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Imaging of Affinity Microcontact Printed Proteins by Using Liquid Crystals Matthew L. Tingey, Sean Wilyana, Edward J. Snodgrass, and Nicholas L. Abbott* Department of Chemical and Biological Engineering, University of Wisconsin, Madison, Wisconsin 53706 Received January 31, 2004. In Final Form: April 19, 2004 This paper reports the design of surfaces on which thermotropic liquid crystals can be used to image affinity microcontact printed proteins. The surfaces comprise gold films deposited onto silica substrates at an oblique angle of incidence and then functionalized with a monolayer formed from 2-mercaptoethylamine. Ellipsometric measurements confirm the transfer of anti-biotin IgG to these surfaces from affinity stamps functionalized with biotinylated bovine serum albumin (BSA), while control experiments performed using anti-goat IgG confirmed the specificity of the IgG capture on the stamp. On these surfaces, anti-biotin IgG caused nematic phases of 4-cyano-4′-pentylbiphenyl (5CB, ∆ ) | - ⊥ > 0) to assume orientations that were parallel to the surfaces (planar anchoring) but with azimuthal orientations that were distinct from those assumed by the liquid crystals on the amine-terminated surfaces not supporting IgGs. Following incubation of these samples for >8 h at 36 °C, we observed that the amine-terminated regions of the surface not supporting IgG cause 5CB to undergo a transition from planar to perpendicular (homeotropic). Because N-(4-methoxybenzylidene)-4-butylaniline (MBBA) (∆ < 0) does not undergo a similar transition in orientation, this transition is consistent with the effects of an electrical double layer formed at the amineterminated surface on the liquid crystal. Following the transition to homeotropic anchoring, the liquid crystals provide high optical contrast between regions of the surface supporting and not supporting IgG. We conclude that amine-terminated surfaces (I) uniformly align liquid crystals when not supporting proteins and (II) have sufficiently high surface free energy to capture proteins delivered to the surface from an affinity stamp, and thus they form the basis of a useful class of surfaces on which affinity microcontact printed proteins can be imaged using liquid crystals.

Introduction Affinity microcontact printing of proteins exploits functionalized elastomeric “stamps” to capture a targeted analyte from solution and then transfer the analyte onto the surface of a solid.1,2 The approach relies on the covalent immobilization of a receptor on the surface of the elastomeric stamp (e.g., formed from poly(dimethylsiloxane) (PDMS)). Following the capture of the analyte by the receptor on the stamp, the stamp is brought into contact with the surface of a solid. Because nonspecific interactions between the analyte on the stamp and the contacting surface can be designed to be stronger than the specific interactions that bind the analyte to the stamp, the analyte can be transferred onto the surface of the solid by contact of the stamp with the solid. By fabricating elastomeric stamps with surfaces that were patterned with a range of receptors, affinity microcontact printing has been demonstrated to be useful for the simultaneous capture and arraying of more than one protein analyte.1,2 Analytical tools that provide rapid and sensitive methods capable of reporting specific protein-protein interactions have the potential to be broadly useful. For example, such tools may enable a better understanding of cellular processes that are regulated by complex pathways of signaling proteins.3,4 * To whom correspondence should be addressed. Phone: (608) 265-5278. Fax: (608) 262-5434. E-mail: [email protected]. (1) Renault, J. P.; Bernard, A.; Juncker, D.; Michel, B.; Bosshard, H. R.; Delamarche, E. Angew. Chem., Int. Ed. 2002, 41, 2320-2323. (2) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Nat. Biotechnol. 2001, 19, 866-869.

In this paper, we report that affinity microcontact printing can be combined with the use of liquid crystals to permit label-free detection of protein analytes in a sample. This advance revolves around the design of surfaces that both orient liquid crystals and permit the capture of proteins from an affinity stamp. Our approach builds from the results of past studies that demonstrated uniform alignment of thermotropic and lyotropic liquid crystals on self-assembled monolayers (SAMs) of organosulfur compounds that were formed on gold films deposited from a vapor at an oblique angle of incidence onto a glass substrate.5-17 In this approach, the long-range (∼1-100 µm) communication between molecules within liquid crystals (mesogens) permits changes in the orientations (3) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 21012105. (4) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (5) Tercero Espinoza, L. A.; Luk, Y. Y.; Schumann, K.; Israel, B. A.; Abbott, N. L. Langmuir 2004, 20, 2375-2385. (6) Luk, Y. Y.; Tingey, M. L.; Hall, D. J.; Israel, B. A.; Murphy, C. J.; Bertics, P. J.; Abbott, N. L. Langmuir 2003, 19, 1671-1680. (7) Tingey, M. L.; Luk, Y. Y.; Abbott, N. L. Adv. Mater. 2002, 14, 1224-1227. (8) Skaife, J. J.; Abbott, N. L. Langmuir 2001, 17, 5595-5604. (9) Skaife, J. J.; Brake, J. M.; Abbott, N. L. Langmuir 2001, 17, 54485457. (10) Skaife, J. J.; Abbott, N. L. Langmuir 2000, 16, 3529-3536. (11) Skaife, J. J.; Abbott, N. L. Chem. Mater. 1999, 11, 612-623. (12) Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B.; Abbott, N. L. Science 1998, 279, 2077-2080. (13) Gupta, V. K.; Abbott, N. L. Science 1997, 276, 1533-1536. (14) Gupta, V. K.; Abbott, N. L. Phys. Rev. E 1996, 54, R4540-R4543. (15) Gupta, V. K.; Abbott, N. L. Langmuir 1996, 12, 2587-2593. (16) Luk, Y. Y.; Abbott, N. L. Science 2003, 301, 623-626. (17) Van Nelson, J. A.; Kim, S. R.; Abbott, N. L. Langmuir 2002, 18, 5031-5035.

