Plant Viral Capsids as Nanobuilding Blocks: Construction of Arrays on

Oct 21, 2006 - ... use the virus-derived building blocks for construction of arrays on solid ..... Hyuk-Seong Seo , Seong-Eun Kim , Jin-Seung Park , J...
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Plant Viral Capsids as Nanobuilding Blocks: Construction of Arrays on Solid Supports Nicole F. Steinmetz,† Grant Calder,‡ George P. Lomonossoff,† and David J. Evans*,† Department of Biological Chemistry and Department of Cell and DeVelopmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom ReceiVed July 21, 2006. In Final Form: September 13, 2006 The virions of Cowpea mosaic Virus (CPMV) can be regarded as programmable nanobuilding blocks with a diameter of approximately 28 nm. The particles display a number of features that can be exploited for nanoscale material fabrication. In this study we use the virus-derived building blocks for construction of arrays on solid supports. Biotinmodified CPMV particles are used with Streptavidin as a linker molecule in order to enable self-assembly of arrays from the surface up by a layer-by-layer approach. CPMV particles with different fluorescent labels, which enable differential detection of each layer, have been immobilized on surfaces and arranged in defined layers. This approach provides novel structured arrays which have the potential for development as functional devices at the nanoscale.

Introduction Self-assembly of (bio)molecules in defined layers on solid supports is highly desirable and an essential part of nanotechnology.1 Such arrays provide a high density of, and equivalent environments for, binding sites for functionalization. Their spontaneous or directed self-assembly relies on cooperative interactionsscovalent or noncovalentsof one or more small components that assemble in a predefined way to produce a larger structure from the bottom-up. Nanostructured arrays or devices are of growing technological interest for the design of new functional materials and find applications in sensors, nanoelectronics, information processing, data storage, optics, and biomedicine.2,3 Biology has played a key role as a source of inspiration for nanosciences. Biological macromolecules are endowed with capabilities for self-assembly that facilitate bottom-up fabrication.3,4 In this study we utilized capsids from the Cowpea mosaic Virus (CPMV) as nanobuilding blocks for the generation of arrays of defined layers of biomolecules on solid supports. The capsids of CPMV have a well-defined icosahedral structure formed by 60 identical copies of the asymmetric unit with a diameter of approximately 28 nm. CPMV particles provide a number of features that make them an ideal nanobuilding block: high quantities can be produced easily, their properties are well characterized,5-7 they are extremely robust, and the capsid surface is multidentate. The feasibility of CPMV as a molecular assembly in nanotechnology has been demonstrated. In previous studies both CPMV wild type (wt) and CPMV mutants have been function-

* To whom correspondence should be addressed. E-mail: [email protected]. † Department of Biological Chemistry, John Innes Centre. ‡ Department of Cell and Developmental Biology, John Innes Centre. (1) Whitesides, G. M.; Grzybowski, B. Science 2002, 295, 2418. (2) Gates, B. D.; Xu, Q.; Stewart, M.; Ryan, D.; Willson, C. G.; Whitesides, G. M. Chem. ReV. 2005, 105, 1171. (3) Lowe, C. R. Curr. Opin. Struct. Biol. 2000, 10, 428. (4) Wu, L. Q.; Payne, G. F. Trends Biotechnol. 2004, 22, 593. (5) Lomonossoff, G. P.; Johnson, J. E. Prog. Biophys. Mol. Biol. 1991, 55, 107. (6) Lin, T.; Johnson, J. E. AdV. Virus Res. 2003, 62, 167. (7) Lin, T.; Chen, Z.; Usha, R.; Stauffacher, C. V.; Dai, J. B.; Schmidt, T.; Johnson, J. E. Virology 1999, 265, 20.

