Manipulation of the Orientational Response of Liquid Crystals to

Seung-Ryeol Kim and Nicholas L. Abbott*. Department of Chemical Engineering, University of Wisconsin, 1415 Engineering Drive,. Madison, Wisconsin 5370...
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Langmuir 2002, 18, 5269-5276

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Manipulation of the Orientational Response of Liquid Crystals to Proteins Specifically Bound to Covalently Immobilized and Mechanically Sheared Films of Functionalized Bovine Serum Albumin Seung-Ryeol Kim and Nicholas L. Abbott* Department of Chemical Engineering, University of Wisconsin, 1415 Engineering Drive, Madison, Wisconsin 53705 Received December 26, 2001. In Final Form: April 16, 2002 This paper reports on the orientational behavior of thermotropic liquid crystals supported on films of biotinylated bovine serum albumin (BSA) that are covalently immobilized on the surface of glass substrates using (3-aminopropyl)triethoxysilane (APES) and disuccinimidyl suberate (DSS). The films of biotinylated BSA are mechanically sheared and then immersed into aqueous solutions of anti-biotin immunoglobulin G (IgG) prior to contact with nematic phases of 4-cyano-4′-pentylbiphenyl (5CB). Using ellipsometry, we found the capacity of the films of biotinylated BSA to bind anti-biotin IgG to be unchanged upon shearing with a polyester cloth. In contrast, we determined the orientational response of nematic phases of 5CB to the presence of bound anti-biotin IgG to be a strong function of the normal pressure applied during shearing, as well as the magnitude of the lateral displacement of the film of biotinylated BSA when in contact with the surface of the polyester cloth. The shear rate, however, appears to have little influence on the response of the liquid crystal to bound IgG. These results demonstrate that it is possible to manipulate the response (sensitivity) of liquid crystals to IgG specifically bound to sheared films of functionalized BSA by changing the manner of shearing of the films of BSA without changing the capacity of the films to bind IgG. We also report a characterization of the nanometer-scale topography of mechanically sheared films of biotinylated BSA using atomic force microscopy (AFM).

Introduction We have previously reported that films of bovine serum albumin (BSA) covalently attached to glass surfaces and then mechanically sheared using a cloth exhibit four properties that make them potentially useful as substrates for biomolecular assays based on liquid crystals.1 First, nematic phases of 4-cyano-4′-pentylbiphenyl (5CB) assume azimuthally uniform and planar orientations on surfaces of sheared films of BSA. Second, sheared films of BSA resist nonspecific adsorption of proteins from aqueous solutions. Third, sheared films of BSA are recognized by anti-BSA immunoglobulin G (IgG) such that the antiBSA IgG binds to these surfaces. Fourth, liquid crystals placed onto sheared films of BSA to which anti-BSA IgG is bound assume orientations that are visually distinct from those assumed by the liquid crystal in the absence of the anti-BSA IgG. More recently, we also reported that sheared films of BSA functionalized with receptors (such as biotin) can be used to detect the presence of proteins that bind to the receptors conjugated to the BSA (such as anti-biotin IgG to biotinylated BSA).2 This result suggests that sheared films of functionalized BSA could be used to form the basis of a general methodology for imaging ligand-receptor interactions using liquid crystals. These two past studies1,2 also suggest a mechanism by which sheared films of protein orient liquid crystals. Because the presence of IgG bound to the sheared films can erase the preferred azimuthal orientation of the liquid crystal observed in the absence of the bound IgG, we conclude from these past studies that the process of * To whom correspondence should be addressed. Fax: 608-2625434. E-mail: [email protected]. (1) Kim, S.-R.; Shah, R. R.; Abbott, N. L. Anal. Chem. 2000, 72, 46464653. (2) Kim, S.-R.; Abbott, N. L. Adv. Mater. 2001, 13, 1445-1449.

