Influence of Molecular-Level Interactions on the Orientations of Liquid

We report an experimental investigation of the role of molecular-level interactions in determining the anchoring of liquid crystals supported on surfa...
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Langmuir 2001, 17, 5595-5604

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Influence of Molecular-Level Interactions on the Orientations of Liquid Crystals Supported on Nanostructured Surfaces Presenting Specifically Bound Proteins Justin J. Skaife and Nicholas L. Abbott* Department of Chemical Engineering, University of Wisconsin, Madison, Wisconsin 53706 Received January 25, 2001. In Final Form: May 30, 2001 We report an experimental investigation of the role of molecular-level interactions in determining the anchoring of liquid crystals supported on surfaces possessing nanometer-scale topography on which immunoglobulins (IgG) are specifically bound to immobilized antigens. Molecular-level interactions are manipulated by using self-assembled monolayers (SAMs) of organosulfur compounds formed on thin films of gold that possess an anisotropic, nanometer-scale topography (corrugation). We compare the orientational response of liquid crystal to the presence of anti-biotin IgG specifically bound to mixed SAMs formed from biotin-(CH2)2[(CH2)2O]2NHCO(CH2)11SH and either CH3(CH2)6SH or CH3(CH2)7SH on the gold films. When using SAMs that contain 70% alkanethiolate, we measure the orientational (and thus optical) response of the liquid crystal to IgG to depend on whether the alkanethiolate within the mixed SAM is CH3(CH2)6S or CH3(CH2)7S. We conclude, therefore, that molecular-level interactions controlled by the structure of the alkanethiolates, in addition to long-range (elastic) interactions that result from the nanometer-scale topography of the gold film, influence the response of liquid crystal to the IgG specifically bound to these surfaces. The influence of the nanometer-scale topography does, however, dominate the response of the liquid crystal. The molecular interactions appear to influence the lifetimes of line defects formed as nematic phases spread across these surfaces: the defects are observed to anneal quickly (∼seconds) on SAMs containing CH3(CH2)7S but slowly (>days) on those containing CH3(CH2)6S. The pinning of defects within the liquid crystal when using SAMs containing CH3(CH2)6S causes these surfaces to be more sensitive to bound IgG than surfaces containing CH3(CH2)7S.

Introduction Recent studies have demonstrated the use of liquid crystals to amplify and transduce biologically relevant binding events (e.g., protein-ligand and protein-protein recognition events) at nanostructured surfaces.1,2 Changes to the nanometer-scale structures of these surfaces that are caused by specific binding of biological species to surface-bound receptors were found to lead to distinguishable orientations of liquid crystals supported on these surfaces.1 These studies also demonstrated that it is possible to quantify the optical response of the liquid crystal as a function of the amount of bound protein by measuring the intensity of polarized light transmitted through the liquid crystal.2 The optical response was observed to be a continuous function of the amount of bound protein.2 In this paper, we report an investigation of the intermolecular interactions between liquid crystals and nanostructured surfaces presenting bound proteins that govern the previously reported response of liquid crystals to bound proteins. This investigation is guided by the proposition that the orientational response (including sensitivity) of the liquid crystals to bound IgG might be manipulated by altering the balance of molecularand long-range interactions between the nanostructured surfaces and the supported liquid crystals. The work reported in this paper is broadly motivated by the need for new principles that will enable the analysis of complex mixtures of proteins, such as the tens of * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 608-262-5434. (1) Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B.; Abbott, N. L. Science 1998, 279, 2077. (2) Skaife, J. J.; Abbott, N. L. Langmuir 2000, 16, 3529.

