Letter pubs.acs.org/NanoLett
Solution Phase Gold Nanorings on a Viral Protein Template Omar Khalil Zahr and Amy Szuchmacher Blum* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 2K6 Canada S Supporting Information *
ABSTRACT: Current studies on materials that exhibit metamaterial properties are mainly focused on lithographygenerated 2D substrates. Here we report the successful fabrication of 22 nm gold nanoparticle rings with and without a central nanoparticle assembled on Tobacco Mosaic Virus coat protein disks. These structures are one of the first examples of nanorings produced independently of a substrate and represent the first steps toward the realization of a solution-phase or coatings-based metamaterial. KEYWORDS: Self-assembly, nanoparticles, biotemplate, metamaterial, Tobacco Mosaic Virus
B
exists between the three phases of helix, disk, and monomer is highly sensitive to small changes in solution conditions.24−26 Here we report the successful fabrication of solution-stable, three-dimensional, gold nanoparticle rings with sub-23 nm diameters utilizing a mutant of TMVcp as a template. TMVcp, when assembled into disk structures, can selectively bind bis(psulfonatophenyl)phenylphosphine (BSPP) passivated gold nanoparticles by electrostatic interaction to produce metallic rings. By utilizing the natural electrostatic surface of the disk as a command surface for self-assembly (Figure 1), gold nanoparticles attach to the top edge of the coat protein disk in a reproducible manner through Coulombic attraction to the arginine residues tracing its outer circumference. We also observe pH dependent binding of a nanoparticle to the center of the nanorings. The interaction involved in binding the central nanoparticle can be reliably switched on and off by altering the pH of the solution. The central nanoparticle binds to a group of arginine residues surrounding the inner pore of the disk only when the ambient pH of the solution allows protonation of the negatively charged carboxyl groups that also surround the disk pore. Gold nanoparticle rings at this size scale are predicted to have a number of interesting spectroscopic properties applicable to next generation optical and electronic devices.27,28 Plasmonic particles have recently been described as artificial atoms with pseudowave functions that couple together much like atoms do to form molecules.29 This perspective has exposed an opportunity to explore the effects of various configurations of plasmonic particles on electromagnetic fields. Several models suggest that rings on this size scale without a central nanoparticle may display a negative permeability in the optical region of the spectrum with a small imaginary component.30,31 In combination with the negative permittivity
ottom-up self-assembly techniques have produced a growing library of nanoscale structures.1,2 These fabrication systems, driven by the practical and theoretical limitations of top-down lithographic techniques at size scales below 50 nm, are a potential route toward next generation nanotechnologies.3,4 Practical bottom-up nanofabrication typically utilizes a template or command surface. In doing so, the choice of an effective template is pivotal to the assembly of functional nanoscale materials.5 Among the many templates explored for the assembly of complex structures, those based on biological systems have attracted a great deal of attention due to their inherent monodispersity and the ease by which they can be manipulated through well-established biochemical protocols in mutation and selective chemical functionalization.6−8 Within the past decade, viruses and virus capsids have proved to be effective candidates for this pursuit due to their monodispersity, robustness, and low cost.9,10 Such advantages are especially apparent in the utilization of viruses and virus capsids as three-dimensional command surfaces for the arrangement of molecules and nanoscale components. To date, these pursuits have produced fixed-diameter nanowires and nanotubes,11−13 light-harvesting assemblies,14,15 and nanoscale breadboards for single-molecule sensing.10,16,17 In several of these studies, Tobacco Mosaic Virus coat protein (TMVcp) proved to be a very versatile template. In its natural form, TMV is a rod-shaped plant virus that is 300 nm in length with an outer diameter of 18 nm and possesses an inner pore that is 4 nm in diameter.18 Of particular note is that the TMV coat protein in isolation is capable of self-assembly into 18 nm diameter stacked disks or helical rods of variable length19 depending on the pH, ionic strength, and temperature of the solution in which it is suspended. While the TMVcp helical rod has received much attention as a template for self-assembly and the in situ reduction of metal salts,20−23 there are fewer examples of the disk phase being utilized in the same manner. One possible reason for this is that the disk phase exists within a relatively small pH region, and the dynamic equilibrium that © 2011 American Chemical Society
Received: September 27, 2011 Revised: December 19, 2011 Published: December 26, 2011 629
dx.doi.org/10.1021/nl203368v | Nano Lett. 2012, 12, 629−633
Nano Letters
Letter
Figure 1. (a) Molecular model of a TMV coat protein disk. Colors denote groups of interest: blue = negatively charged carboxyl group, green = positively charged amine groups. (b) Vacuum Coulombic map of TMV coat protein disk. Blue denotes negative charge, and green indicates positive charge.
