On the Thermal Stability of Surface-Assembled Viral-Metal

pubs.acs.org/Langmuir © 2010 American Chemical Society

On the Thermal Stability of Surface-Assembled Viral-Metal Nanoparticle Complexes Amy K. Manocchi,† Soenke Seifert,‡ Byeongdu Lee,*,‡ and Hyunmin Yi*,† †

Department of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, and ‡X-ray Science Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439 Received November 13, 2009. Revised Manuscript Received January 29, 2010 Biological supramolecules offer attractive templates for nanoparticle synthesis and nanodevice fabrication because of their precise size and shape. Viruses in particular have gained significant attention in nanodevice fabrication for applications such as nanoelectronics, batteries, catalysis, and sensing. However, the performance range of these viral-nanoparticle complexes is not well known because of the lack of fundamental studies on their properties. In this work, we employ in situ grazing incidence small-angle X-ray scattering (GISAXS) to examine the thermal stability of viral-nanoparticle complexes composed of tobacco mosaic virus (TMV) and palladium nanoparticles. Specifically, we show that the stability of the Pd nanoparticles on TMV is significantly enhanced as compared to that of particles on the solid substrate surface. Furthermore, we show that the agglomeration of Pd nanoparticles and the degradation of the TMV templates are coupled and occur simultaneously. These results demonstrate a potent methodology toward the in situ analysis of subtle changes in viral-nanoparticle complexes in dynamic environments. We envision that the results and methodology demonstrated in this study could be applied to better understand the properties and dynamic behaviors of organic-inorganic hybrid materials and nanodevices in various applications.

Introduction Biological supramolecules have recently gained significant attention as nanotemplates for inorganic material synthesis because of their controlled size and structure.1-5 Specifically, viruses ranging in shape from icosahedral6 to filamentous7,8 and tubular structures9,10 have been employed as templates for the assembly of fluorescent markers,11 quantum dots,8,12,13 and *To whom correspondence should be addressed. (B.L.) Telephone: (630) 252-0395. E-mail: [email protected]. (H.Y.) Telephone: (617) 627-2195. Fax: (617) 627-3991. E-mail: [email protected]. (1) Sotiropoulou, S.; Sierra-Sastre, Y.; Mark, S. S.; Batt, C. A. Chem. Mater. 2008, 20, 821–834. (2) Douglas, T.; Young, M. Science 2006, 312, 873–875. (3) Bigall, N. C.; Reitzig, M.; Naumann, W.; Simon, P.; Pee, K.-H. v.; Eychmuller, A. Angew. Chem. Int. Ed. 2008, 47, 7876–7879. (4) Deplanche, K.; Woods, R. D.; Mikheenko, I. P.; Sockett, R. E.; Macaskie, L. E. Biotechnol. Bioeng. 2008, 101, 873–880. (5) Douglas, T.; Young, M. Adv. Mater. 1999, 11, 679–681. (6) Douglas, T.; Young, M. Nature 1998, 393, 152–155. (7) Flynn, C. E.; Lee, S.-W.; Peelle, B. R.; Belcher, A. M. Acta Mater 2003, 51, 5867–5880. (8) Huang, Y.; Chiang, C.-Y.; Lee, S. K.; Gao, Y.; Hu, E. L.; Yoreo, J. D.; Belcher, A. M. Nano Lett 2005, 5, 1429–1434. (9) Knez, M.; Sumser, M. P.; Bittner, A. M.; Wege, C.; Jeske, H.; Hoffmann, D. M. P.; Kuhnke, K.; Kern, K. Langmuir 2004, 20, 441–447. (10) Niu, Z.; Bruckman, M. A.; Li, S.; Lee, L. A.; Lee, B.; Pingali, S. V.; Thiyagarajan, P.; Wang, Q. Langmuir 2007, 23, 6719–6724. (11) Yi, H.; Rubloff, G. W.; Culver, J. N. Langmuir 2007, 23, 2663–2667. (12) Lee, S.-W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892– 895. (13) Mao, C.; Flynn, C. E.; Hayhurst, A.; Sweeney, R.; Qi, J.; Georgiou, G.; Iverson, B.; Belcher, A. M. Science 2003, 100, 6946–6951. (14) Lee, S.-Y.; Choi, J.; Royston, E.; Janes, D. B.; Culver, J. N.; Harris, M. T. J. Nanosci. Nanotechnol. 2006, 6, 974–981. (15) Lee, S.-Y.; Royston, E.; Culver, J. N.; Harris, M. T. Nanotechnology 2005, 16, S435–S441. (16) Knez, M.; Bittner, A. M.; Boes, F.; Wege, C.; Jeske, H.; Mai, E.; Kern, K. Nano Lett. 2003, 3, 1079–1082. (17) Knez, M.; Sumser, M.; Bittner, A. M.; Wege, C.; Jeske, H.; Martin, T. P.; Kern, K. Adv. Funct. Mater. 2004, 14, 116–124. (18) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J. N.; Mann, S. Nano Lett. 2003, 3, 413–417.

