Self-Assembly of Tobacco Mosaic Virus at Oil ... - ACS Publications

and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, ... Xuefei WuQingqing YuanShuyu LiuShaowei ShiThomas P. RussellDong Wan...
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Self-Assembly of Tobacco Mosaic Virus at Oil/Water Interfaces

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Jinbo He,† Zhongwei Niu,‡ Ravisubhash Tangirala,† Jia-Yu Wang,† Xinyu Wei,† :: :: Gagandeep Kaur,‡ Qian Wang,‡ Gunther Jutz,§ Alexander Boker,§,^ Byeongdu Lee, Sai Venkatesh Pingali, Pappannan Thiyagarajan, Todd Emrick,† and Thomas P. Russell*,† †

Department of Polymer Science & Engineering, University of Massachusetts, Amherst, Massachusetts 01003, Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, :: :: Columbia, South Carolina 29208, §Lehrstuhl fur Physikalische Chemie II, Universitat Bayreuth, 95440 :: ^ Bayreuth, Germany, DWI an der RWTH Aachen e.V. and Lehrstuhl fur makromole kulare materialien and :: oberflachen, RWTH Aachen university, 52056 Aachen, Germany, and Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439 )

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Received October 23, 2008. Revised Manuscript Received January 30, 2009 The oil/water interfacial assembly of tobacco mosaic virus (TMV) has been studied in situ by tensiometry and small-angle X-ray and neutron scattering (SAXS and SANS). TMV showed different orientations at the perfluorodecalin/water interface, depending on the initial TMV concentration in the aqueous phase. At low TMV concentration, the rods oriented parallel to the interface, mediating the interfacial interactions at the greatest extent per particle. At high TMV concentrations, the rods were oriented normal to the interface, mediating the interfacial interactions and also neutralizing inter-rod electrostatic repulsion. We found that the inter-rod repulsive forces between TMVs dominated the in-plane packing, which was strongly affected by the ionic strength and the bulk solution but not by the pH in the range of pH = 6-8.

Introduction The self-assembly of nanoscale objects into ordered structures has attracted significant attention for generating novel optical, electronic, and magnetic materials and devices.1-3 The use of liquid interfaces as platforms to assemble inorganic nanoparticles has been reported.1-12 Recently, bionanoparticles, such as ferritin, cowpea mosaic virus (CPMV), and tobacco mosaic virus *Corresponding author. E-mail: [email protected]. (1) Duan, H. W.; Wang, D. A.; Kurth, D. G.; Mohwald, H. Angew. Chem., Int. Ed. 2004, 43, 5639. (2) Reincke, F.; Hickey, S. G.; Kegel, W. K.; Vanmaekelbergh, D. Angew. Chem., Int. Ed. 2004, 43, 458. (3) Aveyard, R.; Binks, B. P.; Clint, J. H. Adv. Colloid Interface Sci. 2003, 100, 503. (4) Binks, B. P. Curr. Opin. Colloid Interface Sci. 2001, 6, 17. (5) Lin, Y.; Boker, A.; Skaff, H.; Cookson, D.; Dinsmore, A. D.; Emrick, T.; Russell, T. P. Langmuir 2005, 21, 191. (6) Lin, Y.; Skaff, H.; Boker, A.; Dinsmore, A. D.; Emrick, T.; Russell, T. P. J. Am. Chem. Soc. 2003, 125, 12690. (7) Lin, Y.; Skaff, H.; Emrick, T.; Dinsmore, A. D.; Russell, T. P. Science 2003, 299, 226. (8) Skaff, H.; Lin, Y.; Tangirala, R.; Breitenkamp, K.; Boker, A.; Russell, T. P.; Emrick, T. Adv. Mater. 2005, 17, 2082. (9) He, J.; Zhang, Q.; Gupta, S.; Emrick, T.; Russell, T. R.; Thiyagarajan, P. Small 2007, 3, 1214. (10) Carbone, L.; Nobile, C.; De Giorg, M.; Sala, F. D.; Morello, G.; Pompa, P.; Hytch, M.; Snoeck, E.; Fiore, A.; Franchini, I. R.; Nadasan, M.; Silvestre, A. F.; Chiodo, L.; Kudera, S.; Cingolani, R.; Krahne, R.; Manna, L. Nano Lett. 2007, 7, 2942. (11) Wang, R. K.; Reeves, R. D.; Ziegler, K. J. J. Am. Chem. Soc. 2007, 129, 15124. (12) Bigioni, T. P.; Lin, X. M.; Nguyen, T. T.; Corwin, E. I.; Witten, T. A.; Jaeger, H. M. Nat. Mater. 2006, 5, 265. (13) Liu, W. L.; Alim, K.; Balandin, A. A.; Mathews, D. M.; Dodds, J. A. Appl. Phys. Lett. 2005, 86, 3. (14) Fonoberov, V. A.; Balandin, A. A. Nano Lett. 2005, 5, 1920. (15) Russell, J. T.; Lin, Y.; Boker, A.; Su, L.; Carl, P.; Zettl, H.; He, J. B.; Sill, K.; Tangirala, R.; Emrick, T.; Littrell, K.; Thiyagarajan, P.; Cookson, D.; Fery, A.; Wang, Q.; Russell, T. P. Angew. Chem., Int. Ed. 2005, 44, 2420. (16) Niu, Z. W.; Bruckman, M. A.; Li, S. Q.; Lee, L. A.; Lee, B.; Pingali, S. V.; Thiyagarajan, P.; Wang, Q. Langmuir 2007, 23, 6719. (17) Lee, B. D.; Lo, C. T.; Thiyagarajan, P.; Winans, R. E.; Li, X. F.; Niu, Z. W.; Wang, Q. Langmuir 2007, 23, 11157. (18) Niu, Z. W.; Bruckman, M.; Kotakadi, V. S.; He, J. B.; Emrick, T.; Russell, T. P.; Yang, L.; Wang, Q. Chem. Commun. 2006, 3019.

