- Open Access
Biotemplating rod-like viruses for the synthesis of copper nanorods and nanowires
© Zhou et al.; licensee BioMed Central Ltd; licensee BioMed Central Ltd. 2012
- Received: 9 January 2012
- Accepted: 1 May 2012
- Published: 1 May 2012
In the past decade spherical and rod-like viruses have been used for the design and synthesis of new kind of nanomaterials with unique chemical positioning, shape, and dimensions in the nanosize regime. Wild type and genetic engineered viruses have served as excellent templates and scaffolds for the synthesis of hybrid materials with unique properties imparted by the incorporation of biological and organic moieties and inorganic nanoparticles. Although great advances have been accomplished, still there is a broad interest in developing reaction conditions suitable for biological templates while not limiting the material property of the product.
We demonstrate the controlled synthesis of copper nanorods and nanowires by electroless deposition of Cu on three types of Pd-activated rod-like viruses. Our aqueous solution-based method is scalable and versatile for biotemplating, resulting in Cu-nanorods 24–46 nm in diameter as measured by transmission electron microscopy. Cu2+ was chemically reduced onto Pd activated tobacco mosaic virus, fd and M13 bacteriophages to produce a complete and uniform Cu coverage. The Cu coating was a combination of Cu0 and Cu2O as determined by X- ray photoelectron spectroscopy analysis. A capping agent, synthesized in house, was used to disperse Cu-nanorods in aqueous and organic solvents. Likewise, reactions were developed to produce Cu-nanowires by metallization of polyaniline-coated tobacco mosaic virus.
Synthesis conditions described in the current work are scalable and amenable for biological templates. The synthesized structures preserve the dimensions and shape of the rod-like viruses utilized during the study. The current work opens the possibility of generating a variety of nanorods and nanowires of different lengths ranging from 300 nm to micron sizes. Such biological-based materials may find ample use in nanoelectronics, sensing, and cancer therapy.
- Tobacco mosaic virus
- M13 phage
- fd phage
- Electroless deposition
- Polyaniline coating
A great interest in gold nanorods is being motivated by their potential applications in various technologies including optical filtering, subwavelength imaging, data storage, and sensor devices [1, 2]. One efficient approach in preparing large quantities of gold nanorods is via seed-mediated synthesis in the presence of a surfactant . Nonetheless, polydispersity and byproducts in the form of nanospheres and nanoplates may be limiting for certain optical applications. Recent progress in purification  and synthesis of gold-nanorods  shows great promise. Moreover, interest in nanorods of various metals had arisen as well. For example, copper oxide nanorods had been produced by Liu et al. and Cheng , who were motivated by potential applications of copper oxide nanorods in solar and electrochemical cells. While their methods have potential for the production of copper oxide nanorods on surfaces, scalable solution-based methods that produce monodisperse and well-dispersed copper (Cu) nanorods is still a topic of significant importance in fundamental studies and commercial applications.
Our strategy for the synthesis of Cu-nanorods is biotemplating. Biotemplating is an attractive method to synthesize nanosize inorganic materials because it takes advantage of the well-defined size and shape of the biological structures and the surface functional groups that can interact with metal atoms leading to nucleation and growth of nanoparticles. It can potentially produce a wide variety of materials for applications in electronics, sensing, optics, and cancer therapy [8–10].
Biomolecules such as DNA, amino acids, peptides, protein cages, and viruses have been used as templates and scaffolds for the synthesis of inorganic nanomaterials including metals and semiconductors . DNA  and fiber-like protein structures like microtubules  have been used as biotemplates for the synthesis of Cu-nanowires. Still the utilization of DNA and microtubules for biotemplating face some challenges. For example, DNA requires specialized techniques for the production of straight nanowires and the aspect ratio of Cu-nanowires from microtubules is difficult to control due to the polydisperse nature of the microtubules.
