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Polypeptide-Mediated Silica Growth on Indium Tin Oxide Surfaces Diana D. Glawe,† Francisco Rodrı´guez,‡ Morley O. Stone,§ and Rajesh R. Naik*,§ Engineering Science Department, Trinity University, San Antonio, Texas, General Engineering Department, University of Puerto Rico, Mayaguez, Puerto Rico, and Air Force Research Laboratory, Materials and Manufacturing Directorate, 3005 Hobson Way, Wright-Patterson Air Force Base, Dayton, Ohio 45433-7702 Received August 13, 2004. In Final Form: October 22, 2004 Herein, we describe the formation of silica structures on indium tin oxide (ITO) surfaces using poly(PLL) to template the condensation of silicic acid. Precisely controlled electrostatic fields were used to preposition PLL onto ITO surfaces. Subsequent polypeptide-mediated silicification resulted in the formation of silica with concentration gradients that followed the pattern of the externally applied electrostatic field used in the deposition of the PLL. The resulting silica structures were securely attached to the ITO surface. The technique described here offers an inexpensive and rapid method for the deposition of polypeptides on surfaces. L-lysine
Introduction Diatom cell walls are considered a paradigm for the controlled production of nanostructured silica. The conventional chemical synthesis of silica-based materials requires harsh conditions such as extreme temperature, pH, and pressure, whereas biosilicification occurs at neutral pH, ambient temperature, and ambient pressure.1,2 The creation of inorganic materials for advanced structures has led to a growing interest in the area of biomineralization. Silicateins isolated from within sponge silica have been shown to catalyze the in vitro polymerization of silica from tetraethoxysilane at neutral pH.3 Similarly, a set of cationic polypeptides, termed silaffins, isolated from diatoms can generate a network of silica nanospheres when added to a solution of silicic acid in vitro.4 Recent studies have shown that the silica morphologies resulting from the participation of various polypeptides in the silicification process can be manipulated using external electric and hydrodynamic fields or by introducing chemical additives.5-7 The external influences affect the transport and conformational states of the polypeptide before or during the reaction with silicic acid, resulting in unique silica morphologies. Other fundamental studies of the biomineralization processes have led to the development of strategies for the synthesis of hybrid structures that could lead to the development of devices based in part on the biomimetic synthesis of silica.8,9 * Corresponding author. E-mail:
[email protected]. Fax: (937) 255-4913. Phone: (937) 255-3808. † Trinity University. ‡ University of Puerto Rico. § Air Force Research Laboratory. (1) Morse, D. E. Biotechnology 1999, 17, 230-232. (2) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 11291132. (3) Cha, J. N.; Katsuhiko, K.; Zhou, Y.; Christiansen, S. C.; Chmelka, B. F.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 26, 361-365. (4) Kroger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14133-14138. (5) Naik, R. R.; Whitlock, P. W.; Rodriguez, F.; Brott, L. L.; Glawe, D. D.; Clarson, S. J.; Stone, M. O. Chem. Commun. 2003, 2, 238-239. (6) Naik, R. R.; Brott, L. L.; Rodriguez, F.; Agarwal, G.; Kirkpatrick, S. M.; Stone, M. O. Prog. Org. Coat. 2003, 47, 249-255. (7) Rodriguez, F.; Glawe, D. D.; Naik, R. R.; Hallinan, K. P.; Stone, M. O. Biomacromolecules 2004, 5, 261-265.
Biomolecules that nucleate and control the growth of inorganic materials are building blocks that can be used in the bottom-up fabrication of nano- and microscale devices. Several methods have been explored to spatially control the deposition of biomolecules on surfaces.10-12 The controlled deposition of biomolecules (capable of inorganic material synthesis) facilitates the growth of structures within a specified location on a device surface. Herein, we describe the deposition of poly-L-lysine (PLL) capable of precipitating silica onto flat indium tin oxide (ITO) surfaces using electrical fields. After deposition of the polypeptides, incubation of the surfaces in the presence of a silicic acid solution resulted in the formation of silica in regions covered with the polypeptide. The method described here is relatively simple, does not require sophisticated instrumentation for depositing peptides on surfaces, and can be used to effectively pattern large areas. The ability to organize materials that have electronic, optical, or magnetic properties (by virtue of using peptides to serve as templates for nucleating and growing these materials) would be beneficial for a wide range of applications. Materials and Methods Materials. Poly-L-lysine (MW 30-70 kDa) and tetramethyl orthosilicate (99%) were obtained from Sigma-Aldrich (St. Louis, MO). Indium tin oxide-coated glass slides were purchased from Colorado Concept Coatings (Boulder, CO). Polypeptide and Silicic Acid Solutions. Poly-L-lysine was dissolved in 0.1 M sodium phosphate buffer, pH 7.5, to obtain a 70 nM PLL solution. Silicic acid solution was prepared as previously described.2 Uniform Field Polypeptide Deposition. A 2.5 cm diameter polished copper disk was used as the anode electrode and a flat conductive surface of indium tin oxide (ITO) was used as the (8) Brott, L. L.; Naik, R. R.; Pikas, D. J.; Kirkpatrick, S. M.; Tomlin, D. W.; Whitlock, P. W.; Clarson, S. J.; Stone, M. O. Nature 2001, 413, 291-293. (9) Luckarift, H.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211-213. (10) Fang, A. P.; Ng, H. T.; Li, S. F. Y. Langmuir 2001, 17, 43604366. (11) Agarwal, G.; Naik, R. R.; Stone, M. O. J. Am. Chem. Soc. 2003, 125, 7408-7412. (12) Bruckbauer, A.; Zhou, D.; Ying, L.; Korchev, Y. E.; Abell, C.; Klenerman, D. J. Am. Chem. Soc. 2003, 125, 9834-9839.
