Langmuir 2002, 18, 9617-9621
9617
Detection of Cholesterol through Electron Transfer to Cholesterol Oxidase in Electrode-Supported Lipid Bilayer Membranes Anando Devadoss and James D. Burgess* Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 Received April 24, 2002. In Final Form: October 3, 2002 Cholesterol oxidase is immobilized in lipid bilayer membranes assembled on tin-doped indium oxide electrode surfaces to sequester cholesterol from solution and to follow cholesterol oxidation via electrochemical reduction of hydrogen peroxide. The inner leaflet of the bilayer is chemically bound to the electrode surface through a thiol functionality at the polar headgroup end of the lipid. The outer lipid leaflet, containing cholesterol oxidase, is formed using a deoxycholate dialysis procedure. Continuous solution flow experiments, where the flow is changed from buffer solution containing no cholesterol to a buffer solution containing cholesterol, show currents for the reduction of hydrogen peroxide generated by the enzyme. The data indicate that cholesterol oxidase is immobilized on the electrode in an active state. The data are also consistent with energetically favored collection of cholesterol from solution by the electrode-supported lipid bilayer membrane.
Introduction Lipid bilayer membranes on solid supports have been the subject of numerous publications,1-3 especially over the past decade.1a-c,e,h,j-l,2a-h,3a,d-q The focus of several review articles1a-j is given in the Supporting Information. Lipid bilayer membranes prepared on various solids by optimized variations of the available deposition chemistries have been shown to accommodate a variety of proteins and enzymes in controlled orientations and in active conformations. The supported bilayer membranes somewhat mimic the native environment of membrane-associated biomolecules. Another attractive feature is that the resulting electrode architecture is thin (i.e., 50 Å) so that the incorporated biomolecules are close to both the electrode surface and the membrane-solution interface.1-4 The electrode-supported lipid bilayer membranes prepared here for immobilization of cholesterol oxidase sequester cholesterol from aqueous solution because equilibration of aqueous phase cholesterol into the hydrophobic environment of the lipid bilayer is energetically favored.5 * To whom correspondence should be addressed. E-mail:
[email protected]. (1) (a) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Buoninsegni, F. T. J. Electroanal. Chem. 2001, 504, 1-28. (b) Sinner, E. K.; Knoll, W. Curr. Opin. Chem. Biol. 2001, 5, 705-711. (c) Sackmann, E. Science 1996, 271, 43-48. (d) Tiede, D. M. Biochim. Biophys. Acta 1985, 811, 357-379. (e) Plant, A. L. Langmuir 1999, 15, 5128-5135. (f) Plant, A. L. Langmuir 1993, 9, 2764-2767. (g) Tien, H. T.; Salamon, Z. Biochem. Bioenerg. 1989, 22, 211-218. (h) Ottova, A. L.; Tien, H. T. Bioelectrochem. Bioenerg. 1997, 42, 141-152. (i) Tien, H. T.; Ollova, A. L. Electrochim. Acta 1998, 43, 3587-3610. (j) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400-1414. (k) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Biochim. Biophys. Acta 1996, 1279, 169-180. (l) Ding, L.; Li, J.; Dong, S.; Wang, E. J. Electroanal. Chem. 1996, 416, 105-112. (2) (a) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651653. (b) Parikh, A. L.; Beers, J. D.; Shreve, A.; Swanson, B. Langmuir 1999, 15, 5369-5381. (c) Ross, E. E.; Bondurant, B.; Spratt, T.; Conboy, J. C.; O’Brien, D. F.; Saavedra, S. S. Langmuir 2001, 17, 2305-2307. (d) Kanzaki, Y.; Hayashi, M.; Minami, C.; Inoue, Y.; Kogure, M.; Watanabe, Y.; Tanaka, T. Langmuir 1997, 13, 3674-3680. (e) Wu, Z.; Wang, B.; Cheng, Z.; Yang, X.; Dong, S.; Wang, E. Biosens. Bioelectron. 2001, 16, 47-52. (f) Gao, H.; Luo, G. A.; Feng, J.; Ottova, A. L.; Tien, H. T. J. Photochem. Photobiol., B 2000, 59, 87-91. (g) Hillebrandt, H.; Wiegand, G.; Tanaka, M.; Sackmann, E. Langmuir 1999, 15, 84518459. (h) Wiegand, G.; Layton, N. A.; Hillebrandt, H.; Sackmann, E.; Wagner, P. J. Phys. Chem. B 2002, 106, 4245-4254. (i) Huang, L. Biochemistry 1985, 24, 29-34.
