Influence of Nanometer-Scale Topography of Surfaces on the

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Influence of Nanometer-Scale Topography of Surfaces on the Orientational Response of Liquid Crystals to Proteins Specifically Bound to Surface-Immobilized Receptors Justin J. Skaife, Jeffery M. Brake, and Nicholas L. Abbott* Department of Chemical Engineering, University of Wisconsin, Madison, Wisconsin 53706 Received December 18, 2000. In Final Form: May 30, 2001 We report procedures based on oblique deposition of gold that lead to the preparation of ultrathin, semitransparent films of gold that possess systematic differences in their nanometer-scale topography. The nanometer-scale topography of these surfaces is controlled by the angle of incidence of the gold during the oblique deposition of each film. The topography is quantified by using atomic force microscopy (AFM) in terms of the azimuthal dependence of the contour length and local curvature of the surface. We use these surfaces to test our hypothesis that control of nanometer-scale topography permits manipulation of the orientational response of liquid crystal to proteins bound to receptors immobilized on surfaces. We measure the orientational response of nematic phases of 5CB to anti-biotin immunoglobulin G (IgG) bound to biotin-terminated self-assembled monolayers to depend strongly on the nanometer-scale topography of the surfaces. The response of the liquid crystal correlates closely with quantitative measures of the surface topography obtained by AFM and thus demonstrates that it is possible to tune the sensitivity of nematic liquid crystals to the presence of specifically bound IgG by manipulating the nanometer-scale topography of surfaces. The surfaces with the smallest local curvatures were found to be the most sensitive to the presence of bound IgG. We also calculate the anchoring energy of liquid crystal on the surfaces by using continuum elastic theory and the topography obtained from the AFM images. Although the sensitivity of the liquid crystal to the bound protein increases with decreasing anchoring energy, it is not possible to provide a complete account of the orientational behavior of the liquid crystal on these surfaces on the basis of continuum elastic theory.

Introduction Past studies, both experimental and theoretical, have established that the orientations assumed by liquid crystals near surfaces can be exquisitely sensitive to the chemical and physical structure of surfaces.1,2 Recently, we reported use of the surface sensitivity of liquid crystals to amplify and transduce biologically relevant (proteinreceptor and protein-protein) binding events at surfaces into optical signals.3-5 Surfaces were fabricated with a nanometer-scale topography such that changes in the structure of the surfaces induced by specific binding of proteins to surface-immobilized receptors resulted in orientations of liquid crystals that were optically distinguishable from the orientations assumed by the liquid crystals in the absence of bound protein.3 We also demonstrated that it was possible to quantify the optical response of the liquid crystal as a function of the amount of bound protein by measurement of the fraction of polarized light that was transmitted through the liquid crystal.4 These results suggest that nanostructured surfaces, when combined with the use of liquid crystals, may provide the basis of label-free methods that permit the imaging of proteins bound to receptors patterned on surfaces.3,5 In this paper, we report an investigation that builds on these past studies by establishing experimental procedures for the preparation and characterization of surfaces that possess systematic differences in nanometerscale topography. We investigate the influence of the * Author to whom correspondence should be addressed. E-mail: [email protected]. Fax: 608-262-5434. (1) Cognard, J. Mol. Cryst. Liq. Cryst. Suppl. 1982, 78, 1. (2) Jerome, B. Rep. Prog. Phys. 1991, 54, 391. (3) Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B.; Abbott, N. L. Science 1998, 279, 2077. (4) Skaife, J. J.; Abbott, N. L. Langmuir 2000, 16, 3529. (5) Kim, S.-R.; Shah, R. R.; Abbott, N. L. Anal. Chem. 2000, 72, 4646.

nanometer-scale topography on the response of liquid crystal to proteins bound to these surfaces. This study is driven by the proposition that the sensitivity of liquid crystals to surface-bound proteins can be manipulated by appropriate design of the nanometer-scale topography of a surface. It has been known for some time that the topography of surfaces can influence the orientations of liquid crystals.1,2,6-15 In some situations (see below), this effect can be captured in theoretical descriptions of the anchoring of liquid crystals that are based on continuum descriptions of the elastic energy that is stored in a liquid crystal distorted by topography of a surface.8-10 Past studies have demonstrated this elastic mechanism of alignment of liquid crystals to depend on the wavelength (Λ) as well as the amplitude (A) of the topographical features of the surface, among other factors.8-10 Only when Λ2/A > R, where R is the coherence length of the liquid crystal (typically ∼15 nm), is it possible to describe the influence of topography on the orientation of liquid crystal using a continuum elastic model based on bulk elastic constants.9,15 In the case of a surface characterized by short-wavelength, (6) Janning, J. L. Appl. Phys. Lett. 1972, 21, 172. (7) Dixon, G. D.; Brody, T. P.; Hester, W. A. Appl. Phys. Lett. 1974, 24, 47. (8) Wolff, U. J.; Greubel, W.; Kru¨ger, H. Mol. Cryst. Liq. Cryst. 1973, 23, 187. (9) Berreman, D. W. Phys. Rev. Lett. 1972, 28, 1683. (10) Skaife, J. J.; Abbott, N. L. Chem. Mater. 1999, 11, 612. (11) Berreman, D. W. Mol. Cryst. Liq. Cryst. 1973, 23, 215. (12) Barberi, R.; Giocondo, M.; Sayko, G. V.; Zvezdin, A. K. J. Phys.: Condens. Matter 1994, 6, A275. (13) Blinov, L. M.; Durand, G.; Yablonsky, S. V. J. Phys. II France 1992, 2, 1287. (14) Bodammer, G.; Gourlay, J.; Vass, D. G.; Hossack, W. J. Proc. SPIE 1995, 2731, 95. (15) De Gennes, P. G.; Prost, J. The Physics of Liquid Crystals; Oxford University Press: Oxford, 1993.

