect ratio bioinorganic nanotubular composites consisting of a TMV central core and external coatings of silica, iron oxides, or lead and cadmium sulfides.21,22 Recently, isolated Ni nanoparticles have been prepared on and within TMV particles by Pd(II)-mediated reduction of Ni(II) salts, although the numbers of metal particles incorporated per virion was small (1 to 3).23 The high surface specificity exhibited in these systems appears to be related to the binding of reaction precursors to amino acids such as glutamic and aspartic acid, arginine, and lysine.24 It should therefore be possible to influence these predominantly electrostatic interactions and hence the spatial organization of the templated inorganic phase by modifications in the surface chemistry of the TMV particles. For example, the exposed functional groups within the cavity are predominantly aspartic and
glutamic acids, the pKa of which are either 1-3 or 6-8 depending on the location within the protein.25 On the other hand, the outer surface contains a significant number of lysine and arginine having pKa values of 11 and 12.5, respectively. Thus, at pH around 3, the surface charge of the inner cavity is close to neutral but the outer surface is positively charged. Adsorption and subsequent reduction of anionic metalcomplex precursors should then occur preferentially on the outer surface of the TMV cylinder. On the other hand, metal cations should be sequestered specifically within the cylindrical nanocavity at neutral pH and above. In this work, we use the above considerations to prepare a range of TMV-metallic nanotubular composites. Wildtype viruses were exposed to gold, platinum or silver precursors prior to chemical or photochemical reduction. We show that anionic metal precursors (e.g., [AuCl4]-, [PtCl6]2-) interact predominantly with the outer surface to form metallic coatings or externally decorated nanoparticles, whereas Ag(I) cations are incorporated inside the TMV central channel to produce a linear array of regularly spaced Ag nanoparticles. In addition, we demonstrate that reduction in the anionic charge of the channel surface by site-directed mutagenesis can significantly influence nucleation, suggesting that ion binding to glutamic and aspartic acid residues is important for site-directed deposition. Our results suggest that recombinant methods could be of general importance in future strategies involving TMV templates. 2. Experimental Section. 2.1. Suspensions of Wild-Type TMV and E95Q/D109N Mutant. Wild-type TMV (wTMV, U1 strain, 30 mg mL-1) was kept in 10 mM sodium phosphate buffer at pH 7. The mutant virus E95Q/D109N, in which amino acid residues glutamate 95 (Glu95) and aspartate 109 (Asp109) were replaced by the corresponding amides, was obtained by site-directed oligonucleotide mutagenesis as described elsewhere.25 The concentration of mutant stock suspension was 4.5 mg mL-1 in 10 mM sodium phosphate buffer at pH 7. 2.2. Platinum Mineralization of wTMV. 10 µL of the stock suspension of wild-type TMV was dispersed into 200 µL of a 10-3 M aqueous hydrogen hexachloroplatinate(IV) (H2PtCl6) solution (pH ) 3). The suspension was stirred at room temperature in the dark for 16 h before adding 100 µL of a 10-3 M hydrazine hydrate solution. 5 µL aliquots were subsequently taken at various times and diluted 10 times with ultrapure water for transmission electron microscopy (TEM) analysis. 2.3. Gold Mineralization of wTMV. Suspensions containing 10 µL of wild-type TMV stock suspension, 500 µL of acetic acid (0.89 M, 5 wt %; pH ) 2.3) and 50 µL of a 10-2 M aqueous hydrogen tetrachloroaurate(III) (HAuCl4) solution (pH ) 2.9) were stirred in the dark for 30 min. Reduction of the gold salt at pH ) 2.3 was carried out by adding 100 µL of a 10-3 M hydrazine hydrate solution. Aliquots were taken and used undiluted for TEM sample preparations. 2.4. SilVer Mineralization of wTMV. A 0.5 mL sample of a 10-3 M aqueous silver benzoate or silver nitrate at pH ) 7 was incubated with 5 µL of the stock suspension of wildtype TMV overnight with continuous stirring in the dark. 414
Figure 1. TEM micrographs of unstained Pt-decorated wild-type TMV at (a) low and (b) high magnifications. Inset: corresponding EDXA spectrum showing the presence of platinum only († indicates copper, ‡ chromium and * iron peaks due to the supporting grid).
