Langmuir 2006, 22, 8891-8896
8891
Assembly of Multilayer Films Incorporating a Viral Protein Cage Architecture Peter A. Suci,#,⊥ Michael T. Klem,†,⊥ Fernando T. Arce,§ Trevor Douglas,*,†,⊥ and Mark Young*,‡,⊥ Department of Chemistry & Biochemistry, Department of Plant Sciences, Department of Physics, Department of Microbiology and Center for BioInspired Nanomaterials, Montana State UniVersity, Bozeman, Montana 59717 ReceiVed May 1, 2006. In Final Form: July 24, 2006 Protein cage architectures such as viral capsids, heat shock proteins, ferritins, and DNA-binding proteins are nanoscale modular subunits that can be used to expand the structural and functional range of composite materials. Here, layer-by-layer (LbL) assembly was used to incorporate cowpea chlorotic mottle virus (CCMV) into multilayer films. Three types of multilayer films were prepared. In the first type, ionic interactions were employed to assemble CCMV into triple layers. In the second type, complementary biological interactions (streptavidin/biotin) were used for this purpose. In a third variation of LbL assembly, complementary biological interactions were employed to produce nanotextured films that exhibit in-plane order over a micron scale without the need to adsorb onto a prepatterned template.
Introduction Protein cage architectures such as viral capsids,1-5 ferritins,6-11 heat shock proteins,12-14 and DNA-binding proteins15 are nanoscale building blocks that can be used to implement biomimetic approaches directed toward fabricating higher order nanostructures similar to those conceived by nature.16,17 Protein cages have been directed to assemble into higher order structures both by exploiting endogenous attributes 1,14 and by inserting functional groups that can participate in complementary * Corresponding author. E-mail:
[email protected] (T.D.);
[email protected] (M.Y.). † Department of Chemistry & Biochemistry. ‡ Department of Plant Sciences. § Department of Physics. # Department of Microbiology. ⊥ Center for BioInspired Nanomaterials. (1) Fowler, C. E.; Shenton, W.; Stubbs, G.; Mann, S. AdV. Mater. 2001, 13, 1266-1269. (2) Mao, C. B.; Flynn, C. E.; Hayhurst, A.; Sweeney, R.; Qi, J. F.; Georgiou, G.; Iverson, B.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 69466951. (3) Douglas, T.; Young, M. Nature 1998, 393, 152-155. (4) Gillitzer, E.; Willits, D.; Young, M.; Douglas, T. Chem. Commun. 2002, 2390-2391. (5) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J. N.; Mann, S. Nano Lett. 2003, 3, 413-417. (6) Allen, M.; Willits, D.; Young, M.; Douglas, T. Inorg. Chem. 2003, 42, 6300-6305. (7) Kramer, R. M.; Li, C.; Carter, D. C.; Stone, M. O.; Naik, R. R. J. Am. Chem. Soc. 2004, 126, 13282-13286. (8) Okuda, M.; Kobayashi, Y.; Suzuki, K.; Sonoda, K.; Kondoh, T.; Wagawa, A.; Kondo, A.; Yoshimura, H. Nano Lett. 2005, 5, 991-993. (9) Scheybani, T.; Yoshimura, H.; Baumeister, W.; Nagayama, K. Langmuir 1996, 12, 431-435. (10) Yamashita, I. Thin Solid Films 2001, 393, 12-18. (11) Gilles, C.; Bonville, P.; Rakoto, H.; Broto, J. M.; Wong, K. K. W.; Mann, S. J. Magn. Magn. Mater. 2002, 241, 430-440. (12) Flenniken, M. L.; Liepold, L. O.; Crowley, B. E.; Willits, D. A.; Young, M. J.; Douglas, T. Chem. Commun. 2005, 447-449. (13) Flenniken, M. L.; Willits, D. A.; Brumfield, S.; Young, M. J.; Douglas, T. Nano Lett. 2003, 3, 1573-1576. (14) McMillan, R. A.; Howard, J.; Zaluzec, N. J.; Kagawa, H. K.; Mogul, R.; Li, Y. F.; Paavola, C. D.; Trent, J. D. J. Am. Chem. Soc. 2005, 127, 2800-2801. (15) Wiedenheft, B.; Mosolf, J.; Willits, D.; Yeager, M.; Dryden, K. A.; Young, M.; Douglas, T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10551-10556. (16) Du, C.; Falini, G.; Fermani, S.; Abbott, C.; Moradian-Oldak, J. Science 2005, 307, 1450-1454. (17) Aizenberg, J.; Weaver, J. C.; Thanawala, M. S.; Sundar, V. C.; Morse, D. E.; Fratzl, P. Science 2005, 309, 275-278.