10.1021/la0017678 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/11/2001

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high-amplitude roughness, Λ2/A < R, the orientational order within the liquid crystal will decrease near the surface and thus cause changes to the local elastic constants of the liquid crystal.15 Past studies have used combinations of advanced lithography and chemical etching to introduce anisotropic topography into surfaces.16-18 Patterned grooves with characteristic dimensions of ∼100 nm or greater have been demonstrated to cause liquid crystals to align near surfaces in orientations that minimize the elastic energy stored in the liquid crystals. In the case of a nematic phase of p-methoxybenzylidene-p-n-butylaniline (MBBA) exposed to a SiO2 substrate having patterned grooves spaced by 320 nm and with depths of 25 nm (Λ2/A > R), the MBBA was aligned with its bulk director parallel to the grooves.8 A second method that has been used to introduce nanometer-scale topography into surfaces is based on the oblique deposition of inorganic materials.17-23 The anisotropic topography is introduced through a “self-shadowing” mechanism whereby localized regions of the surface are blocked to further adatom adsorption by previously deposited material.24 Although the method of oblique deposition creates an anisotropic surface, the alignments of liquid crystals on these surfaces cannot always be predicted by continuum elastic theory. For example, films of evaporated SiOx are typically characterized by shortwavelength (Λ ∼ 50 nm), high-amplitude (A ∼ 20 nm) roughness due to the fact that the mobility of SiOx is low.25 The alignment of liquid crystal on such surfaces, where Λ2/A < R, cannot be predicted using a continuum description of alignment of the liquid crystals.25 Recently, we reported a series of studies on the orientational behavior of liquid crystals supported on films of gold deposited at an angle of incidence of 50° (measured from normal) that were chemically derivatized with organothiol compounds.3-5,10,26-30 We used atomic force microscopy (AFM) to determine the topography of such gold surfaces on spatial scales that would orient liquid crystals via elastic distortions of the liquid crystal.10 We determined the amplitude of the topography to be ∼1-5 nm and the wavelength to be 5-50 nm, the latter being comparable to the grain size of the gold film.10 For a given wavelength, the amplitude of the topography was measured to be greatest in the azimuthal direction that was parallel to the plane of incidence of the gold during its deposition. This type of topography does satisfy the criterion Λ2/A > R, for which the alignment of liquid crystal can be described by a continuum elastic theory. The topography measured by using AFM was used to calculate a topography-induced anchoring energy of ∼0.015 mJ/ m2, with the preferred orientation of the liquid crystal (16) Barberi, R.; Giocondo, M.; Sayko, G. V.; Zvezdin, A. K. Phys. Lett. A 1996, 213, 293. Flanders, D. C.; Shaver, D. C.; Smith, H. I. Appl. Phys. Lett. 1978, 32, 597. (17) van Kranenburg; Lodder, C. Mater. Sci. Eng. 1994, R11, 295. (18) Smith, D. O.; Cohen, M. S.; Weiss, G. P. J. Appl. Phys. 1960, 31, 10. (19) Kakati, K. K. Indian J. Pure Appl. Phys. 1977, 15, 530. (20) Takeda, S. Thin Solid Films 1996, 281, 539. (21) Chopra, K. L.; Randlett, M. R. J. Appl. Phys. 1968, 39, 1874. (22) Kawabata, S.; Ichiji, K. Surf. Sci. 1976, 56, 316. (23) Kakati, K. K.; Wilman, H. J. Phys. D: Appl. Phys. 1980, 13, 1477. (24) Smith, D. L. Thin-Film Deposition: Principles & Practice; McGraw-Hill: New York, 1995. (25) Galatola, P.; Oldano, C.; Rajteria, M.; Barbero, G. Phys. Lett. A 1996, 210, 101. (26) Gupta, V. K.; Abbott, N. L. Langmuir 1996, 12, 2587. (27) Gupta, V. K.; Abbott, N. L. Science 1997, 276, 1533. (28) Gupta, V. K.; Abbott, N. L. Phys. Rev. E 1996, 54, R4540. (29) Gupta, V. K.; Miller, W. J.; Pike, C. L.; Abbott, N. L. Chem. Mater. 1996, 8, 1366. (30) Shah, R.; Abbott, N. L. J. Phys. Chem. B 2001, 105, 4936.