10.1021/la047964e CCC: $30.25 © 2005 American Chemical Society Published on Web 12/18/2004
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Glawe et al. using a Noran Voyager EDS system verified that the observed structures were indeed silica.
Results and Discussion
Figure 1. Experimental setup for uniform deposition. (A) The 2.5 cm diameter copper disk used as the anode. (B) Schematic illustration of the experimental setup, not to scale.
Figure 2. Gradient field deposition experimental setup. (A) SEM micrograph of the tungsten anode with a tip radius of ∼500 nm. (B) Schematic illustration of the experimental setup, not to scale. cathode electrode to create an electrostatic field within the polycationic polypeptide solution (Figure 1). Two finite flat electrodes with a voltage differential (V) and a separation (h), such as the configuration shown in Figure 1, produce an electric field (E ) V/h) that is nominally uniform across the central portion of the electrodes and slightly reduced near the outer perimeter due to edge effects. For the uniform polypeptide deposition results discussed in this paper, an electric potential (V) of 2 V was applied for 20 min across the ∼1 mm space (h) between the electrodes. A 200 µL volume of PLL solution filled the space at the center of the electrodes where the field was expected to be uniform. Gradient Field Polypeptide Deposition. A sharp tungsten pin with a tip radius of ∼500 nm was used as the anode electrode and a flat conductive surface of ITO was used as the cathode electrode to create an electrostatic field within the polycationic polypeptide solution (Figure 2). The gradient electric field (Figure 2) is the strongest between the tip of the pin and the flat electrode surface, where the space between the electrodes is smallest, and decreases radially outward. According to electric field theory, the electric field lines are expected to align perpendicular to each electrode surface. For the configuration shown in Figure 2, this creates a nonlinear electric field gradient along the radius on the surface of the flat electrode. In the gradient field polypeptide deposition experiments, the pin was slowly lowered using a manually controlled translation stage with micrometer resolution. A predetermined clearance (2 µm e h e 10 µm) between the electrodes was established, permitting the generation of a localized high electrostatic field with minimal joule heating effects on the polypeptide solutions, ranging in volume from 1 to 100 µL placed between the electrodes. A voltage (V) in the range 1-4 V was applied between the electrodes for a period of 5-20 min. Poly-L-lysine-Mediated Silica Growth. The deposition of poly-L-lysine on the ITO surface was confirmed by coomassie blue staining and by Fourier transform infrared (FTIR) spectroscopy. For both the gradient field and uniform field peptide deposition tests, once the externally applied electrostatic field was deactivated, the cathode electrode surface was washed with sodium phosphate buffer, pH 7.5, to remove free polypeptides that did not attach to the electrode surface. Silicic acid was then placed onto the flat cathode electrode surface and allowed to react with the polypeptide-patterned surface for 1-2 min. The surface was then washed with double-distilled deionized water prior to imaging. Scanning Electron Microscopy (SEM) and Electron Dispersive Spectroscopy (EDS) of Silica Nanostructures. All SEM micrographs were obtained using a Phillips XL30 FEG environmental scanning electron microscope. Elemental analysis
The effect of the electrostatic field on an individual polypeptide in solution can be described by electrophoresis. Electrophoresis is defined as the motion experienced by a charged particle suspended in an aqueous medium resulting from the force of the electric field strength coupled with the particle charge.13 Factors such as polypeptide concentration as well as hydrodynamic effects influence polypeptide mobility. The average translational speed of the polypeptide, and therefore the rate of deposition, increases with an increase in applied electric field strength. Characterization of PLL transport in solution due to an externally applied electric field has been previously described.14 Several experiments were conducted to investigate the effect of using well-defined electrostatic fields on the deposition of PLL from bulk solution onto an ITO surface and to study the ability of the polypeptides to retain their silica precipitating ability in situ. The externally applied electric field caused a local area of high polypeptide concentration near the surface of the ITO cathode. The distribution of polypeptide on the electrode surface depended on the electrode shape and resulting electric field geometry (i.e., uniform or gradient electric field). The formation of silica on the electrode surface