Langmuir 2003, 19, 6443-6448
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Molecular Orientation Distributions in Protein Films. V. Cytochrome c Adsorbed to a Sulfonate-Terminated, Self-Assembled Monolayer† Yue-Zhong Du and S. Scott Saavedra* Department of Chemistry, University of Arizona, Tucson, Arizona 85721-0041 Received January 30, 2003 Controlling the molecular orientation of protein molecules immobilized on insoluble substrates is now recognized as an important issue in numerous fields such as biosensing. In this study, we have investigated adsorption of horse heart cytochrome c (hcyt c) to a silane-based, self-assembled monolayer (SAM) as a strategy for preparing an oriented protein film. Previous attempts in our laboratory to create oriented protein films using this strategy were unsuccessful because multiple types of adsorptive interactions between the protein and the SAM produced a broad range of adsorbed orientations. In contrast, here we show that when a single type of site-directed interaction between hcyt c molecules and the SAM predominates, an oriented protein film results. Sulfonate-terminated SAMs were prepared by in situ oxidation of thioacetate SAMs. Electrostatically mediated adsorption of positively charged hcyt c produced a film with a porphyrin tilt angle distribution of 41° ( 13°, which is significantly narrower than distributions measured previously for cytochrome c films formed on other types of silane-based SAMs.
Introduction The ligand binding site(s) on a protein typically occupies only a small fraction of the total surface area on the molecule. Immobilization on a solid substrate may therefore impair the native binding activity of the protein if its molecular orientation renders the binding site sterically inaccessible to a dissolved ligand.1 The efficiency of direct electron transfer between an immobilized metalloprotein and a solid electrode may also be strongly influenced by the geometric orientation of the protein on the electrode surface.2 Consequently, the development of methods to control and assess molecular orientation in immobilized protein films has become a focus of considerable research activity in recent years.3-13 This activity level is due in part to the widespread interest in developing molecular †
Part of the Langmuir special issue dedicated to David O’Brien. * To whom correspondence should be addressed. Voice: (520) 621-9761. Fax: (520) 621-8407. E-mail:
[email protected]. (1) (a) Chang, I.-N.; Herron, J. N. Langmuir 1995, 11, 2083-2089. (b) Huang, W.; Wang, J.; Bhattacharyya, D.; Bachas, L. G. Anal. Chem. 1997, 69, 4601-4607. (2) (a) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (b) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877-882. (3) For a review, see: Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochim. Acta 1998, 128, 127-143. (4) (a) Stayton, P. S.; Ollinger, J. M.; Jiang, M.; Bohn, P. W.; Sligar, S. G. J. Am. Chem. Soc. 1992, 114, 9298-9299. (b) McLean, M. A.; Stayton, P. S.; Sligar, S. G. Anal. Chem. 1993, 65, 2676-2678. (c) Firestone, M. A.; Shank, M. L.; Sligar, S. G.; Bohn, P. W. J. Am. Chem. Soc. 1996, 118, 9033-9041. (5) Yeung, C.; Purves, T.; Kloss, A. A.; Kuhl, T. L.; Sligar, S.; Leckband, D. Langmuir 1999, 15, 6829-6836. (6) (a) Pachence, J. M.; Amador, S.; Maniara, G.; Vanderkooi, J.; Dutton, P. L.; Blasie, J. K. Biophys. J. 1990, 58, 379-389. (b) Tronin, A.; Edwards, A. M.; Wright, W. W.; Vanderkooi, J. M.; Blasie, J. K. Biophys. J. 2002, 82, 996-1003. (7) Bos, M. A.; Kleijn, J. M. Biophys. J. 1995, 68, 2573-2579. (8) Edmiston, P. L.; Lee, J. E.; Cheng, S.-S.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 560-570. (9) Wood, L. L.; Cheng, S.-S.; Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 571-576. (10) Edmiston, P. L.; Saavedra, S. S. Biophys. J. 1998, 74, 9991006. (11) Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1998, 120, 1665-1671. (12) Lee, J. E.; Saavedra, S. S. Langmuir 1996, 12, 4025-4032.