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(TMV), have attracted significant interest13-19 for their monodisperse size, the high-yield production of the virus particles, and the versatile functionalization of the protein shell that can be modified by genetic or chemical modification. TMV was the first plant virus discovered and has since been studied extensively.20 TMV is a rigid hollow cylinder about 300 nm in length and 18 nm in diameter. It has ∼2130 protein subunits arranged helically around single stranded RNA, forming an internal channel of 4 nm diameter, with a total molecular weight of ∼ 4.18  107 g/mol. TMV retains structural integrity in the pH range of 3-9 and up to 50 C. TMV has an isoelectric point of 3.4; at neutral pH, the TMV surface is negatively charged with a charge density of ∼ 20 e-/nm2 due to the dissociated amino acid groups.21 It has also been shown that the surface properties of TMV can be manipulated chemically or genetically without affecting the integrity and structure of the TMV.13,14,16,18,22,23 Unlike spherically symmetric colloidal particles or nanoparticles, anisotropic nanoparticles show interesting structures and orientations at interfaces due to their shape.9,10 However, detailed information on the influence of concentration, ionic strength, pH, and aspect ratio of the particle on the characteristics of the assemblies of the protein nanoparticles at the interface is still unknown, but such information is essential for designing hierarchically ordered structures from these nanoscale components. Anisotropic rodlike TMV has a single characteristic diameter which is ideal for studying the self-assembly and order(19) Kalinin, S. V.; Jesse, S.; Liu, W. L.; Balandin, A. A. Appl. Phys. Lett. 2006, 88. (20) Klug, A. Philos. Trans. R. Soc. London, Ser. B 1999, 354, 531. (21) Baus, M.; Rull, L. F.; Ryckaert, J.-P. North Atlantic Treaty Organization. Scientific Affairs, D.; Nato Advanced Study Institute on Observation, P.; Simulation of Phase Transitions in Complex, F.; Kluwer Academic Publishers: Dordrecht, Boston. (22) Miller, R. A.; Presley, A. D.; Francis, M. B. J. Am. Chem. Soc. 2007, 129, 3104. (23) Schlick, T. L.; Ding, Z. B.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 3718.

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ing of these rodlike particles. In this paper, we report a systemic study on the assembly of TMV at liquid/liquid interfaces as a function of TMV concentration and the ionic strength, and pH of the solution.

Experimental Section TMV Purification. TMV was extracted and purified using

covered with TMV were directly used without further washing. The unrinsed emulsions were directly loaded and measured in Suprasil sample cells with a path length of 2 mm. The data for each sample were corrected for the instrumental backgrounds, parasitic scattering, solvent scattering (D2O buffer for dispersed TMV particles; perfluorodecalin for the TMVcoated oil emulsions), and detector nonlinearity. The data were corrected and placed on an absolute scale following established procedures. Small-Angle X-ray Scattering (SAXS). Synchrotron SAXS measurements were performed at the 12-ID beamline at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL). The operating beam energy was 18 keV, with a wavelength of 0.69 A˚. The beam size was 30 μm (height)  50 μm (width). A Mar CCD detector was used to acquire 1024  1024 pixel images with typical exposure times in the range of 1-10 s. The center of the beam was determined by a transmission scan during the experiment. When the beam scans cross the interface, a sharp drop of the transmission intensity shows up. The beam is centered at the halfway point of the sharp transition of the transmitted beam.