of gold nanoparticles in the same optical region, the fundamental requirement of a negative refractive index can be met in these freestanding nanoring structures.32,33 The optical characteristics of these plasmonic nanostructures may have a great impact on sensing, lasing, and nonlinear optics technologies. At the very least, this system of ring fabrication provides a method by which to explore the many theoretical models that have attempted to predict the nontrivial interaction of light with materials possessing nanoscale complexity. Fabrication of the rings is achieved in two steps schematically depicted in Figure 2. BSPP-passivated gold nanoparticles at the
Figure 3. Typical TEM images at each pH condition. Lines illustrate how ring diameter (d) and interparticle distance (a) are measured.
Table 1. Summary of Image Analysis of Rings at each pH Conditiona
a
Figure 2. Schematic illustrating process of ring assembly with and without a central nanoparticle.
pH
outer diameter (nm)
5.5 6.0 6.5 7.0 7.5
22.0 22.4 22.2 22.0 22.7
± ± ± ± ±
2.2 (67) 1.9 (54) 1.8 (59) 1.7 (75) 2.2 (52)
particles per ring 10 10 10 10 9
± ± ± ± ±
2 (67) 1 (54) 1 (59) 1 (75) 1 (52)
interparticle distance (nm) 5.3 5.5 4.7 4.5 5.4
± ± ± ± ±
1.0 (67) 1.1 (54) 1.0 (59) 0.9 (75) 1.0 (52)
rings with central nanoparticle (%) 95 (67) 74 (54) 54 (59) 52 (75) 20 (52)
Ring population size is shown in brackets.
comparison of the ring geometry with the known TMV disk structure. This places the nanoparticles in the same region as the outer ring of arginine residues illustrated as primary amine groups in Figure 1a. There are also an equal number of carboxylate groups in this region, but it seems that the repulsion between the nanoparticles and the carboxylate groups is not strong enough to prevent interaction with the amine groups. This is likely due to differences in the exposure of these groups to the aqueous environment. The interparticle distance measured from the center of adjacent nanoparticles is approximately 5 nm, while that for BSPP nanoparticles on a grid is 7 nm. It seems that interparticle repulsion is alleviated once particles are incorporated into the rings. The reason for this is unclear; however, the interaction of the phosphorus lone pair on BSPP is sterically crowded by the groups that surround it, which may result in the displacement of the ligand as nanoparticles approach each other. TEM image analysis reveals that the proportion of complete rings among the total number of binding events ranges from 46 to 70% depending on pH (see
appropriate concentration are added to a buffered solution of TMVcp at various pH conditions. A good yield of complete rings is obtained after a five day incubation period at room temperature. The progression of the assembly process is monitored in situ by UV/Visible spectroscopy. Transmission electron microscopy (TEM) grids of the reaction solution were prepared after 2 days of interaction and again after 5 days of interaction. An analysis of the TEM images (Figure 3) shows that ring diameter, interparticle distance and the number of particles per ring do not change as the pH is varied (Table 1). This indicates that the groups involved in binding the nanoparticles are not significantly influenced by changes in pH. An average of 10 nanoparticles are observed binding with their centers on the top-face edge of the TMVcp disks. The position of the nanoparticles is determined through observing the binding solution under TEM within the first few hours of binding (see Supporting Information, Figure S5) and through 630
dx.doi.org/10.1021/nl203368v | Nano Lett. 2012, 12, 629−633
Nano Letters
Letter
Figure 4. (a) Increase of the peak absorption at 600 nm over a 5 day period. (b) Spectra of binding solutions at each pH studied. Identical solutions with buffer instead of TMVcp are used for blank correction.