Lang et al.6 have used thiol-functionalized lipid monolayers as the inner lipid leaflet of bilayers formed on gold. In the surface structure reported here, the inner lipid monolayer is similarly fixed to the electrode surface through a thiol functionality at the lipid headgroup. It is noted that this electrode modification step is required to produce membranes that exhibit enzymatic activity (vide infra). The outer lipid leaflet is deposited using the cholate dialysis procedure reported earlier for immobilizing cytochrome c oxidase in electrode-supported lipid bilayer membranes.3m-o,4 Because the inner lipid monolayer is bound to the electrode surface via a thiol functionality prior to dialysis, the resulting bilayer structure is also conceptually similar to the hybrid alkane thiol/lipid bilayer structures described by Plant1f where vesicle spreading produces the outer lipid leaflet on the hydrophobic alkane thiol monolayer. The outer lipid leaflet of such hybrid bilayer structures has been shown to be a reasonable model for the surface of biological lipid bilayers in that membrane-associated proteins can be immobilized in an active (3) (a) Salamon, Z.; Macleod, H. A.; Tollin, G. Biochim. Biophys. Acta 1997, 1331, 131-152. (b) Nakanishi, M. FEBS Lett. 1984, 176, 385388. (c) Salamon, Z.; Tollin, G. Bioelectrochem. Bioenerg. 1991, 25, 447454. (d) Salamon, Z.; Tollin, G. Arch. Biochem. Biophys. 1992, 294, 382-387. (e) Salamon, Z.; Gleason, F. K.; Tollin, G. Arch. Biochem. Biophys. 1992, 299, 193-198. (f) Salamon, Z.; Hazzard, J. T.; Tollin, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6420-6423. (g) Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E. Langmuir 1993, 9, 136-140. (h) Bianco, P.; Haladjian, J. J. Electroanal. Chem. 1994, 367, 79-84. (i) Bianco, P.; Haladjian, J. Electrochim. Acta 1994, 39, 911-916. (j) Zhang, Z.; Nassar, A. E. F.; Lu, Z.; Schenkman, J. B.; Rusling, J. F. J. Chem. Soc., Faraday Trans. 1997, 93, 1769-1774. (k) Pierrat, O.; Lechat, N.; Bourdillon, C.; Laval, J. M. Langmuir 1997, 13, 4112-4118. (l) Naumann, R.; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bunjes, N.; Graber, P. Bioelectrochem. Bioenerg. 1997, 42, 241-247. (m) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877-882. (n) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 2467-2475. (o) Burgess, J. D.; Jones, V. W.; Porter, M. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 6628-6631. (p) Edmiston, P. L.; Saavedra, S. S. Biophys. J. 1998, 74, 999-1006. (q) Edmiston, P. L.; Saavedra, S. S. J. Am. Chem. Soc. 1998, 120, 16651671. (r) Dolfi, A.; Buoninsegni, F. T.; Moncelli, M. R.; Guidelli, R. Langmuir 2002, 18, 6345-6355. (4) Rhoten, M. C.; Burgess, J. D.; Hawkridge, F. M. J. Electroanal. Chem., in revision. (5) (a) Phillips, M. C.; Johnson, W. J.; Rothblat, G. H. Biochim. Biophys. Acta 1987, 906, 223-276. (b) Rothblat, G. H.; Ilera-Moya, M. L.; Afger, V.; Kellner-Weibel, G.; Williams, D. L.; Phillips, M. C. J. Lipid Res. 1999, 40, 781-796. (6) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210.