thousands of proteins found in cells.3-17 Unlike genomic analyses, where a few copies of DNA can be amplified by polymerase chain reaction and then analyzed, experi(3) Van Oss, C. J.; van Regenmortel, M. H. V. Immunochemistry; Dekker: New York, 1994. (4) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Springs Harbor Laboratory: Cold Springs Harbor, NY, 1988. (5) Whereas numerous techniques exist for detection of individual protein species, such as Western blotting, ELISA, radio-immuno assay (RIA), radio-immuno precipitation, HPLC, and ion exchange, very few methods exist for analysis of complex mixtures of proteins (refs 3, 4, and 6-13). Mass spectroscopy is powerful but requires complex and expensive instrumentation and is not widely accessible (refs 14-17). Methods such as ion exchange, HPLC, and two-dimensional (2D) gels for routine analysis of the protein composition of cells are slow, typically requiring several days to complete the sample preparation, measurement, and analysis (refs 3 and 4). Routine gel-based methods are also generally insensitive: standard stains do not permit detection of 18.2 MΩ cm; Millipore, Bedford, MA). Affinity-isolated goat anti-biotin immunoglobulin G (anti-biotin IgG) and rabbit antiFITC immunoglobulin G (anti-FITC IgGs) were obtained from Sigma BioScience (St. Louis, MO). Solutions of the IgG were prepared in 25 mM PBS buffer (pH 7.35) using 100 mM NaCl, 0.01 wt % NaN3 as a preservative, and 0.01 wt % Triton X-100 purchased from Sigma (St. Louis, MO). Vacuum Deposition of Films of Gold. Films of titanium (thickness of 8 ( 1 nm) and gold (thickness of 20 ( 1 nm) were evaporated onto clean glass substrates with an electron beam evaporator (Tekvak, Brentwood, NY). The rates of deposition of the metals were 0.02 ( 0.005 nm/s, and the metals were deposited at an angle of incidence of 45 ( 5° from the normal. These deposition parameters lead to semi-transparent films (∼50% transmission of visible light). A detailed description of these procedures, including procedures for the cleaning of the glass microscope slides, can be found in a previous publication.18 Following the deposition of each batch of gold films, we performed two experiments to confirm the quality of the films. We formed SAMs from C16SH and from C8SH on the surface of the gold films and confirmed that nematic phases of 5CB were anchored uniformly in a direction parallel to the azimuthal direction of deposition of the gold (see below for procedures). Formation of SAMs. Self-assembled monolayers were formed on the surfaces of the gold films by immersion of the films into ethanolic solutions containing either C7SH/BiSH or C8SH/BiSH. The concentrations of C7SH, C8SH, and BiSH were 22, 22, and 44 µM, respectively. After immersion for 15 h at room temperature, the slides were removed, rinsed with ethanol, and then dried under a stream of N2. Binding of Proteins. In our previous work, we demonstrated control of the amount of bound IgG by immersing mixed SAMs containing BiSH in solutions containing increasing concentrations of anti-biotin IgG for a fixed duration (30 min).2 In this paper, we have adopted a different procedure to achieve control over the amount of bound anti-biotin IgG. We immersed the SAMs into solutions containing a fixed concentration of anti-biotin IgG for increasing lengths of time. We chose this method because multiple solutions containing the same concentration of antibiotin IgG can be conveniently prepared. The final concentration of anti-biotin IgG in solution was fixed at 40 nM.39 We used the IgG solutions immediately following their preparation. The binding of anti-biotin IgG was performed by placing mixed SAMs formed from either C7SH/BiSH or C8SH/BiSH into polypropylene vials filled with the aqueous solutions of IgG for various times (without stirring) at 25 °C (Figure 2). Immediately following removal of the SAMs from the IgG solutions, they were rinsed with water for ∼10 s and dried under a stream of N2 (∼10 s). By using ellipsometry, we found that neither rinsing of SAMs for 5-45 s nor drying with a stream of gaseous N2 (5-30 s) affected the amount of bound IgG measured by ellipsometry. Control experiments using a nonspecific IgG (anti-FITC IgG, 500 nM) demonstrated that rinsing of the mixed SAM with water for 10 s was sufficient to remove nonspecifically adsorbed IgG (and Triton X-100). Fabrication of Liquid Crystal Cells. Mixed SAMs supporting bound IgG were assembled into optical cells in order to observe the optical appearance of liquid crystal anchored on the SAMs. The optical cells were fabricated (see Figure 2) by spacing two SAMs (facing each other) apart by ∼10 µm using thin strips of Mylar (DuPont Films, Wilmington, DE). The optical cells were held together with small binder clips. Each cell was placed on (38) Sprinke, J., et al. J. Chem. Phys. 1993, 99, 7012. (39) We note that 3 mL of a 10 nM solution of IgG contains approximately ∼2 × 1013 IgG molecules. Because adsorption of a monolayer of IgG onto a 2 cm2 area of a surface would remove ∼1.3 × 1012 molecules (∼104 molecules per square micrometer) from solution, we conclude that nonspecific adsorption from solutions containing 10 nM or less IgG can potentially deplete the IgG in solution to levels that would influence the extent of binding of IgG to the surface of SAMs used in our experiments. For this reason, we used Triton X-100 to minimize the nonspecific adsorption of IgG to the surface of the vial as well as to the SAMs.