10.1021/la049728+ CCC: $27.50 © 2004 American Chemical Society Published on Web 07/01/2004

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of mesogens near a protein-decorated surface to be amplified into changes in orientations of micrometer-thick films of liquid crystal supported at the surface. Because liquid crystals are optically anisotropic phases, change in orientation of the micrometer-thick films of liquid crystal is easily transduced by using polarized light microscopy. This approach also permits proteins patterned on surfaces on the micrometer-scale to be imaged by using liquid crystals.6,12 To permit the use of liquid crystals for imaging proteins delivered to surfaces by affinity microcontact printing, we sought to design surfaces that possessed three essential properties. First, we sought to design surfaces that were hydrophilic, so as to maximize the transfer of protein from the stamp to the surface.18 The transfer of protein from stamps to hydrophilic surfaces is believed to be driven by the high surface energy of the hydrophilic surfaces (exposed to air) prior to the transfer of the protein to the surface.18 Second, we aimed to design surfaces with a chemical functionality that would not permit desorption of the stamped proteins upon subsequent contact with aqueous buffers or liquid crystal. For example, past studies have demonstrated that ethylene glycol-terminated surfaces are effective at capturing proteins from PDMS stamps.18 However, immersion of these surfaces under aqueous buffer will likely result in the partial or complete desorption of protein into the aqueous solution.19,20 Here, we note that the irreversible capture of the protein by the surface will also permit incubation of the captured protein in a second solution (e.g., to assay for the binding activity of the stamped protein). Third, we sought to design surfaces that would permit liquid crystals to be used to image captured proteins. Our approach exploited the results of our past studies in which we demonstrated that the orientational response of liquid crystals to bound protein can be tuned widely by supporting the proteins on gold films deposited at oblique angles of incidence.7,9 The experiments described in this study revolve around a model system in which elastomeric stamps formed from PDMS were covalently functionalized with biotinylated bovine serum albumin (BSA). The stamp presenting biotinylated BSA was “inked” by incubation in a solution containing anti-biotin IgG (control: anti-goat IgG). The inked stamp was then contacted with a gold substrate that had been decorated with an amine-terminated selfassembled monolayer (SAM). The inked protein was transferred to the gold substrate during stamping. After stamping the protein onto the gold substrate, the immobilized protein was imaged by placement of a micrometer-thick film of liquid crystal on the surface (Figure 1). Materials and Methods Materials. Titanium (99.999%) and gold (99.999%) were obtained from International Advanced Materials (New York, NY). The glass microscope slides were Fisher’s Finest, premium grade slides obtained from Fisher Scientific (Pittsburgh, PA). The nematic liquid crystal 4-cyano-4′-pentylbiphenyl (5CB), manufactured by BDH, was purchased from EM Industries (Hawthorne, NY). The liquid crystal N-(4-methoxybenzylidene)-4butylaniline (MBBA), 3-aminopropyltriethoxysilane (APES), and octyltrichlorosilane (OTS) were purchased from Aldrich (Milwaukee, WI). All aqueous solutions were prepared with highpurity deionized water (18 MΩ cm) using a Milli-Q water purification system (Millipore, Bedford, MA). All protein solutions (18) Tan, J. L.; Tien, J.; Chen, C. S. Langmuir 2002, 18, 519-523. (19) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (20) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167.