The silver salt was photochemically reduced to metallic silver by irradiating the solution with a 254 nm UV lamp (Mineralight UVGL-25) for 2, 5, and 10 min. At each time, aliquots were taken for TEM analysis. 2.5. SilVer Mineralization of Mutant TMV (E95Q/D109N). A 5 µL sample of a 4.5 mg mL-1 stock suspension of mutant TMV E95Q/D109N and 200 µL of a 10-3 M aqueous silver nitrate or silver benzoate were mixed at pH 7 at room temperature and stored at 4 °C for 24 h. After incubation, the silver salt was photochemically reduced by exposing the suspensions to daylight for up to 8 h. Aliquots were then diluted with doubly distilled water to reach a virus concentration of 3 µg mL-1 for TEM sample preparation. 2.6. Methods. TEM observations of TMV samples were made on a JEOL 1200EX electron microscope operated at 120 kV and equipped with an Oxford EDX analyzer. Typically 2 to 5 µL droplets of TMV suspension were deposited on carbon-coated 300 mesh copper grids for 2 min before the excess liquid was wicked off with filter paper. When negative staining was performed, the dried TEM grid was exposed to a 10 µL droplet of a 1 wt % uranyl acetate solution (pH 4) for 10 s. The excess staining solution was wicked off with filter paper. UV-visible absorption spectra were recorded in the 200 to 1100 nm range on a Lambda 11 Perkin Elmer spectrophotometer. Typically, 200 µL of metal-decorated TMV samples were transferred into a 500 µL quartz cuvette with a 5 mm light path. 3. Results and Discussion. Incubation of TMV particles with 10-3 M H2PtCl6 followed by chemical reduction with hydrazine at pH 3 produced intact TMV rods that were decorated with Pt nanoparticles (Figure 1). The nanoparticles were spheroidal, 2.5 to 5 nm in diameter, and attached to the surface of the virus. EDX analysis confirmed the presence of only Pt (Figure 1, inset), and no well-defined reflections were observed by selected aperture electron diffraction (SAED). Interactions between [PtCl6]2- anions and the positively charged lysine and arginine residues on the outer Nano Lett., Vol. 3, No. 3, 2003
Figure 3. (a) TEM micrograph of ∼5 nm silver nanoparticles grown inside the hollow channel of wild-type TMV. Arrows indicates nanoparticles that prevented the stain (uranyl acetate) from penetrating further in the cavity. One of these is magnified in the inset. Scale bar for the main image is 50 nm. (b) EDXA spectrum showing the presence of silver and uranium (stain). Supporting grid: † Cu. (c) UV-visible absorption spectrum displaying a plasmon band at 422 nm, characteristic of metallic silver colloid.
Figure 2. TEM micrographs of gold nanoparticles produced in the presence of wild-type TMV. (a) Low magnification image showing multiple TMV rods with dense external coating of gold nanoparticles; scale bar ) 100 nm. (b) Single TMV rod with dense coating of discrete gold nanoparticles; scale bar ) 50 nm. Inset: corresponding EDXA spectrum showing gold and † Cu (from supporting grid).
surface are favored by the acidic medium, so that reduction and nucleation of the primary particles occurs preferentially on the TMV template rather than in solution. Significantly, when the acid Pt salt was replaced by K2PtCl6, no Pt particles were observed in association with the virus surface due to increase in the pH to a value of 9.9 (data not shown). Similar procedures were followed to prepare gold-TMV nanotubular composites. Incubation of the virus in a solution containing HAuCl4 and hydrazine at pH ) 2.3 resulted in a dense coating of discrete spherical gold nanoparticles, 8.6 ( 3 nm in size, across the entire outer surface of the virus (Figure 2a and 2b). Corresponding SAED patterns of the metalated biostructures showed d spacings at 2.4, 2.1, 1.5, and 1.3 Å, consistent with the [111], [200], [220], and [311] reflections of well-ordered fcc gold structure. Similar experiments at higher pH values did not produce high densities of gold nanoparticles associated with the virus external surface (data not shown). Control experiments undertaken in the absence of TMV showed Au nanoparticles with a significantly larger mean diameter of 12 nm. Experiments were also undertaken to specifically mineralize the inner 4-nm-wide channel of TMV particles. For this, neutral or alkaline conditions were used to reduce the positive charge of the outer surface and increase the negative charge Nano Lett., Vol. 3, No. 3, 2003
associated with acidic amino acids lining the cavity wall, with the expectation that cationic reactants would be preferentially sequestered within the central channel. We also used photochemical rather than chemical reduction to minimize potential problems associated with restricted mass transport of reactants and byproducts in and out of the channel. Using this strategy, we were able to produce linear arrays of Ag nanoparticles specifically organized along the inner channel of the virion by incubation of wild-type TMV with Ag(I) benzoate or AgNO3 in the dark at pH 7.0 followed by UV irradiation (Figure 3). Significantly, spherical nanoparticles with a diameter of less than 5 nm were observed regularly spaced along the axis of the TMV rods. Typically, between two and six Ag nanoparticles were observed along the TMV channel for samples prepared using AgNO3. In contrast, reduction of Ag benzoate generally resulted in a larger number (4 to 10) of entrapped nanoparticles. Some large particles (10-15 nm in size) were also adsorbed on the outer surface (Figure 3), but few unattached nanoparticles were observed, thus confirming the template-directed mechanism of mineralization. Unequivocal evidence that the silver nanoparticles aligned along the rod axis were inside the virus channel was provided by a detailed examination of stained TEM images. These showed that penetration of the uranyl acetate stain into the cavity was often blocked by the presence of nanoparticles near to the ends of the TMV inner channel (arrows in Figure 3a and insert). EDXA and UV absorption spectra confirmed that the particles were made of metallic silver, with a plasmon band centered at 422 nm. Lattice d spacings (2.3 Å [111], 2.0 Å [200], 1.4 Å [220], 1.2 Å [311] and 1.1 Å [400]) measured from SAED patterns were in agreement with a fcc structure for metallic silver. 415
Figure 4. (a-c) TEM micrographs of mutant E95Q/D109N showing rows of 3 ( 2 nm silver particles on the outer surface and in the inner channel. (d) EDXA spectrum showing the presence of silver (supporting grid: ‡ Cr and * Fe). (e) UV-visible absorption spectrum displaying a plasmon band at 407 nm, characteristic of metallic silver colloid.