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Figure 2. Schematic illustration of the experimental procedure used to measure the orientation of a liquid crystal on a SAM presenting specifically bound IgG. (A) Binding of IgG to a mixed SAM formed from C7SH/BiSH or C8SH/BiSH on obliquely deposited gold. (B) IgG specifically bound to SAM after removal from aqueous solution containing IgG. After removal from the aqueous solution of IgG, the SAM was rinsed in water and the excess water was displaced from the surface using a stream of gaseous nitrogen. (C) Liquid crystal cell fabricated from mixed SAMs supporting the IgG. The optical appearance of the liquid crystal was determined by transmission of polarized light through the SAMs and film of liquid crystal sandwiched between the SAMs. a warm surface (at 40 °C) and gently heated with a hot air gun for approximately 10 s. This procedure was used to maintain the temperature of the cell above the nematic-isotropic transition temperature of 5CB during the filling of the cell by 5CB. 5CB was heated into its isotropic phase (>35 °C) in a glass syringe and then introduced into the cell by capillary action (10 s). The cell was allowed to sit on the hot plate for 30 s prior to removal and cooling to room temperature. During cooling, the 5CB changed from its isotropic state to its nematic state. We address the time dependence of the optical appearance of the liquid crystal below. The optical appearance of the sample was observed in transmission by using a polarizing light microscope (under crossed polars). The settings of the microscope (light source and aperture) were kept constant in all experiments (see below). The sample was aligned in the microscope with the azimuthal direction of deposition of the gold film parallel to the axis of the polarizer. This arrangement results in the extinction of light transmitted through the sample in cases where 5CB is uniformly anchored by the mixed SAM.40 Image Capture and Analysis. Optical images of the liquid crystal were captured with a CCD camera (DXC-151A, Sony, Park Ridge, NJ) and frame grabbing software (Mediagrabber, Rasterops Inc., Santa Clara, CA) that was attached to a polarized light microscope (BX60, Olympus, Melville, NY). A quantitative comparison of the textures was made by using computer software (NIH Image, Bethesda, MD) to calculate the average luminance (average pixel value on a scale of 0-255) of the image after conversion of the image from color to gray scale. Consistent settings of the microscope light source (50% of maximum intensity, 50% open aperture, and no condenser) and CCD camera (40) Maximum extinction of the light transmitted through a cell containing uniformly oriented liquid crystal occurs when one of the crossed polars is oriented parallel to the orientation of the liquid crystal.

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(0 dB gain, no auto color correction, and open shutter) were used to permit comparison of values of luminance between samples. The raw luminance of each sample (S) was corrected for the luminance of an image of the liquid crystal supported on the mixed SAM (no bound protein; Smin) and normalized by the corrected, maximum luminance of the images of liquid crystals supported on SAMs on which proteins were bound (Smax).41 Variations in Smin and Smax were found to be small (2-3%) from batch to batch of samples. The equation used to calculate the normalized and corrected luminance (optical output) L is given by

L (%) )

(

)

Smin - S × 100 Smin - Smax

(1)

Ellipsometry. We measured the optical thicknesses of mixed SAMs and IgG bound to mixed SAMs by using ellipsometry. Ellipsometry was performed using thick (∼50 nm) films of gold because these films are optically reflecting. The thick films of gold were deposited on glass microscope slides while rotating the microscope slides to avoid the introduction of anisotropy into the gold films. Obliquely deposited films of gold possess anisotropic optical constants that make interpretation of the ellipsometric measurements more complex than when using gold films deposited without a preferred direction. The procedures used to form mixed SAMs on thick gold and to bind IgG to the mixed SAMs were the same as those used on thin gold films. We measured the ellipsometric constants at three locations on each sample using a Rudolph AutoEL II ellipsometer (wavelength of 632 nm, angle of incidence of 70°; Rudolph Tech., Flanders, NJ). Ellipsometric constants of the bare gold surfaces were determined immediately after removal of the gold films from the evaporator. A simple slab model was used to interpret the ellipsometric constants when a mixed SAM or mixed SAM supporting bound protein was formed on the surface of the gold film. The slab (SAM + protein) was assumed to have an index of refraction of 1.46.

