Polypeptide-Mediated Silica Growth on Indium Tin OxideSurfaces

Diana D. Glawe,† Francisco Rodrı́guez,‡ 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-L-lysine (PLL) to template the condensation of silicic acid. Precisely controlled electrostatic fields wereused to preposition PLL onto ITO surfaces. Subsequent polypeptide-mediated silicification resulted in theformation of silica with concentration gradients that followed the pattern of the externally appliedelectrostatic field used in the deposition of the PLL. The resulting silica structures were securely attachedto the ITO surface. The technique described here offers an inexpensive and rapid method for the depositionof polypeptides on surfaces.

Introduction

Diatom cell walls are considered a paradigm for thecontrolled production of nanostructured silica. The con-ventional chemical synthesis of silica-based materialsrequires harsh conditions such as extreme temperature,pH, and pressure, whereas biosilicification occurs at neu-tral pH, ambient temperature, and ambient pressure.1,2

The creation of inorganic materials for advanced struc-tures has led to a growing interest in the area of biomin-eralization. Silicateins isolated from within sponge silicahave been shown to catalyze the in vitro polymerizationof silica from tetraethoxysilane at neutral pH.3 Similarly,a set of cationic polypeptides, termed silaffins, isolatedfrom diatoms can generate a network of silica nanosphereswhen added to a solution of silicic acid in vitro.4

Recent studies have shown that the silica morphologiesresulting from the participation of various polypeptidesin the silicification process can be manipulated usingexternal electric and hydrodynamic fields or by introducingchemical additives.5-7 The external influences affect thetransport and conformational states of the polypeptidebefore or during the reaction with silicic acid, resultingin unique silica morphologies. Other fundamental studiesof the biomineralization processes have led to the devel-opment of strategies for the synthesis of hybrid structuresthat could lead to the development of devices based inpart on the biomimetic synthesis of silica.8,9

Biomolecules that nucleate and control the growth ofinorganic materials are building blocks that can be usedin the bottom-up fabrication of nano- and microscaledevices. Several methods have been explored to spatiallycontrol the deposition of biomolecules on surfaces.10-12

The controlled deposition of biomolecules (capable ofinorganic material synthesis) facilitates the growth ofstructures 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 ofthe polypeptides, incubation of the surfaces in the presenceof a silicic acid solution resulted in the formation of silicain regions covered with the polypeptide. The methoddescribed here is relatively simple, does not requiresophisticated instrumentation for depositing peptides onsurfaces, 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 peptidesto serve as templates for nucleating and growing thesematerials) would be beneficial for a wide range ofapplications.

Materials and MethodsMaterials. 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 fromColorado Concept Coatings (Boulder, CO).

Polypeptide and Silicic Acid Solutions. Poly-L-lysine wasdissolved in 0.1 M sodium phosphate buffer, pH 7.5, to obtain a70 nM PLL solution. Silicic acid solution was prepared aspreviously described.2

Uniform Field Polypeptide Deposition. A 2.5 cm diameterpolished copper disk was used as the anode electrode and a flatconductive surface of indium tin oxide (ITO) was used as the

* Corresponding author. E-mail: Rajesh.Naik@wpafb.af.mil.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, 1129-

1132.(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.

(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, 4360-4366.

(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.

717Langmuir 2005, 21, 717-720

10.1021/la047964e CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 12/18/2004

cathode electrode to create an electrostatic field within thepolycationic polypeptide solution (Figure 1).

Two finite flat electrodes with a voltage differential (V) anda separation (h), such as the configuration shown in Figure 1,produce an electric field (E ) V/h) that is nominally uniformacross the central portion of the electrodes and slightly reducednear the outer perimeter due to edge effects. For the uniformpolypeptide deposition results discussed in this paper, an electricpotential (V) of 2 V was applied for 20 min across the ∼1 mmspace (h) between the electrodes. A 200 µL volume of PLL solutionfilled the space at the center of the electrodes where the field wasexpected to be uniform.