alized and decorated with a variety of molecules.8-28 Unique amines8,11,24 and carboxylates9 on the solvent-exposed surface of CPMV wt allow chemical modification, and genetically engineered cysteine mutants (CPMVCYS) have been functionalized via thiol-selective chemistry.12 The multiple addressability of the robust CPMV building block makes it an ideal nanoscaffold, and functionalization may lead to development of novel devices and materials. To date, most functionalization studies have been in solution. There are, in addition, a few examples where the virions have been bound to surfaces. The genetically modified capsids of CPMVCYS have been bound to maleimido-functionalized templates, which has enabled the use of dip-pen and scanning probe (8) Steinmetz, N. F.; Lomonossoff, G. P.; Evans, D. J. Small 2006, 2, 530. (9) Steinmetz, N. F.; Lomonossoff, G. P.; Evans, D. J. Langmuir 2006, 22, 3488. (10) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192. (11) Wang, Q.; Kaltgrad, E.; Lin, T.; Johnson, J. E.; Finn, M. G. Chem. Biol. 2002, 9, 805. (12) Wang, Q.; Lin, T.; Johnson, J. E.; Finn, M. G. Chem. Biol. 2002, 9, 813. (13) Wang, Q.; Lin, T.; Tang, L.; Johnson, J. E.; Finn, M. G. Angew. Chem., Int. Ed. 2002, 41, 459. (14) Medintz, I. L.; Sapsford, K. E.; Konnert, J. H.; Chatterji, A.; Lin, T.; Johnson, J. E.; Mattoussi, H. Langmuir 2005, 21, 5501. (15) Portney, N. G.; Singh, K.; Chaudhary, S.; Destito, G.; Schneemann, A.; Manchester, M.; Ozkan, M. Langmuir 2005, 21, 2098. (16) Blum, A. Z.; Soto, C. M.; Wilson, C. D.; Brower, T. L.; Pollack, S. K.; Schull, T. L.; Chatterji, A.; Lin, T.; Johnson, J. E.; Amsinck, C.; Franzon, P.; Shashidhar, R.; Ratna, B. R. Small 2005, 1, 702. (17) Wang, Q.; Raja, K. S.; Janda, K. D.; Lin, T.; Finn, M. G. Bioconjugate Chem. 2003, 14, 38. (18) Binder, W. H. Angew. Chem., Int. Ed. 2005, 44, 5172. (19) Russell, J. T.; Lin, Y.; Bo¨ker, A.; Su, L.; Carl, P.; Zettl, H.; He, J.; 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. (20) Chatterji, A.; Ochoa, W. F.; Ueno, T.; Lin, T.; Johnson, J. E. Nano Lett. 2005, 5, 597. (21) Blum, A. S.; Soto, C. M.; Wilson, C. D.; Cole, J. D.; Kim, M.; Gnade, B.; Chatterji, A.; Ochoa, W. F.; Lin, T.; Johnson, J. E.; Ratna, B. R. Nano Lett. 2004, 4, 867. (22) Raja, K. S.; Wang, Q.; Gonzalez, M. J.; Manchester, M.; Johnson, J. E.; Finn, M. G. Biomacromolecules 2003, 4, 472. (23) Sen Gupta, S.; Raja, K. S.; Kaltgrad, E.; Strable, E.; Finn, M. G. Chem. Commun. 2005, 34, 4315. (24) Chatterji, A.; Ochoa, W. F.; Paine, M.; Ratna, B. R.; Johnson, J. E.; Lin, T. Chem. Biol. 2004, 11, 855. (25) Chatterji, A.; Ochoa, W.; Shamieh, L.; Salakian, S. P.; Wong, S. M.; Clinton, G.; Ghosh, P.; Lin, T.; Johnson, J. E. Bioconjugate Chem. 2004, 15, 807. (26) Raja, K. S.; Wang, Q.; Finn, M. G. Chembiochem 2003, 4, 1348. (27) Meunier, S.; Strable, E.; Finn, M. G. Chem. Biol. 2004, 11, 319. (28) Strable, E.; Johnson, J. E.; Finn, M. G. Nano Lett. 2004, 4, 1385.