Rod-like viruses provide the following advantages for the synthesis of nanorods: (1) well-define shape and dimensions in the nanoscale, (2) stability at broad pH ranges, (3) easy to purify in large scale, (4) mechanically robust, which allows the utilization of ultracentrifugation and sonication techniques during sample processing, and (3) virus particles are intrinsically monodisperse. The rod-like plant virus tobacco mosaic virus (TMV) and bacteriophages like fd and M13 are ideal templates for producing high aspect ratio materials such as nanorods. These viruses also share common favourable characteristics for biotemplates including stability over a wide pH range, and a net negative charge at neutral pH. TMV is a 300 nm long cylindrical rod with an outer diameter of 18 nm and a 4 nm central cavity. Approximately, 2130 identical coat protein (CP) subunits form a right-handed helix around the viral single stranded RNA [14, 15]. Filamentous bacteriophage fd and wild type M13 are structurally identical. They are 880 nm in length and 6.6 nm in diameter . Each phage consists of approximately 2700 CP (pVIII) subunits wrapped around a circular loop of single stranded DNA. fd and M13 differ by one amino acid per CP, which results in a net 30% more negative charge in fd[17, 18]. The M13 phage is a widely-used cloning system as a phage display for expression of small peptides  used to identify amino acid sequences that are specific towards metals, metal oxides , and semiconductor surfaces .
High aspect ratio viral protein structures have been explored to fabricate metallic nanorods. Even though a variety of metals have been deposited on TMV [22–35] and M13 [36, 37], continuous coating have been reported only for Pd , Pt , Co , and Ni [32, 33] on TMV and Ag  and Au/Ag alloy  on M13. Meanwhile, there are not reports to date on the metallization of the fd bacteriophage.
Among the various metals, Cu offers the advantages of high electric conductivity and low cost. If high quality biotemplated Cu-nanorods and Cu-nanowires can be fabricated in large quantities, they may be of utility as interconnects in future nanoscale electronics . Previous strategies for copper incorporation into TMV include photochemical reduction of Cu2+ TMV , direct chemical reduction  of CuCl2, and copper reduction inside the TMV channel . Major issues reported in the literature include sparse and uneven Cu coverage , product aggregation , poor yield and difficulties in controlling the length of the resulting Cu-nanorods.
In the current work, we report the synthesis of straight, continuous and dispersed Cu-nanorods and Cu-nanowires by electroless deposition of Cu on Pd-activated virus outer surfaces. Our solution-based method is performed in aqueous solution and at room temperature, making it amenable for large scale production. The Cu-TMV nanorods were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and a nanoparticle size determination system. Furthermore, we produced Cu-fd and Cu-M13 nanorods and PANI-Cu-TMV nanowires to demonstrate the versatility of this metallization procedure for other biotemplates. The current work opens the possibility of generating a variety of nanorods and nanowires of different lengths ranging from 300 nm to micron sizes.
Electroless deposition of Pd on wild type (WT) TMV
The synthesis of TMV-templated Cu-nanorods was achieved by a two-step electroless deposition on wild type (WT) TMV. In the first step, the virus template surface was activated by seeding small Pd nanoparticles on the surface. In the second step, the Pd nanoparticles served as catalytic sites for the chemical reduction of Cu2+, leading to nanocrystal growth and the formation of continuous Cu coating on the template.
Electroless deposition of Cu on WT-TMV
XPS characterization of Pd-TMV and Cu-TMV
Dispersion of Cu-TMV
The effect of C11-PEG-thiol was further analyzed using a NanoSight nanoparticle size determination system, which uses laser scattering to track the Brownian motion of individual nanoparticles and calculates the hydrodynamic diameter of the nanoparticles. The software Nanosight NTA2.1 was designed to determine the size of spherical particles; therefore, the length of rod-shaped TMV obtained from the analysis cannot be reported quantitatively and was used only for qualitative comparison. Particle size analysis showed multiple peaks and broad distribution in unmodified Cu-TMV (Figure 5c), but a single sharp peak with an average value of 75 ± 3 nm (n = 3) for Cu-TMV-C11 (Figure 5e). The size distribution of Cu-TMV-C11 was comparable to that of WT-TMV, (Additional file 1: Table S1; Figure S1). C11-PEG-thiol treated Cu-TMV remained stable and readily dispersed at RT for months. Due to the amphiphilic nature of the C11-PEG chain, Cu-TMV-C11 was easily suspended in water and organic solvents such as ethanol and DMSO.
