Enzyme Modification of Platinum Microelectrodes for Detection of

Communication—Microelectrode Detection of Cholesterol Efflux from the Human Buccel Mucosa. Xiaochun Yu , Thomas J. Kelley , Hillel J. Chiel , James ...
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Anal. Chem. 2005, 77, 7393-7398

Enzyme Modification of Platinum Microelectrodes for Detection of Cholesterol in Vesicle Lipid Bilayer Membranes Anando Devadoss, M. Simona Palencsa´r, Dechen Jiang, Michael L. Honkonen, and James D. Burgess*

Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-7078

Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content. This group recently reported detection of cholesterol contained in the plasma membrane of a single oocyte using microelectrodes modified with a lipid bilayer membrane containing cholesterol oxidase.1 Cholesterol is an amphipathic lipid molecule that is known to play essential roles in cellular membrane structure and function.2 The cholesterol content of cellular membranes is believed to be tightly regulated through vesicular trafficking pathways that consume energy, and alterations in cell plasma membrane cholesterol content are likely involved in the onset of atherosclerosis.3 The results presented in this article are focused on electrode fabrication and characterization including detection of cholesterol contained in vesicle lipid bilayer membranes that serve as a simple model system for the cell plasma membrane. The electrode-supported thiolipid/lipid bilayer membrane is formed by initially modifying a bare platinum electrode surface with a submonolayer of thiolipid.4 A vesicle fusion method is used to deposit a lipid bilayer structure on the electrode where the thiolipid submonolayer constitutes a portion of the inner lipid * To whom correspondence should be addressed. E-mail: [email protected]. (1) Devadoss, A.; Burgess, J. D. J. Am. Chem. Soc. 2004, 126, 10214-10215. (2) Tabas, I. J. Clin. Invest. 2002, 110, 583-590. (3) Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726. (4) Bokoch, M. P.; Devadoss, A.; Palencsar, M. S.; Burgess, J. D. Anal. Chim. Acta 2004, 519, 47-55. 10.1021/ac051173f CCC: $30.25 Published on Web 10/05/2005

© 2005 American Chemical Society

leaflet anchoring the bilayer to the electrode. Cholesterol oxidase has a natural affinity for interaction with lipid bilayer membranes, and it spontaneously inserts into the electrode-supported lipid bilayer membrane from solution. The structural model for interaction between cholesterol oxidase and the electrode-supported lipid bilayer membrane4,5 is that proposed for natural interaction of the bacterial enzyme with the plasma membrane of mammalian cells.6 Cholesterol oxidase partially inserts in the outer lipid leaflet of the cell plasma membrane undergoing a conformational change that exposes hydrophobic residues and opens a path for movement of cholesterol into the active site pocket directly from the plasma membrane. Based on this background, cholesterol is believed to partition into the electrode-supported lipid bilayer membrane prior to enzymatic oxidation. That is, the electrode-supported lipid bilayer membrane functions as an acceptor of cholesterol efflux from the donor membrane (i.e., vesicle lipid bilayer membrane or cell plasma membrane). Detection of cholesterol contained in the lipid bilayer membrane of giant vesicles involves positioning the electrode-supported lipid bilayer membrane in contact with the vesicle membrane. A thin aqueous layer between the electrode-supported lipid bilayer membrane and the vesicle membrane is predicted due to lipid headgroup solvation. Contacting the vesicle positions the electrodesupported lipid bilayer membrane directly adjacent to the vesicle membrane. Because cholesterol has a finite solubility in the aqueous phase,7 the electrode-supported lipid bilayer membrane containing cholesterol oxidase consumes aqueous-phase cholesterol that is directly adjacent to, and in equilibrium with, the vesicle membrane. This consumption of aqueous-phase cholesterol at the vesicle membrane surface causes further cholesterol efflux from the vesicle membrane so that cholesterol mass transport occurs from the high concentration in the vesicle membrane to the lower concentration of the electrode-supported lipid membrane. This mass-transfer model is based on earlier work by Rothblat and coworkers8 that demonstrates the use of lipid vesicles as an aqueousphase acceptor of cellular cholesterol efflux. (5) Devadoss, A.; Burgess, J. D. Langmuir 2002, 18, 9617-9621. (6) Sampson, N. S.; Vrielink, A. Acc. Chem. Res. 2003, 36, 713-722. (7) Haberland, M. E.; Reynolds, J. A. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 2313-2316. (8) Phillips, M. C.; Johnson, W. J.; Rothblat, G. H. Biochim. Biophys. Acta 1987, 906, 223-276.