10.1021/la0258594 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/29/2002
9618
Langmuir, Vol. 18, No. 25, 2002
state and are able to diffuse laterally in the outer lipid leaflet.1f The cholate dialysis procedure used here reconstitutes cholesterol oxidase in the outer leaflet of the membrane as it is formed (i.e., the enzyme and the outer lipid leaflet are deposited simultaneously). Lipid bilayer membranes are effective collectors of aqueous phase cholesterol.5 Lipid bilayer vesicles have been used to stimulate cholesterol efflux from the lipid monolayer of lipoproteins and from the plasma lipid bilayer of cells. A primary mechanism of cholesterol efflux from “donor” lipid membranes (e.g., lipoproteins and cell plasma membranes) to “acceptor” vesicle lipid bilayer membranes is aqueous diffusion. For a solution (or suspension) of cholesterol donor particles, the equilibrium between cholesterol in the lipid membrane and aqueous phase cholesterol lies largely toward membrane-resident cholesterol. Addition of vesicles containing no cholesterol to the donor particle solution results in cholesterol efflux from the donor particles as aqueous cholesterol is sequestered by the vesicles.5 On the basis of this background, it is reasonable to assume that the electrode-supported lipid bilayer membranes prepared here collect aqueous phase cholesterol from solution. Cholesterol oxidase is a flavin adenosine dinucleotide (FAD) containing enzyme (i.e., flavoenzyme) that catalyzes the isomerization and oxidation of cholesterol. Molecular oxygen is reduced by the FAD moiety generating cholestenone (oxidized cholesterol) and hydrogen peroxide.7 The crystal structures of the oxidase isolated from Brevibacterium sterolicum and Streptomyces species indicate dimensions of ca. 73 Å × 53 Å × 51 Å and 51.3 Å × 73 Å × 63 Å for the monomers, respectively, with the FAD group buried in the hydrophobic interior of the polypeptide.8 The enzyme is widely used for assessing the cholesterol content of cell culture samples.7 Generally, a spectrophotometric detection scheme is used where a dye is produced upon enzymatic generation of hydrogen peroxide. The model for enzymatic oxidation of cholesterol in the plasma membrane of cells has the oxidase inserted in the lipid bilayer through hydrophobic interactions9 such that cholesterol movement occurs directly from the plasma membrane into the enzyme pocket. This model has been proposed by Sampson and co-workers based on kinetic studies for treatment of vesicles containing cholesterol with the oxidase enzyme.9 Acrylodan (flurophore) labeled enzyme was produced using site-directed mutagenesis to probe the depth of protein insertion into the lipid bilayer membrane.9b They have also shown that cholesterol oxidase causes partial disruption of vesicles resulting in defects or leaks in the lipid bilayer.9c Further support for this model is given by studies involving the treatment of cells with cholesterol oxidase.10 Complete lysis of mid-gut epithelial cells10a and morphological changes in the plasma membrane of smooth red muscle cells10b have been reported on exposure to the enzyme. Also, the activity of cholesterol oxidase has been shown to be dependent on the cell type, (7) MacLachlan, J.; Wotherspoon, A. T. L.; Ansell, R. O.; Brooks, C. J. W. J. Steroid Biochem. Mol. Biol. 2000, 72, 169-195. (8) (a) Vrielink, A.; Lolyd, L. F.; Blow, D. M. J. Mol. Biol. 1991, 219, 533-554. (b) Yue, K. Q.; Kass, I. J.; Sampson, N. S.; Vrielink, A. Biochemistry 1999, 38, 4277-4286. (9) (a) Sampson, N. S.; Kass, I. J.; Ghoshroy, K. B. Biochemistry 1998, 37, 5770-5778. (b) Chen, X.; Wolfgang, D. E.; Sampson, N. S. Biochemistry 2000, 39, 13383-13389. (c) Ghoshroy, K. B.; Zhu, W.; Sampson, N. S. Biochemistry 1997, 36, 6133-6140. (10) (a) Greenplate, J. T.; Duck, N. B.; Pershing, J. C.; Purcell, J. P. Entomol. Exp. Appl. 1995, 74, 253-258. (b) Liu, K.; Maddafor, T. G.; Ramjiawan, B.; Kutryk, M. J. B.; Pierce, G. N. Mol. Cell. Biochem. 1991, 108, 39-48. (c) Crockett, E. L.; Hazel, J. R. J. Exp. Zool. 1995, 271, 190-195.