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metal nanoparticles.14-18 The applications of these novel materials range from sensors19,20 and batteries21,22 to nanoelectronic devices21,23-25 and catalysis.26,27 Despite recent research endeavors aimed at understanding the stability of nanoparticles and hybrid nanomaterials,28-30 methods that allow for fundamental studies on the performance and physical properties of nanodevices under various working conditions are lacking, limiting the knowledge of their performance range. Furthermore, commonly employed nanoscale analytical techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) are traditionally ex situ (with some exceptions such as ESEM31 and in situ AFM32 (19) Tan, W. S.; Lewis, C. L.; Horelik, N. E.; Pregibon, D. C.; Doyle, P. S.; Yi, H. Langmuir 2008, 24, 12483–12488. (20) Srinivasan, K.; Cular, S.; Bhethanabotla, V. R.; Lee, S.-Y.; Harris, M. T. In Nanomaterial Sensing Layer Based Surface Acoustic Wave Hydrogen Sensors; Ultrasonics Symposium, 2005 IEEE; pp 645-648. (21) 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, 885–888. (22) Royston, E.; Ghosh, A.; Kofinas, P.; Harris, M. T.; Culver, J. N. Langmuir 2008, 24, 906–912. (23) Tseng, R. J.; Tsai, C.; Ma, L.; Ouyang, J.; Ozkan, C. S.; Yang, Y. Nat. Nano. 2006, 1, 72–77. (24) Bromley, K. M.; Patil, A. J.; Perriman, A. W.; Stubbs, G.; Mann, S. J. Mater. Chem. 2008, 18, 4796–4801. (25) Mao, C.; Solis, D. J.; Reiss, B. D.; Kottmann, S. T.; Sweeney, R. Y.; Hayhurst, A.; Georgiou, G.; Iverson, B.; Belcher, A. M. Science 2004, 303, 213– 217. (26) Avery, K. N.; Schaak, J. E.; Schaak, R. E. Chem. Mater. 2009, 21, 2176– 2178. (27) Yang, C.; Manocchi, A. K.; Lee, B.; Yi, H. Appl. Catal., B 2010, 93, 282– 291. (28) Diallo, A. K.; Ornelas, C.; Salmon, L.; Aranzaes, J. R.; Astruc, D. Angew. Chem., Int. Ed. Engl. 2007, 46, 8644–8648. (29) Winans, R.; Vajda, S.; Ballentine, G.; Elam, J.; Lee, B.; Pellin, M.; Seifert, S.; Tikhonov, G.; Tomczyk, N. Top. Catal. 2006, 39, 145–149. (30) Carino, E. V.; Knecht, M. R.; Crooks, R. M. Langmuir 2009, 25, 10279– 10284. (31) Donald, A. M. Nat. Mater. 2003, 2, 511–516. (32) McPherson, A.; Malkin, A. J.; Kuznetsov, Y. G. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 361–410.