the protocol described by Foster and Taylor.24 The infected tobacco leaves were crushed and blended with 0.01 M potassium phosphate buffer at pH 7.8 with 0.2% β-mercaptoethanol. The mixture was centrifuged at 9000 rpm for 15 min before the supernatant was clarified with CHCl3/n-butanol = 1:1. The aqueous portion was separated by centrifugation, and TMV was precipitated by the addition of 10% PEG 8K and 0.2 M NaCl. The resulting pellet was resuspended in 0.01 M potassium phosphate buffer at pH 7.8. After several rounds (>3) of washing, dispersing, and ultracentrifugation at 42 000 rpm for 2.5 h, purified TMV was dispersed in pure H2O, pure D2O, or 0.01 M potassium phosphate buffer solution (pH = 7.8) for different purposes. Tensiometry. The changes in interfacial tension between the TMV solution and the oil phase (perfluorooctane or perfluorodecalin) were determined at room temperature (∼22 C) using OCA 15 and 20 Dataphysics pendant drop tensiometers. A fast video camera was used for rapid drop image acquisition. The interfacial tension was determined by fitting the droplet shape using the Young-Laplace equation. Pickering Emulsions. Pickering emulsions were produced by shaking the TMV solution with perfluorodecalin (10% volume fraction of the aqueous phase). The emulsions were allowed to stabilize for 4-12 h at ∼4 C and then were repeatedly washed with pure water to remove the excess TMV and the salt in the aqueous phase. The washed emulsions were transferred to clean silicon wafers or a carbon-coated copper grid for examination by scanning force microscopy (SFM) and transmission electron microscopy (TEM) Zeta (ζ) Potential Measurement. Measurements of the zeta potential of TMV in aqueous solutions were made using a NanoZS instrument, model ZEN3600 (Malvern Instruments, U.K.). At least 10 measurements of each sample were carried out to ensure reproducibility. Scanning Force Microscopy (SFM). SFM images were taken on a Digital Instruments Dimension 3100 microscope operated in tapping mode. The standard silicon nitride probes were driven at their resonance frequencies around 300 kHz. Height and phase images were taken at a scanning speed of 4 μm/s. Transmission Electron Microscopy (TEM). TEM images of TMV-stabilized perfluorodecalin droplets after drying on a carbon-covered copper grid were taken with JEOL 200CX transmission electron microscope. TMV was negatively stained for imaging.18,25 Small-Angle Neutron Scattering (SANS). The SANS experiments were carried out at the small-angle neutron diffractometer (SAND) at the Intense Pulsed Neutron Source (Argonne National Laboratory, Argonne, IL) and the low-q diffractometer at the Los Alamos Neutron Science Center (LANSCE, Los Alamos, NM) with a q range of ∼0.0035-0.5 A˚-1, where q is related to the neutron wavelength λ and the scattering angle 2θ by q = 4π sin θ/ λ. The form factor of TMV was measured using a solution of 10 mg/mL TMV in 0.01 M potassium phosphate buffer in D2O. For emulsion measurements, a H2O/D2O mixture with a 0.687 volume fraction of D2O was used as the aqueous phase to contrast match the perfluorodecalin (oil phase) with a neutron scattering length density of 4.18  1010 cm-2. Pickering emulsions of perfluorodecalin droplets

Results and Discussion In general, for all the spherical nanoparticles, the interfacial tension decreased rapidly with time during the early stage of the self-assembly. As the interfacial coverage of nanoparticles increased, the decrease in interfacial tension slowed down and eventually reached a plateau, corresponding to a dynamic equilibrium. As the concentration in the bulk solution was increased, the final equilibrium value decreased, until the interface was saturated.26 Figure 1a shows a series of pendant drop tensiometer measurements of TMV in 0.1 M potassium phosphate buffer solution at pH = 7.8 as a function of its concentration at the water/perfluorooctane interface. Perfluorooctane served as the drop phase. In the case of TMV nanorods, in addition to a similar trend, we observed an additional behavior. At very low concentration (0.0625 mg/mL; number density = 9.00  1011 mL-1), the interfacial tension first decreased rapidly (see Figure 1a), but slowed down for an extended period of time when compared to the spherical systems, and finally reached an equilibrium value. This behavior was only observed for the rod-shaped nanoparticles, which suggested a change in the packing of the TMV at the interface with time due to the anisotropic shape of the particles 15,26 Interestingly, these transitions seemed to be strongly dependent on the bulk TMV concentration in solution as the transition behavior became less distinct above 1 mg/mL. Figure 1b shows the concentration dependence of the final equilibrium interfacial tension of the perfluorooctane/water interface for different bulk TMV concentrations. The first transition in the interfacial tension occurred at a TMV concentration in the range of 0.031 mg/mL (number density = 4.50  1011 mL-1) to 0.1 mg/mL, where it rapidly decreased. At TMV concentration from 0.1 to 0.8 mg/mL, the interfacial tension plateaued around 33 mN/m. The next transition occurred around TMV concentration of 0.8 mg/mL (number density = 1.15  1013 mL-1), whereupon the interfacial tension further decreased to an equilibrium interfacial tension (∼27 mN/m). This did not change over a wide concentration range (above 2.5 mg/mL, number density g 3.6  1013 mL-1), which suggests that the interface became saturated with TMV. This indicates that the bulk concentration strongly influences the packing of anisotropic nanoparticles at the oil/water interfaces in a way that is different from spherical nanoparticles, where only

(24) Foster, G. D.; Taylor, S. C. Plant virology protocols: from virus isolation to transgenic resistance; Humana Press: Totowa, NJ, 1998. (25) Horne, R. W.; Hobart, J. M.; Markham, R. J. Gen. Virol. 1976, 31, 265.

