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Complex Pattern Formation by Cowpea Mosaic Virus Nanoparticles Jiyu Fang,*,† Carissa M. Soto,† Tianwei Lin,‡ John E. Johnson,‡ and Banahalli Ratna† Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375-5438, and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Received August 3, 2001. In Final Form: October 12, 2001 We report the formation of complex patterns by the drying of concentrated cowpea mosaic virus droplets on surfaces. The virus particles can self-organize into parallel and orthogonal lines, forming fingerlike patterns on freshly cleaved mica and crosslike patterns on acid-treated mica. Atomic force microscopy shows that the parallel and orthogonal lines have a uniform width and thickness.
Pattern formation during the simple process of drying a droplet of colloidal particles is a fascinating phenomenon. Complex morphologies such as rings, networks, fractals, and dendrites have been observed over a wide range of particles from inorganic particles1-3 to collagen4,5 and bacteria.6 Therefore, understanding the underlying mechanism that drives the formation of these complex patterns is of interest to both the biological and physical sciences. Technologically it would be very significant if these patterns can be achieved over the macroscopic scale. Here we present one such possibility using a cowpea mosaic virus (CPMV) particle. Parallel and orthogonal lines with a uniform width and thickness are formed on surfaces over hundreds of micrometers by the drying of concentrated CPMV droplets. CPMV was isolated from cowpea plants in yields of 1-2 g/kg of leaves. Its detailed atomic structure determined by X-ray diffraction at a resolution of 2.8 Å shows a picornalike T ) 1 protein shell (Figure 1a) with an asymmetric unit containing three β-sandwich folds formed by two polypeptides.7 A 15 mg/mL stock solution of CPMV was diluted by 10 mM Tris pH 8.0 buffer into different concentrations. Droplets (2 µL) of diluted CPMV solutions were placed on freshly cleaved mica and acid-treated mica. The samples were dried in air at room temperature. The self-assembled patterns of virus particles formed during the drying on surfaces were examined using an Olympus optical microscope and a Nanoscope III atomic force microscope (AFM), which was operated in the tapping mode in air under ambient conditions. At a low concentration (0.0015 mg/mL), individual CPMV particles on freshly cleaved mica can be seen in Figure 1b, which was taken using a carbon nanotube AFM tip. It is known that the average virus diameter is 27.8 * To whom correspondence should be addressed: CBMSE.NRL.NAVY.MIL. † Naval Research Laboratory. ‡ The Scripps Research Institute.
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(1) Ohara, P. C.; Heath, J. R.; Gelbart, W. M. Angew. Chem., Int. Ed. Engl. 1997, 36, 1078. (2) Haidara, H.; Mugin, K.; Schultz, J. Langmuir 2001, 17, 659. (3) Deegan, R. D. Phys. Rev. E 2000, 61, 475. (4) Maeda, H. Langmuir 2000, 16, 9977. (5) Thiele, U.; Mertig, M.; Pompe, W. Phys. Rev. Lett. 1998, 80, 2869. (6) Ben-Jacob, E.; Shochet, O.; Tenenbaum, A.; Cohen, I. Phys. Rev. E 1996, 53, 1835. (7) Lin, T.; Chen. Z.; Usha, R.; Stauffacher, C. V.; Dai, J. B.; Schmidt, T.; Johnson, J. E. Virology 1999, 265, 20.
nm.7 The measured height of virus particles in Figure 1b was found to be 27.0 ( 0.4 nm, which is consistent with the size of the virus particle. This suggests that virus particles are not compressed by the tip force employed for scanning or distorted by the drying. The apparent lateral dimension in Figure 1b is convoluted by the geometrical effect of the AFM tip, therefore the virus particles appear large. Remarkably, virus particles self-assemble into macroscopic structures with distinct morphologies during the drying of concentrated CPMV droplets on surfaces. An AFM image of 0.15 mg/mL CPMV droplet, which was dried on freshly cleaved mica, is shown in Figure 2a. The virus particles organize into sets of parallel and orthogonal lines, forming fingerlike patterns. The high-resolution image in Figure 2b shows more detail of the fingers. There is slight distortion in the width of the fingers near the connections. The height profile (Figure 2c) along the line drawn in Figure 2b shows that these parallel fingers have almost same height of 250 nm, which is close to nine virus layers. The average width of the fingers is about 600 ( 40 nm. The typical distance between the parallel fingers is about 7 µm. The self-assembly of virus particles during the drying can be mediated by surface properties. Freshly cleaved mica was treated with 0.5 M HCl for 2 h followed by a 30 min wash with ultrapure water. It has been demonstrated that OH groups can be created at the acid-treated mica surface.8 Figure 3a shows an optical microscopy image of 0.15 mg/mL CPMV droplets, which were dried on acid treated mica. Unlike the fingerlike structures seen in Figure 2, the virus particles self-assemble into crosslike structures. We also observed the presence of a few spherical aggregates among the crosslike aggregates. These spheres have a diameter of 5-7 µm, the same as the apparent width of the arms of the crosses. The length of straight arms is up to 30 µm, and they are perpendicular to each other. Valuable information can be extracted from the AFM image shown in Figure 3b. Four arms point at the center of the cross, but do not connect with each other. The height profile along the line in Figure 3b is shown in Figure 3c, revealing more details of these crosslike aggregates. The straight arms are 300-450 nm high and appear to arise from the alignment and fusion of spherical aggregates. These results imply that the spheres are (8) Carson, G. A.; Granick, S. J. Mater. Res. 1990, 5, 1745.