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EXPERIMENTAL SECTION Microelectrode Fabrication. Platinum microelectrodes were either purchased from Cypress systems (10-µm diameter) or fabricated in-house (11.5-µm-diameter wire, Goodfellow Corp.) using a modified procedure reported to construct microelectrodes with carbon fiber.9 Glass capillaries (Kimax-51, Kimble products) are pulled in a hydrogen flame, and platinum wire is inserted. One end of the glass capillary containing the platinum wire is placed inside a heated platinum coil, and the other end is connected to a weight (e.g., 10 g) so that the glass is pulled to create a thin insulating layer on the platinum wire. The capillary microelectrodes are polished using a beveling machine (WPI, Inc.) to produce a disk electrode. Conductive epoxy (Chemtronics, USA) was used to make electrical contact between the platinum wire and stainless steel lead (syringe needle) at the open end of the capillary. The outer diameter of the glass platinum electrodes usually lies in the range of 15-20 µm. It is noted that the overall dimension of the electrode needs to be small so that cells and vesicles can be contacted. Instrumentation. Voltammetric and amperometric measurements for solution-phase cholesterol detection were conducted using a three-electrode cell and a potentiostat (CV-50, BAS) coupled with a preamplifier (PA-1, BAS). Amperometric measurements at giant vesicles were conducted using a two-electrode cell and a voltammeter-amperometer (Chem-Clamp, Dagan corp.). The three-pole bessel filter in the voltammeter-amperometer was set to 100 Hz. The filtered response was further processed using a noise-rejecting voltmeter (model 7310 DSP, Signal Recovery Inc.) to digitally filter 60-Hz noise. An Ag/AgCl (1 molar KCl) reference electrode was used for all experiments, and the applied potential was 820 mV versus NHE. Electrode modification procedure. The platinum microelectrodes are immersed in a 40 µM ethanolic solution of 1,2 dipalmitoyl-sn-glycero-3-phosphothioethanol (Avanti Polar Lipids Inc.) (referred to as thiolipid) for 3 h to form a covalently attached submonolayer. The microelectrode modified with a thiolipid submonolayer is immersed in a 1 mM 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC; Avanti Polar Lipids Inc.) vesicle solution (sodium phosphate buffer, 50 mM, pH 7.4) for 15 min to form a lipid membrane consisting of bilayer structures on the electrode. The lipid bilayer-modified electrode is immersed in 2.5 mg/mL cholesterol oxidase (Wako Pure Chemical Industries, Ltd., 3.6 units/mg) solution for at least 18 h to immobilize the enzyme in the lipid bilayer membrane. Quiet Solution Experiments. Quiet solution experiments are conducted in a glass cell. The solutions were introduced into the cell using a six-way (Rheodyne model 5020, Supelco Corp.) valve and two syringes, controlled manually. The electrode is placed in 10 mL of the buffer solution (0.05 M sodium phosphate, pH 6.5, 50 mM cyclodextrin; Cargill Inc.), and a stable background current is established. A 10-mL buffered cholesterol solution (50 µM) is injected into the cell. The injected volume is removed, reinjected, and removed again for complete mixing so that the final concentration is 25 µM. For consecutive dilutions, 10 mL of the buffer is injected, mixed, and removed to decrease the concentration by half. (9) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem. 1986, 58, 2088-2091.

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Figure 1. Ferrocyanide voltammetry for deposition of the thiolipid/ lipid bilayer membrane containing cholesterol oxidase on a platinum electrode. Cyclic voltammetry of potassium ferrocyanide at (A) a bare platinum microelectrode, (B) after deposition of thiolipid, (C) after deposition of DPPC, and (D) after incorporating cholesterol oxidase.

