Amplification of Specific Binding Events between Biological Species

Langmuir 2002, 18, 5031-5035

5031

Amplification of Specific Binding Events between Biological Species Using Lyotropic Liquid Crystals John A. Van Nelson, Seung-Ryeol Kim, and Nicholas L. Abbott* Department of Chemical Engineering, University of Wisconsin, 1415 Engineering Drive, Madison, Wisconsin 53706 Received December 31, 2001 This paper reports the orientational behavior of lyotropic liquid crystals (LCs) formed from 29.4 wt % potassium laurate and 6.6 wt % decanol (remainder water) that are supported on gold films possessing an anisotropic nanometer-scale topography (wavelength ∼10 nm, amplitude ∼2 nm). The gold-coated surfaces are decorated with biotin-functionalized alkanethiols to which anti-biotin immunoglobulin G (IgG) is specifically bound. Similar to the behavior of thermotropic LCs, we observe the anisotropic topography of these surfaces to cause formation of a single domain of uniform planar or near planar aligned nematic phase of lyotropic LC. However, the relaxation of the lyotropic LC to the uniform alignment is slow (∼30 min) compared to thermotropic phases (∼seconds). When the anti-biotin IgG was bound specifically to the surface functionalized with biotin, the optical appearance (using polarized light) of the lyotropic LC was azimuthally nonuniform and easily distinguished from the appearance of the lyotropic phase in the absence of bound IgG. In contrast, exposure of the surface to anti-streptavidin IgG does not perturb the uniform orientation of the lyotropic LC observed prior to exposure to the aqueous solution of IgG. These results provide new methods to align lyotropic LCs and demonstrate that lyotropic LCs are useful for amplification of specific binding events between biological species.

Introduction This paper describes the uniform alignment of lyotropic liquid crystals (LCs) on chemically functionalized gold films that possess anisotropic nanometer-scale topography. We also report the use of lyotropic LC to amplify and transduce the receptor-mediate binding of proteins at these surfaces into easily visualized optical signals. The work described in this paper is motivated by two observations. First, whereas many procedures exist to prepare surfaces that align thermotropic LCs in welldefined orientations,1-6 in comparison, relatively few procedures have been reported to align lyotropic LCs.7-10 * To whom correspondence should be addressed. Fax: 608-2625434. E-mail: [email protected]. (1) Past studies have reported a wide variety of procedures that can be used to prepare surfaces that uniformly orient liquid crystals. The procedures include manipulation of the chemical functionality of a surface2 as well as the topographical structure on the nanometer scale.3. (2) (a) Je´roˆme, B. Rep. Prog. Phys. 1991, 54, 391-451. (b) Castellano, J. A. Mol. Cryst. Liq. Cryst. 1983, 94, 33-41. (c) Aoyama, H.; Yamazaki, Y.; Matsuura, N.; Mada, H.; Kobayashi, S. Mol. Cryst. Liq. Cryst. 1981, 72, 127-132. (d) Geary, J. M.; Goodby, J. W.; Kmetz, A. R.; Patal, J. S. J. Appl. Phys. 1987, 62, 4100-4108. (e) Nakajima, K.; Wakemoto, H.; Sato, S.; Yokotami, F.; Ishihara, S.; Matsuo, Y. Mol. Cryst. Liq. Cryst. Sci. Technol., Sect. B 1990, 180, 223-232. (f) Pidduck, A. J.; Bryan-Brown, G. P.; Haslam, S. D.; Bannister, R.; Kitely, I.; McMaster, T. J.; Boogaard, L. J. Vac. Sci. Technol., A 1996, 14, 1723-1728. (3) (a) Berreman, D. W. Phys. Rev. Lett. 1972, 28, 1683-1686. (b) Berreman, D. W. Mol. Cryst. Liq. Cryst. 1973, 23, 215-231. (c) Wolff, U.; Greubel, W.; Krueger, H. Mol. Cryst. Liq. Cryst. 1973, 23, 187-196. (d) Creagh, L. T.; Kruger, H. Mol. Cryst. Liq. Cryst. 1973, 24, 59-68. Past approaches leading to topographically patterned surfaces include oblique evaporation,4 photolithographic techniques,5 and a recent technique involving polarized photoalignment.6 (4) (a) Janning, J. Appl. Phys. Lett. 1972, 21, 173-174. (b) Goodman, L. A.; Mcginn, J. T.; Anderson, C. H.; Digeronimo, F. IEEE Trans. Electron Devices 1977, ED-24, 795-804. (c) Barberi, R.; Giocondo, M.; Sayko, G. V.; Zvedin, A. K. J. Phys. Condens. Matter 1994, 6, A275A278. (5) Fujita, J.; Watanabe, H.; Ochiai, Y.; Manako, S.; Tsai, J. S.; Matsui, S. Appl. Phys. Lett. 1995, 66, 3065-3067. (6) (a) Kim, M.-H.; Kim, J.-D.; Fukuda, T.; Matsuda, H. Liq. Cryst. 2000, 27, 1633-1640. (b) Shannon, P. J.; Gibbons, W. M.; Sun, S. T. Nature 1994, 368, 532-533. (c) Schadt, M.; Seiberle, H.; Schuster, A. Nature 1996, 381, 212-215. (d) Wu, Y. L.; Demachi, Y.; Tsutsumi, O.; Kanazawa, A.; Shiono, T.; Ikeda, T. Macromolecules 1998, 31, 349354.