Gradient Field Polypeptide Deposition. A sharp tungstenpin with a tip radius of ∼500 nm was used as the anode electrodeand a flat conductive surface of ITO was used as the cathodeelectrode to create an electrostatic field within the polycationicpolypeptide solution (Figure 2).

The gradient electric field (Figure 2) is the strongest betweenthe tip of the pin and the flat electrode surface, where the spacebetween the electrodes is smallest, and decreases radiallyoutward. According to electric field theory, the electric field linesare expected to align perpendicular to each electrode surface.For the configuration shown in Figure 2, this creates a nonlinearelectric field gradient along the radius on the surface of the flatelectrode. In the gradient field polypeptide deposition experi-ments, the pin was slowly lowered using a manually controlledtranslation stage with micrometer resolution. A predeterminedclearance (2 µm e h e 10 µm) between the electrodes wasestablished, permitting the generation of a localized highelectrostatic field with minimal joule heating effects on thepolypeptide solutions, ranging in volume from 1 to 100 µL placedbetween the electrodes. A voltage (V) in the range 1-4 V wasapplied between the electrodes for a period of 5-20 min.

Poly-L-lysine-Mediated Silica Growth. The deposition ofpoly-L-lysine on the ITO surface was confirmed by coomassieblue staining and by Fourier transform infrared (FTIR) spec-troscopy. For both the gradient field and uniform field peptidedeposition tests, once the externally applied electrostatic fieldwas deactivated, the cathode electrode surface was washed withsodium phosphate buffer, pH 7.5, to remove free polypeptidesthat did not attach to the electrode surface. Silicic acid was thenplaced onto the flat cathode electrode surface and allowed toreact with the polypeptide-patterned surface for 1-2 min. Thesurface was then washed with double-distilled deionized waterprior to imaging.

Scanning Electron Microscopy (SEM) and ElectronDispersive Spectroscopy (EDS) of Silica Nanostructures.All SEM micrographs were obtained using a Phillips XL30 FEGenvironmental scanning electron microscope. Elemental analysis

using a Noran Voyager EDS system verified that the observedstructures were indeed silica.

Results and Discussion

The effect of the electrostatic field on an individualpolypeptide in solution can be described by electrophoresis.Electrophoresis is defined as the motion experienced bya charged particle suspended in an aqueous mediumresulting from the force of the electric field strengthcoupled with the particle charge.13 Factors such aspolypeptide concentration as well as hydrodynamic effectsinfluence polypeptide mobility. The average translationalspeed of the polypeptide, and therefore the rate ofdeposition, increases with an increase in applied electricfield strength. Characterization of PLL transport insolution due to an externally applied electric field hasbeen previously described.14

Several experiments were conducted to investigate theeffect of using well-defined electrostatic fields on thedeposition of PLL from bulk solution onto an ITO surfaceand to study the ability of the polypeptides to retain theirsilica precipitating ability in situ. The externally appliedelectric field caused a local area of high polypeptideconcentration near the surface of the ITO cathode. Thedistribution of polypeptide on the electrode surfacedepended on the electrode shape and resulting electricfield geometry (i.e., uniform or gradient electric field). Theformation of silica on the electrode surface occurred inareas where poly-L-lysine was deposited.

GradientPolypeptideDeposition. The silica patternresulting from the gradient field polypeptide depositionsetup was marked by a central spot occurring nearest tothe tip of the anode electrode and a ring at the edge of thepolypeptide solution, as depicted by the SEM image inFigure 3A.

The dominant influence creating the central silica spotis due to the electrostatic field. The dominant influencecreating the outer ringlike structure is attributed toair-liquid interface electrohydrodynamic phenomena.Surfactants, such as proteins, accumulate and deposit onthe surface at the air-liquid-surface boundary along theperimeter of the solution volume.15

The typical central silica spot diameter for the con-figuration described in Figure 3 was on the order of200-400 µm. The central spot delineates the region ofmost intense electric field produced by the voltagedifference between the 500 nm radius anode tip and theflat 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 thelayer of larger, less organized silica structures. The densityof silica structures on the ITO surface decreases radiallyoutward from the center, as seen in Figure 3B-E. Thedensity decreases drastically in the central spot (Figure3B-D) and trails off to be relatively uniform in the regionoutside the central spot and within the boundary of theouter ring (Figure 3E). No silica deposition was observedin areas lacking PLL.