mechanical shearing of covalently immobilized films of BSA introduces anisotropy into these surfaces on a spatial scale that is comparable to the size of the proteins and that the bound IgG can mask this anisotropic structure. Specifically, the process of shearing of the protein film does not lead to a preferred orientation of the liquid crystal through the introduction of topography that is large compared to proteins (e.g., ∼0.1-1 µm scratches) because these types of surface structures would not be erased or masked by the bound IgG. In this paper, we report a study that builds on these past observations by investigating the effects of shear conditions on the structure and properties of the rubbed protein films. The goals of this work were three-fold. First, we aimed to determine whether the capacity of sheared films of biotinylated BSA to bind anti-biotin IgG is influenced by the conditions used to shear the films of biotinylated BSA. Second, we aimed to determine whether the response of the liquid crystal to specifically bound IgG could be manipulated by the choice of the conditions used to shear the covalently immobilized films of biotinylated BSA. We also wanted to determine whether any effects of the shear conditions on the response of the liquid crystal result from (i) changes in the capacity of the surfaces to bind IgG or (ii) changes in the anisotropic structure of these surfaces that lead to the preferred orientation of the liquid crystals. Third, we also sought to use atomic force microscopy (AFM) to characterize the structure of mechanically sheared films of biotinylated BSA before and after binding of anti-biotin IgG. Experimental Section Materials. Glass microscope slides were Fisher’s Finest, Premium Grade, obtained from Fisher Scientific (Pittsburgh, PA). Polished silicon (100) wafers were purchased from Silicon

10.1021/la011844e CCC: $22.00 © 2002 American Chemical Society Published on Web 05/18/2002

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Figure 1. Experimental procedure used to covalently immobilize biotinylated BSA on the surfaces of glass microscope slides: (A) reaction of the ethoxy groups of APES with silanol groups present at the surface of the glass slide to form a siloxane linkage, (B) activation of the aminopropylated substrate by reaction with the homobifunctional cross-linker (DSS), and (C) reaction of the free succinimide ester groups of the activated substrate with the amine residues of biotinylated BSA. Sense (Nashua, NH). (3-Aminopropyl)triethoxysilane (APES) was purchased from Gelest (Tullytown, PA). Disuccinimidyl suberate (DSS) was obtained from Pierce (Rockford, IL). Biotinylated bovine serum albumin (biotinylated BSA, 8 mol of biotin per mole of BSA) and anti-biotin immunoglobulin G (IgG) (polyclonal, developed in goat) were obtained from Pierce (Rockford, IL) and Sigma (St. Louis, MO), respectively, and used as received. All IgG solutions used in this study were prepared in phosphatebuffered saline (PBS) solution at pH 7.4. All aqueous solutions were prepared with deionized water (Milli-Qplus, Millipore, Bedford, MA). The nematic liquid crystal 4-cyano-4′-pentylbiphenyl (5CB), manufactured by BDH, was purchased from EM Industries (Hawthorne, NY). Covalently Immobilized Films of Biotinylated BSA. Glass microscope slides and silicon wafers were cleaned by using “piranha solution” (70% H2SO4/30% H2O2) for 1 h at 80 °C. (Caution: Piranha solution reacts violently with organic materials and should be handled with extreme caution; do not store the solution in closed containers.). After removal from the cleaning solution, the slides were rinsed sequentially with copious amounts of deionized water, ethanol, and methanol and then dried under a nitrogen stream. The cleaned substrates were stored in an oven (120 °C) for at least 3 h before use.

The experimental procedure used to covalently immobilize biotinylated BSA on the surface of glass microscope slides or silicon wafers is shown schematically in Figure 1. Clean glass slides and silicon wafers were aminopropylated by reaction for 3 h at 80 °C with 5% APES in a sodium acetate/acetic acid buffer (10 mM, pH 5.0 prior to addition of APES). The aminopropylated substrates were immersed three times into sodium acetate/acetic acid buffer in a sonication bath for 10 min at 80 °C, rinsed with deionized water, dried with nitrogen flow, and then stored at 120 °C for at least 3 h before they were activated with a succinimide ester cross-linker (DSS) to promote the coupling of the biotinylated BSA to the surface by amide bond formation. The aminopropylated substrate was immersed in anhydrous methanol, and then a 50 mM DSS stock solution in anhydrous DMSO was added to the methanol solution (final concentration of DSS was 1 mM). The substrate was immersed in the solution for 1 h (with stirring), washed with methanol and deionized water, and then immediately coupled to amine groups of biotinylated BSA. The coupling of biotinylated BSA was achieved by immersion of the DSS-activated glass slide in a 0.1 mg/mL solution of biotinylated BSA (PBS, pH 7.4) overnight. Sheared Films of Biotinylated BSA. Sheared films of biotinylated BSA were prepared by sliding a velvet-type cloth