One goal of this study was to determine if recently developed procedures that yield surfaces with well-defined nanometer-scale topographies and chemistries, and which cause uniform alignment of thermotropic LCs, can also be exploited to manipulate the orientations of lyotropic LCs. Here we address this question by using lyotropic nematic phases formed from aqueous mixtures of 29.4% potassium laurate, 6.6% decanol, and 64% water. The phase diagram for this system has been reported in a past study.11 The second observation that motivates this study is the recently reported use of LCs to amplify events of molecular and biomolecular recognition into easily visualized optical signals.12-14 In these studies, the binding of molecules to receptors hosted on nanostructured surfaces led to a change in the orientation of a thermotropic LC supported on the surface. In particular, when using thin gold films that possessed an anisotropic nanometer-scale topography (wavelength ∼10 nm, amplitude ∼2 nm) on which monolayers presenting biotin were formed, anti-biotin immunoglobulin G (IgG) bound to the surface was demonstrated to mask the anisotropic topography of the gold film. This (7) Most past studies have used strong magnetic fields to cause uniform alignment of nematic phases of lyotropic LC.8 Recently, a few studies have reported on the orientational behaviors of nematic phases of lyotropic LCs on rubbed films9 of polymers as well as fluorinated surfaces.10 (8) (a) Oliveira, E. A.; Figueiredo Neto, A. M.; Durand, G. Phys. Rev. A 1991, 44, R825-R827. (b) Vega, L.; Bonvent, J. J.; Barbero, G.; Oliveira, E. A. Phys. Rev. E 1998, 57, R3715-R3718. (c) Turchiello, R. de F.; Oliveira, E. A. Phys. Rev. E 1996, 54, 1618-1624. (d) Oliveira, E. A.; Figueiredo Neto, A. M. Phys. Rev. E 1994, 49, 629-635. (9) Bonvent, J. J.; Bechtold, I. H.; Vega, M. L.; Oliveira, E. A. Phys. Rev. E 2000, 62, 3775-3779. (10) Shahidzadeh, N.; Merdas, A.; Urbach, W. Langmuir 1998, 14, 6594-6598. (11) Yu, L. J.; Saupe, A. Phys. Rev. Lett. 1980, 45, 1000-1003. (12) (a) Skaife, J. J.; Brake, J. M.; Abbott, N. L. Langmuir 2001, 17, 5448-5457. (b) Skaife, J.; Abbott, N. L. Langmuir 2000, 16, 35293536. (c) Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B.; Abbott, N. L. Science 1998, 279, 2077-2080. (13) (a) Kim, S.-R.; Abbott, N. L. Adv. Mater. 2001, 13, 1445-1449. (b) Kim, S.-R.; Shah, R. R.; Abbott, N. L. Anal. Chem. 2000, 72, 46464653. (14) (a) Shah, R. R.; Abbott, N. L. Science 2001, 293, 1296-1299. (b) Shah, R. R.; Abbott, N. L. J. Am. Chem. Soc. 1999, 121, 11300-11310.