Published on Web 02/16/2010

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in static environments) and often require sample preparation and thus are not suitable for in situ examination to monitor subtle and dynamic changes on the nanoscale. Therefore, there is a critical need for dynamic analyses conducted with simple and nondestructive in situ monitoring methodology that is capable of detecting small changes in viral-inorganic hybrid materials or nanodevices on the nanoscale. We exploit tubular viral nanotemplates for the readily controllable synthesis of palladium (Pd) nanoparticles. Wild-type tobacco mosaic virus (TMV) is a rigid tubular plant virus, 18 nm in diameter and 300 nm in length with a 4-nm-diameter inner channel. It is composed of 2130 identical coat proteins helically assembled around a single genomic RNA strand.33 Specifically, we utilize genetically engineered TMV1cys, which displays one cysteine group on the outer surface of each coat protein subunit, as shown in the schematic diagram of Figure 1a. These abundant and precisely spaced thiol functionalities rising from the cysteines enable tunable surface assembly and enhanced Pd nanoparticle formation to form viral-metal nanoparticle complexes, as shown in the schematic diagram of Figure 1b and the AFM images of Figure 1c,d. In a previous study, we employed grazing incidence small-angle X-ray scattering (GISAXS) to examine the nanoparticle size and size distribution of Pd nanoparticles formed on TMV1cys and demonstrated a wide range of nanoparticle sizes that were tunable on the basis of the reducing agent concentration.34 Importantly, GISAXS offers several advantages that make it suitable for the in situ examination of dynamic changes in complex nanoscale objects. First, it is nondestructive to the sample because the measurement does not involve physical contact as in AFM or extensive sample preparation procedures such as the coating of thin metals for SEM. Second, the featuresize range from 1 to 60 nm is easily covered in GISAXS and is useful for a variety of nanoscale objects. Third, abundant information can be gathered in real time in a highly programmable manner with a time resolution of better than 10 ms with the help of a synchrotron radiation X-ray source and modern detector technologies.35 GISAXS produces statistically meaningful measurements of the average properties of the surface by utilizing a sizable sampling area with a large beam size (∼0.5
5 mm2 in this study). Most importantly, small-angle X-ray scattering techniques have been shown to be a very accurate method for examining nanoscale features, as compared to TEM.36,37 These advantages have made GISAXS a powerful tool that has been extensively employed in polymer and material science fields as well as in monitoring the growth of nanoparticles in real time.29,38-44 However, the study of dynamic behavior of (33) Culver, J. N. Annu. Rev. Phytophathol 2002, 40, 287–308. (34) Manocchi, A. K.; Horelik, N. E.; Lee, B.; Yi, H. Langmuir 2010, in press, DOI: 10.1021/la9031514. (35) Macfarlane, R. J.; Lee, B.; Hill, H. D.; Senesi, A. J.; Seifert, S.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 10493–10498. (36) Rieker, T.; Hanprasopwattana, A.; Datye, A.; Hubbard, P. Langmuir 1999, 15, 638–641. (37) Christensen, S. T.; Lee, B.; Feng, Z.; Hersam, M. C.; Bedzyk, M. J. Appl. Surf. Sci. 2009, 256, 423–427. (38) Renaud, G.; Lazzari, R.; Revenant, C.; Barbier, A.; Noblet, M.; Ulrich, O.; Leroy, F.; Jupille, J.; Borensztein, Y.; Henry, C. R.; Deville, J.-P.; Scheurer, F.; Mane-Mane, J.; Fruchart, O. Science 2003, 300, 1416–1419. (39) Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. Nature 2008, 451, 553–556. (40) Leroy, F.; Revenant, C.; Renaud, G.; Lazzari, R. Appl. Surf. Sci. 2004, 238, 233–237. (41) Biswas, K.; Varghese, N.; Rao, C. N. R. Small 2008, 4, 649–655. (42) Winans, R. E.; Vajda, S.; Lee, B.; Riley, S. J.; Seifert, S.; Tikhonov, G. Y.; Tomczyk, N. A. J. Phys. Chem. B 2004, 108, 18105–18107. (43) Lee, B.; Yoon, J.; Oh, W.; Hwang, Y.; Heo, K.; Jin, K. S.; Kim, J.; Kim, K.-W.; Ree, M. Macromolecules 2005, 38, 3395–3405. (44) Gibaud, A.; Dourdain, S.; Gang, O.; Ocko, B. M. Phys. Rev. B 2004, 70, 161403.

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Figure 1. In situ examination of the thermal stability on viralnanoparticle complexes. (a) Chimera simulation of a portion (400 proteins) of genetically engineered TMV1cys. Red dots represent cysteine groups genetically displayed on the outer surface of each coat protein. (b) Schematic diagram depicting TMV1cys assembly onto gold surfaces, followed by the reductive metallization of Pd precursor in a mild reducing agent (sodium hypophosphite) to form Pd nanoparticles on the TMV1cys templates. (c) AFM image of surface-assembled TMV1cys on a gold chip. (d) AFM image of Pd nanoparticles formed on surface-assembled TMV1cys. (e) Schematic diagram of in situ thermal stability examination via GISAXS. The chip sample is irradiated at an incident angle Ri, and the scattered rays are measured as a function of 2θ and Rf.

viral-inorganic complexes via in situ GISAXS remains uninvestigated to date. In this article, we report in situ monitoring of the thermal stability of viral-metal nanoparticle complexes. Specifically, we enlisted in situ GISAXS to examine the scattering patterns of four different complex types: TMV1cys on a gold chip (TMV chip), Pd nanoparticles on a gold chip (Pd-Au chip), TMV1cys with large Pd nanoparticles, and TMV1cys with small Pd nanoparticles (Pd-TMV chips), all assembled on solid substrates. As shown in Figure 1b, we first assembled TMV1cys onto clean gold surfaces and then synthesized large or small Pd nanoparticles34 on the TMV1cys template surface via the reduction of Pd precursor with a mild reducing agent. The samples were heated in a sample cell purged with helium gas, and their scattering patterns were measured throughout the course of heating via GISAXS, as shown in the schematic diagram of Figure 1e. The initial examination of the scattering images showed substantial changes in scattering over the course of heating, which demonstrates the strong potential of the GISAXS technique for in DOI: 10.1021/la904324h

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situ monitoring of dynamic changes in viral-metal nanoparticle complexes. A further in-depth examination showed that smallparticle complexes sinter at a lower temperature than largeparticle complexes. Additionally, we show that the surfaceassembled TMV1cys structure degrades at a higher temperature than reported previously for TMV in liquid solution.45 Finally, we show that the stability of the Pd nanoparticles is significantly enhanced by TMV, as compared to particles formed on the substrate surface. Combined, these results demonstrate, for the first time, the in situ analysis of the thermal stability of viralnanoparticle complexes. We envision that the methodology and results shown in this study could be applied to study dynamic nanoscale behaviors in a wide range of complex nanodevices and hybrid nanomaterials such as viral-nanoparticle complexes.