Solution Flow Experiments. Flow injection experiments were conducted in a Lucite cell constructed in-house. Experiments were conducted using a six-way valve (Rheodyne model 5020, Supelco Corp.) and two syringe pumps (Harvard Apparatus) to direct buffered cyclodextrin or buffered cyclodextrin containing cholesterol over the electrode. Injection times were controlled manually. Giant Vesicle Experiments. The procedure for electroformation of giant lipid vesicles is adopted from a method published by Menger and Angelova.10 L-R-Phosphatidylcholine from soybean (1 mg/ml) and cholesterol (both from Sigma) were dissolved in chloroform/methanol (9:1) to form a lipid mixture. Molar ratios of cholesterol to lipid were 0.33, 0.5, and 0.66 to prepare vesicles with various cholesterol content. A 2-µL aliquot of the lipid mixture was allowed to dry on 250-µm-diameter platinum wires in a vacuum chamber. The platinum wire containing the dried lipid film was placed on a clean glass slide. The dried lipid film was rehydrated using Tris-HCl buffer (2 mM, pH 6.5), under an applied ac voltage (10 Hz) with an initial peak-to-peak amplitude of 0.2 V that was increased to 1 V over ∼15 min. The vesicle growth on platinum was monitored under an inverted microscope. About 50% of the experiments produced vesicles of 20-70-µm diameter. The vesicle bilayer membrane is expected to be unilamellar10 and in the LR state, where the lipids are present in a loosely packed disordered state.11 The microelectrode was initially positioned ∼20 µm from the vesicle membrane and was repositioned for contact where slight vesicle deformation was observed. It was noted that the experiment must be isolated from mechanical vibrations to maintain contact between the electrode and the vesicle. RESULTS AND DISCUSSION Figure 1 (trace A) shows the cyclic voltammogram of ferrocyanide at a bare platinum microelectrode. The steady-state current is slightly higher than that expected for a disk electrode embedded in an infinite insulating plane (e.g., 5.5 nA for reaction of 5 mM ferrocyanide at an 11.5-µm-diameter electrode). The increased limiting current is expected due to the thin glass (10) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789-797. (11) Brown, D. A.; London, E. J. Biol. Chem. 2000, 275, 17221-17224.

capillary and diffusion from the back of the electrode plane.12,13 The limiting current value of 6-7.5 nA is consistent with an outer diameter of the glass capillary of 15-20 µm. This value agrees well with the size of the capillary under optical magnification. Figure 1 (trace B) is the cyclic voltammogram of ferrocyanide after the platinum microelectrode was reacted with thiolipid. The decrease in the limiting current (∼10%) suggests the formation of a submonolayer of thiolipid on the electrode surface. The distribution of thiolipid molecules on the electrode surface is not known, and the structure of the thiolipid submonolayer could change upon subsequent deposition of DPPC. The dependence of electrode response on the degree of thiolipid coverage has not been rigorously characterized. However, it is noted that a nearcomplete monolayer coverage of thiolipid does not allow construction of functional oxidase-modified electrodes. High coverages of thiolipid likely block facile electrooxidation of the hydrogen peroxide that is generated by the enzyme. Figure 1 (trace C) shows ferrocyanide voltammetric data after deposition of DPPC on the thiolipid submonolayer-modified platinum electrode. The further decrease in limiting current suggests deposition of DPPC molecules on the electrode. The positive shift of the half-wave potential after deposition of DPPC suggests a decrease in electron-transfer rate. This is consistent with regions of the electrode containing a lipid coverage that allows reaction of ferrocyanide at an increased distance of closest approach compared to bare platinum. The smaller limiting current measured after DPPC deposition indicates that ∼50% of the platinum surface is blocked from reaction with ferrocyanide. These microelectrode data are similar to those from ferrocyanide studies conducted for constructing this architecture on conventionally sized platinum electrodes.4 Quartz crystal microbalance (QCM) data obtained at conventionally sized (0.2 cm2) platinum electrodes suggest that some regions of the electrode are covered with a lipid multibilayer structure (a multilamellar coating).4 Ferrocyanide characterization studies conducted at the QCM platinum electrodes modified with the thiolipid/lipid bilayer membrane show incomplete blocking, indicating that a fraction of the electrode remains unmodified. However, deposition of the thiolipid/lipid bilayer membrane shows a mass increase that is roughly that expected for a complete bilayer membrane (∼99 ng, footprint of DPPC assumed to be 60 Å2) that covers the entire electrode surface. Taken together, these data suggest that regions of the electrode are modified with a multilamellar structure. It is noted that atomic force microscopy (AFM) images for vesicle fusion on mica show multilamellar structures (manuscript in preparation). It is hypothesized that the platinum microelectrodes are modified with a lipid bilayer structure that consists, in part, of mutlilammellar lipid islands as well as defects where bare platinum remains exposed to solution. Exposing the lipid bilayer-modified electrode to enzyme solution further decreases the limiting current for reaction of ferrocyanide (Figure 1, trace D). The data are consistent with 15-20% of the platinum electrode remaining unmodified and exposed to solution. One possible explanation for this result is adsorption of cholesterol oxidase at defect sites (bare platinum not modified with a lipid bilayer). While some adsorption of enzyme to bare (12) Amphlett, J. L.; Denuault, G. J. Phys. Chem. B 1998, 102, 9946-9951. (13) Shao, Y.; Mirkin, M. V. J. Phys. Chem. B 1998, 102, 9915-9921.