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corresponding to an azimuthal direction that is perpendicular to the plane of the incidence of the gold.10 This orientation is consistent with experimental observations of the orientations of liquid crystals on the obliquely deposited gold films (not supporting self-assembled monolayers (SAMs)) as well as gold films supporting certain types of alkanethiols.10 These results and others3 suggest that the nanometer-scale topography of these surfaces is important in determining the orientations of liquid crystals supported by these surfaces. We also note that the molecular-level structure of the SAM can also influence the orientations of liquid crystals on these surfaces and in some cases cause the liquid crystal to orient in an azimuthal direction that is parallel to the plane of incidence of the gold.10 These latter orientations cannot be understood solely on the basis of continuum elastic theory. By using obliquely deposited films of gold that present receptors for specific proteins, we have determined that proteins bound to these receptors can erase the uniform alignment of liquid crystals observed on these surfaces in the absence of bound protein.3 Because proteins are similar in size (5-20 nm) to the anisotropic topography induced by oblique deposition, their presence on the surface appears to mask or erase the topography responsible for uniform alignment of the liquid crystal in the absence of bound protein. A particular focus of our past work was directed to SAMs formed from two components, CH3(CH2)7SH (C8SH) and biotin-(CH2)2[(CH2)2O]2NHCO(CH2)11SH (BiSH). These SAMs were formed on ∼20 nm thick films of gold that were deposited at an angle of incidence of 50°.3-5 We demonstrated that binding of avidin and antibiotin IgG to the biotinylated SAMs led to an observable change in the alignment of liquid crystal.3 Additionally, we demonstrated the ability to pattern a ∼10 µm region of the surface with receptors and to image (using liquid crystal) protein bound to the region.3,5 The broad goal of the work reported in this paper is to advance our understanding of the role of the nanometerscale topography of gold films in determining the optical response of liquid crystals to specifically bound proteins. We sought to change the nanometer-scale topography of gold films by altering the angle of incidence of gold (measured from normal) during oblique deposition of gold films. The results of a number of past studies using a variety of deposited materials suggest that it should be possible to control the nanometer-scale topography through manipulation of the angle of deposition of the gold films.31-35 By using scanning electron microscopy, Robbie and Brett observed changes in the surface structure of a variety of films (SiO, Mn, Cu, Al, Cr, CaF2) with the angle of deposition.31 They observed columnar structures to form during their glancing angle deposition procedure. These structures were inclined toward the source and became more pronounced as the angle of deposition increased. Several other authors have also measured variations in properties (optical and magnetic) of thin films that are induced by manipulation of the angle of oblique deposition.31-33 These results, when combined, indicate that the level of anisotropic structure within these surfaces increases with the deposition angle. These studies led us to believe that it should be possible to tailor the nanometer(31) Robbie, K.; Brett, M. J. J. Vac. Sci. Technol. 1997, A15, 1460. (32) Ko¨nig, H.; Helwig, G. Optik 1950, 6, 111. (33) Machaggah, S. M.; Kivaisi, R. T.; Lushiku, E. M. Sol. Energy Mater. 1989, 19, 315. (34) Hsieh, Y.-C.; Gadetsky, S.; Suzuki, T.; Mansuripur, M. J. Appl. Phys. 1997, 81, 3555. (35) Lee, Y. E.; Kim, S. G.; Kim, Y. J.; Kim, H. J. J. Vac. Sci. Technol. 1997, A15, 1194.

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scale topography of gold films supporting SAMs by manipulating the angle of deposition of the gold films. We hypothesized that these changes in nanometer-scale topography would change the orientational response of liquid crystal to the presence of protein specifically bound to surface-immobilized receptors. The specific goals of the work reported in this paper were threefold. First, we aimed to establish procedures that would lead to the preparation of gold films with systematic and reproducible differences in their nanometer-scale topography. Second, we aimed to characterize the nanometer-scale topography of these surfaces by using AFM and to use knowledge of this nanometer-scale topography to predict the response of liquid crystals to proteins bound to these surfaces. Third, we aimed to test these predictions by measurement of the optical response of liquid crystal to anti-Bi IgG bound to biotin immobilized on these surfaces. Materials and Methods Materials. Octanethiol (C8SH) and hexadecanethiol (C16SH) were purchased from Aldrich (Milwaukee, WI). The biotinylated thiol, biotin-(CH2)2[(CH2)2O]2NHCO(CH2)11SH (BiSH), was synthesized using procedures reported in the literature.36 The liquid crystal, 4-cyano-4′-pentylbiphenyl (5CB) was obtained from EM Sciences (New York, NY). All aqueous solutions were prepared with high-purity deionized water (18 MΩ cm) using a Milli-Q water filtration system (Millipore, Bedford, MA). Affinity-isolated goat anti-biotin immunoglobulin G (IgG), rabbit anti-FITC immunoglobulin G (anti-FITC IgG), and rabbit anti-goat immunoglobulin G (anti-goat IgG) were obtained from Sigma BioScience (St. Louis, MO). Solutions of 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). Preparation of Films of Gold. Films of titanium (nominal thickness of ∼10 ( 1 nm) and gold (nominal thickness of ∼20 ( 1 nm) were evaporated onto clean glass substrates with an electron beam evaporator (Tekvak, Brentwood, NY). A detailed description of the procedures used to clean the glass microscope slides can be found in a recent publication.10 The thickness of each metal layer deposited in the evaporator was measured by using a quartz crystal microbalance (QCM) oriented normal to the incident vapor flux. The true thicknesses of the gold films deposited on the glass microscope slides, d, are dependent on the angle of deposition, θ, as described by

d ) dQCM cos(θ)

(1)

where dQCM is the thickness reported by the QCM. The nominal thicknesses given above for the titanium and gold layers are the thicknesses that are measured by the QCM (dQCM). Thus, the films used in our experiments that were deposited onto the glass microscope slides at different angles of incidence possess different thicknesses. The consequence of the variation in thickness is addressed below (see Results and Discussion). The rate of deposition of both metals (Au and Ti) was 0.02 ( 0.005 nm/s, and the metals were deposited at angles of incidence of 15°, 30°, 45°, and 60° ( 5° (measured from the surface normal; see Figure 1A). Following the deposition of each batch of gold films, we examined the films by using AFM. Any batch of gold films that supported debris or metal aggregates (>100 nm in size) within randomly chosen 5 µm × 5 µm areas on the surface was discarded. In our past work,10 we have correlated the presence of these aggregates on the surface of the gold films to contaminants in the gold source (probably causing “spitting” of the gold source). Films of gold that have surfaces supporting aggregates have been found to not provide reproducible anchoring of liquid crystal. We found aggregates to be present ∼5-10% of the time. Formation of SAMs. Self-assembled monolayers were formed on the surfaces of the gold films by immersion of the films into ethanolic solutions containing 22 µM C8SH and 44 µM BiSH. (36) Sprinke, J., et al. J. Chem. Phys. 1993, 99, 7012.