(26) Kutuzov, S.; He, J.; Tangirala, R.; Emrick, T.; Russell, T. P.; Boker, A. Phys. Chem. Chem. Phys. 2007, 9, 6351.

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Figure 1. (a) Time dependent pendent drop tensiometer measurements of TMV at different concentrations (0.0625, 1, and 5 mg/mL) in 0.1 M potassium phosphate buffer solutions at pH = 7.8. Perfluorooctane is the oil phase. (b) Concentration dependence of the final equilibrium interfacial tension of the perfluorooctane/water interface with different TMV concentration in the bulk. Black squares: 0.1 M potassium phosphate buffer solution at pH = 7.8. Red dots: 0.01 M potassium phosphate buffer solution at pH = 8. (c) Ionic strength dependence of the final equilibrium interfacial tension of the perfluorooctane/water interface with different TMV concentrations (red dots for 0.25 mg/mL and black squares for 1 mg/mL) in the potassium chloride solution at the same pH= 7. (d) Black squares: zeta potential of TMV as a function of ionic strength in different potassium phosphate buffer concentrations at pH = 7.8. Red dot: zeta potential of TMV in 0.01 M potassium phosphate buffer at pH = 6. Green dot: pH = 7. Blue dot: pH = 8.

a simple decay was observed.26 In 0.01 M potassium phosphate buffer solution at pH = 8 (red dots), as the TMV concentration increased, the final equilibrium interfacial tension showed a similar trend as in 0.1 M potassium phosphate buffer solution with pH = 7.8, except that the final were was higher at similar TMV concentrations. These results show the strong role of ionic strength and pH of the bulk solution on the assembly of TMV at the oil/water interface. Figure 1c shows the ionic strength dependence of the final equilibrium interfacial tension of the perfluorooctane/water interface for two TMV concentrations (red dots for 0.25 mg/mL and black squares for 1 mg/mL) in the potassium chloride solution at pH = 7. When the ionic strength of the solution was low, less than 1  10-4 mol/L, the final equilibrium interfacial tension was between pure perfluorooctane and water (∼54 mN/m). When the ionic strength of the solution was increased to 1  10-3 mol/L, for high concentration of TMV (1 mg/mL), a sharp drop in the interfacial tension was observed, but not at the low TMV concentration (0.25 mg/mL). This difference could be due to different orientations of TMV at the interfaces but not due to the concentration difference. When the Langmuir 2009, 25(9), 4979–4987

ionic strength was increased, the final interfacial tension showed a sharp reduction, even at low TMV concentrations. These results imply that the assembly of TMV at the oil/water interface is strongly dependent on the ionic strength of the solution as well as the bulk TMV concentration. For charged particles such as TMV, it is expected that a change in ionic strength or pH will strongly influence the “effective” surface charge that determines the interparticle interactions. To quantify these effects, zeta potential measurements were carried out, and the results are shown in Figure 1d. At pH 7.8 when the ionic strength of the potassium phosphate buffer solution increases, the zeta potential of TMV decreases dramatically, as indicated by the black squares. The “effective” surface charge decreases, since the charged groups on the TMV surface are better shielded at higher buffer concentration, which is common for the like-charged particles in solution.27 However, the zeta potential of TMV in 0.1 M potassium phosphate buffer solutions did not change as a function of pH from a pH of 6 to 8, as indicated by the colored (27) Onsager, L. Ann. N.Y. Acad. Sci. 1949, 51, 627.

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Figure 2. SFM phase images of the surface of dried emulsions prepared at TMV concentrations of 0.2 mg/mL (a) and 0.8 mg/mL (b) in 0.01 M potassium phosphate buffer solutions at pH = 7.8. (c) Section analysis of the dried emulsions in (b). TEM images on the dried emulsions for (d) 0.2 mg/mL and (e) 0.8 mg/mL.

circles. This suggests that, under the experimental conditions (pH = 6-8), the pH does not influence the “effective” surface charge of TMV and, therefore, the organization at the interfaces. This is consistent with the results of Brenner and Mcquarrie28 where the magnitude of repulsive interactions between the TMV particles reached a saturation value in the vicinity of pH = 7 and a further increase in pH did not change the value significantly. As suggested by Fraden and Purdy, for a highly charged particle such as TMV (∼20 e-/nm), the effective diameter of TMV in aqueous solution is insensitive to the variation of surface charge densities induced by the changes in pH values in this regime.29 Pickering emulsions were generated to capture the self-assembled structures of TMV at the oil/water interfaces by shaking an aqueous TMV solution in 0.01 M potassium phosphate buffer at pH = 7.8 and a 10% volume fraction of perfluorodecalin (PFD). Two TMV concentrations 0.2 mg/mL (number density = 2.88  1012 mL-1) and 0.8 mg/mL (number density = 1.15  1013 mL-1) were investigated. In the absence of salt, no stable emulsions formed, which is consistent with the tensiometry data. The size of stabilized emulsion droplets varies from 10 to 100 μm. In (28) Brenner, S. L.; McQuarrie, Da. J. Colloid Interface Sci. 1973, 44, 298. (29) Purdy, K. R.; Fraden, S. Phys. Rev. E 2004, 70, 8.