10.1021/la0118715 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/22/2002

5032

Langmuir, Vol. 18, No. 13, 2002

masking of the topography prevented the uniform alignment of the thermotropic LC that was observed in the absence of bound IgG.12 Whereas past studies have focused on the use of thermotropic LCs (e.g., 4-cyano-4′-pentylbiphenyl, 5CB), here we aimed to determine if the orientational behaviors of lyotropic LCs could also be used to amplify the specific interaction of an antibody and its antigen into an easily visualized optical signal. Experimental Section Materials. Glass microscope slides were Fisher’s Finest, Premium Grade obtained from Fisher Scientific (Pittsburgh, PA). Lauric acid and decanol were purchased from Aldrich (Milwaukee, WI). Potassium hydroxide was purchased from Fisher Scientific (Fair Lawn, NJ). Octanethiol (C8SH) was purchased from Aldrich, and the biotinylated thiol (BiSH), biotin-(CH2)2(CH2CH2O)2NHCO(CH2)11SH, was synthesized using procedures reported elsewhere.15 The anti-biotin immunoglobulin G (IgG) (polyclonal, developed in goat) and anti-streptavidin IgG (polyclonal, developed in rabbit) were obtained from Sigma (St. Louis, MO). All aqueous solutions were prepared with deionized water having a resistivity greater than 18 MΩ cm (Milli-Q system, Millipore, Bedford, MA). All solutions of IgG used in this study were prepared in aqueous solutions of phosphate-buffered saline (PBS, pH 7.4, 0.005% Triton X-100). Deposition of Films of Gold. Semitransparent films of gold with a thickness of 20 nm (8 nm of titanium as an adhesion layer) were deposited onto clean glass substrates by using an electron beam evaporator (VES-3000-C, Tek-Vac Industries Inc., Long Island, NY). The rate of deposition of each metal was 0.02 nm/s at a pressure less than 1 × 10-6 Torr. Gold films without anisotropic nanometer-scale topography (called hereafter “uniformly deposited gold films”) were deposited onto glass slides mounted on a planetary that rotated the slides in an epicyclic manner with respect to the gold source. The rotation of the samples ensured uniform and planar deposition of gold films. Gold films possessing anisotropic nanometer-scale topography (called hereafter “obliquely deposited gold films”) were deposited without rotation of the substrates. The gold was deposited at an angle of 60° from the normal of the glass substrates. The details of these procedures, including procedures for the cleaning of the glass microscope slides, can be found elsewhere.16 Our past studies have demonstrated that obliquely deposited gold films possess an anisotropic, nanometer-scale topography.12,16 Formation of Self-Assembled Monolayers (SAMs). Selfassembled monolayers were formed on the surfaces of gold films by immersion of the films into ethanolic solutions containing 1 mM C8SH or a mixture of 44 µM BiSH and 22 µM C8SH. After immersion for 2 h at room temperature, the substrates were removed, rinsed with ethanol, and then dried under a gaseous stream of nitrogen. Binding of IgG. Anti-biotin IgG was bound to the biotinylated SAM by preparing a thin poly(dimethylsiloxane) (PDMS) sheet (∼2 mm of thickness) containing rectangular wells (9 mm by 4.5 mm). The sheet of PDMS was placed onto the biotinylated gold substrate. Each well was filled with an aqueous solution of 1 µM anti-biotin IgG (PBS, pH 7.4, 0.005% Triton X-100) and incubated for 2 h. The slides were then rinsed with deionized water, and dried under a gaseous nitrogen stream. Preparation of Lyotropic LC. The desired composition of the lyotropic LC was determined by inspection of a phase diagram reported in a past study.11,17 A composition corresponding to a nematic phase was selected, namely, 29.4 wt % potassium laurate, 6.6 wt % decanol, and 64.0 wt % water. Potassium laurate was prepared by combining equimolar amounts of potassium hydroxide (1.876 g, 87.7 wt % of this weight is KOH, the rest is H2O) and lauric acid (5.874 g) in methanol (∼500 mL). The solids were mixed until they were completely dissolved in the methanol. The methanol and water were evaporated using a Buchi (15) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.; Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019. (16) Skaife, J. J.; Abbott, N. L. Chem. Mater. 1999, 11, 612-623. (17) Ribas, A. M.; Evangelista, L. E.; Palangana, A. J.; Oliveira, E. A. Phys. Rev. E 1995, 51, R5204-R5207.