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Figure 1. Experimental procedure for imaging of affinity microcontact printed proteins using liquid crystals. were made from phosphate buffered saline (PBS), pH 7.4 (Sigma, St. Louis, MO). The 2-mercaptoethylamine, anti-biotin IgG, and anti-goat IgG were also from Sigma (St. Louis, MO). The silicon wafers were from Silicon Sense (Nashua, NH). The PDMS stamps were prepared from Sylgard 184 (Dow Corning, Midland, MI). The liquid crystal cells were held together by mini binder clips (Acco, Lincolnshire, IL). The BS3 (bis(sulfosuccinimidyl) suberate) and biotinylated BSA were from Pierce (Rockford, IL). Cleaning of Substrates. The microscope slides were cleaned sequentially in piranha (70% H2SO4, 30% H2O2) and alkaline solutions (70% KOH, 30% H2O2) using nitrogen to provide agitation (1 h at ∼80 °C). Warning: Piranha solution should be handled with extreme caution; in some circumstances, most probably when it has been mixed with significant quantities of an oxidizable organic material, it has detonated unexpectedly. The slides were then rinsed thoroughly in deionized water (18.2 MΩ cm), ethanol, and methanol and dried under a stream of gaseous N2. The clean slides were stored in a vacuum oven at 110 °C. Preparation of Octyltrichlorosilane (OTS)-Treated Glass Slides. A solution of 10 mM OTS in n-heptane was passed through a column of aluminum oxide to remove any residual water. The piranha-cleaned glass slides were then immersed into the OTS/ n-heptane solution for 30 min. The slides were rinsed with methylene chloride and dried under a stream of gaseous N2. The OTS slides were tested for homeotropic alignment by observing the orientation of 5CB sandwiched between two OTS slides. Any slide not inducing homeotropic alignment was discarded. Uniform Deposition of Gold Films. For ellipsometry, films of gold with thicknesses of ∼500 Å were deposited onto silicon wafers mounted on rotating planetaries by using an electron beam evaporator (VES-3000-C, manufactured by Tek-Vac Industries, Brentwood, NY). The rotation of the substrates on the planetaries ensured that the gold was deposited without a preferred direction of incidence. A layer of titanium (thickness of ∼100 Å) was used to promote adhesion between the silicon wafer and the film of gold. The rates of deposition of gold and titanium were ∼0.2 Å/s. The pressure in the evaporator was 35 °C) within a glass syringe, was dispensed onto the edge of each cell on the warm plate. The 5CB was drawn into the space between the two surfaces by capillary forces. The thickness of the film of liquid crystal (13 ( 2 µm) was measured according to a previously reported procedure.16 The cell was slowly cooled from 36 to 33 °C over a period of ∼1 h. Upon cooling, the 5CB transitioned from the isotropic to the nematic phase. Image Capture. Images of the liquid crystals were captured using a digital camera (Olympus C-2020 Zoom) mounted onto a polarized light microscope (BX60, Olympus, Tokyo, Japan). Consistent settings of the microscope white light source (50% of maximum intensity, 50% open aperture, 10× magnification) and digital camera (f-stop of 11 and shutter speed of 1/100 s) were used. Homeotropic alignment was suspected of any samples observing no transmission of light over a 360° rotation of the stage. Confirmation of homeotropic alignment was achieved by inserting a condenser below the stage and a Bertrand lens above (21) Kim, S. R.; Shah, R. R.; Abbott, N. L. Anal. Chem. 2000, 72, 4646-4653.

Tingey et al. Table 1 surface (see text for details) amine monolayer on gold film amine monolayer treated with 0.1 N HCl amine monolayer treated with 1 N HCl affinity printed anti-Bi IgG on amine monolayer affinity printed anti-Bi IgG on amine monolayer treated with 0.1 N HCl affinity printed anti-goat IgG on amine monolayer (control) affinity printed anti-goat IgG on amine monolayer treated with 0.1 N HCl (control) affinity printed anti-goat IgG on amine monolayer treated with 0.1 N HCl and rinsed with Triton X-100 (control) affinity stamp incubated in PBS

ellipsometric thickness (nm) 1.3 ( 0.2 1.3 ( 0.2 1.0 ( 0.2 7.4 ( 0.4 10.8 ( 0.4 2.7 ( 0.2 2.3 ( 0.2 0.6 ( 0.1 0.5 ( 0.1

the stage and observing an interference pattern consisting of two crossed isogyres.22 Ellipsometry. Ellipsometric measurements were performed to determine the optical thicknesses of the amine-terminated monolayers and films of proteins. The optical thickness reported is the average of three samples, each substrate measured at three different locations. The measurements were performed using a Rudolph Auto EL ellipsometer (Flanders, NJ) at a wavelength of 6320 Å and an angle of incidence of 70°. The gold substrates used for the ellipsometric measurements were uniformly deposited gold films. The ellipsometric thicknesses of the amine-terminated monolayers and immobilized proteins were estimated by using a three-layer model and by assuming a refractive index of 1.46 for both the monolayer and the protein.

Results and Discussion Orientations of Liquid Crystals on Amine-Terminated Monolayers Formed on Gold Films. As described in the Introduction, the investigation reported in this paper was structured around the proposition that affinity microcontact printed proteins could be imaged with liquid crystals by designing surfaces that possess three essential properties: the surfaces should be hydrophilic, so as to maximize the transfer of protein from the stamp to the surface; the surfaces should possess a chemical functionality that leads to irreversible capture of the proteins (the proteins should not desorb upon subsequent contact with aqueous buffers or liquid crystals); and the surfaces should uniformly orient liquid crystals in the absence of bound protein but not in the presence of captured protein. Because past studies have demonstrated that amine-terminated monolayers are hydrophilic and strongly adsorb biomolecules,23,24 we hypothesized that amine-terminated monolayers supported on obliquely deposited gold films would likely possess these properties. In this section, we report the results of a study that aimed to characterize the orientations of liquid crystals on amine-terminated monolayers formed on obliquely deposited gold films. To determine if amine-terminated monolayers uniformly align the nematic liquid crystal 5CB, we assembled an optical cell using two surfaces separated by a thin film of Mylar. One surface was an amine-terminated monolayer formed from 2-mercaptoethylamine on a film of obliquely deposited gold. The other surface was a glass microscope slide functionalized with OTS. We used the OTS-treated slide for the second surface because OTS is known to cause homeotropic (perpendicular) alignment of 5CB.5 The (22) Brake, J. M.; Abbott, N. L. Langmuir 2002, 18, 6101-6109. (23) Harnett, C. K.; Satyalakshmi, K. M.; Craighead, H. G. Appl. Phys. Lett. 2000, 76, 2466-2468. (24) Dulcey, C. S.; Georger, J. H., Jr.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551-554.