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this size range, the interface is essentially flat for TMV particles. After stabilization, TMV-coated emulsions were carefully rinsed with pure water to remove the free virus particles and salts. Washed emulsions were transferred to clean silicon wafers or a carbon-coated copper grid for SFM and TEM measurements. Figure 2a and b shows SFM phase images of the surface of dried emulsions prepared at the TMV concentrations of 0.2 and 0.8 mg/mL, respectively. In the case of 0.2 mg/mL TMV emulsion, individual TMV particles can be seen that are oriented parallel to the substrate with no lateral ordering, that is, an isotropic arrangement. However, in the 0.8 mg/mL TMV emulsion, a totally different morphology is observed (Figure 2b). As indicated by the phase image, individual “disks” of ∼25 nm in diameter with ∼6 nm “pores” are closely packed in a liquidlike manner. Further increase in TMV concentration in the bulk above 0.8 mg/mL shows similar SFM images. This suggests that, at high TMV concentration in the bulk solution, TMV particles at the perfluorodecalin/water interface may orient normal to the interface. Scanning across the edge of isolated dried emulsions by SFM yields a film thickness of about 23.8 nm, as shown in Figure 2c. Considering possible deformation in tapping mode and a double layer structure of the emulsion, the thickness of each layer is less than 20 nm, which is inconsistent with the 300 nm Langmuir 2009, 25(9), 4979–4987

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Figure 3. (a) SANS data of 10 mg/mL TMV in 0.01 M potassium phosphate buffer in D2O. Data are fitted by a core-shell cylinder model. (b) Contrast-match SANS data of emulsions generated at 0.2 mg/mL TMV bulk concentration in 0.01 M potassium phosphate buffer in 68.7% D2O. (c) Contrast-match SANS data of emulsions generated at 0.8 mg/mL TMV bulk concentration potassium phosphate buffer in 68.7% D2O. Red: 0.01 M potassium phosphate buffer. Blue: 0.03 M potassium phosphate buffer. The red curve is shifted 1 order down for clarity. (d) Contrast-match SANS data of emulsions generated at 0.8 mg/mL TMV bulk concentration potassium phosphate buffer in 68.7% D2O. Red: Emulsions generated at 5 mg/mL TMV in 0.01 M potassium phosphate buffer. Blue: Emulsions generated at 5 mg/mL TMV in 0.001 M potassium phosphate buffer. The blue curve is shifted 1 order down for clarity.

length of TMV. We speculate that this is due to the denaturation of TMV nanoparticles right at the oil/water interface during washing with pure water. TEM images on the dried emulsions in Figure 2d (0.2 mg/mL) and e (0.8 mg/mL) are consistent with the SFM results. For TMV at low concentrations, a parallel orientation of the TMV is observed. For higher TMV concentrations, ∼4 nm black dots are seen, which arise from the negative staining of the sample. To investigate the equilibrium structure of the TMV assembled at the perfluorodecalin/water interface, SANS experiments were carried out on Pickering emulsions prepared at different TMV concentrations. Figure 3a shows the scattering profile of 10 mg/mL TMV in 0.01 M potassium phosphate D2O buffer at pH 7.8 (the reported pH values are the as measured values using a pH meter and do not take into account the different ionization potential for D2O and H2O). The scattering profile was fitted using a core-shell cylinder model with an inner core radius of 20.2 ( 0.2 A˚ with a neutron scattering length density of 4.18  1010 cm-2, a shell thickness of 60.0 ( 0.5 A˚, a shell neutron scattering length density of 2.93  1010 cm-2, and a cylinder length of ∼3000 ( 200 A˚. For the emulsion experiments, TMV Langmuir 2009, 25(9), 4979–4987

was dispersed in potassium phosphate H2O/D2O solution at pH = 7.8. The H2O/D2O mixture with a 31.3/68.7 vol % of D2O has an effective scattering length density of 4.18  1010 cm-2, matching that of perfluorodecalin. Under the contrast matching condition, the interface between the aqueous and oil phase is invisible and, hence, the liquid medium is effectively a single phase for SANS, enabling the study of the assembly of TMV rods at the interface. The scattering length density of the TMV is quite low compared to the aqueous sample in D2O shown in Figure 3a. Figure 3b shows the SANS data of emulsions generated at the 0.2 mg/mL TMV concentration in 68.7% D2O 0.01 M potassium phosphate buffer. No correlation between TMV particles is observed due to a very low surface coverage at the oil/water interface, except for the weak form factor resulting from the rodlike shape of individual TMV nanoparticles, consistent with the results from SFM and TEM. The power-law scattering for Q < 0.01 A˚-1 arises from the TMV interface as well as some parasitic scattering that is difficult to subtract at very low contrast conditions. In the case of 0.8 mg/mL TMV emulsion, a correlation peak is seen at qy = 0.0147A˚-1 (Figure 3c), corresponding to an average separation distance of 427 A˚ between the TMV DOI: 10.1021/la803533n