Materials and Methods Reagents. Acetone (HPLC grade), isopropanol, and methanol were used as received (all from Fisher Scientific, Waltham, MA). Sodium tetrachloropalladate(II) (Na2PdCl4) was used as the Pd precursor for Pd nanoparticle formation (Sigma-Aldrich, St. Louis, MO). Precursor reduction was conducted using sodium hypophosphite (Sigma-Aldrich). TMV1cys Surface Assembly on Gold Chips. TMV1cys was generously provided by Dr. James N. Culver at the University of Maryland Biotechnology Institute, Center for Biosystems Research. Gold-coated silicon wafers (Platypus, Madison, WI) were cut into small chips (about 1 cm
2 cm) and cleaned sequentially using acetone, isopropanol, and methanol for 20 min each, with thorough rinsing with deionized water between steps. The chips were then dried in a stream of ultrapure nitrogen gas and etched with plasma for 3 min (Ernest F. Fullam Inc., Clifton Park, NY). Immediately after etching, the chips were incubated in 100 μg/mL TMV1cys in 0.01 M sodium phosphate (pH 7) buffer overnight at room temperature. After TMV1cys binding, the chips (TMV chip) were thoroughly rinsed with deionized water, dried in a stream of ultrapure nitrogen gas, and stored dry at room temperature until metallization or GISAXS studies.

Palladium Nanoparticle Formation on TMV1cys Templates. Palladium nanoparticles were formed on the TMV1cys templates through the reductive metallization of the palladium precursor, Na2PdCl4, in aqueous sodium hypophosphite solutions. The Pd-TMV chips were prepared using different concentrations of reducing agent, which controls the Pd nanoparticle size, as previously reported.34 TMV1cys-bound gold chips (TMV chip) were incubated vertically in 0.5 mM Na2PdCl4 and sodium hypophosphite solution for 20 min in a microcentrifuge tube. The metallized TMV chips (Pd-TMV chips) were then thoroughly rinsed in deionized water for 5 min after metallization and dried in a stream of nitrogen gas. The large nanoparticle sample (15 nm particles on TMV1cys) was formed using 10 mM sodium hypophosphite, and the small nanoparticle sample (4 nm particles on TMV1cys) was formed using 45 mM sodium hypophosphite. The Pd nanoparticle sample formed on gold in the absence of TMV1cys (Pd-Au chip) was formed by reducing 0.5 mM Na2PdCl4 with 15 mM sodium hypophosphite in the presence of a clean gold chip.

In Situ Grazing Incidence Small-Angle X-ray Scattering (GISAXS). GISAXS measurements were conducted at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) BESSRC/XOR 12 ID-C beamline. Samples were mounted in a homemade portable cubic chamber equipped with a boron nitride heater and two window ports transparent to the X-ray beam. The sample cell was purged with helium gas to a pressure of 800 Torr, and the sample was heated from room (45) Price, W. C. Arch. Virol. 1940, 1, 373–386. (46) Guinier, A.; Fournet, G. Small Angle Scattering of X-rays; John Wiley and Sons: New York, 1955.

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temperature to 300
C at a rate of 5
C/min using a LakeShore 340 temperature controller (LakeShore, Westerville, OH). The beam was irradiated on the sample at an incident angle (Ri) of 0.2
for the pure TMV sample and 0.1
for all other samples, as shown in Figure 1e. The scattered X-rays were measured every 20
C and collected on a CCD detector (Rayonix; Mar165), with a sampleto-detector distance of approximately 2 m. Strong scattering and incident beam reflections in the Rf direction were blocked using a vertically mounted beamstop between the sample and detector. The beam energy was 12 keV. Theoretical considerations are articulated in detail in the Supporting Information. Atomic Force Microscopy. Atomic force microscopy (AFM) images shown in Figures 1 and 5 were acquired using a Dimension 3100 series scanning probe microscope (SPM) (Veeco, Woodbury, NY). Images were analyzed using NanoScope software. All AFM measurements were conducted in tapping mode with TAP-Al-50 AFM tips (Budget Sensors, Sofia, Bulgaria). Chimera Simulation of TMV1cys. The Chimera drawing of TMV1cys was produced using the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera) from the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco.48 The TMV structure (PDB ID: 2tmv)49 was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB, http://www.pdb.org).50