10.1021/la0621362 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/21/2006

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nanolithography to create patterned surfaces.29,30 An alternative approach has used CPMV mutants with a poly-histidine tag inserted into the solvent-exposed capsid surface (CPMVHIS). Immobilization of the CPMVHIS mutants utilized biotin-X-NTA to bridge NeutrAvidin on a glass surface with the histidine tag on the virus particles.14 To extend further the utility of CPMV virions as nanobuilding blocks for construction of arrays for their potential use in nanoelectronic devices or multiple biosensors we demonstrate here that mono-, bi-, and multilayers of CPMV virions can be constructed on surfaces in a controlled manner. The CPMV virions were labeled with different fluorescent dyes and decorated with biotin molecules; the dyes enabled differential detection, and the biotin moieties enabled construction of arrays from the bottomup via a layer-by-layer approach. Construction of the layers was achieved using the molecular recognition between Streptavidin (SAv) and biotinylated virions (CPMVBIO). The molecular interaction between biotin and SAv is characterized by an extraordinarily high affinity constant with Ka > 1014 M-1, which gives the system chemical stability; it is the strongest ligandreceptor interaction presently known.31,32 Immobilization of CPMV particles on solid supports was achieved by either binding functionalized CPMVCYS particles to gold or binding CPMVBIO virions to immobilized SAv. The CPMVCYS mutant used in this study was generated by genetic mutagenesis and is described in Wang et al.12 We report here that various arrays ranging from mono- to multilayers consisting of alternating CPMV-SAv molecules can be constructed on Au surfaces. Construction of multilayers of virion building blocks is of particular importance as it provides routes to arrays of multiple addressable subunits that can be functionalized for fabrication of new nanomaterials such as catalysts. Further, multilayers are often required to increase the sensitivity of array-based sensors. Experimental Section Virus Growth and Purification. Propagation and purification of both CPMV wt and CPMVCYS virions were performed by standard procedures.33 Purified virions were stored at 4 °C in 10 mM sodium phosphate buffer pH 7.0 (referred to as buffer in the following text). To minimize aggregation of the CPMVCYS particles by formation of disulfide linkages, 10 mM tris(2-carboxyethyl)phosphine (Aldrich) was added to the buffer. The concentration of purified virions was determined by Bradford assay or photometrically; the molar extinction coefficient of CPMV at a wavelength of λ ) 260 nm is  ) 8.1 mL mg-1 cm-1. UV-visible spectra were recorded using a Perkin-Elmer Lambda 25 UV-visible spectrometer and UVWINLab software. Chemical Modification of Viral Particles. CPMV wt particles were functionalized at solvent-exposed lysines with biotin moieties on one hand and fluorescent labels on the other. Herein, CPMV wt particles were first partially decorated with a succinimide ester reactive biotin, biotin-LC-NHS (Pierce). CPMV particles in buffer were exposed to a 600-fold molar excess of biotin-LC-NHS dissolved in DMSO (Aldrich); the final DMSO concentration was adjusted to 20 vol %. After incubation for 2-3 h at room temperature the reaction mixture was purified via 100 K centrifugal devices (Pall). CPMV wt and CPMVBIO particles were labeled with different fluorescent dyes, AlexaFluor488 carboxylic acid succinimidyl ester and AlexaFluor568 carboxylic acid succinimidyl ester (AF488 and AF568, (29) Smith, J. C.; Lee, K.; Wang, Q.; Finn, M. G.; Johnson, J. E.; Mrksich, M.; Mirkin, C. A. Nano Lett. 2003, 2, 883. (30) Cheung, C. L.; Camarero, J. A.; Woods, B. W.; Lin, T.; Johnson, J. E.; De Yoreo, J. J. J. Am. Chem. Soc. 2003, 125, 6848. (31) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85. (32) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (33) Wellink, J. Methods Mol. Biol. 1998, 81, 205.