first 30 h. The assembly rate decreases subsequently, as it takes more time to bind the last few nanoparticles. This is likely due to the limits placed on their possible size as well as the decreased likelihood that they will find the last unoccupied sites. Interparticle repulsion probably also contributes to this. There are significant differences in the change in spectra at various pH values (Figure 4b). The characteristic growth of a peak at approximately 575 nm is clearly seen at pH 6.0, 7.0, and 7.5. These peaks, which are unique to the binding solutions and absent in control experiments, are likely due to the formation of aggregate modes as the bound nanoparticles interact with each other. The solution at pH 5.5 is not included in this analysis because it showed signs of precipitation at the end of the binding period. Each of the spectra also shows a dip in the 525 nm region. On the basis of measurements relating the dilution of our gold colloid solutions to the change in the absorbance of their associated plasmon, a drop of 0.1 absorbance units represents a drop in particle concentration as large as 0.8 μM. Thus, the observed drop in absorbance cannot be correlated to the number of particles involved in ring formation. Since there is no evidence of nanoparticle precipitation or aggregation, the source of the observed dip is an optical phenomenon that cannot be accounted for without first decoupling the various resonances elicited by these structures and their indiscriminant orientations in solution. It is, however, clear that the shape of the optical profile is highly correlated to the incidence of rings with central nanoparticles and may be the result of interference between the plasmonic resonance of the central nanoparticle and other resonances present. The dip in the absorbance spectrum is largest at pH 6.5. As the phase map in Figure 5 shows, at this pH the population of TMVcp disks is high while remaining far enough below the pKa of the inner carboxylate groups such that many of the rings obtain a central nanoparticle. The absorbance at pH 7.0 is qualitatively similar in line shape due to similar repulsion conditions as at pH 6.5. At pH 6.0, the population of disks (and thus the yield of gold nanorings) is smaller, even though the chance of having a central nanoparticle is larger. This results in a more asymmetric dip in the absorbance. At pH 7.5, there are few disks, and the majority of the nanorings do not contain a central nanoparticle. This results in a highly asymmetric absorbance peak at 575 nm, with a small dip at 525 nm.
Supporting Information, Table S1). If rings that are only missing one nanoparticle are included, this range increases to 79−91%. This suggests that the main barrier to ring completion is kinetic in nature; the last nanoparticle to be incorporated into the ring must be of appropriate size and must interact with the TMVcp disk in a specific orientation and position. The most prominent change observed when varying pH is the number of rings that possess a central nanoparticle. While the majority of the disks have central nanoparticles at pH values below 7.0, the number drops significantly as pH rises above 7.0. A ring of amine groups around the pore is likely responsible for binding the central nanoparticle. It is very likely that the loss of this central nanoparticle above pH 7.0 is due to a significant increase in negative charge in the central pore of the ring. A potential explanation for this is the deprotonation of the ring of carboxylate groups around the central pore of the TMV disk. According to Lu et al. and Wang et al.,34,35 these carboxylate groups possess an anomalously high pKa and are responsible for the disassembly of disks into protein subunits at pH 7.2. Our observation that the population of rings with central nanoparticles falls below 50% in this pH region supports this assertion. It is also observed that as pH is decreased the specificity of binding decreases, especially at pH 5.5. The result is that multiple nanoparticles bind inside the rings creating “clusters” of nanoparticles on top of TMVcp disks. For this reason and due to signs of precipitation in the binding solution, pH 5.5 is considered an impractical condition for ring selfassembly. In order to drive the assembly process to produce a substantial number of complete rings, a very high concentration of nanoparticles is required. The plasmon resonance absorption of the free nanoparticles in solution is often so large that it is difficult to observe changes in the UV−vis spectra of these solutions over time. This can be circumvented by subtracting the spectra of matching nanoparticle controls, containing all the constituents of the binding solution except the protein itself, to reveal previously unobserved spectral characteristics (Figure 4). The most distinct change is the growth of a new peak in the 570−630 nm region of the spectrum. If this peak is tracked over time, two-phase kinetics can be observed (Figure 4a). By taking TEM images at fixed intervals during the binding period, we observe that the majority of each ring is produced within the 631
dx.doi.org/10.1021/nl203368v | Nano Lett. 2012, 12, 629−633
Nano Letters
Letter
duplicate using top-down lithography. These 22 nm gold nanoparticle rings are stable in solution and can be deposited onto substrates of arbitrary shape and composition. In addition, we can integrate a central nanoparticle into the nanorings by manipulating the solution pH and thus tune the shape and intensity of the resulting LSPR spectrum. The ability to alter the coupling between the ring of nanoparticles and the central nanoparticle provides versatility in their opto-electronic properties. Future endeavors in the purification of these solutions will allow further spectroscopic characterization of ring structures at this scale and thus expand their potential applications in nanotechnology. Synthesis of BSPP-Passivated Gold Nanoparticles. As detailed by Murphy et al.,37 tetrachloroauric acid (2.5 × 10−4 M) and sodium citrate (2.5 × 10−4 M) in 20 mL of deionized water was reduced by adding 1 M of sodium borohydride dissolved in 0.6 mL with vigorous stirring to produce 3.5−5.0 nm nanoparticles. After 5 min, BSPP was dissolved in the solution to a concentration of 1 mg/mL. The nanoparticles were then stored at room temperature for 24 h in the dark. When ready to use, the nanoparticle solution was desalted using a 1 mg/mL solution of BSPP and then spin concentrated to 400 μL with a SH-3000BK free bucket rotor at 3000×g.38 Nanoparticle Ring Assembly and Analysis. One hundred microliters of concentrated BSPP passivated gold nanoparticles were added to 900 μL of TMV coat protein solution and stored in the dark at 23 °C for three days. UV−vis spectra in the 200−800 nm region were collected using a Cary 100 Bio instrument at the beginning and after the three day period. Kinetic data was collected for specific conditions by recording the UV−vis spectrum every 10 min for the three day period. Fluorescence spectra were collected using a Cary Eclipse fluorimeter at an excitation wavelength of 280 nm. TEM samples were plated on 200 mesh carbon-coated copper grids for 5 min before wicking using filter paper and stained using 1% uranyl acetate or 2% phosphotungstic acid. Images were collected using a Philips CM200 TEM at 200 kV. Analysis
Figure 5. Diagram illustrating the dominant species at each pH (pH 5.5 = rod, pH 6.5 = disk, pH 7.5 = A-protein). The red curve shows the increase in the ratio of deprotonated carboxylate groups to protonated carboxylate groups responsible for preventing nanoparticle binding in the center of the TMV disk.