Results and Discussion Representative GISAXS Images Throughout Heating. As shown in Figure 2, we first show the suitability of the in situ GISAXS technique for the investigation of the thermal stability of the Pd-TMV complexes. For this, we examined four chips (a TMV chip, a Pd-TMV chip with small nanoparticles, a Pd-TMV chip with large nanoparticles, and a Pd-Au chip) by placing each on a heater and recording their GISAXS images throughout heating (5
C/min). Figure 2 shows representative scattering images at the start of heating, at the temperature where the most drastic change in the pattern starts to occur, and at the end of heating. First, Figure 2a shows GISAXS images of a TMV chip throughout heating. Initially, the TMV scattering image shows strong scattering in the vertical (Rf) direction, which is characteristic of TMV. As heating progresses, the overall intensity decreases. Next, Figure 2b shows representative scattering images of a Pd-TMV chip with small (4 nm) particles. At low temperature, the scattering image shows strong isotropic scattering in the horizontal direction arising from the spherical nanoparticles as well as vertical scattering resulting from the TMV. Throughout heating, the shape of the scattering image becomes narrower and the intensity continues to decrease. Figure 2c shows scattering images of a Pd-TMV chip with large (15 nm) particles. Similar to the small-particle chip shown in Figure 2b, the Pd-TMV chip with large particles initially shows strong horizontal scattering from the spherical nanoparticles, with the scattering image shape becoming narrower as heating progresses. Finally, Figure 2d shows scattering images of a Pd-Au chip, where the initial scattering image shows a narrow scattering shape that becomes narrower and decreases in intensity as heating progresses. (47) Roe, R.-J. Methods of X-ray and Neutron Scattering in Polymer Science; Oxford University Press: New York, 2000. (48) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605–1612. (49) Namba, K.; Pattanayek, R.; Stubbs, G. J. Mol. Biol. 1989, 208, 307–325. (50) Bhat, T. N.; Bourne, P.; Feng, Z.; Gilliland, G.; Jain, S.; Ravichandran, V.; Schneider, B.; Schneider, K.; Thanki, N.; Weissig, H.; Westbrook, J.; Berman, H. M. Nucleic Acids Res. 2001, 29, 214–218.

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Figure 2. Representative GISAXS scattering images of intensity as a function of scattering angle initially, at the temperature of greatest change and at the end of heating for (a) a TMV chip, (b) a Pd-TMV chip with 4 nm Pd particles, (c) a Pd-TMV chip with 15 nm Pd particles, and (d) a Pd-Au chip.

Upon closer examination, the overall intensity of the scattering images decreased for each sample over the course of heating, indicating that the scattering objects in the measurable size range of GISAXS (less than 60 nm in this experiment, dmax =2π/qmin) decreased. For the TMV sample, the decrease in intensity arises from the destruction of the TMVs on the chip surface. In the case of Pd nanoparticle scattering, the width in the horizontal direction of the scattering-image shape generally indicates the size of the nanoparticles, where narrower scattering indicates larger nanoparticles (Supporting Information).46 Initially, the Pd-TMV samples are highest in scattering intensity, as compared to the Pd-Au sample, indicating that there are substantially more Pd nanoparticles present on the Pd-TMV chip than on the Pd-Au chip.27 Additionally, the Pd-Au chips show the narrowest scattering images among all of the chips containing Pd nanoparticles, meaning that the particles on the gold chips are far larger than those on TMV chips. As heating progresses, the scattering image shapes of Pd-TMV chips become narrower, indicating that the nanoparticles agglomerated and increased in size. At the end of heating, the shapes of the scattering images are very narrow with an overall decrease in intensity, indicating that the agglomerated Pd nanoparticles are too large to be measured by GISAXS. In summary, these clear changes in the scattering images throughout the heating experiments demonstrate the applicability of in situ GISAXS to measuring the changes in the viral-nanoparticle complexes. Langmuir 2010, 26(10), 7516–7522

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Analysis of Scattering Curves. Whereas the changes in the GISAXS images in Figure 2 give clear qualitative information on the changes in the system, an in-depth analysis using scattering “curves” acquired from those scattering images provides extensive quantitative information, as shown in Figure 3. For this, scattering curve plots were created by making horizontal line cuts (shown by the black line in Figure 2a) of all scattering images and plotting intensity as a function of scattering vector qxy (qxy = 4π sin θ/λ). Specifically, Figure 3a-d shows overlays of scattering curves from four chip samples throughout heating, allowing us to determine the specific temperature where the scattering pattern undergoes substantial changes, which indicates the onset of major scattering species’ destruction. First, the scattering curves of a TMV chip are shown in Figure 3a. The initial scattering curve of TMV (top red line) shows oscillations at high qxy values (two of the minima are indicated by arrows) characteristic of monodisperse TMV nanotubes.51 Importantly, these oscillations are observed only when the objects are monodisperse in size, where the oscillation frequency is inversely proportional to the diameter of the object measured (Supporting Information).46 As heating reaches 118
C (black dotted line), the oscillations from the TMV monodispersity are no longer present; however, the general curve shape remains the same. This indicates that the TMV tubes are damaged, resulting in TMV cross sections that are not monodisperse, yet the average size of the TMVs remains the same. At 129
C, the curve shape is changed to show a significant signal at even lower qxy, with no oscillations present. The lack of oscillations suggests that the TMVs have lost their short distance structure completely, and the increased signal at lower qxy indicates the division of the long tubuar shape to shorter pieces. The scattering-curve shape changes significantly at 209
C (black dashed line), with the scattering at low qxy decreased, indicating that the TMV nanotubular structure is completely destroyed. TMV is a multiprotein structure assembled via noncovalent interactions among the protein subunits with the genomic RNA.33 Although the thermal decomposition of individual covalent chemical linkages may occur at various temperatures,52-54 we note that these observations via GISAXS represent “morphological” phenomena by the scattering species, which do not appear to occur up to 118
C. Next, Figure 3b shows scattering curves of a Pd-TMV chip with small Pd nanoparticles. The bumplike scattering features (indicated by two arrows) in the top curve in the log-log plot of intensity versus qxy in Figure 3b demonstrate the presence of both TMV and Pd nanoparticles. The first bump, at qxy =0.03 A˚-1, arises from TMV, where the presence of this feature indicates evenly coated nanoparticles on TMV forming a metallic shell. The bumplike scattering at qxy= 0.13 A˚-1 is the characteristic scattering pattern of polydisperse objects, in this case, nanoparticles. Nanoparticle size can be estimated from the position of the bump, qxy*, using the Bragg equation, where d=2π/qxy*.47 The first bump appearing at lower qxy for the Pd-TMV complex indicates a larger cross section due to the Pd shell.34 The TMV scattering feature at low qxy is comparable to the initial TMV curve in Figure 3a; however, the difference is apparent at high qxy where nanoparticle scattering dominates and the characteristic (51) Lee, B.; Lo, C.-T.; Thiyagarajan, P.; Winans, R. E.; Li, X.; Niu, Z.; Wang, Q. Langmuir 2007, 23, 11157–11163. (52) Yablokov, V. A.; Vasina, Y. A.; Zelyaev, I. A.; Mitrofanova, S. V. Russ. J. Gen. Chem. 2009, 79, 1141–1145. (53) Soares, R. M. D.; Scremin, F. F.; Soldi, V. Macromol. Symp. 2005, 229, 258–265. (54) Razvi, A.; Scholtz, J. M. Protein Sci. 2006, 15, 1569–1578.