Figure 1. Representation of chemically modified CPMV particles: (a) CPMVBIO-AF488, (b) CPMVBIO-AF568, (c) CPMVCYS-BIO, and (d) CPMVCYS-AF488′. Molecular Probes; Figure 1a and b), to generate different labeled particles with distinct emission and absorption maxima. The labeling reaction was carried out in a buffered solution containing 20 vol % DMSO; each dye was used in a ratio of 3000 fluorophores to 1 virion. After incubation overnight at 4 °C in the dark, the reaction mixture was purified via 100 K centrifugal devices. CPMVCYS particles were decorated at surface-exposed lysines with biotin (Figure 1c) and solvent-exposed cysteines with a thiol-reactive dye, AlexaFluor488 C5-maleimide (AF488′, Molecular Probes; Figure 1d). Biotin-LC-NHS and AF488′ were used in a 1000-fold excess; the procedure was as described above. Native Gel Electrophoresis. Intact virions were analyzed by native gel electrophoresis in an agarose matrix. Virions (10 µg) were analyzed on a 1.2% agarose gel. The net negative charge of the CPMV capsid causes migration in the electric field toward the anode. Detection of the viral particles was achieved by ethidium bromide staining of the encapsidated RNA and visualization under UV light. Chemical Modification of SAv. SAv was functionalized with thiol groups (SAvSH) in order to allow binding to Au slides. Thiols were introduced by reacting SAv (Aldrich; 1 mg/mL in phosphatebuffered saline (PBS)) with a 50-fold molar excess of iminothiolane (Aldrich; dissolved in Tris-EDTA (ethylenediamintetraacetic acid),

10034 Langmuir, Vol. 22, No. 24, 2006 pH 8.0) overnight at 4 °C. The reaction mixture was purified with 30 K cut off columns (Millipore). SAvSH was labeled also with AF488′ (SAvSH-AF488′); the reaction was carried out in an analogous manner to the labeling reaction of CPMVCYS (see above). A control reaction with unmodified SAv was also performed. Preparation of Au Slides. Au slides were prepared by sputter coating glass cover slips with gold using an Agar high-resolution sputter coater (Agar Scientific Ltd). SAv-functionalized Au slides were prepared by binding SAvSH to the gold. SAvSH was deposited on the surfaces overnight at 4 °C followed by washing with phosphatebuffered saline (PBS) and Millipore water. Dot Blot Studies. Several dot blot systems were used in this study. SAv and chemically modified SAvSH and SAvSH-AF488′ were spotted on Au slides (10 µg in PBS, incubation overnight at 4 °C). After blocking (PBS + 3% Tween20 (Sigma) + 5% milk (Marvel)), the blots were probed with biotin coupled to horseradish peroxidase (HRP; Zymed); signals were visualized by electrochemiluminescence (ECL) using a Curix 60 film processor (Agfa Gevaert). Between each step the surfaces were washed several times with PBS and 3% Tween20. To prove successful biotinylation, unmodified CPMV wt and mutant particles were spotted on nitrocellulose membranes. Detection was performed using a HRP-SAv complex (Upstate) and ECL. To study binding of CPMV, CPMVBIO, CPMVAF488, CPMVAF568,CPMVBIO-AF488,CPMVBIO-AF568,CPMVCYS,CPMVCYS-BIO, and CPMVCYS-AF488′ on Au slides and SAv-functionalized surfaces, a 10 µg sample in buffer was spotted onto each support (incubation overnight at 4 °C). After blocking, the surfaces were probed with polyclonal antibodies raised in rabbits against CPMV particles, followed by a Donkey anti-rabbit IgG coupled to HRP (Amersham Biosciences). ECL was again used for detection. Fluorescence Microscopy of Arrays. Arrays of dye-labeled CPMV particles were constructed and analyzed by fluorescence microscopy. The arrays were constructed as follows: 10 µg of CPMV in buffer or SAv in PBS, respectively, was spotted on the Au sputtercoated surface and allowed to bind to the support or the previous layer for 6 h at room temperature or at 4 °C overnight. Excess nonbound biomolecules were removed by washing with PBS. A Nikon E600 microscope with ×100/1.4 oil lens was used to characterize the fluorescent labels on the virions. AF488 was excited at 480-500 nm, and emitted light was captured at 509-547 nm. AF568 was excited at 541-569 nm and imaged at 580-654 nm. All images were captured using a Hamamatsu ORCA-AG charged coupled device camera; images were analyzed using ImageJ (http:// rsb.info.nih.gov/ij/).