interactions.14,18-22 Protein cages have been organized into liquid crystal mesophases,1 ordered planar arrays,14,19 two-dimensional networks,22 nanowires,20 and nanorings.21 Layer-by-layer (LbL) assembly offers a relatively simple and versatile method for organizing protein cage architectures into higher order structures.23,24 A general appeal of LbL assembly is that multiple functionalities can be easily incorporated into films that can be deposited, even on irregular or flexible supports, with the potential for intermixing various components at the nanoscale.25,26 LbL assembly can be used to make semipermeable membranes, responsive membranes, sensors, electrochromic films, solid-state electrolytic films for photovoltaic or fuel-cell components, and nanomechanical films. Protein cages offer ideal modular nanoplatforms that can be used to expand the range of both structural and functional components that can be integrated into LbL films. For the studies presented here, cowpea chlorotic mottle virus (CCMV) was incorporated into LbL films. The CCMV capsid is a 28 nm diameter spherical protein cage possessing icosahedral (T ) 3) symmetry. CCMV has a number of special attributes that motivate the effort to incorporate it into LbL films. CCMV capsids exhibit reversible pH-dependent gating, which has been exploited to regulate size-constrained biomimetic mineralization.3 The interior surface of CCMV capsids has been genetically engineered to act as a ferritin surrogate that spatially constrains the formation of iron oxide nanoparticles,27 and exterior surfaces have been (18) Strable, E.; Johnson, J. E.; Finn, M. G. Nano Lett. 2004, 4, 1385-1389. (19) Cheung, C. L.; Camarero, J. A.; Woods, B. W.; Lin, T. W.; Johnson, J. E.; De Yoreo, J. J. J. Am. Chem. Soc. 2003, 125, 6848-6849. (20) Huang, Y.; Chiang, C. Y.; Lee, S. K.; Gao, Y.; Hu, E. L.; De Yoreo, J.; Belcher, A. M. Nano Lett. 2005, 5, 1429-1434. (21) Nam, K. T.; Peelle, B. R.; Lee, S. W.; Belcher, A. M. Nano Lett. 2004, 4, 23-27. (22) Li, M.; Mann, S. J. Mater. Chem. 2004, 14, 2260-2263. (23) 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. (24) Yoo, P. J.; Nam, K. T.; Qi, J. F.; Lee, S. K.; Park, J.; Belcher, A. M.; Hammond, P. T. Nat. Mater. 2006, 5, 234-240. (25) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319-348. (26) Hammond, P. T. AdV. Mater. 2004, 16, 1271-1293. (27) Douglas, T.; Strable, E.; Willits, D.; Aitouchen, A.; Libera, M.; Young, M. AdV. Mater. 2002, 14, 415-418.