In order to purify these structures, horizontal gel electrophoresis was used to separate free nanoparticles from the ring constructs (Figure 6). This was carried out on the pH 6.5 condition since it yielded the largest number of rings. The optical plasmon absorption of the nanoparticles allows the visualization of two bands, the slower moving, less concentrated rings and the faster, free nanoparticles in high concentration. Work is currently underway to extract the nanoring structures from the agarose gel without changing the concentration, ionic strength, and pH of the system so as not to disrupt the TMVcp−gold complexes. Analysis of the gel band intensities in conjunction with TEM image analysis indicates that an average of 7.8% of the total gold nanoparticles are incorporated into rings after 5 days at pH 6.5 (see Supporting Information, Figure S3). The presence of the S123C mutation will provide an additional handle to attach a maleimide dye in order to identify proteins that are bound and unbound to nanoparticles in an agarose gel36 and improve our yield calculations as this work progresses. Combining the intrinsic control and precision of a biological process with synthetic self-assembly techniques has produced ring structures on a scale that is prohibitively difficult to
Figure 6. Profile analysis of horizontal agarose gel for purification of binding solution at pH 6.5 applied by converting image to grayscale and plotting grayscale intensity over distance from well in pixels. 632
dx.doi.org/10.1021/nl203368v | Nano Lett. 2012, 12, 629−633
Nano Letters
Letter
(21) Endo, M.; Wang, H.; Fujitsuka, M.; Majima, T. Chemistry (Weinheim an der Bergstrasse, Germany) 2006, 12, 3735−40. (22) Bruckman, M. a; Kaur, G.; Lee, L. A.; Xie, F.; Sepulveda, J.; Breitenkamp, R.; Zhang, X.; Joralemon, M.; Russell, T. P.; Emrick, T.; Wang, Q. ChemBioChem 2008, 9, 519−23. (23) Lim, J.-S.; Kim, S.-M.; Lee, S.-Y.; Stach, E. a; Culver, J. N.; Harris, M. T. J. Colloid Interface Sci. 2010, 342, 455−61. (24) Raghavendra, K.; Salunke, D. M.; Caspar, D. L.; Schuster, T. M. Biochemistry 1986, 25, 6276−9. (25) Kurganov, B. I.; Rafikova, E. R.; Dobrov, E. N. Biochemistry (Moscow) 2002, 67, 525−33. (26) Shire, S. J.; Steckert, J. J.; Adams, M. L.; Schuster, T. M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2745−9. (27) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem 2000, 1, 18−52. (28) Ozbay, E. Science 2006, 311, 189−93. (29) Prodan, E.; Radloff, C.; Halas, N. J.; Nordlander, P. Science 2003, 302, 419−22. (30) Alù, a; Salandrino, A.; Engheta, N. Opt. Express 2006, 14, 1557− 67. (31) Alù, A.; Engheta, N. Phys. Rev. B 2008, 78, 1−10. (32) Smith, D. R.; Pendry, J. B.; Wiltshire, M. C. K. Science (New York, N.Y.) 2004, 305, 788−92. (33) Veselago, V. G. Physics-Uspekhi 1968, 10, 509−514. (34) Lu, B.; Stubbs, G.; Culver, J. N. Virology 1996, 225, 11−20. (35) Wang, H.; Planchart, a; Stubbs, G. Biophys. J. 1998, 74, 633−8. (36) Soto, C. M.; Blum, A. S.; Wilson, C. D.; Lazorcik, J.; Kim, M.; Gnade, B.; Ratna, B. R. Electrophoresis 2004, 25, 2901−6. (37) Gole, A.; Murphy, C. J. Chem. Mater. 2004, 16, 3633−3640. (38) Loweth, C. J.; Caldwell, W. B.; Peng, X.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. 1999, 38, 1808−1812.
of UV−vis and fluorescence data was carried out using MATLAB while TEM images were characterized using ImageJ.