Silica Structures in Solution versus Attached tothe Surface. It is useful to compare the morphology ofthe silica structures formed under static conditions in theabsence of an electrostatic field to the structures formedusing the procedure described herein. Under static condi-tions, 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.

Figure 1. Experimental setup for uniform deposition. (A) The2.5 cm diameter copper disk used as the anode. (B) Schematicillustration 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.

718 Langmuir, Vol. 21, No. 2, 2005 Glawe et al.

spherelike particles (Figure 4A) obtained using a smallmolecular weight PLL to large silica platelets (Figure 4B)for larger molecular weight poly-L-lysines.7 The transitionfrom spherelike particles to hexagonal platelets occurswith PLL with a molecular weight of > ∼12 kDa. Figure4B shows the randomly oriented hexagon-shaped plateletsand small spherical silica produced by a static solution ofPLL. Since commercially available PLL contains a dis-tribution of sizes, the small spherical particles areattributed to the smaller molecular weight PLLs. Overall,the platelets are predominantly the most abundantstructures obtained using larger molecular weight PLLs.Interestingly, electron diffraction analysis showed thatthe platelike structures are amorphous.7

Electric field assisted deposition of PLL (MW 30-70kDa) onto an ITO surface resulted in the formation ofdistinctly shaped and oriented silica structures attachedto the ITO surface. The distinct silica platelets are orientedperpendicular to the surface with their longest sideattached to the ITO surface (Figure 4C). Similar silicaplatelets were also obtained when larger PLLs (MW150-300 kDa) were used.16

The specific shape and orientation of the silica attachedto the surface is likely due to the orientation of thepolypeptide on the cathode electrode. PLL, in solution, ispresumed to predominantly adopt a random coil confor-mation under neutral pH conditions.17 FTIR analysissuggests a predominantly â-sheet conformation when PLLis attached to the ITO surface. When attached to thesurface, the orientation of PLL is restricted and thepolypeptide-mediated reaction creates different silicastructures as compared to the silica structure obtainedwhen the PLL is not confined to a surface. The growth ofthesilicaplatelets is perpendicular to theelectrodesurface,which suggests that the growth continues beyond the

peptide-coated surface. In areas of higher uniform silicadensity, such as that shown in Figure 4D, the platelikestructures oriented perpendicular to the surface fused toform a fairly uniformly distributed network. Ongoingexperiments are being conducted to determine the con-formation of the peptide on the ITO surface.

When the tungsten pin anode electrode was movedparallel to the flat cathode electrode surface while theelectrostatic field was applied, the polycationic polypep-tides deposited along the path of the anode electrode, asevidenced by silica deposition in a line (data not shown).Likewise, an electrostatic field can be applied usingmultiple anode electrodes and a single cathode electrodesurface to produce an array of concentrated silica spots.This further demonstrates the ability to control the localdeposition of PLL and subsequent silica formation on asurface using well-defined electric fields.

Anodic Nature of Indium Tin Oxide (ITO). ITO hasbeen widely used as an electrode for electrochemistry ofbiomolecules because of its transparent and conductiveproperties. Proteins and amines have been shown to attachto ITO surfaces in the absence of an externally appliedelectrostatic field.18,19 The surface hydroxyl groups on ITOare believed to facilitate protein immobilization viahydrogen bonding and van der Waals interactions.20

(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.

(18) Fang, A. P.; Ng, H. T.; Li, S. F. Y. Langmuir 2001, 17, 4360-4366.

(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.