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Figure 2. Optical images (crossed polars) of 5CB sandwiched between an amine-terminated SAM formed on a film of obliquely deposited gold (bottom surface, orange) and an OTS-treated glass slide (top surface, gray). (A) No pretreatment with HCl, annealed for 1 h at 36 °C prior to imaging at room temperature. (B) No pretreatment with HCl, annealed for 18 h at 36 °C prior to imaging at room temperature. (C) Pretreatment with 0.1 N HCl, annealed for 1 h at 36 °C prior to imaging at room temperature. (D) Pretreatment with 0.1 N HCl, annealed for 18 h at 36 °C prior to imaging at room temperature.

thickness of the film of liquid crystal drawn into the cavity between the two surfaces was measured to be 13 ( 2 µm (see the Materials and Methods section). We examined the orientation of the liquid crystal within the optical cell by rotating the optical cell between crossed polars on a microscope stage. We define the orientation of the sample on the microscope stage as the angle between the direction of incidence of gold during oblique deposition of the gold film and the polarizer of the optical microscope. Figure 2A shows an image of the optical cell with an amineterminated SAM on one surface and OTS on the other at room temperature (following incubation for 1 h in an oven held at 36 °C). When the angle between the sample and the polarizer was 0 or 90°, we observed minimal transmission of light through the sample and crossed polarizers. This result indicates that the average azimuthal orientation of the 5CB at the amine-terminated surface was parallel to one of the crossed polars (i.e., parallel or perpendicular to the direction of deposition of the gold). When the angle between the sample and the polarizer was 45°, the birefringence of the liquid crystal rotated the polarization of the incident light, and thus the sample appeared bright when viewed between crossed polars. These observations lead us to conclude that the amineterminated monolayer formed on obliquely deposited gold does lead to a uniform azimuthal orientation of the liquid crystal. The liquid crystal within the optical cell undergoes splay and bend distortions to accommodate the boundary conditions at the two surfaces that define the optical cell (Figure 2A).25 To determine whether 5CB on the amine-terminated monolayer assumed planar or near-planar alignment, a 1 cm × 1 cm square region of an obliquely deposited film of gold was patterned with n-hexadecanethiol by using microcontact printing. The sample was then rinsed with ethanol and dried under a stream of gaseous N2. Self-

assembled monolayers of n-hexadecanethiol formed on the surface of gold films are known to cause planar alignment of 5CB.15,26 The untreated area of the gold film was subsequently covered with an amine-terminated SAM by soaking the surface in a 1 mM solution of 2-mercaptoethylamine for 1 h. The sample was again rinsed with ethanol and dried under a stream of gaseous N2. Visual inspection of the gold surface during rinsing confirmed the regions of the surface treated with n-hexadecanethiol to be hydrophobic. The surrounding regions supporting the amine-terminated SAM were hydrophilic. One hour after forming the optical liquid crystal cell, we observed the interference color to be identical on the regions with n-hexadecanethiol and the amine-terminated SAM (interference colors will be discussed below). Therefore, we conclude that the anchoring of 5CB on the amineterminated surface is identical to the SAM formed from hexadecanethiol. That is, we conclude that the amineterminated SAM causes planar anchoring of 5CB ∼1 h after contact of the liquid crystal with the SAM. Whereas the results above establish the azimuthal orientation of 5CB to be either parallel or perpendicular to the direction of deposition of the gold on the amineterminated monolayer, we determined the absolute orientation of the liquid crystal by making an optical cell in which the thickness of the film of liquid crystal varied across the cell (a “wedge”-shaped cell). Observation of the change in interference colors upon insertion of a quarter wave plate into the optical path permits determination of the azimuthal orientation of a liquid crystal, as described previously.6 Using this method, we determined the azimuthal orientation of the liquid crystal on the amineterminated SAM to be orthogonal to the azimuthal direction of deposition of the gold. Past studies have established that this orientation corresponds to an azi-

(25) Brake, J. M.; Mezera, A. D.; Abbott, N. L. Langmuir 2003, 19, 8629-8637.

(26) Drawhorn, R. A.; Abbott, N. L. J. Phys. Chem. 1995, 99, 1651116515.