Results and Discussion Characterization of Chemically Modified CPMV Particles. CPMV wt particles were functionalized at solvent-exposed lysines with both biotin moieties and fluorescent labels (AF488 and AF568, Figure 1a and b, respectively). CPMVCYS particles were decorated at surface-exposed lysines with biotin (Figure 1c) and solvent-exposed cysteines with a thiol-reactive dye (AF488′, Figure 1d). The integrity of labeled and double-labeled particles after derivatization was confirmed by transmission electron microscopy (data not shown). In all samples the particles are intact and monodisperse with a diameter of approximately 30 nm. The dye-labeled CPMV particles CPMVAF488, CPMVAF568, CPMVBIO-AF488, CPMVBIO-AF568, and CPMVCYS-AF488′ were analyzed by UV-visible spectroscopy. The absorption maxima characteristic for each dye was used as an indicative peak to monitor covalent binding. The UV-visible spectra (see Supporting Information) are consistent with successful decoration of the amines (lysine residues) in the case of wt virions with AF488 and AF568. Also, labeling of thiols on CPMVCYS with AF488′ was achieved. The degree of labeling was quantified using the relevant extinction coefficients: CPMVAF488 and CPMVAF568 were labeled with up to 240 fluorophores per virion

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Figure 2. CPMV particles after separation in an electric field on a 1.2% agarose gel with (+) and without (-) ethidium bromide (EtBr) staining visualized under UV light: Lane 1 ) CPMVAF488, 2 ) CPMVBIO-AF488, 3 ) CPMV, 4 ) CPMVBIO, 5 ) CPMVBIO-AF568, 6 ) CPMV, 7 ) CPMVAF568, 8 ) CPMVCYS-BIO, 9 ) CPMVCYS, 10 ) CPMVCYS-AF488′, 11 ) CPMVCYS.

(total number of lysines is 300 per virion); up to 200 dyes per virion were attached to CPMVBIO-AF568 and CPMVBIO-AF488 particles (the number is lower because the biotin group is bound to the lysines too; this suggest ca. 40 biotin molecules were attached to the virions); CPMVCYS-AF488′ virions were labeled with approximately 30 dyes per particle. The total number of cysteines is 60 per virion. The CPMVCYS-AF488′ particles have, therefore, enough free thiol groups (30) in order to allow immobilization of the particles onto gold via thiol-gold interactions. Further, the number of functional groups attached to CPMV can be controlled by variation of incubation time and the excess of reactants used. For the double-labeling reactions, the reaction conditions for the first step were chosen to ensure that only partially modified particles were produced, while the conditions for the second step were designed to yield fully modified virions (for details see Experimental Section). Native gel electrophoresis of intact chemically modified virions in an agarose matrix indicated the successful covalent decoration of the virus particles with dyes and biotin (Figure 2). Intact virions of CPMV can be separated into two forms: the higher mobility band in the agarose gel represents virions with a cleaved small subunit34,35 whereas the lower mobility band represents virions with a full-length small subunit. All modified virionss biotinylated, dye-labeled, and double-labeledsshow a higher mobility in the electric field compared to unmodified CPMV wt and CPMVCYS particles. This can be explained by a charge effect (34) Taylor, K. M.; Spall, V. E.; Butler, P. J.; Lomonossoff, G. P. Virology 1999, 255, 129. (35) Geelen, J. L.; Rezelman, G.; van Kammen, A. Virology 1973, 51, 279.