10.1021/la0612062 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/08/2006
8892 Langmuir, Vol. 22, No. 21, 2006
functionalized for the multivalent presentation of ligands.4 Furthermore, the density of a multiligand presentation can be tuned by the reassembly of differentially functionalized protein subunits,28 and functional groups can be placed asymmetrically to restrict functionalization to one region of the symmetrical capsid.29 We recently characterized the electrostatically driven adsorption behavior of CCMV on Si and amine-functionalized Si.30 Here we extended this latter approach to fabricate three types of multilayer films, using a monolayer of CCMV electrostatically bound to a functionalized Si surface as a base film. Methods Virus Preparation and Labeling. CCMV was isolated from cowpea plants as previously described. 31 Purity was verified using size-exclusion chromatography (SEC) and dynamic light scattering (DLS).32 Protein concentration was determined using the absorbance at 260 nm.33 CCMV was biotinylated by reaction with a sulfonated N-hydroxysuccinimide (NHS) ester with a 3.05 nm spacer arm, sulfosuccinimidyl-6′-(biotinamido)-6-hexanamido hexanoate (Pierce). The reaction was performed in 50 mM HEPES buffer and 150 mM NaCl at pH 7.0 at room temperature with stirring under two conditions. To achieve less dense labeling, the concentration of the reagent was 0.5 mM, the CCMV concentration was 3.0 mM (1.4 mg mL-1), and the reaction time was 30 min. To achieve more dense labeling, the concentration of the reagent was 1.7 mM, the CCMV concentration was 6.4 mM (2.9 mg mL-1), and the reaction time was increased to 60 min. In both cases, the reaction was terminated by exchange into 1 mM sodium acetate buffer at pH 4.8 using SEC (Superose 6, Amersham Biosciences, Uppsala, Sweden). Fractions eluting from within the CCMV peak, the position of which was predetermined using unlabeled CCMV, were used for the experiments (see Supporting Information, Figure 1S). The labeled virus was further purified by dialysis into 100 mM sodium acetate buffer. The integrity of the labeled virus was confirmed by DLS (see Supporting Information, Figure 2S) and transmission electron microscopy (TEM) (see Supporting Information, Figure 3S) as described previously.32 Association of biotin with the virus was initially tested by a dot blot assay using an alkaline phosphatase-conjugated antibiotin antibody (A-7064, Sigma) before quantification by liquid chromatography/ electrospray mass spectrometry (LC/MS). Liquid Chromatography/Electrospray Mass Spectrometry. LC/MS was performed on a QToF Micro instrument (Waters). CCMV injected at 50-100 µg mL-1 (1-10 µL) was eluted from a C-8 reverse phase column (VYDAC) using an acetonitrile/H2O gradient in 0.1% formic acid. The virus disassembled in the running buffer into monomers that were analyzed by the detector. Mass spectra of the multiply charged ions were deconvoluted using instrument software to produce a representation of monomer mass versus intensity. Monomers from the labeled virus produced clearly separated peaks in these processed spectra, having masses corresponding to the monomer mass and the monomer mass plus between one and three reacted reagent molecules (i.e., reagent molecules minus the sulfo-NHS ester leaving group) (see Supporting Information, Figure 4S). Relative peak height was used to determine the extent of labeling (see Supporting Information, Table 1S). This was converted to the mean number of biotins per CCMV. Attenuated Total Internal Reflection Infrared Spectroscopy. Details of the attenuated total internal reflection infrared spectroscopy (ATR-FTIR) protocols have been described previously.30 Briefly, (28) Gillitzer, E.; Suci, P. A.; Young, M.; Douglas, K. Small, in press. (29) Klem, M. T.; Willits, D.; Young, M.; Douglas, T. J. Am. Chem. Soc. 2003, 125, 10806-10807. (30) Suci, P. A.; Klem, M. T.; Douglas, T.; Young, M. Langmuir 2005, 21, 8686-8693. (31) Bancroft, J. B.; Hiebert, E. Virology 1967, 32, 354. (32) Basu, G.; Allen, M.; Willits, D.; Young, M.; Douglas, T. J. Biol. Inorg. Chem. 2003, 8, 721-725. (33) Bancroft, J. B.; Hiebert, E.; Rees, M. W.; Markham, R. Virology 1968, 34, 224.