Figure 1. Experimental procedure: (A) Evaporation of ultrathin films of gold onto glass microscope slides at angles of incidence of 15°, 30°, 45°, and 60° (angle measured from normal). (B) Formation of mixed SAMs by immersion of obliquely deposited gold films in ethanolic solutions of BiSH and C8SH. (C) Binding of anti-Bi IgG to mixed SAM by immersion of mixed SAM into PBS solutions of anti-biotin-IgG (40-180 nM) containing 0.01% Triton X-100. (D) Optical inspection of liquid crystal sandwiched between two mixed SAMs supporting bound IgG. After 15 h of immersion at room temperature, the slides were removed, rinsed with ethanol, and then dried under a stream of N2 (Figure 1B). By measuring the ellipsometric thickness of the SAMs (see below for procedure), we estimate SAMs (C8SH/BiSH) formed by this procedure to contain ∼30 ( 2% BiSH.37 Binding of Proteins. Aqueous solutions of IgG were prepared by pipetting a volume of reconstituted stock solution of IgG (0.1 mg/mL) into ∼3 mL of PBS buffer (25 °C) containing 0.01 wt % Triton X-100 held in a small polypropylene vial. The final concentration of IgG in solution was varied between 0 and 100 nM. We used the IgG solutions immediately following their dilution. The binding of IgG was performed by placing the mixed SAM formed from C8SH/BiSH into the plastic vial filled with the aqueous solution of IgG for times of either 30, 45, or 60 min (without stirring) at 25 °C (Figure 1C). Immediately following removal of the SAM from the solution, the samples were rinsed with water for ∼10 s. Excess water was displaced from these surfaces by using a stream of N2. By using ellipsometry, we found that rinsing of SAMs for 5-45 s did not change the amount of bound IgG. Control experiments using a nonspecific IgG (antiFITC IgG, 500 nM) demonstrated that rinsing of the mixed SAM 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 liquid crystal cells (Figure (37) The amount of BiSH present in the SAM was estimated by linear interpolation of the ellipsometric thickness of the pure component SAMs (∼4 and ∼1 nm for pure BiSH and C8SH, respectively).

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1D) in order to observe the optical appearance of liquid crystal in contact with the SAMs. The liquid crystal cells were fabricated by spacing two SAMs (facing each other) ∼10 µm apart using thin strips of Saranwrap. The cell was placed on a warm surface (at 40 °C) and gently heated to 40 °C with a hot air gun for approximately 10 s. 5CB, heated into its isotropic phase (∼35 °C), was spontaneously drawn into each cell by capillary action (10 s). The cell was allowed to sit on the hot plate for 30 s after injection and was then removed and cooled to room temperature. During cooling, the 5CB changed from its isotropic state to its nematic state. 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 deposition direction 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 on the mixed SAM.38 Image Capture and Analysis. Images of the optical appearance of the liquid crystal were captured with a digital camera (C-2020Z, Olympus, Melville, NY) that was attached to a polarized light microscope (BX60, Olympus). A quantitative comparison of the textures was made 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 and 25% open aperture) and digital camera (11m f-stop 1/20 shutter speed) 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 a full coverage of proteins were bound (Smax). 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

(2)

Ellipsometry. We measured the optical thickness of the mixed SAMs and IgG bound to the mixed SAMs by using ellipsometry. Ellipsometry was performed using thick (thickness of 50 nm) films of gold because thick gold films are optically reflecting. The thick films of gold were deposited on glass microscope slides (the same used for the obliquely deposited gold films) 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 cause interpretation of the ellipsometric measurements to be more complex than when using gold films deposited without a preferred direction. The procedures used to form mixed SAMs on thick gold films and to bind IgG to the mixed SAMs on thick gold films were the same as those used on thin gold films. We have assumed that the amounts of IgG bound to mixed SAMs supported on thick and thin gold films are the same. This assumption will be tested in future experiments. 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 the mixed SAM or mixed SAM supporting bound protein was formed on the surface of the gold film. The slab (SAM and protein) was assumed to have an index of refraction of 1.46. Atomic Force Microscopy. Tapping mode AFM was performed under ambient conditions (Multi-Mode Nanoscope 3A, Digital Instruments, Santa Barbara, CA). Our procedures, (38) A dark optical texture (between crossed polars) is observed when liquid crystal having a preferred azimuthal alignment is parallel to either the fast or slow axis of the polarizer.

including precautions that are required so as to avoid potential artifacts in measurements of the topography of surfaces, have been published previously.10 Our measurements were performed using silicon tips that possess an average tip radius of ∼10 nm (OTESPA, Digital Instruments). The gold films were sequentially aligned with the gold deposition direction along the fast and then slow axis of the AFM scanner by physical rotation of the sample so that any artifacts due to the orientation of the sample on the scanner could be identified. All samples were examined with a minimum of five different tips. The instrument was kept in a suspended vibration isolation apparatus during imaging, and image capture was not initiated until after 1-2 h of scanning to minimize drift. The images used for the analyses were 500 nm × 500 nm scans obtained using the E-head scanner. All images were captured using 512 lines per scan, with sequential “scanup” and “scan-down” capture to determine whether drift was present. By use of these procedures, artifacts due to drift and scanner orientation have been eliminated from our results.