Figure 2. Amperometric response obtained for an exposure of 25 µM cholesterol solution at (A) an oxidase-modified electrode and (B) the same electrode with thiolipid/lipid membrane prior to immobilization of the oxidase. The up arrow (v) indicates the times of cholesterol exposure.

Scheme 1. Detection Scheme at Cholesterol Oxidase-Modified Platinum Electrode

platinum likely occurs, another possible reason for the increased blocking of ferrocyanide is that a larger fraction of the electrode surface becomes coated with a lipid bilayer structure upon exposure to enzyme. It is noted that direct adsorption of cholesterol oxidase on bare platinum does not produce electrodes that exhibit enzymatic activity. This result indicates that interaction between the enzyme and the electrode-supported lipid bilayer membrane is required for retention of enzymatic activity. It is hypothesized that exposure of the lipid-modified electrode to enzyme solution results in destabilization of multilamellar lipid islands and an increase of the electrode surface area that is coated with a lipid bilayer membrane. This hypothesis is supported by AFM studies that show disruption of multilamellar lipid structures on mica upon exposure to cholesterol oxidase solution. The AFM study for immobilization of the enzyme in lipid bilayer membranes on mica suggests that cholesterol oxidase is immobilized as monomers and aggregates that are 50-100 nm in diameter. It is understood that the lipid bilayer structures formed on mica may not be relevant to those formed on platinum electrodes initially modified with a submonolayer of thiolipid. Nevertheless, an electrode architecture is proposed where the surface is covered predominately with a lipid bilayer membrane containing islands of immobilized cholesterol oxidase. Figure 2, trace A, is the amperometric response observed at an oxidase-modified electrode upon exposure to cholesterol solution where a quiet buffer solution is spiked with an aliquot of buffered cholesterol and allowed to become quiet. The control experiment (Figure 2, trace B) was conducted at the same microelectrode prior to immobilization of cholesterol oxidase. Scheme 1 shows the membrane reaction for enzymatic oxidation of cholesterol by molecular oxygen and generation of hydrogen peroxide (labeled Membrane), and the electrode reaction for electrochemical oxidation of hydrogen peroxide regenerating molecular oxygen (Electrode). It is hypothesized that unmodified regions of the electrode surface (i.e., 15-20%) provide sites where hydrogen peroxide predominantly reacts. In an earlier report from this group, it was demonstrated that the limiting current observed at conventionally sized cholesterol Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

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Figure 3. Amperometric responses obtained at an oxidase-modified electrode for exposure to cholesterol solution and sequential dilutions. The up arrow (v) indicates the time of cholesterol exposure, and the down arrows (V) indicate the times of buffer dilution.