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Figure 3. log-log scattering curves of intensity vs scattering vector qxy for (a) a TMV chip, (b) a Pd-TMV chip with small (4 nm) Pd nanoparticles, (c) a Pd-TMV chip with large (15 nm) Pd nanoparticles, and (d) a Pd-Au chip.

TMV oscillations are no longer visible. The important difference between TMV and the Pd-TMV complex is its thermal behavior as heating progresses. There is no significant change in scattering from the cross section of the Pd-TMV complex, which is observed at low qxy, up to 217
C, which is substantially higher than the temperature where the pure TMV structure begins to degrade. The Pd nanoparticle scattering at high qxy also does not change significantly up to 217
C. At 240
C (black dashed line), both signals from the cross section of the complex and nanoparticles disappear and the scattering curve resembles a straight line in the log-log plot. This straight line represents typical Porod scattering of polydisperse particles that are larger than the detection limit, which is 60 nm in this work.47 Additionally, further Guinier analysis shows that the nanoparticle diameter is constant up to 240
C (dotted line), when the scattering image changes significantly and the overall structure of the Pd-TMV complex changes drastically (data not shown). Importantly, the disappearance of the TMV feature and the nanoparticle feature occurs at roughly the same temperature, possibly indicating that nanoparticle agglomeration is coupled with TMV structure degradation. 7520 DOI: 10.1021/la904324h

Next, Figure 3c shows GISAXS scattering curves for a Pd-TMV chip with large Pd nanoparticles. Initially, the scattering curve (top red line) shows a bump at qxy=0.04 A˚-1 (indicated by an arrow) corresponding to 15-nm-diameter particles (Supporting Information).34 Scattering from the cross section of the Pd-TMV complex is not observable for this sample because nanoparticle scattering dominates and the TMV scattering features are not observable. This indicates that Pd nanoparticles that are as large as the cross section of TMV are randomly located on the TMV surface and that their distribution does not appear to form a homogeneous shell shape. If the nanoparticles were forming a homogeneous shell on TMV, then characteristic TMV scattering features such as oscillations would be observable.34 The shapes of the scattering curves measured throughout heating do not change at all until 279
C, indicating that 15 nm particles are present until this temperature. Above 279
C, the signal is no longer present, indicating that the 15 nm particles have agglomerated to a size larger than is measurable with this GISAXS setup. Importantly, further Guinier analysis (Supporting Information)46 shows that the change in nanoparticle diameter is negligible up to 279
C (dotted line), when the Langmuir 2010, 26(10), 7516–7522

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Figure 4. Plot of normalized intensity at the nanoparticle bumplike scattering location, qxy*, vs temperature for the 15 nm particles (red circles), the 4 nm particles (blue diamonds), and the particleTMV shell structure for the small-particle Pd-TMV chip (green squares).