Suci et al. infrared spectra were acquired with a Nicolet 740 Fourier transform infrared (FTIR) spectrophotometer equipped with a liquid N2-cooled, medium-range MCT detector (5000-580 cm-1). The flow system and flow cell for ATR-FTIR measurements were similar to those used in previous experiments. 34,35 For adsorption, the surface of a trapezoidal (Si〈100〉) internal reflection element (IRE) (Harrick Scientific Corp., Ossining, NY) was exposed to solutions of either CCMV at 20 µg mL-1 or 50 µg mL-1, poly-L-lysine (Sigma Chemical Company) at 1 mg mL-1, or streptavidin (Sigma Chemical Company) at 50 µg mL-1. All adsorptions were performed in 100mM sodium acetate buffer (pH 4.8). Solutions were pumped into the flow cell for 150 s, and then flow was discontinued. After each adsorption period, the surface was rinsed for 30 min in sodium acetate buffer. Before each series of adsorptions, Si IREs were cleaned and functionalized with an amine. Si IREs were cleaned in a 1:1 mixture of concentrated sulfuric acid and hydrogen peroxide and then rinsed in ultrapure water, ethyl alcohol, and chloroform.30 Si IREs were amine functionalized by exposure to 1% (v/v) (aminopropyl)triethoxysilane (Sigma Corp., St. Louis, Mo) in hexanes for 2 h.30 The evanescent field that samples the interfacial region in ATRFTIR has been well-characterized. 36 The field intensity decays exponentially with distance away from the interface, with a decay rate that is determined by the relative refractive indices of the liquid and the IRE and the wavelength of the light. For the Si-water interface, the penetration depth (inverse of the decay rate) is 254 nm for the amide II region (1550 cm-1) used to follow the adsorption process. Modeling of ATR-FTIR Data. Modeling of ATR-FTIR data was performed as described previously.30 Briefly, the diffusion of a virus through the boundary layer was described by Fick’s law in one dimension, and adsorption at the interface was modeled by an empirical generalized Langmuir process.37 Commercially available software (AQUASIM, 2.0, Peter Reichert, Computer Systems Sciences Department, EAWAG, Switzerland) was used to simulate data curves and to provide estimates of kd and θrel,pro by a leastsquares criterion optimization. Preparation of Films in Microtiter Wells and Light Microscopy. For microscopic analysis, multilayers were formed in polyL-lysine-coated 96-well microtiter plates (Biocoat Poly-D-Lysine Cellware) (Becton Dickinson, Bedford, MA). Epifluorescence images were acquired through a G-2E/C TRTIC filter block at 100× using a Nikon Eclipse E600 coupled to an Olympus Camedia camera. Neutral density filters and exposure times were adjusted so that pixel intensity was within the dynamic (linear) range of the camera. The average pixel intensity of gray-scaled images was evaluated using Scion image software (Scion Corp.). For adsorption, wells were exposed to 50 µL of virus or streptavidin (Sigma Chemical Co.) at 50µg mL-1 in 100 mM sodium acetate buffer (pH 4.8) for 53 min. Between different adsorptions, the wells were rinsed twice with 200 µL of the sodium acetate buffer. Between rinses, liquid was removed from the wells by aspiration with a Pasteur pipet inserted near the edge of the well. Labeling with propidium iodide (PI) was performed by exposing adsorbed virus to freshly prepared 20 µg mL-1 PI in the sodium acetate buffer for 5 min and then rinsing twice as above. The wells were inverted and observed microscopically immediately after the second rinse. Atomic Force Microscopy (AFM). Si〈100〉 wafers (Virginia Semiconductor Inc., Fredericksburg, Virginia) were cut into small squares and then cleaned by ultrasonication in ultrapure water (Barnstead water purification system, Dubuque, IA) (4×), 100% ethyl alcohol, and chloroform, and finally by exposure to ozone for 40 min. Ethyl alcohol and chloroform were HPLC grade. Si IREs were amine functionalized by exposure to 1% (v/v) (aminopropyl)(34) Suci, P. A.; Siedlecki, K. J.; Palmer, R. J.; White, D. C.; Geesey, G. G. Appl. EnViron. Microbiol. 1997, 63, 4600-4603. (35) Suci, P. A.; Geesey, G. G.; Tyler, B. J. J. Microbiol. Methods 2001, 46, 193-208. (36) Knutzen, K.; Lyman, D. J. In Surface and Interfacial Aspects of Biomedical Polymers: Surface Chemistry and Physics; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, pp 197-247. (37) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 4401-4409.
Multilayer Films of Protein Cage Architectures
Figure 1. Schematic representation of three types of multilayer films: (a) polylysine interlayer between adlayers of CCMV (CCMV cryo-reconstruction is shown above); (b) streptavidin interlayer between adlayers of CCMV-B; (c,d) partial second adlayer induced to form by adsorbing from a mixture of CCMV-B and nonbiotinylated CCMV to form a base adlayer of CCMV-B in a matrix of CCMV (c) followed by the addition of streptavidin (d), and a second adlayer (e). triethoxysilane (Sigma Corp., St. Louis, MO) in 100% methanol for 5 min. Film preparation was in small wells bored in a Teflon plate. The adsorption protocol was the same as that for adsorption in microtiter plates (described above). After the last adsorption step, the wafers were rinsed two times in environmental grade water (Fisher Scientific, Pittsburgh, PA) (pH 5.8) for 1 min to remove salts and dried in a stream of nitrogen. AFM images were acquired in air in tapping mode using a Nanoscope III Multimode SPM (Digital Instruments, Inc., Santa Barbara, CA) with Tap300 cantilevers (NanoDevices, Santa Barbara, CA). The estimate of the expected number of clusters was made by assuming a CCMV coverage of 55% in the base film.30 This yields an area of 616 nm2/0.55 per CCMV or 893 virus particles per 1 × 1 µm2 area in the base film. Dividing by 75 to account for the dilution of biotinylated virus in the matrix of nonbiotinlyated CCMV yields 12 CCMV per 1 × 1 µm2 area and 298 CCMV per 5 × 5 µm2 area. Image-Pro Plus (MediaCybernetics) software was used to count clusters. Clusters were discriminated and identified as contiguous areas having an area greater than 1.6 times the cross-sectional area of a CCMV particle based on its crystal structure (616 nm2 or 10 pixels). The threshold was set by determining the maximum height of a monolayer region in the 5 × 5 µm2 image of the base film.