occurred in areas where poly-L-lysine was deposited. Gradient Polypeptide Deposition. The silica pattern resulting from the gradient field polypeptide deposition setup was marked by a central spot occurring nearest to the tip of the anode electrode and a ring at the edge of the polypeptide solution, as depicted by the SEM image in Figure 3A. The dominant influence creating the central silica spot is due to the electrostatic field. The dominant influence creating the outer ringlike structure is attributed to air-liquid interface electrohydrodynamic phenomena. Surfactants, such as proteins, accumulate and deposit on the surface at the air-liquid-surface boundary along the perimeter of the solution volume.15 The typical central silica spot diameter for the configuration described in Figure 3 was on the order of 200-400 µm. The central spot delineates the region of most intense electric field produced by the voltage difference between the 500 nm radius anode tip and the flat cathode. Within the densest region of the central spot (Figure 3B), a glimpse of a network of silica structures, similar to those in Figure 3C, can be seen beneath the layer of larger, less organized silica structures. The density of silica structures on the ITO surface decreases radially outward from the center, as seen in Figure 3B-E. The density decreases drastically in the central spot (Figure 3B-D) and trails off to be relatively uniform in the region outside the central spot and within the boundary of the outer ring (Figure 3E). No silica deposition was observed in areas lacking PLL. Silica Structures in Solution versus Attached to the Surface. It is useful to compare the morphology of the silica structures formed under static conditions in the absence of an electrostatic field to the structures formed using the procedure described herein. Under static conditions, silica structures shift from a network of fused (13) Pohl, H. A. Dielectrophoresis; Cambridge University Press: Cambridge, U.K., 1978. (14) Rodriguez, F. Ph.D. Dissertation, University of Dayton, 2003. (15) Carey, V. P. Liquid-Vapor Phase-Change Phenomena; Taylor and Francis: 1992.
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Figure 3. Growth of silica on an ITO surface created by a 2 V potential applied for 20 min across a 2 µm gap between electrodes occupied by PLL polypeptide solution, followed by incubation in silicic acid solution. (A) The structures on the ITO surface span from the central spot region b to the outer region e (B-E). (B) Loosely organized large structures atop a dense network of fused silica indicated by the arrows. (C) Dense network of fused silica structures resulting in a layer of nearly uniform height. (D and E) Less dense silica distribution where individual platelets become apparent.
spherelike particles (Figure 4A) obtained using a small molecular weight PLL to large silica platelets (Figure 4B) for larger molecular weight poly-L-lysines.7 The transition from spherelike particles to hexagonal platelets occurs with PLL with a molecular weight of > ∼12 kDa. Figure 4B shows the randomly oriented hexagon-shaped platelets and small spherical silica produced by a static solution of PLL. Since commercially available PLL contains a distribution of sizes, the small spherical particles are attributed to the smaller molecular weight PLLs. Overall, the platelets are predominantly the most abundant structures obtained using larger molecular weight PLLs. Interestingly, electron diffraction analysis showed that the platelike structures are amorphous.7 Electric field assisted deposition of PLL (MW 30-70 kDa) onto an ITO surface resulted in the formation of distinctly shaped and oriented silica structures attached to the ITO surface. The distinct silica platelets are oriented perpendicular to the surface with their longest side attached to the ITO surface (Figure 4C). Similar silica platelets were also obtained when larger PLLs (MW 150-300 kDa) were used.16 The specific shape and orientation of the silica attached to the surface is likely due to the orientation of the polypeptide on the cathode electrode. PLL, in solution, is presumed to predominantly adopt a random coil conformation under neutral pH conditions.17 FTIR analysis suggests a predominantly β-sheet conformation when PLL is attached to the ITO surface. When attached to the surface, the orientation of PLL is restricted and the polypeptide-mediated reaction creates different silica structures as compared to the silica structure obtained when the PLL is not confined to a surface. The growth of the silica platelets is perpendicular to the electrode surface, which suggests that the growth continues beyond the (16) Glawe, D. D.; Rodriguez, F.; Stone, M. O.; Naik, R. R. Mater. Res. Soc. Symp Proc. 2004, 823, W4.17. (17) Greenfield, N.; Fasman, G. D. Biochemistry 1969, 8, 4108.