Results Formation and Characterization of Mixed SAMs. The first goal of our experimental investigation was to establish a procedure that would lead to the formation of mixed SAMs from C7SH/BiSH or C8SH/BiSH that possessed two characteristics in common. First, the procedure needed to yield mixed SAMs that possessed the same number density of biotin groups so as to permit a comparison of the anchoring of liquid crystals on these SAMs after binding of the IgG. Second, the mixed SAMs needed to uniformly align liquid crystal in the absence of bound IgG. Past studies of mixed SAMs of alkanethiols have established that for the same solution composition, the compositions of the resulting SAMs depend on the relative chain lengths of the alkanethiols and the time of formation of the mixed SAMs.36 A mixed SAM is enriched in the long-chain component, and the level of enrichment increases with the time of formation of the SAM. Because the difference between C7SH and C8SH is only one methylene group, we speculated that the difference in compositions of the mixed SAMs formed from solutions containing the same composition of C7SH/BiSH and C8SH/BiSH would be minimal. Our first experiments tested this proposition by using ellipsometry to estimate the compositions of the mixed SAMs. First, we measured the ellipsometric thicknesses (effective optical thicknesses) of the single-component SAMs formed from BiSH, C7SH, and C8SH to be 4.9 ( 0.2, 0.9 ( 0.1, and 1.1 ( 0.1 nm, respectively. Second, we measured (41) We chose the average brightness as a measure of uniformity in the optical textures because it is simple to calculate. Other measures of the appearance of the liquid crystal will likely serve as useful indices of the amount of bound IgG.

Figure 3. The ellipsometric thickness of anti-Bi IgG specifically bound to mixed SAMs formed from C7SH/BiSH (solid circles) and C8SH/BiSH (open circles) plotted as a function of time of immersion of the SAMs in 40 nM solutions of IgG. The dashed and solid lines are drawn to guide the eye.

ellipsometric thicknesses of the mixed SAMs formed from C7SH/BiSH and C8SH/BiSH (formed for 15 h) to be 2.2 ( 0.1 and 2.4 ( 0.1 nm. By linear interpolation, we estimate the mole fraction of BiS within the SAMs formed from C7SH/BiSH to be 30 ( 2%. Similarly, the amount of BiS within the SAMs formed from C8SH/BiSH was also calculated to be 30 ( 2%. We conclude, therefore, that the amount of biotin present within SAMs formed from both C7SH/BiSH and C8SH/BiSH for 15 h is 30 ( 2%. We also explored the effect of time of formation of the mixed SAMs on their composition. Self-assembled monolayers formed for short times (∼1 h) were found have the same composition when using C7SH/BiSH and C8SH/ BiSH. Both types of SAMs possessed slightly less biotin (25 ( 4%) than SAMs formed for 15 h (see above). However, the sample-to-sample variations in ellipsometric thicknesses were measured to be larger when using SAMs formed for 1 h ((0.2 nm) than when using SAMs formed for 15 h ((0.1 nm). When using long formation times (∼24 h), we measured the amount of biotinylated species within the mixed SAMs to increase to ∼40% for both types of mixed SAMs. Whereas the orientation of the liquid crystal was measured to be uniform on the mixed SAMs formed for 1 and 15 h, the mixed SAMs formed for 24 h were measured to give rise to nonuniform orientations of liquid crystals. In summary, the mixed SAMs formed for either C7SH/ BiSH or C8SH/BiSH for 15 h were found to uniformly orient liquid crystal and to possess reproducible compositions that were indistinguishable (30 ( 2% of the biotinylated species) from each other. Thus, the experiments described below utilize these types of mixed SAMs. Measurement of the Amount of Bound IgG. The second goal of our investigation was to prepare mixed SAMs (as described above) that supported known and systematically increasing amounts of bound IgG. In particular, we aimed to determine if, for the same IgG binding conditions, the amount of IgG bound to the mixed SAM formed from C7SH/BiSH differed from that bound to a mixed SAM formed from C8SH/BiSH. We also mention that our past studies2 have established that the procedures described in the methods section of this paper lead to very low levels of binding of nonspecific IgGs (e.g., anti-FITC IgG). Here, we controlled the amount of IgG specifically bound to the mixed SAMs by varying the time of immersion of the SAMs in aqueous solutions containing 40 nM anti-