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Figure 3. Presentation of results from dot blot studies. (A) CPMV particles spotted on Au slides; detection was achieved with polyclonal antibodies against CPMV particles. A dark signal shows binding of the sample to the Au slide. (B) CPMV spotted on nitrocellulose; detection via Streptavidin-labeled horseradish peroxidase (SAv-HRP). A dark signal shows successful biotinylation. (C) CPMV particles spotted on SAv-functionalized Au slides; detection was achieved with polyclonal antibodies against CPMV particles. A dark signal shows binding of the sample to the SAv slide. (D) SAv and thiol-modified SAv spotted on Au slides; detection via a biotin-labeled HRP; a dark signal proves binding.

arising from the attached chemical moieties. Further, the separated virions could be visualized by UV light with and without ethidium bromide staining. The fluorescence of the dye-labeled particles in the gels without ethidium bromide staining confirms covalent decoration with the fluorophores. Binding of CPMV Particles on Au Slides. Binding of the labeled/unlabeled CPMV wt and mutant particles to Au sputtercoated slides was monitored using dot blot techniques. CPMV, CPMVBIO,CPMVAF488,CPMVAF568,CPMVBIO-AF488,CPMVBIO-AF568, CPMVCYS, CPMVCYS-BIO, and CPMVCYS-AF488′ were spotted on Au slides; polyclonal antibodies against CPMV particles and ECL were used for detection. We found that none of the CPMV wt particles (CPMV, CPMVBIO, CPMVAF488, CPMVAF568, CPMVBIO-AF488, CPMVBIO-AF568) were adsorbed on the Aucovered slides. This is expected as CPMV wt does not display any cysteines on its outer capsid surface. CPMVCYS particles, in contrast, display 60 cysteines on the solvent-exposed capsid surface per particle, allowing binding to gold via the strong sulfur-gold interaction. Dot blot tests confirmed binding of all CPMVCYS samples (CPMVCYS, CPMVCYS-BIO, CPMVCYS-AF488′) to Au slides (Figure 3a). To prove successful biotinylation, CPMV wt and mutant particles were spotted on nitrocellulose membranes; detection was performed using a HRP-SAv complex. The results showed successful biotinylation and also that the biotin group when covalently attached to CPMV capsids still has the ability to interact with SAv (Figure 3b). Further, dot blots on SAv-coated surfaces analyzing wt particles (CPMV, CPMVBIO, CPMVAF488, CPMVAF568, CPMVBIO-AF488, CPMVBIO-AF568) demonstrated that biotinylated samples only were bound to the surface. Unspecific adsorption of non-biotinylated CPMV wt particles was not

detected; the biotinylated virions bind specifically to the slides via the strong interaction between biotin and SAv (Figure 3c). The dot blot binding studies indicated that binding of the building blocks, CPMV and SAv, occurred in a controlled manner based on a sulfur-gold interaction (CPMVCYS-Au and SAvSHAu) and on specific interactions between biotin-bound CPMV and SAv; nonspecific adsorption was not detected. This enables the use of the building blocks for directed deposition on surfaces and fabrication of arrays by a layer-by-layer approach. Construction of Mono- and Multilayers Consisting of CPMV Particles. Three different monolayers were studied: CPMVCYS-AF488′ bound onto Au slides and CPMVBIO-AF488 and CPMVBIO-AF568 on SAv-functionalized Au slides. First, the coverage of SAvSH on the Au surface was investigated. SAvSH was labeled with the thiol-reactive dye AF488′; a control reaction with unmodified SAv was also performed. The UV-visible spectra (see Supporting Information) are consistent with successful decoration of SAvSH with the dye. As expected, SAv did not react with the thiol-selective dye as there are no cysteinethiols available. The degree of labeling was calculated using the extinction coefficient of the dye; the SAvSH was decorated with approximately one dye per molecule. The ability of modified SAvSH to bind to Au slides was confirmed using dot blot tests with biotin-HRP for detection; the SAvSH was shown to be attached to the surface (Figure 3D). Immobilization of SAvSH-AF488′ onto Au slides was analyzed by fluorescence microscopy. Imaging demonstrated that SAvSH-AF488′ was evenly distributed over the whole surface (Figure 4). Fluorescence microscopy imaging of immobilized CPMV monolayers (Figure 4)sCPMVCYS-AF488′, CPMVBIO-AF488, and CPMVBIO-AF568 (not shown)swas consistent with successful