oxidase-modified electrodes is significantly diminished under anaerobic conditions.4 Diminished responses are also observed for the microelectrodes under anaerobic conditions, and these data suggest that Scheme 1 is a dominant mechanism involved in the passage of anodic current. A number of factors may contribute to the limiting current observed (e.g., mass transfer of cholesterol to the electrode, rate of cholesterol partitioning into the membrane, lateral diffusion rate of cholesterol and possibly of the enzyme in the membrane, kinetics of the enzymatic reaction, buildup of cholestenone (oxidized cholesterol) in the electrode-supported lipid bilayer membrane, and fraction of the generated hydrogen peroxide that is oxidized at the electrode surface). However, in the range of current densities where response correlates with cholesterol concentration (∼0.1-2 µA/cm2), it has been demonstrated that electrode response is strongly dependent on the amount of enzyme immobilized on the electrode.1 Ferrocyanide characterization studies and experiments for reaction of cholesterol at the electrodes indicate that complete immobilization of enzyme requires exposure to enzyme solution for more than 10 h (data not shown). On the basis of these data, it is hypothesized that the rate of enzymatic oxidation of cholesterol is rate limiting (i.e., the reaction is not limited by mass transfer of cholesterol to the electrode). The steady-state response (over 15 min) suggests that cholestenone (oxidized cholesterol) does not significantly accumulate in the electrode-supported lipid bilayer membrane and affect the rate of enzymatic catalysis. Under steady-state turnover, cholestenone is likely released from the membrane into the cyclodextrin solution. It is noted that cholestenone is known to efflux at a faster rate than cholesterol from lipid membranes due to weaker interaction with lipids.14 The dependence of electrode response on cholesterol concentration is demonstrated by initially spiking the bulk solution with an aliquot of cholesterol solution and subsequently diluting by spiking with buffer (Figure 3). The steady-state responses indicate a clear correlation with cholesterol concentration (1.6-12.5 µM) where higher cholesterol concentration produces larger responses. The concentration dependence of electrode response to cholesterol is also shown for flow injection-type experiments (Figure 4), where a continuous solution flow is changed from buffered cyclodextrin to buffered cyclodextrin containing choles(14) Ohvo, H.; Slotte, J. P. Biochemistry 1996, 35, 8018-8024.

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Figure 4. Flow injection data at an oxidase-modified electrode for exposure to three different cholesterol concentrations. The up arrows (v) indicate the times of cholesterol injection, and the down arrows (V) indicate the times where the flow is reverted to buffer.

Figure 5. Photographs showing an electrode (A) positioned ∼15 µm away from a giant lipid vesicle and (B) contacting the giant lipid vesicle.

terol and back to buffered cyclodextrin. Data for three cholesterol concentrations (100, 50, 25 µM) are shown along with a replicate exposure to 25 µM. These data also indicate a clear correlation between response and cholesterol concentration. The flow injection data demonstrate baseline resolution between the individual cholesterol exposures. The response rise time and decay time are likely influenced by hydrodynamic factors. Due to its low aqueous solubility, cholesterol in biological systems largely resides in lipid membranes, (e.g., the cell plasma membrane and lipid monolayer shell of lipoproteins). Here, vesicle lipid bilayer membranes were employed as a simple model of the cell plasma membrane. The optical micrographs shown in Figure 5 demonstrate the ability to contact a vesicle with a microelectrode (contact is defined as the electrode position that slightly deforms the spherical shape of the vesicle). It is noted that on contacting the vesicle of interest (Figure 5, image B) other vesicles in the vicinity move, which brings a different set of vesicles in focus (compare Figure 5A and B). Various electrode responses for contacting vesicles with a 0.5 cholesterol-to-phopholipid ratio are shown in Figure 6. Figure 7 shows two control experiments (trace A, response of a oxidase modified electrode at vesicle containing no cholesterol; trace B, response of a bare platinum electrode at vesicle containing 0.5 cholesterol-to-phospholipid ratio). The anodic current responses observed at the oxidase-modified electrodes (Figure 6) are assigned to detection of cholesterol present in the vesicle lipid bilayer membrane. It is not yet known if cholesterol present in the inner leaflet of the vesicle lipid bilayer membrane significantly contributes to electrode response. The rate of cholesterol flip-flop (transbilayer

Figure 8. Amperometric responses of an oxidase-modified electrode for two consecutive contacts at the same giant vesicle formed with 0.66 cholesterol-to-lipid ratio. The up arrows (v) indicate the times of contact, and the down arrows (V) indicate the times of withdrawal.