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muthal direction of minimum roughness on obliquely deposited gold films.11 Inspection of Figure 2A also reveals the presence of many line defects (disclinations) within the liquid crystal. We attempted to remove these defects by thermal annealing of the sample. However, following the incubation of the samples at 36 °C (the clearing temperature of 5CB is 35 °C) in an oven for 18 h, we observed the roomtemperature alignment of the liquid crystal to be homeotropic on both surfaces (Figure 2B). The homeotropic alignment of our samples was confirmed by conoscopy.22 We note that previous studies have also observed homeotropic alignment of 5CB on amine-terminated surfaces.5,27 Subsequent experiments revealed that samples heated at 36 °C for ∼8 h transitioned from planar anchoring to homeotropic anchoring. In contrast, samples incubated at room temperature transitioned from planar to homeotropic anchoring after ∼6 days. Past studies have shown that electrical double layers formed within liquid crystals following contact of the liquid crystals with a surface presenting a salt can lead to homeotropic alignment. For example, contact of nematic 5CB (| - ⊥ > 0, where | is the dielectric constant parallel to the director of 5CB and ⊥ is the dielectric constant perpendicular to the director of 5CB) with surfaces presenting sodium carboxylate leads to homeotropic alignment of 5CB.28 The electric field within the electrical double layer aligns 5CB in a homeotropic alignment because this orientation maximizes the polarization of the 5CB. This conclusion was supported by the observation that a liquid crystal possessing a negative dielectric anisotropy (MBBA, | - ⊥ < 0) did not assume a homeotropic alignment on the same surfaces.28 To determine if the homeotropic alignment of 5CB on the amineterminated monolayers (as described above) may result from formation of an electrical double layer within the liquid crystal, we examined the orientation of MBBA in optical cells formed from surfaces presenting amineterminated monolayers and OTS. In contrast to 5CB, after 21 days of heating of the samples above the clearing temperature of MBBA (39 °C), the alignment of MBBA upon cooling to room temperature was parallel to the surface (not homeotropic). This result supports our hypothesis that the transition from planar to homeotropic alignment observed with 5CB on amine-terminated monolayers is caused by the formation of an electrical double layer within the liquid crystal. We hypothesized further that the electrical double layer formed with the 5CB was associated with the presence of ammonium species on the surface. We tested the role of ammonium groups by pretreating the surface with hydrochloric acid prior to contact with the liquid crystal. By using ellipsometry, we determined the ellipsometric thickness of the amine-terminated monolayer with no pretreatment, pretreated with 0.1 N HCl, and pretreated with 1 N HCl to be 1.3 ( 0.2, 1.3 ( 0.2, and 1.0 ( 0.2 nm, respectively. These results suggest that the ellipsometric thickness of the amine-terminated monolayer does not change following pretreatment with HCl, presumably because excess HCl is volatile at room temperature. Figure 2C shows an optical micrograph of the 5CB within an optical cell formed using an amine-terminated monolayer that was pretreated with 0.1 N HCl. The image was obtained after the sample was incubated for 1 h at 36 °C and then cooled to room temperature. Comparison of parts

A and C of Figure 2 reveals that pretreatment of the amineterminated monolayer with 0.1 N HCl reduces the number of defects in the liquid crystal. The decrease in the number of defects following pretreatment of the amine-terminated monolayer with 0.1 N HCl is consistent with a reduction in the anchoring energy of the liquid crystal.29 We note here that an electrical double layer will reduce the anchoring energy of the liquid crystal with a positive dielectric anisotropy. We also observed samples pretreated with 0.1 N HCl to cause homeotropic alignment of 5CB upon incubation at elevated temperatures for >8 h (Figure 2D). The results above, when combined, demonstrate that amine-terminated monolayers supported on obliquely deposited gold films do provide a uniform alignment of nematic 5CB. Immediately following contact of the liquid crystal with the surface, the orientation of the 5CB on the amine-terminated monolayer is planar. Following incubation at elevated temperatures for ∼8 h, the orientation transitions to homeotropic alignment. In contrast, we observed a liquid crystal with a negative dielectric anisotropy (MBBA, | - ⊥ < 0) to not undergo a transition. These results suggest the influence of an electrical double layer on the alignment of the liquid crystals. Finally, we observe that pretreatment of the amine-terminated monolayer with 0.1 N HCl lowers the number of defects observed in the initial alignment of the liquid crystal, which is also consistent with the influence of an electrical double layer on the anchoring of the liquid crystal (lower anchoring energy). Capture of Proteins on Amine-Terminated Monolayers When Using Affinity Microcontact Printing. The results above demonstrate that amine-terminated monolayers provide surfaces that uniformly orient liquid crystals. Next, we aimed to determine the extent of transfer of proteins from affinity stamps to surfaces presenting amine-terminated monolayers. We focused on amineterminated monolayers pretreated with 0.1 N HCl because this pretreatment provided fewer defects in the initial alignment of the liquid crystal (see above). We used ellipsometry as an independent measure of the extent of transfer of proteins to the amine-terminated monolayers. First, we prepared affinity stamps using PDMS surfaces that did not possess topography (relief) by covalently attaching biotinylated BSA. The stamps were inked by placing drops of anti-biotin IgG (1 mg/mL in PBS) on the surfaces of the stamps for 5 h and then rinsing the stamps with water for 15 s. The stamps were then contacted with the amine-terminated monolayers for 30 s. We measured the ellipsometric thickness of the material delivered to the surface by contact of the affinity stamps with the surfaces to be 10.8 ( 0.4 nm. Past studies have reported a similar change in ellipsometric thickness (10 nm) to accompany binding of anti-biotin IgG to immobilized biotinylated BSA.30 To determine if the increment in ellipsometric thickness accompanying contact of the affinity stamps with the amine-terminated monolayers was anti-biotin IgG specifically captured on the surfaces of the stamps, we performed a control experiment in which a biotinylated BSA affinity stamp was inked by placing a drop of nonspecific antibody (anti-goat IgG, 1 mg/mL in PBS) on the stamp surface for 5 h. After rinsing in PBS, the anti-goat IgG inked stamp was then printed in the same manner as the stamp inked with anti-biotin IgG. The change in ellipsometric thickness accompanying the