Letters
Figure 1. Idealized structure of the electrode-supported lipid bilayer membrane containing cholesterol oxidase.
suggesting significant interaction between the enzyme and the cell surface.10c In this work, a method is described for forming lipid bilayer membranes containing cholesterol oxidase on tindoped indium oxide (ITO) electrodes. The procedure produces cholesterol oxidase modified electrodes that show stable enzymatic activity for days. The model of the electrode architecture (Figure 1) has the enzyme partially inserted in the outer leaflet of the bilayer as has been proposed by others for the interaction of the enzyme with the plasma membrane of cells.9 Our proposed mechanistic model for detection of cholesterol at the cholesterol oxidase modified electrode is energetically favorable equilibration of cholesterol into the electrode-supported lipid bilayer membrane from aqueous solution followed by lateral diffusion of cholesterol within the bilayer to enzyme sites. Though no evidence for lateral diffusion of cholesterol is presented in this report, lateral diffusion of species within the outer leaflet of hybrid bilayer membranes has been shown.11 Lateral diffusion coefficient values for lipids and steroids in monolayers lie in the range of 10-7 to 10-8 cm2/s. The lateral diffusion in a solid-supported lipid bilayer is dependent on the phase of the bilayer, the solid support, and the type of monolayer that is attached to the surface.1c,d,11c The outer lipid monolayer in this system is expected to be present in a liquid crystalline state under the experimental conditions.12 It is noted that the cholesterol content affects the rigidity of lipid bilayer membranes.5a Hydrogen peroxide generated by the enzyme is electrochemically reduced at the ITO electrode surface for detection of cholesterol. Scheme 1 shows the enzyme-catalyzed reaction and the electrode reaction.
Scheme 1. Reaction Sequence Membrane: cholesterol + O2 f cholestenone + HOOH Electrode: HOOH + 2H+ + 2e- f 2H2O The method described here builds on other strategies for preparing electrodes modified with cholesterol oxidase13 by demonstrating the potential advantages discussed above of using an electrode-supported lipid bilayer membrane as the host for immobilization of the enzyme. A chronological history and brief descriptions of the studies that have been published on the topic of electrochemical detection of cholesterol are given in the Supporting (11) (a) Fahey, P. F.; Koppel, D. E.; Barak, L. S.; Wolf, D. E.; Elson, E. L.; Webb, W. W. Science 1977, 195, 305-306. (b) Stroeve, P.; Miller, I. Biochim. Biophys. Acta 1975, 157-167. (c) Gyorvary, E.; Wetzer, B.; Sleytr, U. B.; Sinner, A.; Offenhausser, A.; Knoll, W. Langmuir 1999, 15, 1337-1347. (12) Lewis, R. N. H.; Sykes, B. D.; McElhaney, R. N. Biochemistry 1988, 27, 880-887.
Letters
Langmuir, Vol. 18, No. 25, 2002 9619
Figure 2. Dual-chambered electrochemical dialysis cell. The dimensions of the cell are 1 cm × 5 cm × 5 cm. The electrode is mechanically clamped in the sample chamber, and the dialysis membrane is sandwiched between the flow chamber and the sample chamber.