Results and Discussion Both electrostatic and complementary biological interactions were used to form multilayer structures incorporating CCMV (Figure 1). Conventionally, polyelectrolyte LbL films are constructed by alternately exposing an appropriate surface to a cationic and anionic polymer.25 Here, CCMV, carrying a net negative surface charge at the pH of the buffer,30 was substituted for the anionic polymer. In a previous study, carnation mottle virus was incorporated into LbL films using a number of alternating layers of anionic and cationic polymers that were deposited between each viral layer.38 In our case, we found that one interlayer of the cationic polymer (polylysine) was sufficient to promote the binding of successive layers of CCMV to the film. Incorporating functional groups into protein cages that can participate in specific complementary biological interactions (38) Lvov, Y.; Haas, H.; Decher, G.; Mohwald, H.; Mikhailov, A.; Mtchedlishvily, B.; Morgunova, E.; Vainshtein, B. Langmuir 1994, 10, 4232-4236.
Langmuir, Vol. 22, No. 21, 2006 8893
Figure 2. ATR-FTIR characterization of triple layer formation: (a-d) polylysine interlayer; (e-h) streptavidin interlayer. (a) (i) Kinetics of formation of the first adlayer of CCMV, (ii) change in absorbance upon exposure of the adlayer to additional bulk CCMV before exposure to polylysine; (b) kinetics of polylysine adsorption onto the first adlayer; (c) kinetics of formation of the second adlayer after exposure to polylysine; (d) increase in the relative amide II band area associated with CCMV adlayer formation for three adsorption cycles (polylysine interlayer contribution excluded): filled circles, uncorrected for evanescent field distribution; empty circles, corrected for evanescent field distribution; (e) (i) kinetics of formation of the first adlayer of CCMV-B, (ii) change in absorbance upon exposure of the adlayer to additional bulk CCMV before exposure to streptavidin; (f) kinetics of streptavidin adsorption onto the first adlayer; (g) kinetics of the formation of the second adlayer of CCMV-B after exposure to streptavidin; (h) increase in the relative amide II band area associated with CCMV-B adlayer formation for three adsorption cycles (streptavidin interlayer contribution excluded): filled circles, uncorrected for evanescent field distribution; empty circles, corrected for evanescent field distribution. CCMV adsorptions were from either a 25 µg mL-1 solution (a-d) or a 50 µg mL-1 solution (e-h).
expands the possibilities for fabricating higher order structures beyond that attainable by using electrostatic interactions alone. We used streptavidin to couple biotinylated CCMV (CCMV-B) into multilayers using this alternative approach. The kinetics of LbL film formation was characterized using ATR-FTIR. This technique was used to follow temporal changes in the area of the amide II band that originates from the peptide linkages of the protein (Figure 2). This surface-sensitive technique detects protein in the interfacial region located within about 500 nm of the surface. The base film was formed by electrostatic binding to the amine-functionalized silicon (Si) surface. The surface was rinsed with buffer between each cycle of adsorption. During these rinses, there was negligible decrease in signal, indicating that adsorption was irreversible, similar to previous results.30 Our previous AFM measurements indicated that CCMV particles maintained their integrity upon adsorption to the aminefunctionalized Si and that surface coverage saturated near the limit predicted by the random sequential adsorption model (approximately 55% coverage).30 Figure 2a shows that exposure of the surface to CCMV for approximately 1 h was sufficient to occupy most of the cationic adsorption sites. Consequently, re-exposure of this base film to CCMV resulted in only a small increase in the IR signal. Driven by electrostatic interactions, polylysine adsorbs rapidly to the negatively charged CCMV base
8894 Langmuir, Vol. 22, No. 21, 2006
Suci et al.