Figure 4. SEM micrographs of silica structures. Silica structures obtained under static conditions using (A) smaller MW PLLs (MW 1-4 kDa) and (B) larger MW PLLs (MW 30-70 kDa). (C) Silica structures obtained after electric field assisted deposition of larger MW PLLs on an ITO surface. (D) Fused silica platelets in regions of higher polypeptide densities on an ITO surface.
peptide-coated surface. In areas of higher uniform silica density, such as that shown in Figure 4D, the platelike structures oriented perpendicular to the surface fused to form a fairly uniformly distributed network. Ongoing experiments are being conducted to determine the conformation of the peptide on the ITO surface. When the tungsten pin anode electrode was moved parallel to the flat cathode electrode surface while the electrostatic field was applied, the polycationic polypeptides deposited along the path of the anode electrode, as evidenced by silica deposition in a line (data not shown). Likewise, an electrostatic field can be applied using multiple anode electrodes and a single cathode electrode surface to produce an array of concentrated silica spots. This further demonstrates the ability to control the local deposition of PLL and subsequent silica formation on a surface using well-defined electric fields. Anodic Nature of Indium Tin Oxide (ITO). ITO has been widely used as an electrode for electrochemistry of biomolecules because of its transparent and conductive properties. Proteins and amines have been shown to attach to ITO surfaces in the absence of an externally applied electrostatic field.18,19 The surface hydroxyl groups on ITO are believed to facilitate protein immobilization via hydrogen bonding and van der Waals interactions.20 (18) Fang, A. P.; Ng, H. T.; Li, S. F. Y. Langmuir 2001, 17, 43604366. (19) Oh, S. Y.; Yun, Y. J.; Kim, D. Y.; Han, S. H. Langmuir 1999, 15, 4690-4692. (20) Fang, A. P.; Ng, H. T.; Su, X.; Li, S. F. Y. Langmuir 2000, 16, 5221-5226.
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Figure 5. SEM micrograph of silica attached to an ITO surface created in the absence of an electric field for polypeptide deposition (A and B) and with a 2 V differential applied across a 1 mm gap between flat electrodes, using the uniform deposition setup, with 200 µL of PLL solution (C and D). The SEM images in parts B and D were captured at a 30° tilt from the perpendicular to the surface.
Experiments performed in the absence of an externally applied electric field showed that PLL attached to the ITO surface within minutes and formed silica when exposed to silicic acid (Figure 5A and B). The silica structures obtained, using PLL, in the absence of an applied electrical field were similar in morphology to the structures formed using PLL deposited by the applied electric field. Therefore, the silica structures obtained on ITO surfaces with PLL suggest that the orientation of polypeptide is directed by the interaction with the ITO surface and is not a result of the externally applied electric field. The electric field acts primarily to transport and concentrate the PLL at the cathode surface. Uniform Polypeptide Deposition. The silica pattern resulting from the uniform field polypeptide deposition setup (Figure 1) was marked by a relatively uniform deposition of silica across the surface in direct contact with the polypeptide solution during deposition (Figure 5C and D). The homogeneity of silica structure shape, and to some extent the size, when attached to a surface as compared to the variety of shapes and sizes resulting from a static reaction in solution (Figure 4B) was consistent throughout the uniform deposition experiments. The structures were almost exclusively platelike structures attached to the surface. Few to no spherelike particles
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were observed among the platelets on the ITO surface. Assuming the spheres are due to smaller poly-L-lysines7 present in the MW distribution, the results suggest that the larger PLLs preferentially adsorbed to the surface21 or that the smaller PLLs were unable to securely adhere to the ITO surface during the buffer rinse. The denser PLL coverage achieved using an externally applied electric field resulted in a broader range of silica structure sizes (Figure 5D) as compared to the generally larger structures achieved in the absence of an electric field (Figure 5B) under similar conditions. This effect could be attributed to PLL with a wider MW range attaching to the surface under the influence of an electric field, thereby mediating the formation of a wider range of silica structure sizes under these test conditions. Conclusions In summary, we report the deposition of polycationic polypeptides (PLLs), capable of precipitating silica, on ITO surfaces using an externally imposed electrostatic field. The concentration of the resulting silica on the surface followed the patterns of the applied electrostatic field used to deposit the polypeptides. The morphology of the distinctly shaped silica platelets resulting from exposure of the polypeptide-coated surface to silicic acid was noticeably different from the morphology resulting from a static mixture. Further development of the electrode design and experimental conditions could improve the resolution and organization of polypeptide deposition on surfaces. Smaller silica spot diameters, on the order of several microns, would be expected with a more sophisticated injection-based system, such as an ionic current nanopipet, used to deposit the polypeptide.12 The precisely controlled creation of an inorganic material, such as silica, will allow for the future development of bioinspired microand nanodevices for specific applications. Acknowledgment. This research was supported by funding from the Air Force Office of Scientific Research. LA047964E (21) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapmann & Hall: London, 1993.