Information. Many groups have reported electrodes that respond linearly with cholesterol concentration, and the lifetimes of the various electrodes range from hours to months. Experimental Section The cyclic voltammetric and amperometric experiments were conducted using a Bioanalytical Systems CV-50 potentiostat. The atomic force microscope images were acquired using a Molecular Imaging picoscan instrument. The ITO (on glass) (Information Products, Inc.) electrodes were cleaned by successive sonication in aqueous Alconox for 60 min, in ethanol for 60 min, and in water for 60 min. Sodium phosphate aqueous solution (0.01 M) adjusted to pH 6.5 by addition of sodium hydroxide was used as a buffer. Cholesterol (Sigma) solutions were prepared by dissolving cholesterol in a solution of 100 mM hydroxypropyl β-cyclodextrin (Cerestar USA, Inc.) and 0.01 M sodium phosphate (Sigma), pH 6.5. The cyclodextrin is used to solubilize cholesterol.14 The inner lipid leaflet was formed by sonicating the electrode in an ethanolic solution of the 1,2-dipalmitoyl-sn-glycero-3phosphothioethanol (Avanti Polar Lipids Inc.) for 90 min at 50 °C. The dual-chambered electrochemical dialysis cell (Figure 2) and dialysis conditions have been described earlier.3n One exception is that the dialysis solution contained 0.3 mM DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (Avanti Polar Lipids) (earlier work3n used a mixture of DOPE and dioleoyl phosphatidylcholine). In brief, the dual-chambered electrochemical dialysis cell is composed of two separate pieces of Lucite. One forms the sample chamber including the wall jet inlet and the reference and auxiliary electrodes. The other piece forms the (13) (a) Satoh, I.; Karube, I.; Suzuki, S. Biotechnol. Bioeng. 1977, 19, 1095-1099. (b) Bertrand, C.; Coulet, P. R.; Gautheron, D. C. Anal. Lett. 1979, 12, 1477-1488. (c) Karube, I.; Hara, K.; Matsuoka, H.; Suzuki, S. Anal. Chim. Acta 1982, 139, 127-132. (d) Wollenberger, U.; Kuhn, M.; Scheller, F.; Deppmeyer, V.; Janchen, M. Bioelectrochem. Bioenerg. 1983, 11, 307-317. (e) Masoom, M.; Townshend, A. Anal. Chim. Acta 1985, 174, 293-297. (f) Kajiya, Y.; Tsuda, R.; Yoneyama, H. J. Electroanal. Chem. 1991, 301, 155-164. (g) Motonaka, J.; Faulkner, L. R. Anal. Chem. 1993, 65, 3258-3261. (h) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-1076. (i) Kumar, A.; Malhotra, R.; Malhotra, B. D.; Grover, S. K. Anal. Chim. Acta 2000, 414, 43-50. (j) Gobi, K. V.; Mizutani, F. Sens. Actuators, B 2001, 80, 272-277. (k) Ropers, M. H.; Bilewicz, R.; Stebe, M. J.; Hamidi, A.; Miclo, A.; Rogalska, E. Phys. Chem. Chem. Phys. 2001, 3, 240-245. (l) Bongiovanni, C.; Ferri, T.; Poscia, A.; Varalli, M.; Santucci, R.; Desideri, A. Bioelectrochemistry 2001, 54, 17-22. (m) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849-856. (n) Vidal, J. C.; Ruiz, E. G.; Castillo, J. R. Electroanalysis 2001, 13, 229-235. (14) Haynes, M. P.; Phillips, M. C.; Rothblat, G. H. Biochemistry 2000, 39, 4508-4517.