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nanoparticles. Interestingly, the power-law scattering, of the form I(q)  q-2.25(0.17, in the low Q region is consistent with a monolayer sheet of TMV at the oil/water interface. It is well established that an infinitely thin sheet will produce a power-law scattering ∼ q-2 at qRg , 1. For emulsions prepared at 0.8 mg/mL TMV bulk concentration in aqueous solution, as the ionic strength of the potassium phosphate buffer is increased from 0.01 to 0.03 M (pH = 7.8), Figure 3c shows a power-law dependence of the form I(q)  q-1.895(0.07 with a correlation peak shifted to qy = 0.017 A˚-1 (average separation distance of 369 A˚). Upon increasing the bulk TMV concentration to 5 mg/mL in 0.01 M potassium phosphate buffer, SANS of the emulsions in Figure 3d shows a correlation peak at qy = 0.0215 A˚-1 (average separation distance of 292 A˚). When the ionic strength of the potassium phosphate buffer is decreased from 0.01 to 0.001 M (pH = 7.8), this correlation peak shifts to lower qy = 0.0125 A˚-1 or a larger separation distance of 502 A˚. Therefore, as the concentration of TMV in the solution increases, with more TMV present at the interface, the average separation distance of TMV at the perfluorodecalin/water interface decreases, which indicates a more densely packed structure at the interface. Changes of the ionic strength in the buffer solution also greatly affect the structures of TMV at the oil/water interface, which is consistent with the tensiometry results. However, in situ SANS experiments on the TMV-coated perfluorodecalin droplets did not provide any information of the orientation of TMV at the interface. To gain insight on the TMV orientation at the oil/water interface, in situ SAXS experiments were carried out at a flat perfluorodecalin/water interface in a setup shown in Figure 4a. A solution of 15 mg/mL TMV in 0.01 M potassium phosphate buffer (pH = 7.8) was placed on top of the perfluorodecalin layer. The SAXS pattern (Figure 4b) taken through the upper aqueous phase showed only isotropic rings, which is due to the TMV rods with no preferential orientations in the solution. The qy line-cut of the data is presented in Figure 4e (red curve), and this is similar to the SANS data in Figure 3a. After 30 min assembly, a SAXS pattern (Figure 4c) was taken right at the flat perfluorodecalin/water interface. Besides the isotropic rings, two strong peaks perpendicular to the qy direction were observed, which suggests that the TMV nanoparticles are preferentially oriented parallel to the interface30 (qy linecut analysis in Figure 4e, blue curve). The isotropic pattern mainly arises from the dispersed TMV nanoparticles in the solution, since the X-ray beam has a height of ∼30 μm that is much larger than the width of the perfluorodecalin/water interface. Another SAXS pattern (Figure 4d) was taken at the perfluorodecalin/water interface after 3 h of assembly (qy line-cut analysis in Figure 4e, green curve). A transition of the two strong scattering peaks changing from perpendicular to parallel to the qy direction was observed, which implies a rotation of TMV nanoparticles from being oriented preferentially parallel to preferentially perpendicular to the oil/water interface. By subtracting the background arising from the solution, an average separation distance of ∼21 nm of TMV at the interface was seen, as shown in Figure 4e (black curve). The in situ SAXS experiments clearly indicate that (1) at the early stage of TMV self-assembly at the perfluorodecalin/ water interface TMV is parallel to the interface and (2) as more TMV is driven to the interface, a rotation of TMV from parallel orientation to the perpendicular orientation at the interface occurs, which confirms the ex situ SFM and TEM results. This conclusion is also supported by the X-ray reflectivity data shown (30) Glatter, O.; Kratky, O. Small angle x-ray scattering; Academic Press: London, New York, 1982.