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Figure 4. The optical appearance (crossed polars; ∼1 mm × 1 mm) of liquid crystal supported on SAMs formed from either C7SH/BiSH or C8SH/BiSH after immersion of the SAMs in aqueous solution containing 40 nM solution of anti-Bi IgG. The time of immersion of each SAM is shown in the inset in each figure.

biotin IgG. The amount of bound IgG was determined by using ellipsometry (Figure 3). The amount of IgG bound to the surface was measured to increase with the time of immersion for both types of mixed SAMs and approached an asymptotic value of 5 ( 0.2 nm after 24 h. The results in Figure 3 also indicate that the amount of IgG bound to the mixed SAMs as a function of time is similar for both types of mixed SAMs. There is, however, a slight (∼0.2 nm) but consistently greater amount of protein bound to the mixed SAMs formed from C7SH at times below 3 h.

Qualitative Optical Appearance of Liquid Crystal. The third goal of our investigation was to compare the influence of bound IgG on the orientations of liquid crystals supported on the two types of mixed SAMs. By comparing and contrasting the response of the liquid crystal to the C7S and C8S species within the mixed SAM, we aimed to test the role of molecular-level interactions between the liquid crystal and SAM on the response of the liquid crystal to bound IgG. The optical textures of the liquid crystals placed between crossed polars and illuminated by polar-

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ized white light are shown in Figure 4. As previously mentioned, to compare the optical textures of the liquid crystals, all cells were oriented with the deposition direction of the gold film aligned parallel to the axis of the polarizer. Aligned in this manner, a dark texture indicates uniform alignment of the liquid crystal since linearly polarized light passing through the liquid crystal remains linearly polarized along the same axis. If, however, the alignment of the liquid crystal is nonuniform, the polarization of light passing through the sample will be rotated and a texture with bright regions will be observed.42 The optical textures in Figure 4 were recorded 1 min after cooling the 5CB into its nematic state. The time dependence of the images is addressed below. In the absence of bound IgG, the mixed SAM formed from C7SH/BiSH uniformly aligned the liquid crystal, as indicated by the black texture at t ) 0 in Figure 4. We also determined that mixed SAMs immersed into aqueous buffer (free of IgG) for 24 h caused uniform alignment of the liquid crystal. For IgG binding times that are less than 30 min, the optical textures of liquid crystal placed on the C7SH/BiSH surfaces remain largely uniform but possess an increasing number of disclination lines. After ∼90 min of immersion in the IgG solution, the appearance of the liquid crystal on the mixed SAM formed from C7SH/BiSH becomes increasingly nonuniform. For times of immersion that exceed ∼135 min, the extent of nonuniformity on C7SH/BiSH surfaces appears to plateau. We attribute the overall transition from uniform to nonuniform optical textures to be the result of masking of the nanometer-scale topography of the obliquely deposited gold film by the bound IgG. However, as revealed below, molecular-scale interactions also appear to play a role in determining the way in which this transition from uniform to nonuniform alignment occurs as a function of the amount of bound IgG. Inspection of the optical textures of the liquid crystals on the mixed SAMs formed from C8SH/BiSH with increasing amounts of bound IgG reveals an overall transition in the liquid crystal from uniform to nonuniform alignment, similar to the mixed SAM formed from C7SH/BiSH. However, for times of immersion of the mixed SAMs in the IgG solution that are less than ∼150 min, we do observe differences in the appearance of the liquid crystals on the two types of mixed SAMs. In contrast to the mixed SAMs formed from C7SH/BiSH, the optical textures of liquid crystals supported on mixed SAMs formed from C8SH/BiSH remain uniform with few disclinations until ∼75 min. The optical textures of C8SH/BiSH begin to exhibit highly nonuniform textures at ∼150 min, with the extent of nonuniformity reaching a plateau above ∼180 min. In summary, the amount of bound anti-biotin IgG required to induce nonuniformity (defects) into the alignment of the liquid crystal is substantially less when using mixed SAMs formed from C7SH/BiSH than C8SH/BiSH. The initial transition from uniform alignment of the liquid crystal to nonuniform alignment in the case of C7SH/BiSH is associated with the increasing appearance of disclination lines within the liquid crystal. In contrast, the appearance (42) Interference colors result from the transmission of particular wavelengths of light when using a white light source of illumination. The extent of transmission of a particular wavelength of light depends on the retardation, ∆nd, where ∆n is the birefringence and d is the thickness of the birefringent film. Light propagating through the liquid crystal will be extinguished under conditions where ∆nd ) jξ, j ) 0, 1, 2,..., where ξ is the wavelength of light. Interference colors are divided into orders according to the magnitude of the retardation: 0-550 nm (first order), 550-1100 nm (second order), and 1100-1650 nm (third order).