Figure 3. Growth of silica on an ITO surface created by a 2V potential applied for 20 min across a 2 µm gap betweenelectrodes occupied by PLL polypeptide solution, followed byincubation in silicic acid solution. (A) The structures on theITO surface span from the central spot region b to the outerregion e (B-E). (B) Loosely organized large structures atop adense network of fused silica indicated by the arrows. (C) Densenetwork of fused silica structures resulting in a layer of nearlyuniform height. (D and E) Less dense silica distribution whereindividual platelets become apparent.

Figure 4. SEM micrographs of silica structures. Silicastructures obtained under static conditions using (A) smallerMW PLLs (MW 1-4 kDa) and (B) larger MW PLLs (MW 30-70kDa). (C) Silica structures obtained after electric field assisteddeposition of larger MW PLLs on an ITO surface. (D) Fusedsilica platelets in regions of higher polypeptide densities on anITO surface.

Polypeptide-Mediated Silica Growth on ITO Surfaces Langmuir, Vol. 21, No. 2, 2005 719

Experiments performed in the absence of an externallyapplied electric field showed that PLL attached to theITO surface within minutes and formed silica whenexposed to silicic acid (Figure 5A and B). The silicastructures obtained, using PLL, in the absence of anapplied electrical field were similar in morphology to thestructures formed using PLL deposited by the appliedelectric field. Therefore, the silica structures obtained onITO surfaces with PLL suggest that the orientation ofpolypeptide is directed by the interaction with the ITOsurface and is not a result of the externally applied electricfield. The electric field acts primarily to transport andconcentrate the PLL at the cathode surface.

Uniform Polypeptide Deposition. The silica patternresulting from the uniform field polypeptide depositionsetup (Figure 1) was marked by a relatively uniformdeposition of silica across the surface in direct contactwith the polypeptide solution during deposition (Figure5C and D). The homogeneity of silica structure shape,and to some extent the size, when attached to a surfaceas compared to the variety of shapes and sizes resultingfromastatic reaction insolution (Figure4B)wasconsistentthroughout the uniform deposition experiments. Thestructures were almost exclusively platelike structuresattached to the surface. Few to no spherelike particles

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 thatthe larger PLLs preferentially adsorbed to the surface21

or that the smaller PLLs were unable to securely adhereto the ITO surface during the buffer rinse. The denserPLL coverage achieved using an externally applied electricfield resulted in a broader range of silica structure sizes(Figure 5D) as compared to the generally larger structuresachieved in the absence of an electric field (Figure 5B)under similar conditions. This effect could be attributedto PLL with a wider MW range attaching to the surfaceunder the influence of an electric field, thereby mediatingthe formation of a wider range of silica structure sizesunder these test conditions.

ConclusionsIn summary, we report the deposition of polycationic

polypeptides (PLLs), capable of precipitating silica, onITO surfaces using an externally imposed electrostaticfield. The concentration of the resulting silica on thesurface followed the patterns of the applied electrostaticfield used to deposit the polypeptides. The morphology ofthe distinctly shaped silica platelets resulting fromexposure of the polypeptide-coated surface to silicic acidwas noticeably different from the morphology resultingfromastaticmixture.Furtherdevelopmentof theelectrodedesign and experimental conditions could improve theresolution and organization of polypeptide deposition onsurfaces. Smaller silica spot diameters, on the order ofseveral microns, would be expected with a more sophis-ticated injection-based system, such as an ionic currentnanopipet, used to deposit the polypeptide.12 The preciselycontrolled creation of an inorganic material, such as silica,will allow for the future development of bioinspired micro-and nanodevices for specific applications.

Acknowledgment. This research was supported byfunding from the Air Force Office of Scientific Research.

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(21) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.;Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapmann & Hall:London, 1993.

Figure 5. SEM micrograph of silica attached to an ITO surfacecreated in the absence of an electric field for polypeptidedeposition (A and B) and with a 2 V differential applied acrossa 1 mm gap between flat electrodes, using the uniform depo-sition setup, with 200 µL of PLL solution (C and D). The SEMimages in parts B and D were captured at a 30° tilt from theperpendicular to the surface.

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