(27) Uchida, T.; Ishikawa, K.; Wada, M. Mol. Cryst. Liq. Cryst. 1980, 60, 37. (28) Shah, R. R.; Abbott, N. L. J. Phys. Chem. B 2001, 105, 49364950.

(29) Kleman, M. Points, Lines, and Wallss In Liquid Crystals, Magnetic Systems, and Various Ordered Media; John Wiley & Sons: New York, 1983. (30) Kim, S. R.; Abbott, N. L. Langmuir 2002, 18, 5269-5276.

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control experiment was 2.3 ( 0.2 nm. By rinsing the stamp in PBS containing 0.01% Triton X-100 for 15 s (following inking with anti-goat IgG), this increment in ellipsometric thickness for the control experiment was reduced to 0.6 ( 0.1 nm. These results lead us to conclude that nonspecific adsorption of anti-goat IgG on the affinity stamp was largely responsible for the ellipsometric thickness of 2.3 nm measured without the rinse with Triton X-100. We also measured a biotinylated BSA affinity stamp inked in PBS for 5 h and then contacted with the amine-terminated monolayer to cause an increment in ellipsometric thickness of 0.5 ( 0.1 nm. We believe this increment of 0.5 nm is likely the result of some transfer of biotinylated BSA or PDMS from the affinity stamp during the stamping process.2,31-34 The results above demonstrate that affinity microcontact printing onto amine-terminated monolayers (pretreated with 0.1 N HCl) leads to the delivery of anti-biotin IgG with an ellipsometric thickness of ∼10 nm. Using the same incubation conditions of IgG (1 mg/mL, incubation time of 5 h), we compared the amount of protein transferred to the amine-terminated monolayers by affinity microcontact printing with the amount of IgG delivered to the same surface by adsorption from bulk solution. The change in ellipsometric thickness measured to accompany adsorption of IgG from bulk solution was 5.4 ( 0.2 nm. Incubation of samples for 24 h did not result in the delivery of additional IgG to the surface. This result demonstrates that affinity microcontact printing can lead to the delivery of a greater areal density of an antibody than direct adsorption. We speculate that the greater increase in ellipsometric thickness achieved by using affinity microcontact printing is the result of the higher packing density of the anti-biotin IgG on the affinity stamp compared to that of the anti-biotin IgG adsorbed directly to the amineterminated monolayer. This suggestion is supported by the observation that microcontact printing of anti-biotin IgG from the PDMS stamp (not decorated with biotinylated BSA) onto an amine-terminated monolayer (pretreated with 0.1 N HCl) leads to an increment in ellipsometric thickness of 5.5 ( 0.7 nm. We examined the role of pretreatment of the amineterminated monolayer in determining the ellipsometric thickness of antibody captured on the surface. We varied the concentration of acid used in the pretreatment and measured the resulting ellipsometric thickness of IgG captured on the surface. The ellipsometric thickness of the affinity microcontact printed anti-biotin IgG on the amine-terminated monolayers with no pretreatment and 0.1 N HCl pretreatment was 7.4 ( 0.4 and 10.8 ( 0.4 nm, respectively. The ellipsometric thickness of the affinity microcontact printed anti-goat IgG (control) on the amineterminated monolayers with no pretreatment and 0.1 N HCl pretreatment was 2.7 ( 0.2 and 2.3 ( 0.2 nm, respectively. This result suggests that pretreatment of the amine-terminated monolayers with HCl increases the efficiency of capture of protein from the affinity stamps (perhaps by promoting ionic interactions between the ammonium groups on the surface and on the protein). (31) Bohm, I.; Lampert, A.; Buck, M.; Eisert, F.; Grunze, M. Appl. Surf. Sci. 1999, 141, 237-243. (32) Glasmastar, K.; Gold, J.; Andersson, A. S.; Sutherland, D. S.; Kasemo, B. Langmuir 2003, 19, 5475-5483. (33) Graham, D. J.; Price, D. D.; Ratner, B. D. Langmuir 2002, 18, 1518-1527. (34) Yang, Z. P.; Belu, A. M.; Liebmann-Vinson, A.; Sugg, H.; Chilkoti, A. Langmuir 2000, 16, 7482-7492. (35) Bloss, F. D. An Introduction to the Methods of Optical Crystallography; Holt, Rinehart and Winston: New York, 1961.