Letters Rotavapor (Fischer Scientific) leaving solid potassium laurate. The potassium laurate (1.815 g) was then combined with water (3.95 mL) and decanol (0.491 mL). Ultrasonification was used to mix the components. The components were mixed for 30 min, allowed to stand overnight, and then mixed for another 30 min. The lyotropic LC was stored at room temperature in a glass, screw top scintillation vial in its nematic phase. Optical Cells. We observed the alignment of the lyotropic LC on the surfaces of the gold films by assembling the gold films into optical cells. Optical cells were fabricated by pairing two glass slides, each of which supported gold films. The substrates were aligned (facing each other) such that the directions of deposition of the gold films were parallel within each cell. The substrates were kept apart by inserting thin polyester film (thickness of ∼10 µm, Saran wrap, Dow Brands L. P., Indianapolis, IN) between the surfaces of the films. The cells were held together by using “bulldog” clips placed along the edge of the glass microscopic slides. The cells and syringe containing the lyotropic LC were heated to ∼40 °C by placing them on a hot plate. We also used a hot air gun to warm the air around the cells to ∼40 °C. A drop of lyotropic LC was placed onto the edge of each cell on the hot plate. The lyotropic LC was drawn into the cavity between the two surfaces by capillary forces. Occasionally, it was necessary to put a drop of the lyotropic LC on the opposite edge of the cell in order to completely fill the cell by capillary action. The cell was immediately placed into a Petri dish containing water-soaked tissue papers. The water-soaked tissue paper was used to saturate the air inside the Petri dish with water, so as to minimize evaporation of the water from the lyotropic LC. Analysis of Optical Textures. A polarized light microscope (BX60, Olympus, Tokyo, Japan) was used to observe the optical textures formed by light transmitted through the optical cells filled with lyotropic LC. All images were obtained using a 10× objective lens with a 1.0 mm field of view between crossed polarizers. An image of each lyotropic LC cell was captured with a digital camera (C-2020 Z, Olympus America Inc., Melville, NY) that was attached to the polarized light microscope. The images were obtained using a resolution of 1600 × 1200 pixels, aperture of f10 and a shutter speed of 1/20 s. Ellipsometric Thickness. All ellipsometric measurements were performed using thick (10 nm of Ti/50 nm of Au) and uniformly deposited gold films. The details of the measurements are described elsewhere.12 Briefly, ellipsometric thicknesses were measured at three points on each sample by using a Rudolph Auto EL ellipsometer (Flanders, NJ) at a wavelength of 6320 Å and an angle of incidence of 70°. To interpret the ellipsometric thickness of bound protein, we used a simple two-layer model (organic layer/effective substrate of gold-coated glass substrate) by assuming the refractive index to be 1.46 for the organic layers (SAMs/anti-biotin IgG).18

Results and Discussion Orientations of Lyotropic LC on Obliquely and Uniformly Deposited Gold Films. We first compared the optical textures of lyotropic LCs anchored on obliquely deposited and uniformly deposited gold films. Each gold film supported a SAM formed from C8SH. Our past studies have established that obliquely deposited gold films possess a nanometer-scale topography (wavelength ∼10 nm, amplitude ∼2 nm) whereas uniformly deposited films do not possess an anisotropic topography.12,16 The images of the lyotropic LCs shown in Figure 1 were obtained 30 min after introduction of the lyotropic LC into each optical cell. We did not observe the alignment of the lyotropic LC to change further after 30 min of equilibration (other than by the effects of evaporation, as discussed below). We note that this slow relaxation of the nematic phase of the lyotropic LC contrasts with the fast equilibration of nematic phases of thermotropic LCs such as 5CB on these same surfaces. (18) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir Blodgett to Self-Assembly; Academic Press: Boston, MA, 1991.