■
ASSOCIATED CONTENT
S Supporting Information *
Detailed methods, expression of TMV coat protein, synthesis of gold nanoparticles, nanoparticle control UV−vis spectra, and analysis of ring yield. This material is available free of charge via the Internet at http://pubs.acs.org.
■ ■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
ACKNOWLEDGMENTS We thank Dr. M. Francis for the expression plasmid pTMVPC123S and Ki Yin Michael Chan for aiding in its expression and purification. We also thank Dr. R. B. Lennox, Dr. Paul Goulet, Dr. C. M. Soto, and Dr. B. R. Ratna for helpful discussions. We gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI) and the Centre for Self-Assembled Chemical Structures (CSACS).
■
REFERENCES
(1) Ariga, K.; Hill, J. P.; Lee, M. V.; Vinu, A.; Charvet, R.; Acharya, S. Sci. Technol. Adv. Mater. 2008, 9, 014109. (2) Tan, S. J.; Campolongo, M. J.; Luo, D.; Cheng, W. Nat. Nanotechnol. 2011, 6, 268−276. (3) Whitesides, G. M.; Boncheva, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4769−74. (4) Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418−21. (5) Kovtyukhova, N. Mater. Sci. Eng., C 2002, 19, 255−262. (6) Schlick, T. L.; Ding, Z.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 3718−23. (7) Dedeo, M. T.; Duderstadt, K. E.; Berger, J. M.; Francis, M. B. Nano Lett. 2010, 10, 181−6. (8) McMillan, R. A.; Paavola, C. D.; Howard, J.; Chan, S. L.; Zaluzec, N. J.; Trent, J. D. Nat. Mater. 2002, 1, 247−52. (9) Nam, K. T.; Kim, D.-W.; Yoo, P. J.; Chiang, C.-Y.; Meethong, N.; Hammond, P. T.; Chiang, Y.-M.; Belcher, A. M. Science 2006, 312, 885−8. (10) Blum, A. S.; Soto, C. M.; Wilson, C. D.; Brower, T. L.; Pollack, S. K.; Schull, T. L.; Chatterji, A.; Lin, T.; Johnson, J. E.; Amsinck, C.; Franzon, P.; Shashidhar, R.; Ratna, B. R. Small 2005, 1, 702−6. (11) Balci, S.; Bittner, a; Hahn, K.; Scheu, C.; Knez, M.; Kadri, a; Wege, C.; Jeske, H.; Kern, K. Electrochim. Acta 2006, 51, 6251−6257. (12) Tsukamoto, R.; Muraoka, M.; Seki, M. Chem. Mater. 2007, 2389−2391. (13) Górzny, M. Ł.; Walton, A. S.; Evans, S. D. Adv. Funct. Mater. 2010, 20, 1295−1300. (14) Miller, R. a; Presley, A. D.; Francis, M. B. J. Am. Chem. Soc. 2007, 129, 3104−9. (15) Ma, Y.-Z.; Miller, R. a; Fleming, G. R.; Francis, M. B. J. Phys. Chem. B 2008, 112, 6887−92. (16) Blum, A. S. A. S.; Soto, C. M. C. M.; Wilson, C. D. C. D.; Cole, J. D. J. D.; Kim, M.; Gnade, B.; Chatterji, A.; Ochoa, W. F. W. F.; Lin, T.; Johnson, J. E. J. E.; Ratna, B. R. Nano Lett. 2004, 4, 867−870. (17) Sapsford, K. E.; Soto, C. M.; Blum, A. S.; Chatterji, A.; Lin, T.; Johnson, J. E.; Ligler, F. S.; Ratna, B. R. Biosens. Bioelectron. 2006, 21, 1668−73. (18) Klug, A. Philos. Trans. Royal Soc. London, Ser. B 1999, 354, 531− 5. (19) Kegel, W. K.; van der Schoot, P. Biophys. J. 2006, 91, 1501−12. (20) Knez, M.; Sumser, M.; Bittner, A. M. a. M.; Wege, C.; Jeske, H.; Martin, T. P. T. P.; Kern, K. Adv. Funct. Mater. 2004, 14, 116−124. 633
dx.doi.org/10.1021/nl203368v | Nano Lett. 2012, 12, 629−633