Orientational Response of Liquid Crystals to Proteins (90% polyester/10% Spandex, Logantex Inc., New York) across biotinylated BSA-coated substrates using a strip chart recorder (model SR-255 A/B, Heath Company). The details of this procedure are described elsewhere.1 Briefly, the cloth was attached to the chart paper using double-sided tape, and the biotinylated BSA-immobilized substrates were placed face down on the cloth. The film of biotinylated BSA was sheared by the movement of the cloth beneath the substrate. The shear speed (mm/s) and length of cloth translated across the film of biotinylated BSA were controlled by changing the feeding speed of the chart recorder and the displacement of the chart paper, respectively. The normal pressure applied to the film of biotinylated BSA during shearing was controlled by the placement of aluminum blocks of different weights onto the substrate. Binding of Anti-Biotin IgG. Sheared films of biotinylated BSA were immersed into aqueous solutions of anti-biotin IgG (PBS, pH 7.4) for 90 min. During incubation, the solution of IgG was stirred using a magnetic stirring bar coated with Teflon. After removal from the solution of anti-biotin IgG, each substrate was rinsed with deionized water and dried under a stream of gaseous nitrogen. Ellipsometric Thickness. All ellipsometric measurements were performed using silicon wafers covered with films of native oxide as the substrates. The details of the measurements are described elsewhere.1 Briefly, films of biotinylated BSA on the wafers were prepared by the same procedure used to prepare films of BSA on the glass microscope slides. Ellipsometric thicknesses were measured at three points on each sample using a Rudolph Auto EL ellipsometer (Flanders, NJ) at a wavelength of 6320 Å and an angle of incidence of 70°. To interpret the ellipsometric thickness of bound protein, we used a simple twolayer model (organic layer/effective substrate of SiO2/Si) by assuming the refractive index to be 1.46 for the organic layers (aminopropylsilane/biotinylated BSA/anti-biotin IgG).3 Optical Cells. We observed the alignment of nematic liquid crystal (5CB) on the surfaces of the sheared films of the biotinylated BSA by assembling the films into optical cells. Optical cells were fabricated by pairing two glass slides, each of which supported a sheared film of biotinylated BSA. The sheared films were aligned (facing each other) such that the directions of shearing were parallel within the cell. The sheared films were kept apart by inserting thin polyester film (∼10 µm thickness of Mylar, DuPont Films, Wilmington, DE) between the surfaces of the films. The cells were held together by using “bulldog” clips placed along the edge of the glass microscopic slides. The cells were heated to ∼40 °C by placing them on a hot plate. We also used a hot air gun to warm the air around the cells to ∼40 °C. The 5CB was heated into its isotropic phase (∼35 °C) within a glass syringe. A drop of 5CB was placed onto the edge of each cell on the hot plate. The 5CB was drawn into the cavity between the two sheared surfaces by capillary forces. Once filled with 5CB, the cell was removed from the hot plate and cooled in air to room temperature. Upon cooling, the isotropic phase of 5CB transformed to the nematic state. Analysis of Optical Textures. A polarized light microscope (BX60, Olympus, Tokyo, Japan) was used to observe the optical textures formed by light transmitted through the optical cells filled with nematic 5CB. All images were obtained using a 10× objective lens with a 1.0-mm field of view between crossed polarizers. Images of the optical appearance of each liquid crystal cell were captured with a digital camera (C-2020 Z, Olympus America Inc., Melville, NY) that was attached to a polarized light microscope. The images were obtained using a resolution of 1600 × 1200 pixels, an aperture of f11, and a shutter speed of 1/160 s. The optical images were analyzed using Photoshop software (Adobe Systems Incorporated, San Jose, CA) to calculate the average luminance (average pixel value on a scale of 0-255) of each image after conversion of the image from color to gray scale. Images by Atomic Force Microscopy (AFM). Images of sheared films of biotinylated BSA were obtained by AFM using a Digital Instruments Nanoscope III instrument (Santa Barbara, CA) operating in tapping mode. Samples were imaged under (3) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir Blodgett to Self-Assembly; Academic Press: New York, 1991.

Langmuir, Vol. 18, No. 13, 2002 5271 ambient conditions using silicon nitride tips with an average radius of ∼10 nm (OTESPA, Digital Instruments). Images were acquired at a scan rate of 1.0 Hz with 512 sample points per line.