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Figure 5. The optical response (see text for details) of liquid crystal supported on SAMs formed from either C7SH/BiSH (solid circles) or C8SH/BiSH (open circles) after immersion of the SAMs in aqueous solution containing 40 nM solution of anti-Bi IgG. The amount of IgG bound to each mixed SAM is reported as an ellipsometric thickness of IgG. The square data points indicate the optical response curve reported by us in a past study (ref 2) using gold films (and SAMs formed from C8SH/BiSH) that possessed a nanometer-scale topography that differs from the topography of the gold films used in the study reported in this paper (see text for discussion).

of the liquid crystal on surfaces supporting large amounts of bound IgG is indistinguishable. These differences in the optical appearance of the liquid crystal are quantified and investigated further in the sections below. Quantitative Analysis of the Optical Appearance of Liquid Crystal. To quantify and directly compare the optical responses of the liquid crystals on the two types of mixed SAMs, we have evaluated the normalized optical brightness of each of the optical textures, as defined in the methods section. The optical response shown in Figure 5 was calculated from the optical textures in Figure 4 and the amount of anti-biotin IgG bound to the mixed SAMs, as measured by ellipsometry (Figure 3). Consistent with the appearance of the liquid crystal in Figure 4, the optical textures of the liquid crystal supported on mixed SAMs formed from C7SH/BiSH exhibit modestly larger values of normalized optical brightness at low amounts of bound IgG (6 h, the disclinations remain unchanged on C7SH/BiSH surfaces, indicating that the C7SH/BiSH surface pins the disclinations and prevents their annihilation. Although we do not yet understand the details of the mechanisms that lead to formation and pinning of the defect lines on the mixed SAMs supporting the bound IgGs, the presence of the defects on the mixed SAMs formed from C7SH/BiSH leads to an optical response of

Liquid Crystals on Nanostructured Surfaces

Figure 7. (A-C) Optical appearance of liquid crystal on SAMs formed from C8SH/BiSH to which anti-Bi IgG is bound (ellipsometric thickness of ∼2 nm). (A) Anti-Bi IgG bound to a mixed SAM by immersion of the mixed SAM into a 40 nM solution of anti-Bi IgG for 80 min. (B) Anti-Bi IgG bound to a mixed SAM by immersion of the mixed SAM into a 100 nM solution of anti-Bi IgG for 30 min. (C) Optical appearance of liquid crystal reported in a past study (ref 2) in which anti-Bi IgG was bound to a mixed SAM formed from C8SH/BiSH by immersion of the mixed SAM into a 100 nM solution of anti-Bi IgG for 30 min. The graph shown below the images plots the normalized optical outputs calculated from the images in A-C on the response curves measured in the present study (Figure 5) and our past study when using mixed SAMs formed from C8SH/BiSH (ref 2).

the liquid crystal that can be readily quantified. On these surfaces, the scattering of light from the line defects provides an optical response of the liquid crystal that increases in a relatively linear fashion with increasing amounts of bound IgG. This result suggests that there may exist a proportional relationship between the amount of bound IgG and the total length of disclination line on the surface. We note that for quantitative assays of proteins at surfaces, the linear optical response of the liquid crystal may be preferred over the more abrupt response of the liquid crystal that is measured on the mixed SAMs formed from C8SH/BiSH. The overall optical response of the liquid crystal measured on the mixed SAMs formed from C8SH/BiSH (Figure 4) is qualitatively similar to results of our past studies (sigmoidal response curve). However, a quantitative comparison (Figure 7) reveals a substantial difference in the amount of bound IgG that causes a nonuniform orientation of the liquid crystal. In our past work, we reported a 50% optical signal when ∼1.6 nm of IgG was bound to the mixed SAM, whereas here we report a 50% optical signal when ∼3.0 nm of IgG is bound to the mixed SAM (Figure 7). Below, we identify the origin of this difference. We first point out several differences that exist between the experimental procedures used in our past studies and those used in this investigation. First, the obliquely deposited films of gold used in the two studies were prepared using different electron beam chambers. Al-