devices and biomaterials that rely on the bioactivity of immobilized proteins. Spectroscopic methods that probe electronic transitions provide a convenient means to assess molecular orientation in protein films, provided that a chromophore with suitable structural and photophysical properties is bound to the protein. Heme-containing proteins meet this criterion.4,6-12 In previous reports from this laboratory, we described the development of a spectroscopic approach to determine the molecular orientation distribution of porphyrin tilt angles in a hydrated heme protein film.8-11 Measuring absorbance linear dichroism and fluorescence emission anisotropy in a total internal reflection geometry provides two parameters from which both the mean porphyrin tilt angle and the angular distribution about the mean are recovered by modeling the orientation distribution as a Gaussian function.14 This approach (referred to as the IOW-ATR+TIRF technique) has been used to assess molecular orientation in heme films formed by noncovalent adsorption, site-directed covalent bonding, and biospecific binding to a variety of substrate materials.8-12 Of particular relevance to this work are previous studies of cytochrome c films deposited on silane-based, selfassembled monolayers (SAMs).8,9 Adsorption of horse heart cytochrome c (hcyt c) and yeast cytochrome c (ycyt c) on thiol-terminated SAMs was found to generate disordered protein films, as characterized by a broad tilt angle distribution. In both cases, it was clear that several types of adsorptive interactions between the protein and the SAM were operative; the result was adsorption in multiple geometric orientations. In contrast, hcyt c adsorption to the carboxy termini of an arachidic acid Langmuir-Blodgett film produces a highly oriented (13) (a) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (b) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706-1708. (14) Other combinations of polarized spectroscopic techniques can also be employed to determine the molecular orientation distribution in a thin film of chromophores. See: Simpson, G. J.; Westerbuhr, S. G.; Rowlen, K. L. Anal. Chem. 2000, 72, 887-898.
10.1021/la034167s CCC: $25.00 © 2003 American Chemical Society Published on Web 05/16/2003
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protein film.8 This result was attributed to the predominance of a site-directed, electrostatic interaction between the cationic protein and the anionic substrate. The same mechanism is also thought to predominate when hcyt c is adsorbed to a carboxy-terminated, alkanethiolate SAM, producing an orientation that allows direct electrochemical communication between the porphyrin moiety and the Au electrode.2a Thus electrostatic adsorption appears to be a viable strategy to create an oriented hcyt c film on a silane-based SAM. An assessment of this strategy is presented in this study. Sulfonate-terminated SAMs were prepared and used as substrates for adsorption of hcyt c films of submonolayer thickness. A variety of surface analytical techniques was employed to characterize both the SAMs and the protein films. In contrast to previous results obtained with other types of SAMs, adsorption to a sulfonate SAM produces a well-ordered hcyt c film, which is attributed to the site-directed nature of the electrostatic interaction. Experimental Section SAM Preparation. A hexadecyl, thioacetate-terminated trichlorosilane (Cl3SiC16H32SCOCH3) was synthesized as described by Balachander and Sukenik.15 Dicyclohexyl (99%) and chloroform (99.9%) were purchased from Aldrich, and ethanol (200 proof) was purchased from Equistar. These materials were used as received. Water used in this study was Type I Reagent Grade deionized (DI) water, obtained from a Barnstead Nanopure apparatus. Si wafers (single crystal, 0.43 mm thick, 〈111〉 orientation with p-type resistivity of 5-15 Ω/cm, polished on one side) were used as substrates for ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) measurements. Fused silica slides (2.5 cm × 7.5 cm × 1 mm thick, Dynasil) were used as substrates for total internal reflection fluorescence (TIRF) and epifluorescence measurements. Uranium oxide doped, silica-titania planar integrated optical waveguides (IOWs) were fabricated by a sol-gel dip-coating process16 and used as substrates for attenuated total reflection (ATR) measurements. All substrates were cleaned in an identical manner: soaked in a Chromerge bath for 40 min at 60-80 °C, sonicated in DI water for 10 min, and dried under a nitrogen stream. It was assumed that identical SAMs and protein films were formed on all substrates. Thioacetate-terminated SAMs were deposited on Si substrates, IOWs, and fused silica slides at room temperature (22 ( 4 °C). Silane solutions were prepared by dissolving the thioacetate silane in dicyclohexyl at a concentration of 1 µL/mL. Substrates were immersed into the silane solution for 40 min without stirring. Upon withdrawal, the substrates were sonicated sequentially in chloroform, ethanol, and DI water for 5, 7, and 10 min, respectively, to remove weakly bound silane molecules. Substrates were then blown dry under a nitrogen stream. A detailed examination of thioacetate SAMs prepared using this procedure is presented elsewhere.17 Sulfonate SAMs were generated by in situ oxidation.18 Thioacetate-SAM-coated substrates were immersed in a saturated solution of oxone (Dupont; the active ingredient is KHSO5), prepared in DI water, for 2 h at room temperature without stirring, followed by extensive rinsing with DI water. Protein Film Deposition. Solutions of hcyt c (Sigma, 99%) were prepared by dialysis against, and subsequent dilution in, 50 mM phosphate buffer, pH 7.2, at concentrations ranging from 1 to 70 µM. The hcyt c concentration was determined using a molar absorptivity of 8770 M-1 cm-1 at 514.5 nm.19 Adsorbed hcyt c films were adsorbed on sulfonate SAMs at room temperature without stirring using an incubation time of 30 min. (15) Balachander, N.; Sukenik, C. N. Langmuir 1990, 6, 1621-1627. (16) (a) Yang, L.; Saavedra, S. S.; Armstrong, N. R.; Hayes, J. Anal. Chem. 1994, 66, 1254-1263. (b) Edmiston, P. L.; Saavedra, S. S. Chem. Mater. 1997, 9, 2599-2603. (17) Du, Y.-Z.; Wood, L. L.; Saavedra, S. S. Mater. Sci. Eng., C 2000, 7, 161-169. (18) Collins, R. J.; Sukenik, C. N. Langmuir 1995, 11, 2322-2324. (19) Margoliash, E.; Frohwirt, N. Biochem. J. 1959, 71, 570-572.