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Figure 4. Monolayers of fluorescent-labeled Streptavidin (SAvSH-AF488′) and biotinylated (bio) and/or fluorescent-labeled CPMV particles on Au slides. Fluorescence microscopy images (left), and diagrammatic representation of layer structures (right). The green flag shows the AlexaFluor dye AF488. The gray cross depicts thiol-modified Streptavidin. The scale bar is 10 µm.

Figure 5. Bilayers and a mixed monolayer of biotinylated (bio) and fluorescent-labeled CPMV particles on Au slides imaged via fluorescence microscopy (left), and diagrammatic representation of layer structures (right). The green and red flags show the AlexaFluor dyes AF488 and AF568, respectively. The black cross depicts Streptavidin (SAv); the gray cross shows a thiol-modified SAv. The scale bar is 10 µm. (A) bilayer of CPMVBIO-AF488 and CPMVBIO-AF568, CPMVBIO-AF488 in the first and CPMVBIO-AF568 in the second layer; merge shows the overlaid images from the first and second layer. (B) Bilayer of CPMVBIO-AF568 and CPMVBIO-AF488. (C) Mixed monolayer of CPMVBIO-AF488 and CPMVBIO-AF568. Table 1: Measured Intensities (I) of Fluorescent CPMV Particle Arrays (see Figures 4-6) Measured by Fluorescence Microscopya array CPMVCYS-AF488′ on Au slide CPMVBIO-AF488 on SAv slide CPMVBIO-AF568 on SAv slide CPMVBIO-AF488 on Au slide CPMVBIO-AF568 on Au slide CPMVBIO-AF488 on Au slide after treatment with SAv CPMVBIO-AF568 on Au slide after treatment with SAv bilayer of CPMVBIO-AF488 and CPMVBIOAF568 mixed monolayer of CPMVBIO-AF488 and CPMVBIOAF568 CPMVBIO-AF568 added onto CPMVCYS-AF488′-BIO on Au slide

IAF488

IAF568

1230 830 1080 10-100 30-70 20-160 1440 1240 890

60-130 1070 530 20

a Data for AlexaFluor dye AF568 were recorded at 100 ms and for AF488 at 1000 ms. The values measured for Au slides without samples were set to zero.

binding of the building blocks to the solid support. Further, it was found that the fluorescent viral particles were spread over the whole surface and that dense coverage was achieved. This is consistent with a uniform monolayer of CPMV particles being deposited on the surface. The surface was covered with particles having a size range of approximately 0.3-0.5 µm. Due to the limitations of the resolution of the microscope, single CPMV particles cannot be visualized. The appearance of these larger spots could be a consequence of the roughness of the Au slide. Scanning microscopy studies of the Au surfaces showed that these were covered with gold nanoparticles of various sizes (data not shown). These nanoparticles are derived from the sputter coating process and explain the roughness of the surface. It is possible also that the imaged spots are derived from CPMV particles. The dots appear about a factor of 10 bigger than the CPMV particles, which can be explained by the cone effect of the emitted light. In a few samples larger aggregates were detected;

these could be aggregates of viral particles (especially CPMVCYS) or more likely the aggregates are derived from unevenness of the surface itself. The following negative controls were chosen for fluorescence microscopy characterization: CPMVBIO-AF488 (Figure 4) and CPMVBIO-AF568 (not shown) applied directly onto the Au surface; in addition, the samples were added to Au surfaces after applying unmodified SAv (not shown). The intensities of fluorescence (I) measured via the fluorescence microscopy studies for both CPMVBIO-AF488 and CPMVBIO-AF568 applied directly onto the Au surface were only about 1-10% of the I measured when particles were bound to the surface in a controlled manner (Table 1). The values of I increased slightly to 5-15% when SAv was added to the slides first. The differences of measured I of controlled binding versus unspecific adsorption are significant; this is in good agreement with the dot blot data, showing that unspecific adsorption does not, or only to a small degree, occur.