Results and Discussion Characterization of Gold Films by AFM. We first used AFM to characterize the nanometer-scale topography of gold films deposited at angles of incidence (measured from surface normal) of 15°, 30°, 45°, and 60°. We make four observations from the images and cross-sectional profiles of the gold films shown in Figure 2. First, we note that each of the images contains dark areas that have the appearance of “holes” in the gold films. However, the cross-sectional profiles reveal that the holes do not penetrate more than a few nanometers into the gold films. In a past study, we also established (by X-ray photoelectron spectroscopy) that gold films deposited at an angle of incidence of ∼50° do not expose the underlying glass substrate or Ti adhesion layer.10 Second, inspection of Figure 2 reveals that the apparent size of the gold grains within the films of gold varies with the angle of deposition. We have quantified the sizes of the grains in each type of gold film by analyzing at least 10 cross sections per image, each taken at various azimuthal angles and locations on the film. We determined the size of the grains by counting the number of peaks within each cross-sectional profile and by assuming each peak to represent a grain. These measurements were averaged using five different samples for each angle of incidence. Grain sizes estimated by using this procedure are given in Table 1. From these measurements (and by inspection of Figure 2), we conclude that the lateral size of the grains decreases from ∼36 ( 6 to 14 ( 8 nm with increasing angle of deposition of the gold. As mentioned above, the thickness of the gold films depends on the angle of deposition with larger angles of deposition leading to thinner films. It is well-known that the sizes of grains within metallic films increase with the thicknesses of the metallic films.10 Thus, from the results above it is not obvious if the change in size of the gold grains is principally due to the variation in the thickness of the gold films or the angle of deposition. This issue was addressed by depositing a gold film at 45° such that the actual thickness of the film was equal to that of a gold film deposited at 30° (12 nm Ti/24 nm Au as measured by QCM). We found the effects on grain size of variation in thickness to be very small over the range of thicknesses relevant to this study. This result suggests that the variation in grain size seen in Figure 2 is largely due to the change in the angle of deposition of the gold and not due to variation in the thickness of the gold films. The third observation that we extract from Figure 2 is related to the roughness of the gold films. We evaluated the root-mean-square (rms) roughness of the gold films using each of the 500 nm × 500 nm images (Table 1). The gold films deposited at 30° possess the highest rms

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Figure 2. Atomic force micrographs (left column, 500 nm × 500 nm) and cross-sectional profiles (right column) of obliquely deposited films of gold. The angle of incidence of the gold during deposition of the gold film is shown in the top left corner of each micrograph. The arrows indicate the direction of the gold deposition, and the scale bar is 100 nm (all images are presented using the same z-scale). Table 1. Average Grain Size, RMS Roughness, and Calculated Elastic Contribution to Anchoring Energy of a Nematic Liquid Crystal When Using Gold Films Deposited at Angles of Incidence of 15°, 30°, 45°, and 60° (Angle Measured from Normal) angle of deposition of gold film

15°

30°

45°

60°

average grain size [nm] rms roughness [nm] anchoring energy [mJ/m2]

36 ( 6 0.57 ( 0.02 0.004 ( 0.005

32 ( 5 1.55 ( 0.03 0.013 ( 0.010

22 ( 6 1.10 ( 0.04 0.014 ( 0.005

14 ( 8 1.04 ( 0.02 0.021 ( 0.005

roughness (1.55 nm), followed by gold films deposited at 45° (1.10 nm), 60° (1.04 nm), and 15° (0.57 nm). The gold films deposited at 15° have the largest grains but the smallest rms roughness; otherwise, the trend is that surfaces with large grains also have high values of rms roughness. As is discussed below, we find that the values of the rms roughness do not predict the relative anchoring energy of the liquid crystal (see below) because rms roughness does not measure the anisotropy in the topography of the surface. The fourth and final observation that we make regarding Figure 2 is that visual inspection of the AFM images does

not obviously reveal the presence of elongated gold grains (or any other form of anisotropy). As reported previously,10 we are able to identify anisotropy within the obliquely deposited gold films only by performing quantitative analyses of the contour lengths and curvature of the topography as measured by AFM (as discussed in following sections). RMS Contour Lengths of the Surface. Using crosssectional profiles of obliquely deposited gold films obtained by AFM, we first searched for anisotropy within the gold films by evaluating the contour lengths of the crosssectional profiles as a function of the sampling interval

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Figure 4. Anisotropy in contour length (A) and rms curvature (B) of the nanometer-scale topography of obliquely deposited gold films, measured as a function of sampling interval. The angle of incidence of the gold during deposition of the gold film is shown in each plot. Figure 3. Contour lengths calculated from cross-sectional profiles of the nanometer-scale topography of ultrathin gold films deposited by oblique evaporation at angles of incidence of 15° (open triangles), 30° (filled circles), 45° (filled triangles), and 60° (open circles). The contour lengths are shown for sampling intervals of 5, 12, and 20 nm. The azimuthal angle of the cross-sectional profile is defined to be 90° when the crosssectional profile is parallel to the direction of incidence of the gold onto the surface.

and azimuthal direction. The contour length was evaluated as 2

conti,λ )