Letters

Langmuir, Vol. 18, No. 13, 2002 5033

Figure 1. Optical images (crossed polarizers) of lyotropic LCs anchored on SAMs formed from C8SH. The SAMs were formed on (A) uniformly deposited films of gold (without anisotropic nanometer-scale topography) or (B) obliquely deposited films of gold (with anisotropic nanometer-scale topography). The left-side image in (B) was obtained by aligning the direction of deposition of the gold with one of the polarizers. The right-side image was obtained by rotation of the optical cell shown on the left by 45°. The horizontal length of each image is 1.4 mm, and the white arrow indicates the direction of deposition of the gold films.

Inspection of Figure 1A reveals that the lyotropic LCs supported on the uniformly deposited films of gold do not possess a preferred azimuthal orientation. The existence of strong, in-plane birefringence also reveals that the orientation of the nematic phase is planar or close to planar. In contrast, the lyotropic LC supported on the SAM formed on the obliquely deposited gold is uniformly orientated over the entire sample. When the direction of deposition of the gold film was oriented parallel to one of the polarizers, the optical appearance of the lyotropic LCs was dark (Figure 1B). By rotation of the optical cell between crossed polarizers, the textures were observed to clearly modulate between a dark and bright appearance. These results, when combined, indicate that the anisotropic topography of the gold film that is introduced by oblique deposition leads to a uniform azimuthal orientation of the lyotropic LC. We also note that the orientation of the lyotropic LC is parallel or nearly parallel to the surface of the gold film. Dynamic Properties of the Lyotropic LC Supported on the Obliquely Deposited Gold Substrates. Whereas the results described above reveal that it is possible to orient lyotropic LCs using SAMs formed from C8SH on the surfaces of obliquely deposited gold films, here we report a study of the dynamic properties of the lyotropic LCs on SAMs formed from mixtures of C8SH and BiSH for two reasons. First, the use of these surfaces for amplification of biomolecular interactions requires introduction of receptors into the surfaces. Second, the results above suggested to us that the dynamics of the orientational behavior of the lyotropic LCs is slow, especially when compared to thermotropic LCs. Figure 2 shows the time-dependence of the optical appearance of the lyotropic LC supported on a SAM formed by coadsorption of a mixture of C8SH and BiSH. In our past studies, we determined the composition of the mixed SAM to be ∼70% of C8SH and ∼30% of BiSH.12 Immediately following the introduction of the lyotropic LC into the optical cell, the optical appearance of the lyotropic LC was highly nonuniform (Figure 2A). The influence of shear on the alignment of the lyotropic LC was evident

in some of the images captured at short times. Within 30 min, however, we observed the relaxation of the lyotropic LC to a uniform alignment that was indistinguishable from that observed on the SAMs formed from C8SH. This observation indicates that the introduction of BiSH into the SAM does not measurably change the orientation of the lyotropic LC as compared to the case of the C8SH SAM in Figure 1. We observed this orientation to be stable for up to ∼80 min. As shown in panels E and F of Figure 2, at times of ∼80 min and longer, our observations were interrupted by the effects of evaporation. Although the rate of evaporation of the water from the lyotropic LC could be slowed by increasing the humidity of the atmosphere of the Petri dish, we were unable to eliminate the effects of evaporation from our observations over times greater than ∼80 min. Response of Lyotropic LC to IgG Specifically Bound to a Biotinylated SAM Supported on an Obliquely Deposited Gold Film. Our past studies using thermotropic LCs have revealed that IgG, when bound to a surface with topography comparable to the size of the IgG, can influence the orientation of a thermotropic LC supported on the surface. The effect of the IgG on the orientation of thermotropic LC is easily visualized.12 Because the results above reveal that the nanometerscale topography of a surface can also change the orientational behavior of lyotropic LCs, we hypothesized that it may also be possible to use lyotropic LCs to report the presence of proteins bound to nanostructured surfaces. Figure 3 shows the optical appearance of the lyotropic LC formed from potassium laurate and decanol on a biotinylated SAM to which IgG was bound (30 min after introduction of the lyotropic LC into the cell). We confirmed the presence of the bound IgG by using ellipsometry (Table 1). The ellipsometric thickness of the IgG bound to the SAM was determined to be 3.8 ( 0.2 nm. Inspection of Figure 3A, and comparison of its optical appearance to Figure 2C, reveals that the presence of the bound IgG has a striking effect on the orientational behavior of the lyotropic LC on the surface. In contrast to Figure 2C, the presence of the bound IgG causes the azimuthal orienta-