Results and Discussion Optical Response of Nematic 5CB to IgG Bound to Films of Biotinylated BSA Sheared under Different Conditions. In our past studies of sheared films of biotinylated BSA,2 we sheared the covalently immobilized films of biotinylated BSA by displacement of a microscope slide supporting the film of biotinylated BSA across a cloth for a distance of ∼127 mm at a speed of ∼2.1 mm/s. A normal pressure of 1000 Pa was applied to the back surface of the microscope slide during the translation of the slide across the cloth. Figure 2 shows the optical appearances of nematic 5CB supported on films of biotinylated BSA that were sheared using the abovedescribed conditions (Figure 2A) and two other shear conditions (normal pressure of 250 Pa in Figure 2B and normal pressure of 250 Pa and displacement of 51 mm in Figure 2C). The images were obtained after the samples had been immersed into aqueous solutions of PBS containing increasing concentrations of anti-biotin IgG. Inspection of Figure 2 reveals that the optical appearance of nematic 5CB becomes complex and nonuniform with increasing concentration of IgG for all three types of surfaces. At low concentrations of IgG, the uniform alignment of nematic 5CB is first disrupted by the appearance of disclination lines (linear defects) within the liquid crystal. Even though the number of disclination lines increases with the concentration of IgG, these samples, when rotated between crossed polarizers, still exhibit a strong modulation in the intensity of light transmitted through the cells. At high concentrations of IgG, highly nonuniform regions of liquid crystal are observed throughout each cell. These samples, when rotated between crossed polarizers, did not exhibit a substantial modulation in the intensity of light transmitted through the cell. A comparison of parts A and B of Figures 2 reveals the effect of the normal pressure (1000 versus 250 Pa) applied during shearing of the films of biotinylated BSA on the response of nematic 5CB. The reponse is plotted as a function of the IgG concentration in the PBS used to deliver the IgG to the surface. With decreasing normal pressure, we observed the onset of the nonuniform alignment of nematic 5CB to occur at lower IgG concentrations. For example, the sample sheared at a pressure of 1000 Pa (Figure 2A) caused a largely uniform orientation of nematic 5CB when immersed into solutions containing 28 nM of IgG, whereas the sample sheared at 250 Pa was nonuniform in appearance when exposed to 20 nM of IgG. A comparison of parts B and C of Figures 2 reveals the effect of the length of cloth displaced across the surface of the film of biotinylated BSA on the response of nematic 5CB to IgG concentration. This comparison reveals that films of biotinylated BSA that are sheared by displacement of the cloth over shorter distances exhibit a greater sensitivity to the concentration of IgG in the PBS. Finally, we make one comment regarding the samples shown in Figure 2 that were immersed into buffer (0 nM). Although they are largely uniform in appearance, we observed that samples sheared at low normal pressures and short displacements do tend to exhibit defects with frequencies that are higher than those found in samples prepared at high normal pressures and large displacements. The main conclusion that we extract from the results in Figure 2 is that the sensitivity of the liquid crystal to the IgG concentration can be manipulated by varying the

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Figure 2. Optical textures of nematic 5CB (between crossed polarizers) sandwiched between glass microscope slides supporting sheared films of biotinylated BSA as a function of the concentration of anti-biotin IgG in solution. The shear speed, displacement length, and normal pressure applied during shearing are as follows; (A) ∼2.1 mm/s, ∼127 mm, ∼1000 Pa; (B) ∼2.1 mm/s, ∼127 mm, ∼250 Pa; and (C) ∼2.1 mm/s, ∼51 mm, ∼250 Pa. For each concentration of IgG, the optical appearance of the liquid crystal was recorded with (left) the shear direction parallel to the polarizer and (right) the shear direction at an angle of 45° from the polarizer. The white arrows indicate the shear direction, and the horizontal dimension of each image is 500 µm.