Metallization of fd and M13 bacteriophages
After Cu plating, the phage surfaces showed darker contrast in TEM images (Figures 6e-f and 7e-f) and a higher Cu to Pd ratio in the EDS analysis (Additional file 1: Figures S5 and S6) indicating a successful copper coating. The average diameter of the copper coated nanorods was 35 ± 8 nm for Cu-fd Y21M and 24 ± 4 nm for Cu-M13. The Cu-fd Y21M nanorods formed 2–3 μm clusters and the Cu-M13 nanorods had aggregates larger than 10 μm. Overall, based on TEM images the coating on Cu-fd nanorods (Figure 6e) was more uniform in comparison to Cu-M13 (Figure 7e). It is important to note that this is the first time that any metallization is reported for fd-Y21M.
Synthesis of Cu-nanowires
This is the first time that PANI-TMV is coated with Pd and Cu (Figure 8) which opens the possibility of adding a variety of metals that will impart higher conductivity than the inherent conductivity that PANI-TMV has by the fact that polyaniline is a conducting polymer . We had coated PANI-TMV with Cu as shown in Figures 8c and 8f. Differences in the coating can be appreciated in the TEM images (Figures 8d-f). PANI-TMV (Figure 8d) was stained with uranyl acetate for imaging purposes while PANI-Pd-TMV and PANI-Cu-TMV owe the contrast in TEM images to the metallic nature of the corresponding coating. Further experiments are in progress to: increase the thickness of the copper layer on PANI-Cu-TMV, fractionate samples for isolation of 1 μm to 2 μm size nanowires, and perform conductivity measurements.
Reaction conditions described in the current work resulted to be effective in the biotemplating of a series of rod-like viruses for the fabrication of Cu-nanorods and Cu-nanowires. Cu has been successfully deposited onto the outer surfaces of TMV, fd, and M13 viruses, with the Cu-TMV nanorods being the straightest in the series owing to the stiff nature of the biotemplate. Furthermore, reaction conditions were developed to synthesize Pd- and Cu-nanowires of sizes longer than 1 μm by metallization of polyaniline coated WT-TMV. Excellent dispersion of individual Pd- and Cu-nanorods using complexing and capping agents has been demonstrated. By faithfully replicating the shape and dimension of the rod-like biotemplates and separating the aggregates into individual nanostructures, we had achieved an important step towards synthesizing and processing high quality Cu-nanorods and Cu-nanowires by scalable methods.
All chemicals were obtained from U.S.A. sources and used as received. Sodium tetrachloropalladate (II) (Na2PdCl4), Copper (II) sulfate (CuSO4), dimethylamine borane (DMAB), ethylenediaminetetraacetic acid (EDTA), Triton X-100, Sarkosyl, tetracycline, ampicillin, RNase A, and DNase I were purchased from Sigma-Aldrich (St. Louis, MO). Thiotic acid (TA), glucose, potassium phosphate, sodium chloride (NaCl), and polyethylene glycol (PEG, MW = 8000) were purchased from Fisher Scientific (Pittsburgh, PA). Kanamycin was obtained from EMD Chemicals Inc. (Philadelphia, PA), Luria Broth Base media (LB) from Invitrogen (Carlsbad, CA), and XL1-Blue Escherichia coli strain from Agilent Technologies Genomics (Santa Clara, CA). (1-mercaptoundec-11-yl)tri(ethylene glycol) (C11-PEG-thiol) was synthesized in our laboratory . M13 and fd Y21M were grown and purified in house as described in Additional file 1. Purified wild type-tobacco mosaic virus (WT-TMV) was provided by Prof. Qian Wang’s laboratory at the University of South Carolina. All dialysis was performed against Milli-Q water at room temperature (RT) using 20 kDa molecular weight cut off dialysis cassettes from Fisher Scientific.
Electroless deposition of Pd on WT-TMV
Pd deposition was performed by reduction of Pd2+ onto the TMV surface functional groups. The method was optimized for our application from a previously described protocol . Briefly, WT- TMV stored in 100 mM phosphate buffer, pH 8 was dialyzed against water overnight. 2–4 μg/ml TMV was incubated with an aqueous solution of 0.4-0.8 mM Na2PdCl4 at 50°C for 30 min. This procedure produced a loosely packed brown precipitate of Pd-coated TMV (Pd-TMV).
In order to obtain individually dispersed Pd-TMV, 0.5 mM EDTA was mixed with the suspension containing Pd-TMV, and then sonicated for up to 1 hour at RT (Branson Ultrasonic Cleaner 2510). The suspension was centrifuged at 6000 rpm (3300 rcf, Eppendorf centrifuge 5415R) for 15 min at RT. After discarding the supernatant, the brown pellet containing Pd-TMV was resuspended in water. The suspension was mostly uniform in color without visible aggregation.