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in Figure 5. This nontraditional X-ray reflectivity is done on a perfluorodecalin/water interface with 1 mg/mL TMV in 0.01 M potassium phosphate buffer solution (pH = 7.8) after a 12 h assembly. As shown in Figure 5, two series of interference fringes were observed which qualitatively indicate a double-layer structure at the oil/water interface. The average thicknesses of these two layers are ∼200 and ∼20 nm, respectively. These results suggest that TMV particles are oriented normal to the perfluorodecalin/water interface. For the interaction of particles at the liquid interfaces, there has been a long-standing debate about the dominant force. Pieranski and Hurd suggest that the dominant force is the long-range repulsion corresponding to effective dipole-dipole interactions resulting from the asymmetry of the particles’ ionic atmosphere in the aqueous phase.31,32 The force fdipole has a form as shown in eq 1:32,33 fdipole 

6εOil qwater 2 εwater 2 K2 R4

ð1Þ

where εOil and εwater are the dielectric constants of oil and water, respectively, qwater is the effective charge on the particle contributing to the dipole-dipole interaction, κ-1 is the Debye screening length, and R is the average separation distance of the particles. However, the result from Binks and co-workers shows that the dominant long-range repulsion at the liquid interfaces arises from the direct Coulomb repulsion between the unbalanced residue electric change at the particle/oil interface.33 The force fdirect has a form as shown in eq 2:33,34 ( ) qOil 2 1 R pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi fdirect  - 3=2 εOil R2 4δ2 þ R2

ð2Þ

where qOil is the residual charge on the particle/oil interface and δ is the distance of qOil to the interface. Since this direct Coulomb repulsion interacts through the oil phase, fdirect is independent of the ionic strength in the aqueous phase. So changing the ionic strength in the aqueous phase does not affect the separation distance of the particles at the liquid interfaces.33 Recent results from Weitz and co-workers indicate that, in addition to the longrange repulsive force, there is a long-rang attractive force which helps maintain the long-range order of the particles at the liquid/ liquid interface.35 This attractive force is a capillary force induced by the distorted liquid/liquid interface which is caused by the unbalanced residual charge on the particle/oil interface.36 The possible interaction between TMVs at the perfluorodecalin/water interface is shown in Figure 6. For repulsive forces, there could be a long-range dipole-dipole repulsion, the direct Coulomb repulsion through the oil phase, and the screened Coulomb repulsion arising from the interaction of the particle double layers through the water phase, which is the dominant force in the bulk phase between TMV particles.37 For a rod-shaped particle such as TMV with length L much larger than the average separation distance R, the screened Coulomb repulsion fscreened per unit (31) Pieranski, P. Phys. Rev. Lett. 1980, 45, 569. (32) Hurd, A. J. J. Phys. A: Math. Gen. 1985, 18, 1055. (33) Aveyard, R.; Binks, B. P.; Clint, J. H.; Fletcher, P. D. I.; Horozov, T. S.; Neumann, B.; Paunov, V. N.; Annesley, J.; Botchway, S. W.; Nees, D.; Parker, A. W.; Ward, A. D.; Burgess, A. N. Phys. Rev. Lett. 2002, 88. (34) Aveyard, R.; Clint, J. H.; Nees, D.; Paunov, V. N. Langmuir 2000, 16, 1969. (35) Nikolaides, M. G.; Bausch, A. R.; Hsu, M. F.; Dinsmore, A. D.; Brenner, M. P.; Weitz, D. A.; Gay, C. Nature (London) 2002, 420, 299. (36) Nikolaides, M. G.; Bausch, A. R.; Hsu, M. F.; Dinsmore, A. D.; Brenner, M. P.; Gay, C.; Weitz, D. A. Nature (London) 2003, 424, 1014. (37) Parsegian, V. A.; Brenner, S. L. Nature (London) 1976, 259, 632.

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Figure 4. (a) Setup for the in situ SAXS experiment. 15 mg/mL TMV in 0.01 M potassium phosphate buffer solution (pH = 7.8) on top of perfluorodecalin layer. (b) SAXS data of 15 mg/mL TMV in 0.01 M potassium phosphate buffer (pH = 7.8). (c) SAXS data taken at the flat perfluorodecalin/water interface after a 30 min assembly. (d) SAXS pattern taken at the flat perfluorodecalin/water interface after a 3 h assembly. (e) qy line-cut analysis of the SAXS data in (b) red, (c) blue, and (d) green. Black curve: Difference between the data in the blue curve and the red curve.

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Figure 6. Scheme of TMV rods at the oil/water interface and the interactions that contribute to their preferred orientations. (top) TMV rods are perpendicular to the interface. (bottom) TMV rods are parallel to the interface. R is the separation distance between TMV rods. L is the length of TMV rods. Red spheres represent negative charge on TMV rod surface.

Figure 5. (a) X-ray reflectivity pattern of a perfluorodecalin/water interface with 1 mg/mL TMV in 0.01 M potassium phosphate buffer solution (pH = 7.8) after a 12 h assembly. (b) X-ray reflectivity profile of the pattern shown in (a). The dashed red line is used to guide the eyes.

length has the following form:38 e -KR fscreened  ξðK, a, λ, εÞ pffiffiffiffiffiffiffi KR

ð3Þ

where √ξ (κ, a, λ, ε) is the prefactor function which is determined by κ = I/0.304 nm-1 (I is the ionic strength in the aqueous solution at room temperature), a is radius of the rod, λ is the line charge density of the rod, and ε is the dielectric constant of the medium. For attractive forces, there could be the van der Waals attraction and the capillary attraction.35,39 The tensiometry data and in situ SANS results clearly show that the ionic strength of the aqueous phase strongly influences the average separation distance of TMV at the interface, so as to the surface coverage of the interface. These results indicate that (1) the attraction forces are not the dominating forces even if they are present and (2) the direct Coulomb repulsion force is also not the dominating force since the average separation distance of TMV is affected by the ionic strength in the water phase. Since fscreened decays exponentially (38) Brenner, S. L.; Parsegia, Va. Biophys. J. 1974, 14, 327. (39) Reincke, F.; Kegel, W. K.; Zhang, H.; Nolte, M.; Wang, D. Y.; Vanmaekelbergh, D.; Mohwald, H. Phys. Chem. Chem. Phys. 2006, 8, 3828.