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though the nominal angle (45 ( 5°), thickness (20 ( 2 nm), and rate of deposition of gold (0.02 ( 0.01 nm/s) used in these two studies were designed to be similar, geometric parameters such as the source-to-substrate distance were different. The geometry of the evaporator determines factors such as the dispersion in the angle of deposition of the gold onto the substrate that, in turn, can influence that nanometer-scale topography of the gold. A second difference between the two studies was the procedure used to bind the IgGs to the mixed SAMS. Although the compositions of the mixed SAMs used in the past and present studies were designed to be similar, our past work varied the concentration of IgG in solution (with a constant time of binding) to control the amount of IgG bound to the SAM, whereas in the present study we varied the time of binding (at constant bulk concentration of IgG) to control the amount of bound IgG. Because the manner of binding of the IgG could influence its lateral distribution on the surface as well as its bound (conformational) state, we considered it possible that differences in the state of bound IgG were responsible for the differences in the optical response of the liquid crystals seen in Figure 7. For example, it is well-known that some proteins such as human serum albumin adsorbed onto hydrophobic surfaces undergo time-dependent conformational changes (flatten into pancake-type states) that occur over periods of hours or longer.43 To test whether differences in our past and present procedures used to bind the IgGs do influence the response of liquid crystal to bound IgG, we performed an additional set of binding experiments. First, we used ellipsometry to establish conditions based on the two different binding procedures that lead to the same amount of bound IgG. In our previous work (with gold films of the same thickness reported here), we controlled the amount of bound IgG by changing the concentration of IgG in solution and by keeping the binding time at 30 min. By using ellipsometry, we determined that ∼2 nm of IgG bound to C8SH/BiSH SAMs within 30 min (identical to our earlier work) when using a solution containing 100 nM of IgG. By using ellipsometry, we also determined that binding of IgG from a 40 nM solution for 80 min leads to ∼2 nm of bound IgG. Inspection of Figure 7 reveals that the appearance of the liquid crystal is independent of the procedure used to bind the IgG in these experiments. We conclude, therefore, that the differences in the optical response curves seen in our present and past work are not a consequence of the procedures used to bind the IgG to the surface. We infer from this result that the likely origin of the differing optical responses is variation in the nanometer-scale topography of gold films used in the two studies. This inference is supported by results of a separate study in which we have systematically changed the nanometer-scale topography of the gold surface and characterized its substantial influence on the response of the liquid crystal to bound IgG.37 Conclusion The principal conclusion of the work reported in this paper is that the response of liquid crystal to IgGs specifically bound to antigens presented at surfaces is determined by both the molecular-level structure and nanometer-scale topography of the surface. Specifically, we have measured the response of nematic liquid crystals of 5CB to differ when anti-Bi IgG is bound to mixed SAMs formed from either C7SH/BiSH or C8SH/BiSH on obliquely (43) Sheller, N. B.; Petrash, S.; Foster, M. D. Langmuir 1998, 14, 4535.

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deposited films of gold. The principal difference in the response of the liquid crystal is the number density of linear defects that form within the liquid crystal when the IgG is bound to the surface: few defects form when a partial monolayer of IgG with an ellipsometric thickness of ∼2 nm or less is bound to a SAM formed from C8SH/ BiSH. In contrast, when the ellipsometric thickness of IgG bound to a SAM formed from C8SH/BiSH is less than ∼1 nm, we observed substantial densities of linear defects within the liquid crystal. These differences in defect densities were traced to (I) processes of defect generation occurring at the advancing nematic front during the cooling of 5CB into its nematic state and (II) processes of defect annihilation that occurred after passage of the nematic front. When the ellipsometric thickness of bound IgG was greater than ∼3 nm, the optical response of the liquid crystal to bound IgG was measured to be indistinguishable when using the two types of SAMs, presumably because the amount of bound IgG was sufficient to

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mask the influence of the underlying SAMs on the liquid crystal. Finally, we note that although the results reported in this paper do demonstrate that molecular-scale interactions mediated by the structure of the SAM can have a measurable influence on the response of liquid crystal to bound IgG, we also point out that the molecular-level effects reported here are small compared to the measured influence of changes in the nanometer-scale topography of the surfaces on the response of liquid crystal to bound IgG. The dominant effects of nanometer-scale topography will be reported elsewhere.37 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.) and the Center for Nanostructured Interfaces (NSF-DMR 9632527) at the University of WisconsinsMadison. LA010132L