Electroless deposition of Cu on Pd-TMV
Freshly prepared Pd-TMV suspension was mixed with a Cu plating solution containing 1 μg/ml TMV, 1 mM CuSO4, and various concentrations of dimethylamine borane (DMAB: 1 mM, 1.5 mM, 2 mM, and 3 mM) and EDTA (0.03 mM, 0.5 mM, 1 mM, and 1.5 mM) in separate experiments which were performed for optimization purposes (data not shown, optimum procedure is described herein). The pH of the plating bath was about 5. The reaction was allowed to proceed for 12 min for reaction progress study while in subsequent preparations the reaction was stopped at 6 min by diluting the reaction mix 1:1 with water. EDTA (0.5 mM in water) and TA (1 mM in ethanol : H2O = 8:2) were added to the suspension and centrifuged at 6000 rpm for 15 min. After discarding the supernatant, water was added to resuspend the Cu-TMV pellet. In order to further disperse the Cu-TMV, 1 μl of C11-PEG-thiol  was added to the suspension and incubated at RT overnight.
Electroless deposition on fd and M13 phages and polyaniline-TMV
fd mutant Y21M was used for metallization for the first time. Mutant fd Y21M was selected as opposed to WT-fd since it has a higher persistence length in comparison to the WT-fd. We utilized the same procedure and concentrations that produced straight and continuously covered TMV for the metallization of the phages. Namely, the Pd reactions consisted of 2 μg/ml of phage and 0.4 mM Na2PdCl4. The Cu plating baths contained 1 μg/ml of viruses coated with Pd, 1 mM CuSO4, 1 mM DMAB, and 0.5 mM EDTA.
PANI-Pd-TMV and PANI-Cu-TMV nanowires were synthesized by coating WT-TMV with polyaniline via aniline polymerization on self-assembled TMV rods as previously described . After polymerization the sample was dialyzed against water overnight. Metallization reactions of PANI-TMV were performed using a slightly modified procedure found to be optimum for the metallization of WT-TMV. Briefly, the Pd reactions consisted of 2 μg/ml of PANI-TMV and 0.6 mM Na2PdCl4. The Cu plating baths contained 1 μg/ml of PANI-Pd-TMV, 2 mM CuSO4, 2.5 mM DMAB, and 1.0 mM EDTA.
Electron microscopy analysis
Suspensions of all samples were drop casted on acid-cleaned silicon wafers and air dried in the hood. Scanning electron microscopy (SEM) of metalized TMV was performed using a Leo Supra 55 (Cal Zeiss SMT AG).
Transmission electron microscopy (TEM) was obtained from samples deposited onto a 300-mesh formvar carbon coated nickel grid (Ted Pella Inc. Redding, CA). A LIBRA-120 EFTEM equipped with an EDS detector was used for qualitative analysis of metal content (see Additional file 1 for data and discussion). Diameters of the metalized-TMV were measured and analyzed from the TEM images using Image J software (version: 126.96.36.199, copyright 1993–2006, Broken Symmetry Soft). One or two measurements were taken from each nanorod. Thirty to eighty TMV-nanorods were measured for each sample.
X-ray photoelectron spectroscopy (XPS)
XPS was performed with a Thermo Scientific Model K-Alpha spectrometer, using the monochromatized AlKα line at 1486.6 eV. The base pressure was 1.5 x 10-8 Torr. The x-ray spot size was 400 μm. Argon ion sputtering for depth profiles was performed over an area of roughly 2 mm x 2 mm, using a beam energy of either 500 or 1000 eV. The data were background-subtracted and then smoothed using a Savitzky-Golay algorithm.
The authors thank Q. Wang from University of South Carolina for providing the WT-TMV and Z. Dogic from Brandeis University for providing the fd Y21M mutant. JCZ and MAB thank the National Research Council for postdoctoral fellowships. CMS wants to thank M Swain, S. Walper, A. Lee, and E. Goldman for M13 protocols and cells stocks. EB acknowledges support from NSF-MRSEC-0820492 and ACS PRF-50558-DNI7. Authors want to thank the Office of Naval Research for financial support and J. Liu and W. Dressick for reviewing the manuscript.
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