Table 1. Relative Rates of Adsorption and Projected Surface Coverages for Adsorption Cycles 2 and 3 interlayera p-lys StAv/B
cycleb
kads,relc
θ pro, reld
2 3 2 3
1.16 0.26 0.84 0.42
1.15 2.49 1.08 1.33
a p-lys: polylysine interlayer between CCMV adlayers; StAv/B: streptavidin interlayer between CCMV-B adlayers. b Adsorption cycle. c Rate of adsorption normalized to rate of adsorption of the first CCMV adlayer. d Projected surface coverage for infinite time relative to the surface coverage of the first CCMV adlayer (not corrected for evanescent field distribution).
layer (Figure 2b). The polylysine interlayer promotes strong adsorption of a second layer of CCMV (Figure 2c). Figure 2d shows that trilayers can be constructed by repeating these steps, suggesting that the process could be continued to fabricate higher order multilayers. The ordinate values in Figure 2d are represented as the cumulative amount of protein relative to the protein in the first layer. The evanescent field that produces the IR signal decays exponentially away from the surface, and thus the signal from protein located further from the interface is reduced. By assuming that the multilayer structure is lamellar, with an interlayer spacing determined by the diameter of the virus (28 nm), a correction for this signal reduction was made. Figure 2d shows the estimated relative cumulative protein with and without correction for the exponential decay of the field. The corrected values suggest that each cycle of adsorption adds approximately the same amount of CCMV to the multilayer film. LbL films coupled through complementary interactions exhibit deposition behavior similar to that of those associated via electrostatic interactions. Figure 2e-h are ATR-FTIR data for experiments that parallel those presented in Figure 2a-d, except that streptavidin, instead of polylysine, was used as an interlayer to couple CCMV-B. The data in Figure 2h, corrected for the exponential decay of the field, and the incorporation of a small additional factor to account for the size of streptavidin (5 nm), indicate that viral coverage in successive layers can exceed the coverage of the base layer. This may be because the linkage to the CCMV-B film allows more mobility of CCMV-B incorporated into successive films than that present in the base film, which was bound by electrostatic interactions to the amine-functionalized Si. Previously, we developed a model to describe the adsorption behavior of CCMV.30 We applied the model here to predict projected surface coverage for long times and to provide a more quantitative analysis of the kinetics of film deposition measured by ATR-FTIR (see Supporting Information for model fits to the data, Figure 5S). The rate of adsorption of CCMV was reduced for the experiments presented in Figure 5a-c compared to those in Figure 5d-f (Supporting Information). This is because 20 µg mL-1 CCMV was used in the former experiments, and 50 µg mL-1 bulk CCMV was used in the latter experiments. We previously showed that the rate of adsorption of CCMV is approximately proportional to the bulk concentration for the electrostatically driven adsorption of the base film.30 Table 1 presents the rate of adsorption, relative to the rate of adsorption of the base film, for successive adsorption cycles, and the relative projected surface coverage at infinite time predicted from the model. For both types of multilayers, the rate of adsorption of the third layer was substantially slower than that of previous layers, but the projected surface coverage for long adsorption times was higher than the surface coverage of the base film. Since the bulk composition was identical for all adsorptions,
Table 2. Relation between the Mean Abundance of the Biotin Functional Group Presentation and Dual Layer Composition expa
biotins/CCMV-Bb
StAv:CCMV-B(1)c
CCMV-B(2): CCMV-B(1)d
1,2 3 4
209 68 46
62 (65)e 4 10
0.83 (0.89) 1.00 0.86
a Experiment number. b Mean number of biotins per CCMV-B (n in Figure 1, top right). c Molar ratio of streptavidin to CCMV-B in the first adlayer. d Molar ratio of CCMV-B in the second adlayer to CCMV-B in the first adlayer (not corrected for evanescent field distribution). e Results of the replicate experiment (2) are in parentheses.