flow chamber. The ITO electrode is clamped to the sample chamber, and a dialysis membrane is sandwiched between the two cell pieces separating the sample chamber from the flow chamber. The sample chamber is filled with deoxycholate (40.2 mM), lipid, and the cholesterol oxidase enzyme (Wako Pure Chemical Industries, Ltd.). Buffer is passed through the flow chamber to remove deoxycholate from the sample chamber. This procedure drives the formation of the outer lipid leaflet containing the oxidase enzyme on the inner lipid leaflet on the ITO electrode. Dialysis was performed for 12 h, and the sample chamber of the dialysis cell was then flushed with buffer for 12 h at a flow rate of 10 µL/min to remove remaining deoxycholate, lipids, and enzyme from the sample chamber. Continuous sample flow experiments for exposure of the electrodes to cholesterol were conducted using a six-way valve and two syringe pumps (one containing buffer and the other a buffered cholesterol solution) at a flow rate of 0.450 mL/min. Cholesterol exposure times that allowed the current responses to approach a steady-state value were controlled manually. The electrode potential was 120 mV versus normal hydrogen electrode (NHE), and the reference electrode was silver/silver chloride (1 M KCl). At this electrode interface, this potential is sufficient to reduce hydrogen peroxide and it does not result in large oxygen reduction background currents. All potentials are reported versus NHE.
Results and Discussion Contact mode atomic force microscopic images of the bare ITO surface (Figure 3) show a topography that closely resembles other reported images of ITO on glass.15,16 However, it is noted that these structures can vary.16 Relatively flat plateaus with lateral dimensions on the order of tens of nanometers are observed. It is hypothesized that the bilayer may form on these regions with some two-dimensional continuity. The regions of the electrode surface containing steep edges may not contain a complete lipid layer coverage. Hydrogen peroxide produced by the enzyme reaction may be electrochemically reduced at the potentially bare regions of the electrode surface (vide infra). The inner leaflet of the bilayer is attached to the ITO surface presumably through sulfur-metal bonds. Alkane thiols have been shown to form thiolate bonds at ITO surfaces.15,17 However, it is not known if the thiolate bond is with the tin, the indium, or both of the metal centers. The fact that hydrogen peroxide generated by the enzyme is electrochemically reduced at these electrode surfaces (vide infra) suggests that the inner lipid monolayer is not tightly packed or that it contains a substantial defect density (i.e., portions of the ITO surface are not covered by the lipid layer). The voltammetry of potassium ferricyanide (1 mM potassium nitrate) before (Figure 4A) and after deposition of the inner lipid leaflet (Figure 4B) indicates deposition of the thiol-functionalized lipids. The increase in the peak potential separation indicates a slower heterogeneous electron transfer rate. Slowed electron transfer kinetics contributes to the smaller peak currents. It is possible that regions of the electrode surface are completely blocked from ferricyanide in solution. Voltammograms conducted in 1 mM potassium nitrate indicate an electrode capacitance for the bare indium oxide surface of ca. 22 µF/cm2 decreasing to 17 µF/cm2 after formation of the inner lipid monolayer (Supporting Information). The lipids may be dispersed evenly over the surface with a relatively large nearest neighbor distance. Alternately, regions of the surface could be covered by more tightly (15) Yan, C.; Zharnikov, M.; Golzhauser, A.; Grunze, M. Langmuir 2000, 16, 6208-6215. (16) Donley, C.; Dunphy, D.; Paine, D.; Carter, C.; Nebesny, K.; Lee, P.; Alloway, D.; Armstrong, N. R. Langmuir 2002, 18, 450-457. (17) Kondo, T.; Takechi, M.; Yukari, S.; Uosaki, K. J. Electroanal. Chem. 1995, 381, 203-209.
9620
Langmuir, Vol. 18, No. 25, 2002
Letters
Figure 4. Cyclic voltammetry of 5 mM potassium ferricyanide at (A) bare ITO, (B) ITO modified with the thiolipid, and (C) ITO after deposition of the outer lipid leaflet.