5034

Langmuir, Vol. 18, No. 13, 2002

Letters

Figure 2. Optical images of lyotropic LCs (between crossed polarizers) anchored on SAMs formed from C8SH/BiSH as a function of time: (A) 5, (B) 10, (C) 30, (D) 60, (E) 80, and (F) 90 min. The gold films were obliquely deposited. The right-side images were obtained after rotation of the optical cell shown on the left by 45°. The horizontal length of each image is 1.4 mm, and the white arrow indicates the direction of deposition of the gold film.

Letters

Langmuir, Vol. 18, No. 13, 2002 5035

Figure 3. Optical images of lyotropic LCs (crossed polarizers) anchored on SAMs formed from C8SH/BiSH after immersion into aqueous solutions containing 1 µM of IgG for 1 h: (A) anti-biotin IgG and (B) anti-streptavidin IgG. The right-side images were obtained after rotation of the optical cell shown in the left images by 45°. The horizontal length of each image is 1.4 mm, and the white arrow indicates the direction of deposition of the gold film. Table 1. Ellipsometric Thicknesses of IgGs Bound to SAMs Formed from BiSH/C8SH on Uniformly Deposited Thick Films of Golda

a

IgG

ellipsometric thickness of bound IgG (nm)

anti-biotin IgG anti-streptavidin IgG

3.8 ( 0.1 -0.1 ( 0.2

The thickness of the BiSH/C8SH SAM was 1.6 ( 0.1 nm.

tion of the lyotropic phase to be highly nonuniform. We conclude, therefore, that the bound IgG masks the anisotropic topography of the gold films that is responsible for the uniform alignment of the lyotropic LC in Figure 2. Finally, we confirmed that the optical appearance of the lyotropic LC in Figure 3A is the consequence of specific binding of the anti-biotin IgG to the biotinylated surface. For example, Figure 3B shows the largely uniform appearance of the lyotropic LC on a SAM that was immersed into an aqueous solution of anti-streptavidin IgG prior to contact with the lyotropic LC. We also confirmed by ellipsometry that no measurable antistreptavidin IgG was bound to the biotinylated surface (Table 1).

Conclusions The main conclusions of this paper are 2-fold. First, we have demonstrated that lyotropic LCs can be aligned uniformly using SAMs formed on gold films that possess anisotropic nanometer-scale topography. In contrast, the azimuthal orientations of the lyotropic LCs are nonuniform when supported on the same SAMs formed on gold films that do not possess an anisotropic nanometer-scale topography. Second, we have demonstrated that the sensitivity of lyotropic LCs to the nanometer-scale topography of surfaces can be used to report the presence of proteins specifically bound to receptors immobilized on these surfaces. In a separate publication, we will report on the use of water-based lyotropic LCs as solvents for biological molecules and the use of these solvents for the delivery of targeted molecules to nanostructured 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., N00014-99-1-0250), Materials Research and Engineering Center on Nanostructured Materials and Interfaces (NSF-DMR-0079983) at University of Wisconsin, and the Biophotonics Partnership Initiative of NSF (ECS-0086902). LA0118715