Figure 6. Amperometric responses obtained at three different oxidase-modified electrodes for contacting three different giant lipid vesicles prepared with 0.5 cholesterol-to-phospholipid ratio. The up arrows (v) indicate the times of contact.

Figure 7. Control experiments for (A) contacting a giant vesicle formed with no cholesterol with an oxidase-modified electrode and (B) contacting a giant vesicle with 0.5 cholesterol-to-phospholipid ratio with a bare platinum electrode. The up arrows (v) indicate the times of contact, and the down arrows (V) indicate the times of withdrawal.

movement) across the lipid bilayer membrane is not known and discrepancies are found in the literature.15 However, sum frequency generation experiments for tracking transbilayer movement of phospholipid16 and cholesterol (John C. Conboy, private communication) suggest that cholesterol flip-flop occurs with a t1/2 of less than 1 min. This notion is consistent with work by others for gauging cholesterol flip-flop in cell plasma membranes.15 Therefore, it is proposed that cholesterol is replenished to the electrode contact site by both transbilayer movement and lateral diffusion. The amount of cholesterol predicted to be contained in the region of the outer lipid leaflet that is in contact with the platinum disk electrode (100 amol of cholesterol in the electrode footprint region of the outer lipid leaflet) corresponds to 20 pC (assuming (15) Haynes, M. P.; Phillips, M. C.; Rothblat, G. H. Biochemistry 2000, 39, 45084517. (16) Liu, J.; Conboy, J. C. J. Am. Chem. Soc. 2004, 126, 8376-8377.

2 electrons/molecule of cholesterol and complete oxidation of the generated hydrogen peroxide).1 The data shown in Figure 6, trace A, indicate oxidation of ∼35% of this amount of cholesterol (100 amol corresponds to 20 pC,1 and 0.35 pA over 20 s corresponds to 7 pC). The lack of a continuous response decay (i.e., the steadystate responses) suggests that the cholesterol content of the vesicle membrane at the electrode contact site is not significantly depleted of cholesterol. It is speculated that the apparent steadystate responses reflect lateral diffusion of cholesterol in the vesicle membrane to the electrode contact site as well as transbilayer movement of cholesterol from the inner lipid leaflet to the outer lipid leaflet. Assuming replenishment of cholesterol to the electrode contact site through only lateral diffusion in the outer lipid leaflet, (in the equation for diffusion to a cylinder electrode, area of the electrode is replaced with circumference and concentration with mol/cm2)17 a flux that would sustain a ∼9-pA response is predicted (assuming a cholesterol content of 0.5 cholesterol/ phospholipid ratio and a lateral diffusion coefficient of 10-7 cm2/ s). Because cholesterol flip-flop and lateral diffusion likely both contribute to mass transport for replenishment of cholesterol to the electrode contact site, it is reasonable to state that the electrode responses are not mass transport limited. Again, it has been demonstrated that response magnitude depends on the amount of cholesterol immobilized on the electrode surface,1 and this result suggests that the rate of cholesterol exchange between the vesicle membrane and the electrode-supported lipid bilayer membrane is not rate limiting. It is proposed that the responses observed at vesicles are limited by enzyme turnover rate. Sequential experiments for contacting a vesicle with the same oxidase-modified electrode show responses of the same magnitude (Figure 8). This response stability is significant in that it allows evaluation of electrode response at vesicles created with different cholesterol content. Data collected at vesicles containing three different ratios of cholesterol to phospholipid are shown in Figure 9. These data qualitatively show larger responses for increased cholesterol content of the vesicle membrane (0.33-0.66 ratio of cholesterol to phospholipids). This result is consistent with data collected at oocytes showing a dependence of response on plasma membrane cholesterol content.1 However, the 0.33 cholesterol/ phospholipids ratio does not show a discernible response, and the data are basically indistinguishable from control experiments where the vesicle membrane contains no cholesterol. Radhakrishnan and McConnell have shown that the rate of cholesterol efflux from lipid membranes into cyclodextrin solution (17) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980.

