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Comments Comments on Complex Pattern Formation by Cowpea Mosaic Virus Nanoparticles
In a recent paper Fang et al. described regular structures that they ascribed to self-assembly of cowpea mosaic virus (CPMV) on a mica surface.1 The essential step of the procedure consisted of evaporating a small quantity of solution containing the CPMV. The resultant surface structures were analyzed by optical microscopy and atomic force microscopy (AFM)sthe latter operated in the tapping (or intermittent contact) mode. The structures described by Fang et al. are reminiscent of those observed during an AFM study of tobacco mosaic virus (TMV).2 However, careful analysis revealed that those structures were due to precipitation and growth of salt crystals on the mica surface. Indeed, great care must be taken in order to identify and exclude artifacts when investigating biological material (especially evaporated from solution) by scanning probe microscopy (SPM). A biocompatible fluid is usually near, sometimes above, saturation limits for species in solution (especially salts). Thus, evaporative losses will lead to precipitation. Time lapse imaging will in some cases reveal the evolution of processes such as crystal growth.3 This Comment reports the null experiment, identical to that of Fang et al. in every respect but without CPMV. The results of the null experiment show that the surface structures are due to precipitate salt formation. Thus, there is no compelling evidence for self-assembly of CPMV. Results from current studies are reported and discussed below and are compared to those of Fang et al. Results and Discussion Parallel and Orthogonal Line Structures. An image obtained by Fang et al. is reproduced in Figure 1 as a reference. The image shows well formed linear structures of varying widths, for example, the locations labeled x and y. That image was obtained after placing a stock solution of CPMV diluted (dilution factor of 100) with 10 mM Tris pH 8.0 buffer onto mica, followed by evaporation of the aqueous phase. The original stock solution was comprised of a 0.1 M potassium phosphate buffer4 (pH 7) with a 15 mg/mL concentration of virus. Figure 2 shows an AFM image and an optical image of structures arising from evaporation in air of a solution of 0.1 M potassium phosphate buffer diluted (dilution factor of 100) with 10 mM Tris pH 8.0 onto a freshly cleaved mica surface at 23° (but with no biospecies in solution). There are obvious similarities between the structures shown in Figure 2 and those reported by Fang et al.: (1) The patterns are organized in sets of parallel and orthogonal lines. (2) Fingerlike structures are evident. * E-mail:
[email protected]. (1) Fang, J.; Soto, C. M.; Lin, T.; Johnson, J. E.; Ratna, B. Langmuir 2002, 18, 308. (2) Bushell, G. R.; Watson, G. S.; Holt, S. A.; Myhra, S. J. Microsc. 1995, 180, 174. (3) Bushell, G. R.; Cahill, C.; Clarke, F. M.; Gibson, C. T.; Myhra, S.; Watson, G. S. Cytometry 1999, 36, 254. (4) Private communication with the authors.
Figure 1. Data from Fang et al.1 (a) Tapping mode AFM image of structure obtained by evaporation of a 0.15 mg/mL CPMV solution onto mica. (b) An enlarged image of (a), showing a fingerlike pattern. (c) Height profile obtained along the line indicated in (b).
(3) The widths of the fingerlike structures are similar, as shown by comparison of the line contours. (4) The heights of the fingerlike structures are similar (∼250 nm was quoted by Fang et al.). (5) Spacings between parallel lines are comparable. Dilution of the phosphate solution by a further factor of 100 (with the Tris buffer) resulted in no formation of patterned structures. That outcome is consistent with the observation of Fang et al. where no pattern formation occurred with low concentrations of their stock solution. Further investigation showed that solution conditions identical to those producing the structures in Figure 2 resulted in near-identical patterns on glass substrates, as shown in Figure 3 (again with no biospecies present). Similar structures were also observed on diamond-like carbon (DLC) substrates, arising from evaporation of a higher initial quantity of solution. In addition, it was found that evaporation of a stock solution of phosphate-buffered saline solution (PBS) in air onto a fleshly cleaved mica surface also resulted in the same pattern formation as that shown in Figure 2. Crosslike and Spherical Structures. Fang et al. also reported on the formation of crosslike structures on acidtreated mica, again attributing these features to the assembly of CPMV. Spherical structures were also observed which the authors presumed were precursors of
10.1021/la0205046 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/14/2002
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Figure 4. (a) Data from Fang et al.1 showing crosslike structures obtained by evaporation onto acid-treated mica of a 0.15 mg/mL stock CPMV solution. (b) Optical image of crosslike and spherical structures obtained by evaporation onto acidtreated mica (same solution chemistry as used in Figure 2). Figure 2. (a) Contact mode AFM image (obtained from the null experiment) showing a pattern arising from evaporation of a 0.1 M potassium phosphate buffer (pH 7) diluted (dilution factor of 100) with 10 mM Tris pH 8.0 onto freshly cleaved mica (∼2 µL of solution). (b) Typical height profile obtained along the fingerlike projections. (c) Optical image of the salt patterns.
Figure 5. Optical images showing crosslike and spherical structures resulting from evaporation (same solution chemistry as used in Figure 2) onto glass slides. The highlighted square shows the formation of a partially formed crosslike structure.
Figure 3. Optical images showing patterns taken at two different locations arising from solution evaporation (same solution chemistry as used in Figure 2) onto glass slides.
the crosslike features. The optical image from their paper is shown in Figure 4a. Acid-treated mica surfaces in combination with identical solution conditions to those
accounting for the outcomes in Figure 2 sometimes resulted in crosslike structures that were indistinguishable from those of Fang et al. (see Figure 4b). Both crosslike and spherical structures were also evident on glass substrates, where they occurred in close proximity (usually at the periphery) to patterns of parallel and orthogonal line structures (see Figure 5). The heights of the crosslike and spherical structures were typically in the range 200-600 nm. Partially formed crosslike structures (like those shown by Fang et al.) were also observed,
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as highlighted by the square in Figure 5. The size of the crosslike structures varied considerably, as shown in Figure 5. In conclusion, the ability to reproduce the data presented by Fang et al. with the same solution chemistry and conditions but without the virus casts doubt on whether the patterns are comprised of CPMV. The balance of evidence shows that results purported to demonstrate novel self-assembly of CPMV on a mica substrate are more likely to arise from precipitation of salt from evaporation. That conclusion is based on the null experiment. The characteristic patterns can be generated for a range of substrates and solution conditions. If the CPMV do form
Comments
the patterns as suggested by the authors, then it seems plausible that they are directed to follow specific paths as dictated by the salt crystallization. Acknowledgment. The author thanks Dr. Sverre Myhra and Jolanta Blach for helpful discussions. Gregory S. Watson*
School of Science, Griffith University, Nathan, Queensland 4111, Australia Received May 29, 2002 LA0205046