Du and Saavedra Ellipsometry and XPS measurements were performed on protein films that were rinsed in phosphate buffer and then dried under a stream of nitrogen. All other measurements on protein films were performed without removing the film from buffer. For measurements of protein surface coverage by surfactant desorption, protein films were formed by incubating the substrate in a 50 µM solution of Zn-substituted hcyt c (Zn-hcyt c). Zn-hcyt c was prepared and assayed as described elsewhere.10,20 For measurements of absorbance linear dichroism, protein films were formed by incubating the substrate in a 50 µM solution of hcyt c. For measurements of TIRF anisotropy and desorption behavior, protein films were formed by incubating the substrate in a solution of Zn-hcyt c and horse heart ferrocytochrome c (ferrocyt c), in which the molar ratio of Zn-hcyt c to ferrocyt c was 1:9 and the total protein concentration was 50 µM; the ferrocyt c was prepared as described previously.8 Surface Analysis. Sessile water contact angles were measured by digital photography. A droplet (20 µL) was applied to a sample surface, allowed to settle for 10-20 s, and photographed with a charge-coupled device (CCD) through a 50 mm camera lens. Image analysis software (IPLab, Signal Analytics, Vienna, VA) was used to determine the contact angle. Measurements were performed on at least three samples and were made at a minimum of two different spots on each sample. A Gaertner Scientific L116C ellipsometer was used to measure film thickness. The oxide layer thickness on a freshly cleaned Si wafer is 20 ( 2.0 Å (n ) 56). This value was subtracted from the thickness values measured on Si wafers coated with SAM and/or protein layers. The refractive indices of SAMs and protein films were assumed to be 1.46. All thickness measurements were performed on at least three samples, and the thickness of each sample was measured at 6-10 different locations. XPS was performed using a VG ESCALab Mark II using an Al anode in the constant analyzer pass energy mode. Spectra were acquired at a takeoff angle of about 15° from the surface, using energies of 100 eV for survey spectra and 20 eV for highresolution spectra. Peak positions were determined by assigning C1s at 284.6 eV and linearly shifting the other peaks accordingly. AFM was performed with a Nanoscope IIIa Multimode Scanning Probe Microscope (MMSPM) manufactured by Digital Instruments, Inc. All samples were imaged in tapping mode, using Nanoprobe SPM TESP tips. The target amplitude was set at 0.5 V to minimize tip-sample interactions. The scan frequency was set at 1 Hz. Three sets of each type of sample were examined, and each was imaged at three or more physically different locations to ensure that the data obtained were consistent. Furthermore, samples were scanned from larger to smaller x-y scales and then back to larger scales to ensure that tip-induced film deformation did not occur. Protein Adsorption Measurements. Isotherms for hcyt c adsorption to sulfonate SAMs were measured using planar integrated optical waveguide attenuated total reflection spectrometry (IOW-ATR) as described in ref 12. A waveguide substrate coated with a sulfonate SAM was sealed in a liquid flow cell, and the flow cell was mounted on a rotary stage (New England Affiliated Technologies). The 514.5 nm beam from an Ar ion laser (Coherent Innova 70) was launched into the waveguide using an integral SF6 coupling prism (Karl Lambrecht). An input polarizer allowed excitation of the TE0 or TM0 mode. The resultant guided mode “streak” was photographed through the flow cell window using a CCD camera (Princeton Instruments) oriented normal to the waveguide plane. Attenuation curves were generated by plotting the logarithm of the vertically averaged pixel intensity against horizontal propagation distance and fit to log[I(x)] ) Rx + C, where x is the propagation distance in cm, I(x) is the intensity as a function of distance, R is the loss coefficient in cm-1, and C is a constant. Experiments were initiated by injecting buffer solution into the flow cell. An image of the guided mode streak was then acquired in both TE0 and TM0 modes to measure the blank loss coefficients. The procedure was repeated with a series of protein solutions of increasing concentration. The loss coefficients due to adsorbed protein were recovered by subtracting the blank loss coefficient. (20) Vanderkooi, J. M.; Adar, F.; Erecinska, M. Eur. J. Biochem. 1976, 64, 381-387.
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Table 1. Contact Angle and Ellipsometry Data for SAMs and Adsorbed hcyt c Films sessile water contact angle (deg)
film/surface substrate (Si/SiO2) thioacetate SAM) sulfonate SAM hcyt c adsorbed on sulfonate SAM a