(zi+λ - zi)2 + λ2 λ2

(3)

where zi is the height of the ith pixel of the surface and λ is the sampling interval. The rms contour length was calculated from the square root of the average of the square of the contour length and is given by

contλ )

x

n

conti,λ2 ∑ i)1 n

(4)

where contλ is the rms contour length estimated using a given value of the sampling interval, λ. A detailed description of this method of analysis has been reported in our past work.10 Plots of the rms contour lengths of the gold films deposited at angles of incidence of 15°, 30°, 45°, and 60° are shown in Figure 3. The contour lengths were measured using sampling intervals of 5, 12, and 20 nm. The results in Figure 3 show that in general, the gold films do possess anisotropic topography when characterized by using contour lengths. The contour length on each type of gold film is measured to be maximal when the contour possesses an azimuthal orientation that is parallel to the direction of deposition of the gold films (azimuthal angle of 90°). This result indicates that the topography of

the gold films (as measured by contour length) is greatest in a direction that is parallel to the direction of deposition of the gold. We make three additional observations regarding Figure 3. First, inspection of Figure 3 reveals that the gold films deposited at an angle of incidence of 30° possess the longest contour lengths. This result is consistent with measurements of the rms roughness reported in Table 1. Second, we note that there is almost no measurable anisotropy in the contour length of the samples deposited at 15°. This observation is consistent with the measured behavior of liquid crystals on these surfaces (see below). Liquid crystals do not uniformly orient on the gold films deposited at 15°. Third, we note that the level of anisotropy in the contour length for a given sample does decrease with increasing sampling interval (Figure 4A). Interestingly, however, the relative ranking of the four types of films changes with the sampling interval. At short wavelengths, 5 nm for instance, the gold films deposited at 60° possess the largest values in contour length. This is due to the fact that the gold films deposited at 60° are composed of grains that are smaller than the grains found in other surfaces (Figure 2). In contrast, the gold films deposited at 30° exhibit the highest magnitudes in contour lengths at larger sampling intervals (>10 nm), presumably due to the large sizes of the grains found in these films. RMS Curvatures of the Topography. Whereas the measurements of contour lengths unambiguously identify the presence of anisotropic, nanometer-scale topography that changes with the angle of deposition of the gold films, it is the anisotropic, topographical curvature of surfaces that enters continuum elastic descriptions of the interactions of liquid crystals with surfaces possessing topography. Therefore, by using AFM images of the type shown in Figure 2, we have calculated the average curvature of each type of surface as a function of sampling interval and azimuthal direction. A detailed description of this method has also been reported in our earlier work.10 The equation used to calculate the curvature of the surface corresponding to a specific value of the sampling interval,

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surfaces cannot be described on the basis of continuum elastic theory. The topography of the surface and the molecular structure of the SAM are found to influence the liquid crystal through mechanisms that are not captured by the continuum elastic theory. However, we find that the anchoring energy calculated on the basis of the nanometer-scale topography does form a useful index that predicts (for the results reported here, at least) the sensitivity of liquid crystals to protein bound to the surfaces with nanometer-scale topography. Anchoring Energy Based on Continuum Elastic Theory. We used the AFM measurements described above to calculate an anchoring energy induced by the elastic distortion of the liquid crystal over the nanometer-scale topography present on each type of surface. The anchoring energy was calculated from the curvature measurements for each type of surface, as described above. The method used to calculate the anchoring energy, g, has been described in our earlier work10 and is given by

g)

K3 2

∑j ∑k

[ ][ ] { [ ]} { [ ]} Cjλj2 Ckλk2

(2π)2 (2π)2 λj2λk2 cos x

Figure 5. Root-mean-square curvature of cross-sectional profiles of the nanometer-scale topography of ultrathin gold films measured as a function of the azimuthal orientation of the cross-sectional profile (see caption of Figure 3). The gold films were deposited at angles of incidence of 15° (open triangles), 30° (filled circles), 45° (filled triangles), and 60° (open circles).

λ, is given by9-11 2

curvi,λ )

(zi+λ - 2zi + zi-λ)2 λ2

(5)

The rms curvature, curvλ, is calculated from

curvλ )

x

n

curvi,λ2 ∑ i)1 n

(6)

Inspection of Figure 5 reveals that all of the obliquely deposited films of gold possess values of rms curvature that vary with the azimuthal sampling angle. The measured curvature of the surface also decreases rapidly with the sampling interval used to characterize the topography of the surface. At sampling intervals that are less than 20 nm, the gold films deposited at 60° exhibit the largest curvature, consistent with the fact that the gold films deposited at 60° possess the smallest grains Figure 4B. The 30° and 45° surfaces exhibit the second largest values in curvature anisotropy at sampling intervals less than 15 nm. Below, we calculate the contribution of the topography to the anchoring energy of a liquid crystal by using the values of the curvatures described above. We note in advance that all aspects of the orientational behavior of the liquid crystal on these