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manner in which the protein films are sheared. Below, we explore the question of whether this variation in sensitivity reflects a change in the amount of IgG bound to the sheared surface or a change in the structure of the sheared film prior to binding of IgGs (without a change in the amount of bound IgG). This analysis is based on a quantification of the optical appearance of the liquid crystal, which is described first below. Quantitative Analysis of the Optical Appearance of Nematic 5CB Induced by the Binding of IgG. The changes in optical appearance of nematic 5CB shown in Figure 2 can be quantified by measuring the average luminance of the sample (see details in the Experimental Section). We use the luminance to evaluate a corrected and normalized optical signal given by

normalized signal )

S - SMin SMax - SMin

(1)

where S is the maximum luminance ratio obtained by rotating the cell (LMin/LMax) between crossed polarizers. That is, LMin is the average luminance of the sample when the direction of shear is parallel to a polarizer, and LMax is the value obtained by rotating the sample by 45° from that orientation. We also normalize the luminance using two reference values, SMax and SMin, that are obtained using the films of biotinylated BSA with and without shearing, respectively (no bound IgG). Although this definition of the optical signal is not unique (many others can be imagined), we have found this index of the appearance of the liquid crystal to provide a useful means of quantifying the appearance of liquid crystals.2 Figure 3A shows the normalized optical signal obtained from the images in Figure 2. Inspection of Figure 3A confirms and quantifies the trends discussed above by visual inspection of the images in Figure 2, namely, that a decrease in normal pressure or displacement length leads to an increase in the sensitivity of the liquid crystal to the IgG concentration in the PBS used to deliver the IgG to the films of biotinylated BSA. Ellipsometric Measurement of Anti-Biotin IgG Bound to Sheared Films of Biotinylated BSA. To determine whether the effects of shear conditions on the response of nematic 5CB to IgG is due to a change in the amount of IgG bound to each sheared film, we used ellipsometry to measure the amount of IgG bound to the sheared films. Figure 3B shows that the ellipsometric thickness of the bound IgG is not measurably changed by variations in the shear conditions. We determined the ellipsometric thickness of the bound IgG to be approximately 10 nm. Given that the size of an IgG molecule is approximately 4 nm × 10 nm × 15 nm,4 these saturation coverages are high, approximately corresponding to monolayer coverage. This result reveals that the binding capacity of these surfaces is not compromised by the process of shearing the films of biotinylated BSA with a cloth (of the type described in the Materials section). It is possible that the lack of effect of shear conditions on the amount of bound IgG might reflect our use of biotinylated BSA with high biotin-to-BSA ratios (8:1 biotin/BSA). Because the BSA presents many lysine groups at its surface, it is not uncommon for hapten-to-BSA ratios to be high in such conjugates. By combining the data plotted in parts A and B of Figures 3, it can be seen that the response of nematic 5CB to bound IgG depends strongly on the shear conditions (4) Silverton, E. W.; Navia, M. A.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5140-5144.

Figure 3. (A) Optical response (normalized signal) of nematic 5CB (between crossed polarizers) as a function of the concentration of anti-biotin IgG in the solution in contact with the sheared film of biotinylated BSA. (B) Ellipsometric thickness of anti-biotin IgG bound to sheared films of biotinylated BSA as a function of the concentration of anti-biotin IgG in the solution used to pretreat the sheared films. (C) Optical response (normalized signal) of 5CB as a function of the ellipsometric thickness of anti-biotin IgG bound to the sheared film of biotinylated BSA. The shear speed, displacement length, and normal pressure are as follows; (open circles) ∼2.1 mm/s, ∼127 mm, ∼1000 Pa; (filled circles) ∼2.1 mm/s, ∼51 mm, ∼250 Pa; and (open triangles) ∼2.1 mm/s, ∼51 mm, ∼250 Pa.

(Figure 3C). When the normal pressure was decreased from 1000 to 250 Pa, the threshold ellipsometric thickness of bound IgG (defined as the ellipsometric thickness corresponding to 50% of the normalized signal) required to disrupt the orientation of nematic 5CB decreased from around 5.5 to 4 nm. A decrease in the length of cloth displaced across the film of biotinylated BSA from 127 to 51 mm leads to a further decrease in the threshold amount of bound IgG required to disrupt the uniform orientation of nematic 5CB (∼4 to ∼1 nm). We also note here that our previous studies have demonstrated that sheared films of BSA or biotinylated BSA resist the adsorption of nonspecific proteins including nonspecific IgGs.1,2 In particular, ellipsometric measurements of nonspecific proteins indicate that the amounts of protein bound to the surfaces are very small even at high concentrations (e.g., ∼0.2 nm for 100 nM antistreptavidin IgG). Influence of Shear Conditions on the Response of Nematic 5CB to Bound IgG. The results above demonstrate that the response of nematic 5CB to bound IgG depends strongly on the manner in which the chemically