major change in the scattering pattern, and therefore the overall structure and particle size, begins (data not shown). Finally, Figure 3d shows scattering curves of Pd nanoparticles formed on gold chips in the absence of TMV. The first scattering curve (top red line) shows a very broad bump at approximately qxy = 0.03 A˚-1 and power-law-type scattering (logarithmic intensity decays linearly with logarithmic q) indicating that the sizes of some particles are in the range of 20 nm with high polydispersity and a large fraction of the rest have an even larger size. As heating progresses, the bump feature shifts to lower qxy, indicating the growth of the nanoparticles. By 214
C (dotted black line), the bump feature has disappeared, indicating that the Pd nanoparticles whose size was in the range of 20 nm have grown outside the measurable range. It is clear from the scattering curves in Figure 3 that the TMV structure degrades at a significantly lower temperature than the structure of the Pd-TMV complex. We also observe that the larger nanoparticles formed on TMV are more thermally stable than the smaller particles. Additionally, the Pd particles formed on the gold surface are larger and more polydisperse than both samples of TMV-templated particles but agglomerate at a lower temperature (214
C) than the TMV-templated particles (240 and 279
C for small and large particles, respectively), suggesting that the nanoparticle stability is enhanced by TMV. In summary, the GISAXS analysis using scattering curves in Figure 3 shows that we are able to pinpoint the specific temperature where substantial degradation of the complexes is initiated. Changes in the Scattering Intensity at the Nanoparticle Scattering Locations. In addition to the line-cut plots that provide information about the particle size, the relative number of particles can also be examined by plotting the scattering intensity at the bump location, qxy*, as a function of temperature, as shown in Figure 4. For this, the scattering intensity at the nanoparticle bump location for each series of scattering curves for the two Pd-TMV chips (small and large particles) was extracted at qxy*= 0.13 and 0.04 A˚-1, respectively, and at the metallic shell location (qxy*=0.03 A˚-1) for the small-particle chip for the data in Langmuir 2010, 26(10), 7516–7522

Figure 5. AFM images of (a) a TMV chip, (b) a Pd-TMV chip with small nanoparticles, (c) a Pd-TMV chip with large nanoparticles, and (d) a Pd-Au chip after heating.

Figure 3b,c. The normalized scattering intensity with respect to the initial intensity was plotted as a function of temperature. The normallized intensity in Figure 4 is thus proportional to the number fraction of nanoparticles, which remained constant. The data for the small-particle sample shows fluctuations in intensity because the signal at high qxy is weak and noisy, as seen in Figure 3b. The disappearance of both particle sizes and the metallic shell structure occurs rapidly at 200
C, which is consistent with the destruction temperature of TMV (shown in Figure 3a). This result further confirms the outcome of the analysis of line-cut plots in Figure 3 and suggests the coupling of particle degradation with the destruction of TMV. Importantly, the sintering kinetics of the particles on TMV appears to be quite unique as compared to that for particles on solid substrates reported in other studies, where the thermal stability of Pd nanoparticles ranged from 100
C to over 300
C.55-57 We observe slightly higher thermal stability for the larger particles on TMV than for the smaller particles on TMV. The scattering intensity of the smaller particles decreases earlier in the heating than does the scattering intensity of the larger particles. In other words, the scattering intensity arising from the smaller particles decreases to half of the normalized intensity earlier during heating than for the larger particles, indicating that more small particles agglomerate at a lower temperature than larger particles. In general, larger particles are more thermodynamically stable, as explain by the Gibbs-Thomson relation that shows that the melting temperature is proportional to particle size (eq 1, where TM is the melting temperature, TM(¥) is the bulk melting temperature, FS is the density of the particle, ΔHF is the latent (55) Sullivan, J. A.; Flanagan, K. A.; Hain, H. Catal. Today 2009, 145, 108–113. (56) Heemeier, M.; Stempel, S.; Shaikhutdinov, S. K.; Libuda, J.; Baumer, M.; Oldman, R. J.; Jackson, S. D.; Freund, H.-J. Surf. Sci. 2003, 523, 103–110. (57) Campbell, C. T.; Parker, S. C.; Starr, D. E. Science 2002, 298, 811–814. (58) Kim, M. H.; Lee, B.; Lee, S.; Larson, C.; Baik, J. M.; Yavuz, C. T.; Seifert, S.; Vajda, S.; Winans, R. E.; Moskovits, M.; Stucky, G. D.; Wodtke, A. M. Nano Lett 2009, 9, 4138–4146.

DOI: 10.1021/la904324h

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heat of fusion, d is the particle diameter, and σ is the liquid-solid interface energy).58 ΔTM ¼ TM -TM ð¥Þ ¼