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In summary, the results above demonstrate that amineterminated monolayers are effective in capturing proteins from affinity stamps, particularly when pretreated with 0.1 N HCl. These results, when combined with our observation that amine-terminated monolayers formed on obliquely deposited gold can cause uniform orientations of liquid crystals, led us to investigate the use of amineterminated monolayers for imaging of affinity microcontact printed proteins by using liquid crystals. Orientations of Liquid Crystals on Amine-Terminated Monolayers Presenting Affinity Microcontact Printed Proteins. To investigate the orientations of liquid crystals on proteins that were deposited onto amine-terminated monolayers by affinity microcontact printing, we used PDMS stamps that possessed a relief consisting of an array of square pegs (each peg being 300 µm × 300 µm). Using the methods described above, we immobilized biotinylated BSA on the surfaces of stamps and incubated the stamps with either antibiotin IgG or anti-goat IgG (control) solutions. The stamps were then contacted with the amine-terminated monolayers. Optical cells comprising the affinity microcontact printed surface and an OTS-treated glass slide were prepared and filled with liquid crystal. Figure 3A shows an optical image (crossed polars) of liquid crystal supported on a surface stamped with antibiotin IgG. The sample was cooled from 36 to 33 °C over 1 h prior to imaging to minimize the density of the defect lines within the liquid crystal. When the direction of deposition of the gold is parallel to one of the polarizers (orientation of the sample of 0°), the square regions of the amine-terminated monolayer supporting anti-biotin IgG appear green. In contrast, the regions of the amineterminated monolayer that do not support anti-biotin IgG are dark (no transmission of light through the sample and crossed polars). When the sample is rotated at an angle of 45° from the crossed polars, some regions supporting IgG appear dark and the IgG-free regions of the surface appear green. Figure 3B shows an optical image of the sample in Figure 3A after it was annealed in a 36 °C oven for 8 h and then cooled to room temperature. In Figure 3B, the orientation of the liquid crystal on the regions supporting IgG is similar to that in Figure 3A, appearing green at a 0° sample orientation and darker when the sample is rotated at an angle of 45° from the crossed polars. However, the regions of the amine-terminated SAM not covered in protein appear dark at all sample orientations. The dark appearance at all sample orientations is indicative of homeotropic alignment. We confirmed the homeotropic alignment of the regions not supporting protein by conoscopy.22 From these results, we conclude that the amine-terminated SAM on gold caused the 5CB to change to a homeotropic alignment and that the presence of protein on the amineterminated SAM inhibits the change to homeotropic alignment. Next, we investigated the nature of the planar-tohomeotropic transition by recording images of the liquid crystal as a function of the time of annealing at 36 °C. As shown in Figure 4, after 5 and 7 h of annealing at 36 °C, several different interference colors are observed on the amine-terminated, IgG-free regions of the surface. We hypothesized that the change in interference color was caused by a lowering of the birefringence due to a tilt of the liquid crystal toward homeotropic alignment. We used the interference colors to calculate the tilt angle of the liquid crystal at the amine-terminated monolayer using a method reported previously.25

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Figure 3. Optical images (crossed polars) of 5CB in contact with an amine-terminated SAM on which IgGs were affinity microcontact printed in areas having lateral dimensions of 300 µm × 300 µm. The amine-terminated SAM was supported on an obliquely deposited gold film (bottom surface, orange), and the second surface confining the 5CB was an OTS-treated glass slide (top surface, gray). (A) Anti-biotin IgG, annealed for 1 h at 36 °C prior to imaging at room temperature. (B) Sample from part A, annealed for 8 h at 36 °C prior to imaging at room temperature. (C) Anti-goat IgG (control), annealed for 1 h at 36 °C prior to imaging at room temperature. (D) Sample from part C, annealed for 8 h at 36 °C prior to imaging at room temperature.

Figure 4. Optical images (crossed polars) of 5CB in contact with an amine-terminated SAM on which IgGs were affinity microcontact printed in areas having lateral dimensions of 300 µm × 300 µm. The amine-terminated SAM was supported on an obliquely deposited gold film, and the second surface confining the 5CB was an OTS-treated glass slide. The images were taken at room temperature after annealing for 1, 3, 5, 7, and >10 h at 36 °C.