1

1

λj

-

1

λk

×

exp -z

1

λj

+

1

λk

(7)

where K3 is the bend elastic constant (10-8 mN), Cn is the curvature, λn is the wavelength, x is the location along the surface, and z is the height from the surface. Integration of eq 7 over z and x yields the calculated values of the anchoring energies that are given in Table 1. Inspection of Table 1 reveals that the calculated values of the anchoring energy increase with the angle of deposition from 0.004 ( 0.005 mJ/m2 at 15° up to 0.021 ( 0.005 mJ/m2 at 60°. Alignment of Liquid Crystal on SAMs Formed From C8SH and C16SH. We first determined whether liquid crystals were uniformly oriented by the gold films described above. These experiments were performed by forming SAMs from either C16SH or C8SH for 2 h on the surface of the gold films. These surfaces were then assembled into liquid crystal cells. Figure 6 shows the optical appearance of liquid crystal supported on the gold films deposited at 15°, 30°, 45°, and 60°. Inspection of Figure 6 reveals that the nanometer-scale topography induced by deposition of the gold film at an angle of 15° from the normal is not sufficient to uniformly orient the liquid crystal. The appearance of the liquid crystal is similar to that observed on films of gold deposited without a preferred direction of incidence of the gold (not obliquely evaporated). As discussed above, the observation of nonuniform anchoring of the liquid crystal is not surprising in view of the low values of the azimuthal anchoring energies calculated from the AFM images (0.004 ( 0.005 mJ/m2). Inspection of Figure 6 also reveals that the gold films deposited at angles of incidence of 30°, 45°, and 60° cause uniform alignment of the liquid crystal. Although the alignment of the liquid crystal is azimuthally uniform on each of these gold films, we note that the azimuthal orientation assumed by the liquid crystal depends on the structure of the SAM and the structure of the gold film (as controlled by oblique deposition). For example, on the films of gold deposited at 30° and 45°, the azimuthal orientation of the liquid crystal is parallel to the direction of the gold deposition when the SAMs are formed from C16SH and C8SH. In contrast, SAMs formed from C15SH cause the alignment of the liquid crystals to be in an azimuthal direction that is perpendicular to the direction

Orientational Response of Liquid Crystals

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Figure 6. The optical textures (cross-polarizers, ∼1 mm × 1 mm) of nematic phases of 5CB supported on SAMs formed from either C16SH (A, C, E, and G) or C8SH (B, D, F, and H) on gold films deposited at angles of incidence of 15° (A and B), 30° (C and D), 45° (E and F), and 60° (G and H). The direction of deposition of the gold film was aligned parallel to one of the polarizers.

of incidence of the gold. On gold films deposited at 60°, the orientation of the liquid crystal is perpendicular to the direction of the gold deposition when the gold films support SAMs formed from C16SH and C8SH. While the azimuthal orientation of the liquid crystal on the gold films deposited at 15° and 60° can be reconciled on the basis of the anchoring energies calculated in the previous section from continuum elastic theory, it is not possible to account for the orientation of the liquid crystals on the gold films deposited at 30° and 45° on the basis of this type of theory. Our past studies have demonstrated that subtle changes in the structures of molecules within SAMs, when supported on obliquely deposited films of gold, can have a substantial influence on the orientational behavior of liquid crystals observed on these surfaces.26-29 For example, alkanethiols formed from odd and even numbers of carbons can give rise to orthogonal orientations of liquid crystals.26-29 While the effect of the molecular structure of the SAMs (as described above) on the orientation of the liquid crystal is interesting, in a separate study39 we have shown that the effect of the molecular structure of the SAM on the response of liquid crystal to the bound protein is small. Optical Textures of Liquid Crystal on Surfaces Supporting Bound Protein. We next measured the response of liquid crystal to anti-Bi IgG specifically bound to mixed SAMs formed from BiSH/C8SH. The SAMs were prepared on each type of obliquely deposited film of gold. The optical appearances of nematic phases of 5CB as a function of increasing amount of bound IgG are shown in Figure 7. These optical textures were obtained by illumination with polarized white light, and the colors result from the interference of different wavelengths of the light passing through the liquid crystal. The inset in each image indicates the ellipsometric thickness of IgG bound to each SAM prior to contact of the liquid crystal with the SAM. We make three observations from Figure 7. First, we note that the liquid crystal supported on the gold films deposited at 15° is nonuniformly aligned in the absence of bound protein and in the presence of bound IgG. There is little change in the appearance of the liquid crystal as a function of increasing amounts of IgG bound to these surfaces. Second, we note that the appearance of the liquid crystal supported on the gold films deposited at angles of 30° and (39) Skaife, J. J.; Abbott, N. L. Langmuir, in press.

45° changes from uniform to nonuniform as a function of increasing amount of bound IgG. Whereas the appearance of the liquid crystal on the films deposited at 30° is substantially nonuniform when the films support an ellipsometric thickness of ∼0.8 nm of IgG, the appearance of liquid crystal on the gold films deposited at 45° is largely uniform until ∼2.8 nm of IgG is bound to the surface. Below, we quantify these different responses of the liquid crystal to bound IgG. Third, we observe that the appearance of the liquid crystal on the gold films deposited at 60° is largely uniform even when the surfaces support as much as ∼6.5 nm of bound IgG. In an effort to achieve a nonuniform alignment of the liquid crystal on the gold films deposited at 60°, we used a secondary IgG (anti-goat IgG) that binds to the anti-biotin IgG. Although the secondary binding step does appear to increase the density of disclination lines within the liquid crystal, the orientation of the liquid crystal is largely uniform even when the surface supports as much as ∼12 nm of bound IgG. Finally, we performed a series of control experiments to determine the role of nonspecific binding of IgG in the results shown in Figure 7. These experiments were performed by incubation of each SAM in 500 nM antiFITC IgG for 30 min. The textures are shown in Figure 8. The results in Figure 7 reveal the alignment of liquid crystal on these surfaces to be uniform. These results led us to conclude that the change in orientation of the liquid crystal observed in Figure 8 is caused by specific binding of anti-Bi IgG to the mixed SAM (and not nonspecific adsorption). Optical Response of Liquid Crystal to Bound Protein. We have quantified the optical appearance of the liquid crystals shown in Figure 7 so as to permit a comparison of the influence of topography on the response of liquid crystal to bound IgG. The method (see Materials and Methods) that we use here to quantify the images is the average brightness of the image. We chose this index because it is straightforward to calculate. We point out, however, that it does not necessarily represent the optimal measure by which to index the response of the liquid crystal to bound IgG. It is possible to propose various measures of the optical appearance. We do not yet know which methods of quantification represent optimal ones for interpretation of the amount of bound IgG. Figure 9 shows the average brightness of the optical appearance of the