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proportional to the separation distance between the particles, at large separation distances, fscreened can be neglected.32,39 With the experimental conditions used (κR . 1), only the particle surface charge within a Debye length from the three-phase contact line effectively contributes to the long-range dipole-dipole interaction.33 The effective value of qwater in eq 1 is qwater = lκ-1σ, where l is the length of the three-phase contact line and σ is the area charge density on the particle surface. As can be seen from Figure 6, in the case of orientation normal to the liquid/liquid interface, the TMV particles have a much shorter three-phase contact line than that in the case of parallel orientation. Thus, the dipole-dipole repulsion can be reduced significantly. The behavior of nanorods at the oil/water interface has been briefly explained in our previous report.9 TMV particles segregate to the perfluorodecalin/water interface to minimize the interfacial energy, as suggested by Pieranski.31 The concentration of nanorods in solution limits the total number of nanorods that can assemble at the interface, which is determined by the chemical potential difference associated with having the nanorods at the interface or in the bulk solution. At nanorod concentrations in solution, individual nanorods orient parallel to the plane of the interface to maximize the interfacial coverage per particle.9,40-43 This can be confirmed by the in situ SAXS experiments performed at the oil/water interface. Since the surface coverage is low and the separation distance of TMV is large, the parallel geometry is favorable. At higher TMV (40) Basavaraj, M. G.; Fuller, G. G.; Fransaer, J.; Vermant, J. Langmuir 2006, 22, 6605. (41) Dong, L. C.; Johnson, D. T. Langmuir 2005, 21, 3838. (42) Lewandowski, E. P.; Searson, P. C.; Stebe, K. J. J. Phys. Chem. B 2006, 110, 4283. (43) Bresme, F.; Oettel, M. J. Phys.: Condens. Matter 2007, 19, 33.

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concentration, the interfacial tension decreases until the oil/water interface is saturated with randomly packed TMV, oriented parallel to the interface, where a plateau is found in the interfacial tension measurements. Further increasing the TMV concentration increases the volume fraction of TMV at the interface and leads to increased dipole-dipole repulsion between TMV rods until they are forced to orient normal to the oil/water interface. This reorientation is revealed by in situ SAXS and SANS experiments. In this configuration, not only can the TMV particles relieve the strong dipole-dipole repulsion but they can also effectively minimize the interfacial energy, since there is a large excess of free particles in the bulk solution. The interfacial tension starts to decrease again and, with a further increase of the TMV concentration in the bulk solution, more TMV particles assemble at the interface assuming an orientation normal to the interface until the interface is again saturated. Thus, another plateau in the interfacial tension is reached. Changing the ionic strength of the buffer solution will effectively change the interactions between TMVs at the interface, which will result in a different separation distance, surface coverage, and final equilibrium interfacial tension. When the buffer solution is replaced by pure water during the washing procedures used for the SFM and TEM measurements, a strong Coulomb repulsion is induced between the TMV particles, which causes the cleavage of TMV particles at the oil/water interface. This behavior is similar to that seen by Mann and co-workers.44 (44) Fowler, C. E.; Shenton, W.; Stubbs, G.; Mann, S. Adv. Mater. 2001, 13, 1266.

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Conclusion In summary, the orientation and structure of TMV nanorods at the oil/water interface have been studied in situ by tensiometry, SANS, and SAXS. Different orientation of the nanorods at the liquid/liquid interface can be obtained by controlling the concentration of nanorods in the bulk. Repulsive forces dominate the interfacial assembly behavior, which is strongly affected by the ionic strength of the bulk solution but not by pH over the range of 6-8. Subsequent removal of the buffer solution can cause a cleavage of TMV nanorods at the oil/water interface. Acknowledgment. This work was supported by the U.S. Department of Energy, Office of Basic Energy Science, the Army Research laboratory through the MURI program, the NSF supported MRSEC at the University of Massachusetts Amherst, and an NSF CAREER Award. We thank Rex Hjelm and Monika Hartl for assistance with the SANS experiments and Greg Grason at the University of Massachusetts Amherst for the help of calculation. Work benefited from the use of IPNS and APS supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC0206CH11357, and the LQD funded by DOE-BES. A.B. and G.J. acknowledge the support of the Lichtenberg-Program of the Volkswagen Stiftung.

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