By using the methods described above, we evaluated the change in optical appearance of the liquid crystal in Figure 4 to correspond to a change in the effective birefringence from ∼0.09 (t ) 1 h) to ∼0.055 (t ) 5 h) to ∼0.025 (t ) 7 h). This corresponds to a change in the tilt angle of the liquid crystal relative to the surface normal from ∼90° (t ) 1 h) to ∼60° (t ) 5 h) to ∼40° (t ) 7 h) on the amine-terminated SAM. For times greater than 10 h at 36 °C, the alignment of the liquid crystal was homeotropic (tilt angle of 0°) in the regions that did not support IgG. As discussed above, we believe that the transition from planar to homeotropic alignment on the amineterminated SAM is caused by the formation of an electrical double layer. The results in Figures 3A and B and 4 suggest that liquid crystals can be used to image affinity microcontact printed IgGs on amine-terminated monolayers pretreated with 0.1 N HCl. We confirmed that the orientational response of the 5CB was due to the capture of IgG specifically bound to the affinity stamp by performing a control experiment in which anti-goat IgG was inked on

the affinity stamp. Figure 3C shows the results obtained with the control experiment (incubation of the stamp in anti-goat IgG). In brief, the regions of the amineterminated monolayers that were contacted by the stamp caused the same alignment of the liquid crystal as that of those regions that were not contacted by the affinity stamp. This result supports the conclusion that the contrast seen in Figure 3A and B is due to anti-biotin IgG printed on the surface. Figure 3D shows the optical image of the sample in Figure 3C after heating in an oven at 36 °C for 8 h. Again, we observed a transition from planar to homeotropic alignment, as confirmed by conoscopy. Imaging Microcontact Printed Proteins on AmineTerminated Monolayers Formed on Glass Substrates. The observation of homeotropic anchoring of 5CB on amine-terminated monolayers (as reported above) led us to hypothesize that amine-terminated monolayers formed on surfaces other than obliquely deposited gold films could be used to image affinity microcontact printed proteins using liquid crystals. Here, we report the results of an investigation of the orientations of liquid crystals on

Imaging of Affinity Microcontact Printed Proteins

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Figure 5. Optical images (crossed polars) of 5CB sandwiched between an OTS-treated glass slide and an APES-treated glass slide on which an array of anti-biotin IgG (300 µm squares) was microcontact printed. (A) Annealed for 1 h at 36 °C prior to imaging at room temperature. (B) Annealed for 14 days at 36 °C prior to imaging at room temperature.

Figure 6. Optical images (crossed polars) of 5CB in contact with an amine-terminated SAM on which IgGs were affinity microcontact printed in areas having lateral dimensions of 300 µm × 300 µm. The amine-terminated SAM was supported on an obliquely deposited gold film, and the second surface confining the 5CB was an OTS-treated glass slide. (A) First use of stamp. (B) Second use of stamp. (C) Third use of stamp.

microcontact printed proteins on amine-terminated monolayers prepared on glass surfaces using silane chemistry (APES).27 We measured the response of liquid crystals to microcontact printed anti-biotin IgG on these surfaces. At short times (20 h), the contrast between the regions of surface supporting stamped protein became pronounced because the protein-free regions transitioned to homeotropic alignment (Figure 5B). The rate of the transition to homeotropic alignment, however, was much slower when using the glass surfaces than the gold surfaces (∼8 days vs ∼8 h). We conclude that amine-terminated monolayers formed on glass substrates also form the basis

of useful surfaces on which to image microcontact printed proteins by using liquid crystals. Reusing Affinity Stamps. Prior work has shown that affinity stamps can be reused.2 To demonstrate reuse of biotinylated BSA affinity stamps in combination with liquid crystals, we inked and stamped anti-biotin IgG from a stamp three times. Figure 6 shows the response of the liquid crystal to the first, second, and third use of the affinity stamp. Note that the different interference colors observed in these cells are the result of small differences in thickness of the liquid crystal in the optical cell (13 ( 2 µm). We also measured the amount of anti-biotin IgG delivered to the surface by using ellipsometry. We measured the change in ellipsometric thickness for the first, second, and third use of the stamp to be 10.8, 8.5, and 8.3 ( 0.4 nm, respectively. For a control experiment with anti-goat IgG (no rinsing with Triton X-100), the change in ellipsometric thickness for the first, second, and third use of the stamp was 2.3, 1.5, and 1.2 ( 0.2 nm, respectively. These results also indicate that the amount

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of nonspecifically transferred protein is reduced after the first use of the stamp, an observation that is consistent with a prior study.2 Conclusion In summary, we report the design of surfaces on which thermotropic liquid crystals can be used to image affinity microcontact printed proteins. Gold films were functionalized with amine-terminated monolayers to provide surfaces that (I) uniformly aligned liquid crystals when not supporting proteins and (II) were sufficiently high in energy to capture proteins delivered to the surface from an affinity stamp. We also observed a transition in the alignment of liquid crystals from planar to homeotropic on the amine-terminated regions not supporting IgGs after incubation for several hours at 36 °C. The homeotropic

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orientation of 5CB is consistent with the effects of an electrical double layer formed at the amine-decorated interface in contact with the liquid crystal. The transition to homeotropic alignment provides high optical contrast and enables the use of liquid crystals for the imaging of microcontact printed proteins on glass substrates possessing amine-terminated monolayers. Acknowledgment. This research was supported by funding from the Materials Research Science and Engineering Center on Nanostructured Materials and Interfaces (NSF-DMR-0079983) at University of Wisconsin, the Biophotonics Partnership Initiative of the NSF (ECS0086902), and a Biotechnology Training Program Fellowship to M.L.T. (NIH 5 T32 GM08349). LA049728+