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Figure 7. Influence of the amount of bound IgG on the optical texture (crossed polarizers, ∼1 mm × 1 mm) of nematic phases of 5CB supported on gold films that possess different nanometer-scale topographies. The gold films were deposited at 15°, 30°, 45°, or 60° (indicated at top of each column of images) and support mixed SAMs formed from BiSH and C8SH to which various amounts of IgG are bound. The amount of bound IgG is characterized by an ellipsometric thickness that is indicated in the bottom right corner of each image. Note that the optical textures in each row of images do not correspond to the same amount of bound IgG.

liquid crystal plotted as a function of the ellipsometric thickness of IgG bound to the SAMs. The results in Figure 9 reveal that the response of the liquid crystal to bound

IgG depends strongly on the nanometer-scale topography of the surface. As can be seen from inspection of Figure 7, the optical appearance of the liquid crystal supported

Orientational Response of Liquid Crystals

Figure 8. Control experiment: optical textures (crossed polarizers, 1 mm × 1 mm) of liquid crystal supported on mixed SAMs formed from BiSH and C8SH that were immersed into aqueous solutions (PBS) containing 500 nM of anti-FITC IgG. (A) Gold film deposited at an angle of incidence of 30°. (B) Gold film deposited at an angle of incidence of 45°. (C) Gold film deposited at an angle of incidence of 60°.

Figure 9. The normalized optical response (see text for details) of nematic phases of 5CB to anti-Bi IgG specifically bound to mixed SAMs formed from BiSH and C8SH on films of gold obliquely deposited at angles of 15° (open triangles), 30° (closed circles), 45° (closed triangles), and 60° (open circles).

on the gold films deposited at 15° is nonuniform in the absence of bound protein and thus the optical response (normalized optical output) is saturated before any protein is bound at the surface. In contrast, the liquid crystal supported on the gold films deposited at 30° and 45° shows a sigmoidal optical response to bound IgG. For the films deposited at 30°, a response that is 50% of the maximum response is measured when ∼0.8 nm of IgG is bound to the SAM, whereas films deposited at 45° show a 50%

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response when ∼3.0 nm of IgG is bound to the surface. Finally, we note that the liquid crystal supported on the film deposited at 60° does not exhibit a significant increase in normalized optical output as the amount of IgG bound to the SAM increases to as much as ∼12 nm. Although a few disclination lines can be seen in Figure 7 when the films deposited at 60° support high loadings of IgGs, the contribution of these disclinations to the calculated average brightness of the image is small. The results above, when combined with our measurements of the change in nanometer-scale topography of the gold films with angle of deposition, lead us to two main conclusions. First, our results demonstrate that structural changes to the gold films that are induced by manipulation of the angle of deposition do cause substantial (and likely useful) variations in the response of the liquid crystal to bound IgG. That is, it is possible to tune the response of liquid crystal to the amount of bound IgG by varying the angle of deposition of the gold films. Second, although the initial orientation of the liquid crystal on the mixed SAM (without bound protein) cannot be reconciled solely on the basis of continuum elastic theory, there is a close correspondence between the variation in the azimuthal anchoring energy of the liquid crystal (as calculated from the nanometer-scale topography of the gold films using continuum elastic theory) and the response of the liquid crystal to bound IgG. Thus, we conclude (I) that the elastic distortion of the liquid crystal over the nanometer-scale topography of these gold films is likely important in determining the response of the liquid crystal to bound protein and (II) that the calculated anchoring energy forms the basis of a useful index with which to predict the response of the liquid crystal to bound protein. Conclusions We conclude that it is possible to prepare ultrathin gold films that possess systematic variations in their nanometer-scale topography by oblique deposition of gold in an electron beam evaporator. By using AFM, we have characterized the nanometer-scale topography of the gold films in terms of surface contour lengths and local, anisotropic curvature of the surface topography. We have also measured the influence of the nanometer-scale topography of the gold films on the response of supported liquid crystals to the presence of proteins specifically bound to receptors hosted on these surfaces. We have demonstrated that it is possible to tune the response of the liquid crystal over a wide range by manipulation of the nanometer-scale topography. The azimuthal anchoring energy calculated from the nanometer-scale topography is a found to be good index with which to predict the response of the liquid crystal to the presence of bound IgG, although it is not possible to account for the orientations of liquid crystals on the gold films in the absence of bound protein on the basis of the azimuthal anchoring energies calculated from continuum elastic theory. These gold films are straightforward to prepare in large quantities and, when combined with use of liquid crystals, may provide the basis of labelfree methods for the imaging of biomolecular interactions on surfaces. 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.), the Center for Nanostructured Interfaces (NSFDMR 9632527) at the University of Wisconsin, and the Biophotonics Partnership Initiative of NSF (ECS-0086902). LA0017678