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Figure 4. Dependence of optical response (normalized signal) of nematic 5CB (between crossed polarizers) on the shear conditions (open circles) and the corresponding ellipsometric thicknesses of bound IgG (filled circles): (A) effect of normal pressure (∼2.1 mm/s and ∼127 mm), (B) effect of displacement length (∼2.1 mm/s and ∼1000 Pa), and (C) effect of shear speed (∼127 mm and ∼1000 Pa).

immobilized film of biotinylated BSA is mechanically sheared. The ellipsometric measurements also suggest that the capacity of the surface to bind IgG does not change with the manner of shearing of the films of biotinylated BSA. Thus, we conclude that the effect of a change in shear conditions is to change the structure of the sheared film of biotinylated BSA that anchors the liquid crystal in a manner that does not affect the amount of bound IgG. Whereas the results above are based on three shear conditions, below we describe experiments in which we systematically varied the shear rate, magnitude of displacement of the cloth, and normal pressure applied during shearing so as to test the generality of these conclusions. This study also reveals the parameters of the shearing process that provide the greatest level of control over the structure of the sheared film of protein (as characterized by the response of the liquid crystal). Figure 4 shows the normalized optical signal and the ellipsometric thickness of bound anti-biotin IgG obtained with films of biotinylated BSA sheared at different normal pressures (Figure 4A), displacement lengths of cloth (Figure 4B), and shear speeds (Figure 4C). Inspection of these results confirms that the amount of IgG bound to each sheared film of biotinylated BSA is independent of the shear conditions (for all conditions investigated to date). In contrast, the optical appearance of nematic 5CB supported on the sheared films to which IgG is bound was found to be affected strongly by the choice of shear conditions. In particular, variations of the length of cloth

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displaced across the surface and the applied normal pressure both lead to remarkable changes in the response of nematic 5CB to the bound IgG. In contrast, we observed that the shear rate, as shown in Figure 4C, has little measurable effect on the optical appearance of nematic 5CB. Topographical Analysis of Sheared Films of Biotinylated BSA Using AFM. We measured the nanometer-scale topography of films of biotinylated BSA with and without shearing, both before and after binding of anti-biotin IgG (when sheared). Figure 5A shows a topographical image and cross-sectional profiles of a film of biotinylated BSA prior to shearing. The effect of shear on the topographical structure of these surfaces can be seen by inspection of Figure 5B and C. Inspection of Figure 5B and C reveals that shearing has introduced an anisotropic topographical structure into the films of biotinylated BSA. The topographical structure is more pronounced in Figure 5C (see Figure 4B, displacement of 508 mm, for shear conditions) than Figure 5B (see Figure 4B, displacement of 25 mm, for shear conditions). Both of these sheared surfaces caused a uniform alignment of nematic 5CB prior to contact with anti-biotin IgG. The effect of immersion into an aqueous solution of antibiotin IgG is shown in Figure 5D and E. Comparison of parts D and E of Figures 5 with parts B and C of the same figure reveals that the bound IgG masks/erases the anisotropic topography that is measured by AFM in the absence of bound IgG. Although an anisotropic topography is not measurable by AFM after binding of the IgG, liquid crystals placed onto the surface shown in Figure 5E are, in fact, uniformly aligned (the shear conditions correspond to a displacement of 508 mm in Figure 4B). In contrast, liquid crystals placed on to surface shown in Figure 5D are not uniformly aligned (displacement of 25 mm in Figure 4B). Our conclusions from the above AFM measurements are two-fold. First, it is clear that shearing films of biotinylated BSA introduces anisotropic topography into these surfaces. We also observed systematic differences in the topography, depending on the conditions used to shear the films of biotinylated BSA. Second, although our AFM measurements also reveal that the bound IgG masks the anisotropic topography that is measurable by AFM after shearing, this result does not obviously correlate with the response of the liquid crystal to the bound IgG. This result suggests that either (i) the structure identified by AFM is not the anisotropic structure responsible for the orientational behavior of the liquid crystal on these surfaces (see below) or (ii) the anisotropic structure in Figure 5E is too subtle to be detected by our AFM measurements. We note that the above conclusions are not surprising in light of past studies of sheared films of synthetic polymer films (e.g., polystyrene or polyimide). Past studies have established that (i) AFM can be used to measure anisotropic topography on these surfaces5-10 and (ii) the anisotropic topographical structure does not define the (5) Mahajan, M. P.; Rosenblatt, C. J. Appl. Phys. 1998, 83, 76497652. (6) Zhu, Y.-M.; Wang, L.; Lu, Z.-H.; Wei, Y.; Chen, X. X.; Tang, J. H. Appl. Phys. Lett. 1994, 65, 49-51. (7) Devlin, C. L. H.; Glab, S. D.; Chiang, S.; Russell, T. P. J. Appl. Polym. Sci. 2001, 80, 1470. (8) Huang, J. Y.; Li, J. S.; Chen, S.-H. Mol. Cryst. Liq. Cryst. 1995, 270, 77-84. (9) Pidduck, A. J.; Bryan-Brown, G. P.; Haslam, S. D.; Bannister, R.; Kitely, I.; McMaster, T. J.; Boogaard, L. J. Vac. Sci. Technol. A 1996, 14, 1723-1728. (10) Pidduck, A. J.; Bryan-Brown, G. P.; Haslam, S. D.; Bannister, R. Liq. Cryst. 1996, 21, 759-763.