Viral Nanobuilding Blocks

CPMV bilayers comprised of Au-SAvSH-CPMVBIO-AF488SAv-CPMVBIO-AF568 and vice versa (Figure 5) were fabricated and analyzed. In both cases fluorescence microscopy measurements confirmed the presence of both building blocks (CPMVBIO-AF488 and CPMVBIO-AF568) immobilized and ordered on the Au slide. The fluorescent-labeled particles of both layers were evenly distributed on the surface; a dense coverage was achieved. The overlay of the images demonstrated that the individual images line up well, indicating that the virions are sitting on top of each other. To further support these observations, CPMVBIO-AF488 and CPMVBIO-AF568 particles were mixed and applied into the same layer (Figure 5). In this case, the particles compete for the same binding sites, which resulted in less dense and less evenly distributed coverage. The merged image shows that the individual images do not line up, consistent with the particles occupying the same layer and competing for the same binding sites, e.g., the black holes in the CPMVBIO-AF568 layer match with high densities of CPMVBIO-AF488. Further, the values of measured I are less when compared to the I values of the mono- or bilayers (Table 1). The comparison of the overlaid image from the bilayers to that of the mixed layer further supports the successful controlled fabrication of a bilayer consisting of different fluorescent CPMV particles. Construction of a triple layer was also achieved using CPMVCYS-BIO in the first layer and CPMVBIO-AF488 and CPMVBIO-AF568 in subsequent layers (Figure 6). In this arrangement the first layer could not be imaged as the building blocks used were not fluorescent. However, the presence of the second and third layer was confirmed (these would not be present if the first layer was not formed). Again, the overlaid image indicated the particles sitting on top of each other. As a control the second CPMV layer, here CPMVBIO-AF568, was added to the immobilized CPMVCYS-BIO without adding the intermediate SAv layer; imaging via fluorescence microscopy showed that no unspecific adsorption of the particles occurred (see Table 1).

Conclusion We demonstrated the feasibility of CPMV particles as nanobuilding blocks for the controlled fabrication of arrays on

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Figure 6. Triple layer of biotinylated (bio) and/or fluorescentlabeled CPMV particles on Au slides imaged via fluorescence microscopy (left), and diagrammatic representation of layer structures (right). The green and red flags show the AlexaFluor dyes AF488 and AF568, respectively. The black cross depicts Streptavidin. The scale bar is 10 µm. Triple layer of CPMVCYS-BIO, CPMVBIO-AF568 (image second layer), and CPMVBIO-AF488 (image third layer).

solid supports. The multiaddressability of the virions allowed decoration with fluorescent dyes in addition to functionalization with linker molecules (biotin), which enabled directed selfassembly via a layer-by-layer approach. Introduction of functional groups, such as redox-active moieties, in these arrays is currently under investigation and expected to provide potential for development of novel functional devices with interesting electrochemical properties and possible applications in nanoelectronic devices or nanosensors. Acknowledgment. Professor Tianwei Lin (Scripps Research Institute) is thanked for providing the CPMV cysteine mutant. Work was funded by the BBSRC and EU Grant MEST-CT2004-504273. Supporting Information Available: UV-visible spectra of unbound AlexaFluor dyes and dyes bound to Cowpea mosaic Virus particles and Streptavidin. This material is available free of charge via the Internet at http://pubs.acs.org. LA0621362