these differences in adsorption rates are not due to transport limitations. It seems likely that, as more layers are added, the complexity of the interface increases and that, due to steric effects, longer periods are required for interfacial virus particles to approach an equilibrium binding configuration, for example, to form a critical number of electrostatic linkages, or to assume the appropriate orientation for a secure biotin/streptavidin linkage to form. Multilayer protein cage films that incorporate additional biomolecules in the interlayer can be considered to be heteronanostructures. In our case, streptavidin was used only as a coupler but the interlayer could also be endowed with an additional functional role. Table 2 shows that the coverage of the streptavidin interlayer was regulated by the presentation of biotin functional groups in the base film. This provides an additional means to manipulate the multilayer structure. The abundance of biotin groups attached to individual particles was estimated using LC/ MS. The molar ratio of streptavidin to CCMV-B was determined from the IR data and the known molecular weights. The data in Table 1 indicate that altering the coverage of the streptavidin interlayer has a negligible effect on the amount of virus in the second layer, implying that the density of the biotin presentation in the base film is not limiting the coverage of the second layer of CCMV-B. We developed a fast throughput method for characterizing the LbL assembly of CCMV-B that allows experiments to be performed more efficiently and with considerably less material than the ATR-FTIR technique. The wild-type CCMV used for these experiments carries single-stranded RNA as cargo that can be labeled with the nucleic acid stain, PI. This enabled the viral films to be visualized using epifluorescence microscopy. Figure 3 shows results in which this method was used to characterize adlayer formation. Negative controls confirm that multilayer formation is in fact promoted by the complementary biological interaction (streptavidin-biotin). In addition to fabricating LbL films in which the density of the streptavidin interlayer was varied in a controlled fashion (see Table 2), we were also able to control the coverage of the second CCMV-B adlayer by forming the base layer from a mixture of CCMV-B and (nonbiotinylated) CCMV (shown schematically in Figure 1c). The negatively charged, nonbiotinylated CCMV formed a passive (nonadhesive) matrix for the dispersion of CCMV-B. Both IR and microscopic data showed that, as the proportion of CCMV-B in the base layer was increased, the CCMV-B bound via the streptavidin interlayer became more abundant (Figure 4). The relationship measured by both methods was similar and was nonlinear. The shapes of the curves, which were concave downward, suggested that the relationship between the CCMV-B abundance in the second layer and the CCMV-B in the first layer was greater than one to one when the functionalized virus in the base layer was diluted, indicating that the second layer consisted of clusters of CCMV-B (Figure 1e).
Multilayer Films of Protein Cage Architectures
Figure 3. Epifluorescence microscopy of PI-labeled CCMV adlayers adsorbed from 50 µg mL-1 bulk solutions of CCMV onto the surface of polylysine-coated microtiter well plates: (a) series of epifluorescence images for wells exposed to only rinse steps (blank), propidium iodide (PI), CCMV (+), CCMV followed by CCMV (+/+), CCMV followed by CCMV-B (+/B), CCMV exposed to streptavidin and then CCMV-B (+/SA/B), CCMV-B (B), and CCMV-B exposed to streptavidin and then CCMV-B (B/SA/B); (b) data from two experiments quantified in terms of pixel brightness; (c) relation between pixel brightness for adlayers of CCMV-B coupled via a streptavidin interlayer and adsorption cycles.
Figure 4. Relation between the coverage of the second adlayer in dual layers at the end of the adsorption period and the molar ratio of CCMV-B to total CCMV in the bulk mixture used to form the first adlayer: (a) ATR-FTIR data (area of the amide II band is proportional to viral protein); (b) epifluorescence microscopy data (pixel brightness originating from PI is proportional to viral RNA).