4σTM dΔHF FS

ð1Þ

On the basis of this explanation, it is expected that the smaller nanoparticles supported on TMV will agglomerate at a lower temperature than in the larger-particle case. In conclusion, the observed dynamic changes shown above suggest the coupling of the nanoparticle stability with the stability of the viral nanotemplates. It appears that the thermal stability of the TMV structure is enhanced by the Pd nanoparticles, as shown in Figure 3b where the degradation temperature of TMV is increased to 240
C from 209
C. Similarly, the stability of the Pd nanparticles is greatly enhanced by the presence of TMV, as shown by the higher thermal stability of the particles on TMV (Figures 3b,c and 4) as compared to that of the larger (and theoretically more thermally stable) particles on gold (Figure 3d). Note that 20 nm Pd on bare gold has sintered by 217
C whereas 15 nm Pd on TMV is stable above that temperature. This indicates that preventing the diffusion of Pd nanoparticles by anchoring them onto TMV could improve the stability of the Pd nanoparticles against sintering. In other words, the viral-metal nanoparticle complexes start to degrade at the TMV structure’s degradation temperature as shown in Figure 4, but the stability of both the TMV structure and the nanoparticles is significantly enhanced by the viral-metal complex: 240 and 279
C in Figure 3b,c, respectively, versus 209 and 214
C in Figure 3a,d respectively. AFM Examination of Degraded Surfaces. Next, we physically examined the destruction of the Pd-TMV complex and the aggregation of Pd nanoparticles via AFM to confirm the final observations made via GISAXS, as shown in Figure 5. For this, all chips were examined using tapping-mode AFM after heating was complete without further rinsing or sample treatment. First, Figure 5a shows an AFM image of a TMV chip after heating. The surface resembles a clean gold chip, where the mildly rough granular curvature of the gold surface is visible. Importantly, the clear tubular structures of the surface-bound TMVs are no longer visible after the heating experiment, in contrast to the samples prior to heating (Figure 1c) indicating the disappearance of the TMVs through heating. Next, Figure 5b,c shows the surfaces of the Pd-TMV chips with small and large particles, respectively, after heating to 300
C. Both surfaces are densely covered with small and large randomly organized particles. Importantly, there are no distinct nanotubular structures of TMVs visible on either surface as in Figure 1d before heating, clearly indicating the destruction of the Pd-TMV complexes. It is evident from these AFM results that all viruses and viralnanoparticle complexes are destroyed below 300
C. Finally, Figure 5d shows an AFM image of a Pd-Au chip after heating. The surface of the Pd-Au chip resembles the surface of a clean gold chip, with a few clusters of large particles randomly dispersed on the surface. The surface of the Pd-Au chip appears to have significantly fewer agglomerated particles than the Pd-TMV chips, as expected by the significantly fewer Pd nanoparticles initially formed on the chip as compared to the number on the Pd-TMV chips.27 Importantly, it is clear that in all Pd samples the nanoparticles agglomerated during heating, further confirming the GISAXS results and the disappearance of scattering species in the GISAXS-measurable range. In conclusion, the AFM images in Figure 5 indicate the thermal degradation of 7522 DOI: 10.1021/la904324h

the virus and viral-nanoparticle complexes, confirming the GISAXS-based results in Figures 2-4.

Conclusions In this article, we examined the thermal stability of viralnanoparticle complexes assembled on solid substrates via in situ GISAXS to monitor subtle dynamic changes on the nanoscale. The GISAXS images clearly showed that the samples changed significantly over the course of the heating experiments and that these changes were readily detectable with GISAXS. The GISAXS curves allowed us to pinpoint the temperatures at which each sample was destroyed. First, it was observed that the TMV structure degraded at a higher temperature (about 200
C) than previously reported for TMV in aqueous solution, and its thermal stability was further enhanced by the Pd nanoparticles formed on the TMV surface. Similarly, it was shown that the thermal stability of the Pd nanoparticles was greatly enhanced by TMV, as the degradation temperature of the particles were increased from 214
C on gold chips to 240 and 279
C for small and large particles on TMV, respectively (Figure 3). Also, the degradation of the TMV and Pd nanoparticles appeared to be coupled because the particles begin to agglomerate at the TMV degradation temperature. This is shown by the drop in intensity at the nanoparticle bump location in the Pd-TMV scattering-curve plots when the samples were heated to the TMV degradation temperature, which was 209
C in this case (Figure 4). Further ex situ AFM examination of the degraded samples (Figure 5) showed the clear disappearance of TMV’s nanotubular structures and the presence of large particles, confirming our GISAXSbased observations. Whereas we focused on reporting major structural changes in this study, a detailed nanoparticle size change can also be readily examined via Guinier analysis.34,46 Specifically, no notable change in the particle diameter on the nanometer scale was observed until the major structural changes started to occur in all of the samples examined. Combined, these results demonstrate the applicability of the in situ GISAXS method in monitoring subtle dynamic changes in the Pd-TMV complexes over the course of heating and provide insights into the thermal stability of such hybrid nanomaterials. We believe that the methodology and results reported here represent a significant first step torward understanding the dynamic behavior of viralinorganic hybrid materials and nanodevices, and we envision future endeavors in nanobiological and functional hybrid materials research for a wide range of applications such as batteries, sensors, catalysts, and nanoelectronics. Acknowledgment. We gratefully acknowledge Dr. James N. Culver at the University of Maryland Biotechnology Institute, Center for Biosystems Research for his generous gift of TMV1cys. The in situ GISAXS work conducted at Argonne National Laboratory was supported by the U.S. Department of Energy, BES-Chemical Sciences and BES-Scientific User Facilities under contract DE-AC-02-06CH11357 with UChicago Argonne, LLC, operator of Argonne National Laboratory. Partial funding for this work was provided by the Wittich Family Fund for Energy Sustainability. Supporting Information Available: A detailed introduction into SAXS and GISAXS techniques and detailed theoretical considerations for the interpretation of the results presented in this work. This material is available free of charge via the Internet at http://pubs.acs.org. Langmuir 2010, 26(10), 7516–7522