Figure 3. (A) Atomic force microscopy images of an indiumtin oxide surface (contact mode). (B) A zoomed portion of the scan from (A) showing a cross section.
packed domains of lipid (i.e., islands of lipid on the plateau regions of the surface). In either case, the regions of the surface that are modified with the thiol-functionalized lipid do evidently provide a hydrophobic template so that the second leaflet of the bilayer containing cholesterol oxidase is deposited during the cholate dialysis step. Performing the dialysis step on bare ITO electrodes (the ITO is not pretreated with the thiol-functionalized lipid) does not produce interfaces that exhibit enzymatic activity upon exposure to cholesterol. Cholesterol oxidase and presumably the outer leaflet of the bilayer are deposited on the lipid monolayer modified ITO surface during dialysis. It is also possible that the dialysis procedure results in deposition of additional lipids with the polar headgroups associated with the hydrophilic ITO surface. This could occur at regions of the electrode surface that are not initially modified with thiolipid. This possibility would be consistent with the model for bilayer deposition on submonolayer converages of alkane thiol on silver where deoxycholate dialysis was used to reconstitute cytochrome c oxidase.3m-o,4 The ferricyanide characterization studies
(Figure 4C) show a further decrease of the electron transfer rate and possibly increased blocking after dialysis. However, the procedure apparently does not result in lipid deposition over the entire electrode surface as indicated by the voltammetric waves shown in Figure 4C. The electrode capacitance is also further decreased by 31% after dialysis (Supporting Information). The structure of the lipid membrane containing the enzyme is not known. However, the data could reflect formation of a lipid bilayer structure on regions of the electrode that initially contained a coverage of the thiol-functionalized lipid. The possibility that lipid multilayer aggregates are formed on regions of the electrode surface cannot be ruled out. Tapping-mode atomic force microscopy did not reveal the existence of such structures. This indicates that either the structures do not exist or the stability of the structures is insufficient to allow imaging under the experimental conditions (i.e., images resemble that shown in Figure 3). Again, electrochemical reduction of the generated hydrogen peroxide may occur at regions on the electrode surface that are not modified with the bilayer. Control experiments for hydrogen peroxide reduction for this system in the absence of cholesterol also show smaller currents after deposition of the bilayer compared to those for bare ITO (Supporting Information). Additional evidence for bilayer deposition is given by voltammetric data that are consistent with partitioning of FAD (hydrophobic probe molecule) into the electrode-supported membrane (Supporting Information). Amperometry using a wall jet configuration under continuous sample flow conditions was used to detect cholesterol at the cholesterol oxidase modified electrodes. Figure 5 shows the current measured for changing the flow from buffer to buffer containing cholesterol and for changing the flow back to buffer. Exposure of three different cholesterol concentrations to the electrode is shown, where the last exposure is a replicate of the first. The data are consistent with electrochemical reduction of hydrogen peroxide generated by the enzyme upon exposure to cholesterol. The relatively slow approach to a steadystate current is attributed to nonideal wall jet hydrodynamics for the cell geometry used18 (an initial experiment using a thin layer electrochemical cell showed a much faster approach to steady state, e.g., 20 s). The current responses depend on the cholesterol concentration with the higher concentrations yielding larger responses (Fig(18) Elbicki, J. M.; Morgan, D. M.; Weber, S. G. Anal. Chem. 1984, 56, 978-985.
Letters
Figure 5. Amperometric responses for exposure of cholesterol to a cholesterol oxidase modified electrode and to a lipid bilayer modified electrode containing no cholesterol oxidase (control). The electrode area is 0.2 cm2.