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states. Small cholesterol-rich domains (∼10-40 nm rafts) are believed to exist in the plasma membrane of cells.19 While no direct concrete evidence for the existence of rafts in the plasma membrane of living cells has been provided, cholesterol has been shown to phase separate to form large domains in vesicle lipid bilayer membranes of appropriate composition.20 The vesicles used in this work are believed to contain cholesterol in only one phase (i.e., no rafts are expected). However, it is not yet known how cholesterol raft formation may affect the correlation between electrode response and the net cholesterol content of the vesicle membrane. Figure 9. Amperometric responses obtained at an oxidase-modified electrode for contacting three different vesicles containing cholesterolto-lipid ratios of (A) 0.66, (B) 0.5, and (C) 0.33. The arrow (v) indicates the times of contact.

is nonlinear with respect to membrane cholesterol content.18 Their data indicate a sharp increase in the rate of cholesterol efflux when the cholesterol content is increased above the 0.5 cholesterol/ lipid ratio. McConnell proposed a stoichiometric complex where one cholesterol molecule associates with two phospholipids. The activity of cholesterol in the lipid membrane increases sharply when the cholesterol content is increased above this stoichiometric ratio (i.e., 0.5 cholesterol/phospholipid). This behavior may explain the inability to detect vesicle membrane cholesterol at low cholesterol-to-phospholipids ratios (e.g., 0.33). As stated above, it is proposed that cholesterol oxidation at the electrode is limited by the rate of enzymatic oxidation of cholesterol. This hypothesis implies that the rate of cholesterol exchange between the vesicle membrane and the electrodesupported lipid bilayer membrane is fast relative to the rate of cholesterol oxidation. In this case, an increase in cholesterol content of the vesicle membrane results in a higher concentration of cholesterol in the electrode-supported lipid bilayer membrane and, thus, a faster rate of enzymatic cholesterol oxidation. This model assumes that slow enzymatic consumption of cholesterol in the electrode membrane does not significantly alter the cholesterol content of the electrode membrane. It is further noted that cholesterol contained in each leaflet of the vesicle lipid bilayer membrane may be present in two distinct (18) Radhakrishnan, A.; McConnell, H. M. Biochemistry 2000, 39, 8119-8124. (19) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (20) Kahya, N.; Scherfeld, D.; Bacia, K.; Schwille, P. J. Struct. Biol. 2004, 147, 77-89.

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CONCLUSIONS Platinum microelectrodes modified with a lipid bilayer membrane incorporating cholesterol oxidase allow detection of cholesterol in vesicle lipid bilayer membranes. The ferrocyanide characterization studies show that the platinum electrodes modified with a lipid bilayer membrane containing cholesterol oxidase have ∼15% of the platinum electrode surface unmodified and exposed to solution. The unmodified regions of the platinum electrode may provide sites for facile electrooxidation of the enzymatically generated hydrogen peroxide. Experiments for exposure of the electrodes to cholesterol solution show steadystate responses that are believed to be limited by the rate of enzyme turnover. The data indicate that electrode response correlates with the cholesterol content of the vesicle membrane where higher cholesterol content yields larger responses. This behavior suggests that the electrodes will be useful in characterizing predicted changes in the cholesterol content of the macrophage cell plasma membrane during biosynthesis of highdensity lipoprotein (i.e., HDL). Additional experiments using lipid vesicles as a model of the cell plasma membrane are planned to study lateral diffusion of cholesterol in lipid bilayer membranes and interbilayer and transbilayer movement of cholesterol. ACKNOWLEDGMENT This work was supported by the National Institute of Health (5 R21 EB003925) and the Department of Chemistry, Case Western Reserve University. Helpful discussions with Professors Dan Scherson and Barry Miller are also acknowledged. Received for review July 1, 2005. Accepted August 25, 2005. AC051173F