To determine whether the second layer of CCMV-B indeed consisted of clusters, as suggested by the data in Figure 4, a film was prepared on a Si wafer for observation using AFM. A base layer was prepared by adsorbing from a solution with a 1:75 ratio of CCMV-B to total CCMV, rinsed, and then exposed to a solution of CCMV-B. The stoichiometry of CCMV-B to total CCMV in the solution was chosen to optimize the chances that clusters would be well dispersed in the second layer and thus be visible using AFM. Clusters were counted by using image analysis software (see Supporting Information, Figure 6S). The number of clusters in the 1 × 1 µm2 and 5 × 5 µm2 images (Figure 5b and d, respectively) was determined to be 13 and 220, respectively. These are close to the estimated values (12 and 298, respectively), especially considering that the estimate was based upon a 55% areal coverage of the base film predicted by the random sequential adsorption model, and the actual coverage is somewhat less than this.30 Our results demonstrate a number of different ways of forming and controlling the composition of multilayer films that incorporate CCMV using LbL assembly. Protein cages such as CCMV can be viewed as scaffolds supporting independently addressable residues that can be used both to incorporate the cage into an LbL film and to impart individual functions to the LbL-incorporated cages. The interior surfaces of the protein cages have been modified to promote the size-constrained formation
Langmuir, Vol. 22, No. 21, 2006 8895
Figure 5. AFM images of CCMV on amine-functionalized silicon (Si): (a,c) base film; (b,d) clusters of CCMV-B that form on the streptavidin-activated base film. Panels a and b are 1 × 1 µm2, and panels c and d are 5 × 5 µm2. The scale bar indicates a height range of 0-50 nm.
of a variety of inorganic materials, including silver7 and cobalt platinum alloys,39 used to encapsulate preformed metal nanoclusters40 and inorganic catalysts41-43 and functionalized for controlled drug release. 12 One of the most promising short-term applications of LbL films is for use as solid-state electrolytes.26 In this respect, their use is primarily limited by moderate ion mobility and low numbers of dissociable ions. Protein cages with a somewhat sequestered interior cavity such as CCMV could be used to introduce a high density of dissociable ions contained in a region of high dielectric constant, thus enhancing both ion mobility and availability. At elevated pH, CCMV swells, opening 60 pores per cage, 2 nm in diameter, located at the pseudo-3-fold axes.44 Thus, incorporation of CCMV in LbL films might be used to produce a pH-gated semipermeable membrane. This reversible pH gating capability of CCMV has been used to trap paratungstate nanoparticles inside the cage cavity.3 This implies that the incorporation of CCMV into LbL films could be used to disperse insoluble high molecular weight polymers in LbL films. LbL films incorporating functionalized protein cages should be useful for nanostructuring semiconductors for applications such as photovoltaic cells45 and for fabricating controlled-release coatings.46,47 We showed that the surface of an LbL film can be textured at the nanoscale with clusters of protein cages. Although protein cage architectures can be organized into films exhibiting exquisite long-range in-plane order by prepatterning the surface using “top down” approaches,19 these methods are relatively labor intensive. Our method produces (39) Klem, M. T.; Willits, D.; Solis, D. J.; Belcher, A. M.; Young, M.; Douglas, T. AdV. Funct. Mater. 2005, 15, 1489-1494. (40) Chen, C.; Kwak, E. S.; Stein, B.; Kao, C. C.; Dragnea, B. J. Nanosci. Nanotechnol. 2005, 5, 2029-2033. (41) Ueno, T.; Suzuki, M.; Goto, T.; Matsumoto, T.; Nagayama, K.; Watanabe, Y. Angew. Chem., Int. Ed. 2004, 43, 2527-2530. (42) Ensign, D.; Young, M.; Douglas, T. Inorg. Chem. 2004, 43, 3441-3446. (43) Kim, I.; Hosein, H. A.; Strongin, D. R.; Douglas, T. Chem. Mater. 2002, 14, 4874-4879. (44) Speir, J. A.; Munshi, S.; Wang, G. J.; Baker, T. S.; Johnson, J. E. Structure 1995, 3, 63-78. (45) Gratzel, M. Nature 2003, 421, 586-587. (46) An, Z. H.; Lu, G.; Mohwald, H.; Li, J. B. Chem.sEur. J. 2004, 10, 5848-5852. (47) Ai, H.; Jones, S. A.; de Villiers, M. M.; Lvov, Y. M. J. Controlled Release 2003, 86, 59-68.
8896 Langmuir, Vol. 22, No. 21, 2006
films exhibiting a measure of long-range in-plane order without the need for prepatterning the surface. Acknowledgment. This work was funded by grants from NIH (R01 EB00432) and the Office of Naval Research for support of the Center for BioInspired Nanomaterials (19-00-R0006).
Suci et al.
Supporting Information Available: Results of the SEC, DLAS, and TEM characterization of CCMV-B preparations, LC/MS data for CCMV-B preparations, fits of the model ATR-FTIR data curves, and an analysis of the AFM images. This material is available free of charge via the Internet at http://pubs.acs.org. LA0612062