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Figure 5. AFM images of sheared films of biotinylated BSA; (A) prior to shearing, (B-C) sheared films, (D-E) after incubation of sheared films in aqueous solutions of anti-biotin IgG (20 nM). (B, D) and (C, E) correspond to sheared films prepared using different displacement lengths (25 and 508 mm, respectively) at ∼2.1 mm/s and ∼1000 Pa. The black arrows indicate the shear direction, and the two profiles in each image correspond to the cross-sectional views (red and blue lines in the AFM images).

dominant interactions between the liquid crystal and surface that dictate the orientation of the liquid crystal.9-13 (11) Geary, J. M.; Goodby, J. W.; Kmetz, A. R.; Patal, J. S. J. Appl. Phys. 1987, 62, 4100-4108. (12) Dubois, J. C.; Gazard, M.; Zann, A. J. Appl. Phys. 1975, 47, 1270-1274. (13) Kikuchi, H.; Logan, J. A.; Yoon, D. Y. J. Appl. Phys. 1996, 79, 6811-6817.

Instead, oriented polymer chains and molecular-level interactions are generally believed to dictate the orientations of liquid crystals on sheared polymer surfaces.14-18 (14) Je´roˆme, B. Rep. Prog. Phys. 1991, 54, 391-451. (15) de Gennes, P. G. The Physics of Liquid Crystals; Clarendon Press: Oxford, U.K., 1974. (16) Nakajima, K.; Wakemoto, H.; Sato, S.; Yokotani, F.; Ishihara, S.; Matsuo, Y. Mol. Cryst. Liq. Cryst. 1990, 180, 223-232.

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For example, nematic 5CB orients across the “scratches” on films of sheared polystyrene.14-16 Conclusions The main conclusions of this paper are two-fold. First, we demonstrated that the response of liquid crystals to IgG specifically bound to sheared films of functionalized BSA can be manipulated over a wide range by varying the conditions used to shear the films (overall displacement and normal pressure). Second, we established that this variation in the response of the liquid crystal to bound IgG does not reflect a variation in the amount of IgG bound to the sheared films. The binding capacity of biotinylated BSA appears to be independent of the shear history of the (17) Castellano, J. A. Mol. Cryst. Liq. Cryst. 1983, 94, 33-41. (18) Aoyama, H.; Yamazaki, Y.; Matsuura, N.; Mada, H.; Kobayashi, S. Mol. Cryst. Liq. Cryst. 1981, 72, 127-132.

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film. Instead, the variation in the response of the liquid crystal to bound IgG reflects changes in the interaction between the liquid crystal and the sheared protein film. Although AFM measurements reveal anisotropic topography in sheared films of biotinylated BSA, we do not yet understand the role of this topography (if any) in determining the response of the liquid crystals to IgG bound to the sheared films. Ongoing investigations are addressing this issue and will be reported elsewhere. Acknowledgment. This research was supported by funding from the Office of Naval Research (Presidential Early Career Award for Science and Engineering to N.L.A., N00014-99-1-0250), the Center for Nanostructured Interfaces (NSF-DMR-0079983) at University of Wisconsin, and the Biophotonics Partnership Initiative of NSF (ECS-0086902). LA011844E