Finally, we studied the effect of reducing the anionic charge associated with the inner cavity of TMV tubules on silver nanoparticle formation. We prepared a E95Q/D109N mutant with anionic residues Glu95 and Asp109 replaced by their noncharged amide derivatives. Reducing the electrostatic repulsion between these carboxylic groups on the inner surface is known to stabilize the virus particles at higher pH without change in the structure.25 Rod-shaped particles of the E95Q/D109N mutant were incubated in silver nitrate or silver benzoate at neutral pH prior to exposure to light and photoreduction to metallic silver. TEM micrographs of individual unstained mutant viruses prepared from AgNO3 showed Ag nanoparticles on and within the TMV particles (Figure 4). EDX spectra (Figure 4d) and SAED (d spacings: 2.4 Å (111), 2.1 Å (200), 1.2 Å (220), and 1.3 Å (311)) confirmed that the nanoparticles were well-ordered fcc crystals of metallic silver, and UV absorption spectra (Figure 4e) showed a narrow Gaussian-shaped plasmon band with a maximum at 407 nm, consistent with metallic nanoparticle with high particle size monodispersity. The particles were 2-4 nm in size and externally located around the tubes rather than on their surface, suggesting that capillary forces associated with air-drying during sample preparation might be responsible for the observed organization. Moreover, nanoparticles within the channel were preferentially located toward the ends of the TMV tubes compared with the more regular distribution observed for the wild-type viroids, consistent with a capillary-driven mechanism. Overall, the number of Ag nanoparticles encapsulated within the TMV channel was similar to that observed for the wild-type virus 416
exposed to photoactivated AgNO3 solutions. In contrast, reduction of Ag benzoate in the presence of the E95Q/D109N mutant gave no nanoparticles associated with the TMV rods under the conditions employed. The results indicate that silver mineralization of the wild type virus is preferentially located within the central channel, and that this site-specificity is reduced in the mutant TMV. The large negative charge associated with the wild-type channel surface facilitates sequestration of Ag(I) ions with the consequence that nucleation occurs at specific sites followed by autocatalytic growth within the cavity. It is notable that for a bulk silver nitrate concentration of 10-3 M, the solution volume required to produce 10 intrachannel Ag nanoparticles, 4 nm in size, is approximately 104 times the volume of the TMV inner cavity. Significantly, the influence of the channel wall glutamate and aspartate residues is more apparent in the presence of Ag benzoate rather than AgNO3, probably because the rate of nucleation is lower in the former, with the consequence that surface-directed processes become competitive with solution-based mechanisms. The lower redox couple of Ag-carboxylate salts (e.g., E°/Ag benzoate ) +0.52 V; E°/Ag acetate ) +0.63 V, and E°/Ag+ ) +0.80 V)26 is consistent with reduced levels of nucleation, and hence an increase in the number of encapsulated nanoparticles observed for wild-type TMV in the presence of Ag benzoate compared with AgNO3. In contrast, deletion of the channel carboxylate ligands in the mutant TMV reduces ion binding and surface-induced Ag nucleation. However, this can be offset in the presence of nitrate, but not benzoate, by a higher nucleation level in general, such that differences in site-specific activity between the wild type and mutant TMV rods are masked by overriding solution conditions. 4. Conclusion. The preparation of metal-virus tubular nanocomposites has been achieved by chemical or photochemical reduction of platinum, gold, and silver precursors on the external surface or within the 4-nm central channel of tobacco mosaic virus. By chemically controlling the surface charge of the virus, metallization was selectively performed on the outer cylindrical surface or in the inner capillary-like cavity. The biological nature of the substrate opens up the possibility of using site-directed recombinant technology to design the surface properties of TMV and trigger biomineralization inside the nanodimensional cavity. In both approaches, anisotropic assembly of spherical metallic nanoparticles can be achieved by mineralizing a onedimensional organic template. In the future, it should be possible to grow continuous metallic nanowires in the TMV and align them in large arrays by ordering the virus in its nematic liquid crystal phase.22 These perspectives are currently being explored in our laboratory Acknowledgment. The authors thank Drs. A. Seddon, W. Shenton, and C. Fowler for fruitful discussions and Dr. S. A. Davis for assistance in high resolution TEM. E.D. acknowledges the European Union for a Marie Curie Individual Fellowship (HPMF-CT-1999-00254). C.P. thanks the University of Bristol for a postgraduate studentship. Nano Lett., Vol. 3, No. 3, 2003
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