ure 5). Control experiments for lipid bilayer modified electrodes that contain no cholesterol oxidase do not show current responses upon exposure to cholesterol (Figure 5, control). As expected, cholesterol exposure experiments conducted under reduced oxygen concentrations (i.e., purged solutions) show ca. 40% smaller current responses. These data are consistent with cholesterol oxidase being immobilized on the ITO surface in an active state and with the proposed reactions shown in Scheme 1. The responses are reproducible (within 10%) for 3-5 days at a given electrode, and the lifetime of the electrodes has not been fully characterized. The current for injection of a given cholesterol concentration varies between electrodes. The data shown in Figure 5 were collected at the electrode that yielded the largest responses for exposure to cholesterol. A different electrode that yielded the smallest responses observed (data not shown) showed currents that were ca. 30% of those shown in Figure 5. The differences in response between electrodes are likely due to variations in the rate of hydrogen peroxide reduction between electrodes and/or disparity in the amount of oxidase immobilized. The applied potential (120 mV) does not reduce hydrogen peroxide at a mass transfer controlled rate at these electrode surfaces (Supporting Information). While the limited data presented for the cholesterol concentration dependence do not suffice for evaluation of mass transport and kinetically controlled regimes, the data do suggest that the electrode-supported lipid bilayer effectively sequesters cholesterol that exists in solution as a cyclodextrin-cholesterol complex14 (see Experimental Section). These attributes may allow these electrodes to extract cholesterol from the lipid monolayer of lipoproteins (e.g., low-density lipoproteins, LDL). At the applied potential, steady-state oxygen reduction current (e.g., 4 nA) is observed during the flow experiments. For data presentation, the nonzero baseline current due to oxygen reduction was subtracted from the data shown in Figure 5. The reaction catalyzed by the enzyme is a two-electron oxidation of cholesterol and a two-electron reduction of molecular oxygen producing hydrogen peroxide which is detected at the electrode. Some of the generated hydrogen
Langmuir, Vol. 18, No. 25, 2002 9621
peroxide could diffuse out of the lipid membrane into solution, and this may be a function of the electrode preparation. Detection of cholesterol by electrochemical oxidation of the generated hydrogen peroxide would eliminate nonzero baseline currents due to oxygen reduction. However, mass transfer controlled hydrogen peroxide oxidation requires potentials as positive as ca. 1.32 V at ITO electrodes in this medium and experiments for detection of cholesterol by electrochemical oxidation of hydrogen peroxide have so far not yielded reproducible current responses. Evidence for accumulation of cholestenone (oxidized cholesterol) in the bilayer inhibiting responses for sequential cholesterol exposures has not been observed (Figure 5). Lipid bilayer membranes containing cholesterol oxidase formed on platinum electrodes are currently being investigated. Using platinum as the substrate allows electrochemical oxidation of the generated hydrogen peroxide.19 Another approach to overcome kinetic and structural inconsistencies between electrode preparations (described above) is to implement a coulometric detection scheme for quantitative analysis of cholesterol in a known volume of sample. Conclusions This method for immobilizing cholesterol oxidase in electrode-supported lipid bilayer membranes allows detection of cholesterol through electrochemical reduction of hydrogen peroxide generated by the enzyme. The data show that the enzyme remains active upon immobilization in the lipid bilayer. The data also suggest that cholesterol partitions into the lipid bilayer membrane from solution due to hydrophobic interactions between cholesterol and the lipid tails. A current goal is to coulometrically quantify the amount of cholesterol present in small sample volumes by using a thin layer electrochemical cell in conjunction with the cholesterol oxidase modified electrode. Acknowledgment. Anando Devadoss acknowledges a student fellowship through Eveready Battery Company, Inc., and the Ernest Yeager Center for Electrochemical Sciences. Helpful discussions with Professors Barry Miller and Daniel A. Scherson are also acknowledged. Supporting Information Available: Summary of reports describing supported lipid bilayer membranes, discussion of systems for immobilization of cholesterol oxidase on solid supports, capacitance data (Figure S1) for formation of the lipid bilayer membrane, voltammetry indicating adsorption of FAD in the lipid membrane (Figure S2), and voltammetry for hydrogen peroxide reduction at a lipid bilayer modified electrode (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. LA0258594 (19) (a) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1998, 43, 579-588. (b) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1998, 43, 2015-2014. (c) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1999, 44, 2455-2462. (d) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1999, 44, 4573-4582. (e) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 2000, 45, 3573-3579. (f) Bokoch, P. M.; Palencsar, M. S.; Devadoss, A.; Burgess, J. D. Michaelis-Menten Kinetics for Oxidation of Cholesterol by Cholesterol Oxidase Immobilized in Lipid Membranes on Platinum. Manuscript in preparation.