10.1021/la0112357 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/27/2001
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Figure 1. (a) Image of cowpea mosaic virus particle reconstructed from X-ray crystal structure data. (b) Tapping mode AFM image of individual CPMV particles on freshly cleaved mica. This image was taken with a carbon nanotube tip (Piezomax Technologies, Inc.) in air under ambient conditions.
Figure 2. (a) Tapping mode AFM image of self-assembly from 0.15 mg/mL CPMV solution dried on freshly cleaved mica, where CPMV particles self-assemble into parallel and orthogonal lines over a large area. (b) An enlarged tapping mode AFM image of (a), where the self-assembled lines show a fingerlike pattern. (c) Height profile obtained along the line indicated in (b).
precursors of the crosslike structures. This is further supported by the observation that the spherical aggregates are formed along the periphery of the drop as the solvent line recedes, whereas the crosses are formed inside where the effective concentration of the virus particles in the drop has increased due to evaporation (not shown). The spherical aggregates coalesce into lines. It is important to point out that these self-assemblies occur while concentrated virus solutions are dried on surfaces and not in solutions. The droplet evaporation triggers the driving force for the self-assembly of virus particles on surfaces. For freshly cleaved mica, exposed aluminosilicate six-ring sites with the amount of aluminum ions give a negatively charged surface. While for the acid-treated mica, the surface charge is mainly determined by OH groups. At pH 8.0, the OH groups on the acidtreated mica are fully protonated. Therefore the effective charge on the acid-treated mica is smaller than that on the freshly cleaved mica. On the other hand, no change is observed by AFM in the topography and by water contact angle in the hydrophilicity of mica surface after the acid
treatment. These suggest that the electrostatic interaction between the charged virus with the surface dominates the outcome of the assembly of virus particles on surfaces.9 The length scale of the lines formed on freshly cleaved mica (Figure 2) are orders of magnitude larger than those formed on acid-treated mica (Figure 3). The creation of inhomogeneous OH groups on acid-treated mica inhibits the growth of the extended orthogonal line pattern that is observed on freshly cleaved mica. The inhibition of extended growth of patterns by chemically inhomogeneous surfaces is also reported by Haidara and co-workers.2 They find that the growth of multibranched patterns of Au nanoparticles observed on chemically homogeneous self-assembled monolayers (SAMs) is inhibited on heterogeneous SAMs. Though there is no theoretical understanding of the mechanism behind the length selection of the self-assembled patterns, it has been suggested that the pattern growth of colloid particles (9) The pKa of the virus is around 5.0. Therefore it is charged at pH 8.0.
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Figure 3. (a) Optical microscopy image of self-assembly from 0.15 mg/mL CPMV solution dried on acid-treated mica, where CPMV particles self-assemble into orthogonal lines which form crosses. (b) Tapping mode AFM image of the crosslike structure. (c) Height profile obtained along the line indicated in (b).
during the drying of droplets is a result of the competition between pinning and dewetting forces.3 The beautiful selfsimilar patterns of CPMV particles observed here shows that a drying drop is a rich and unexplored phenomenon with a lot of experimental and theoretical possibilities. Also these patterns exhibited by the virus particles are not only interesting, because they open up new insights into the self-assembly of complex biological structures in concentrated solutions, but also have technological im-
plications since they provide new prospects in the field of patterning and structuring on surfaces. Acknowledgment. This work was supported by ONR. Dr. Soto acknowledges a NRC Postdoctoral Fellowship. We thank Professor C. M. Knobler and Dr. J. Selinger for helpful discussions. LA0112357
