Anal. Chem. 2000, 72, 6030-6033
Correspondence
Ion Channel Behavior of Supported Bilayer Lipid Membranes on a Glassy Carbon Electrode Zhengyan Wu, Jilin Tang, Zhiliang Cheng, Xiurong Yang,* and Erkang Wang*
Laboratory of Electroanalytical Chemistry and National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China
A new kind of solid substrate, a glassy carbon (GC) electrode, was selected to support lipid layer membranes. On the surface of the GC electrode, we made layers of didodecyldimethylammonium bromide (a synthetic lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We studied the ion channel behavior of the supported bilayer lipid membrane. In the presence of perchlorate anions as the stimulus and ruthenium(II) complex cations as the marker ions, the lipid membrane channel was open and exhibited distinct channel current. The channel was in a closed state in the absence of perchlorate anions. The ion channel is a very important system in biological activities, e.g., conversion of extracellular events into intracellular signals via hormones, transfer of information in the nervous system, and others. The unique feature of ion channels in a biological cell membrane is a selective recognition of substrates and the following amplification of its information by channel switchings: The selective binding of substrates with receptors triggers the opening of an ion-specific channel which allows the permeation of a great amount of ions across the membrane following an electrochemical potential gradient. The property of a ion channel can be utilized as a model for the construction of devices useful for monitoring a large number of biochemicals of clinical, environmental, and agricultural interest or for uses in food and pharmaceutical analysis.1-3 Up to now, the study of ion channel behavior in a biomembrane has aroused the interest of more scientists. A biomembrane is an essential element of all living organisms. All biomembranes are composed of a lipid bilayer intercalated with other constituents such as proteins, carbohydrates, and their complexes of lipid. Simplified models of biomembranes have been the subject of intense study. Many kinds of model membranes have been used in research, including lipid vesicles or liposomes, bilayer lipid membranes (BLMs), Langmuir-Blodgett layers, and * To whom correspondence should be addressed: (fax) 86-431-5689711; (email)
[email protected]. (1) Nikolelis, D. P.; Krull, U. J. Electroanalysis 1993, 5, 539-545. (2) Nikolelis, D. P.; Krull, U. J. Anal. Chim. Acta 1994, 288, 187-192. (3) Nikolelis, D. P.; Siontorou, C. G. Anal. Chem. 1995, 67, 936-944.
6030 Analytical Chemistry, Vol. 72, No. 24, December 15, 2000
so on. The BLM system has been employed extensively as an experimental model of biomembranes,4 since the reconstituted bilayer lipid membrane that separates two solutions was first reported in 1962.5 Studies on the BLM model system have laid the foundations for its potential applications in the field of specific electrodes,6 biosensor devices,7 biomolecular electronic devices8 and solar energy transduction.9 Although BLM systems formed by the conventional method proved to be very useful, they have one major deficiency, namely, stability. Due to the nature of the thin membrane and the conventional BLM apparatus providing very unstable support, the lipid layer had a high sensitivity to mechanical disturbances in the laboratory environment and could unexpectedly be ruptured at any time. Therefore, it is of limited use for protracted studies and for practical applications. There is a stringent demand to develop systems that reinforce such a fragile structure without interfering with its functional aspects. Recently, it was demonstrated that some BLMs supported on solid substrates, such as metal,10-12 hydrogel,13 Si, and polymer,14 had the requisite dynamic properties and mechanical stability. The mechanical stability of the supported BLM (s-BLM) was greatly improved by several orders of magnitude and its dynamic properties remain intact for a long time.10 Because the s-BLM has been proved to be very useful and easy to work in the field of membrane research and has solved the shortcomings of the conventional BLM, supported bilayer lipid membranes on solid substrates will be an ideal model for investigating the ion channel behavior of biomembranes. In the present study, we select a new kind of solid substrate, the glassy carbon (GC) electrode, to support lipid layer membranes because the GC electrode is widely used in electrochemical (4) Tien, H. T. Bilayer Lipid Membranes (BLM): Theory and Practice; Marcel Dekker: New York, 1974. (5) Muller, P.; Rudin, D. O.; Tien, H. T.; Wescott, W. C. Nature 1962, 194, 979-980. (6) Milazzo, G. Bioelectrochemistry and Bioenergetics; Wiley: New York, 1983; Vol. 5, p 157. (7) Zviman, M.; Tien, H. T. Biosens. Bioelectron. 1991, 6, 37-42. (8) Kutnik, J.; Tien, H. T. Photochem. Photobiol. 1987, 46, 413-419. (9) Tien, H. T. Prog. Surf. Sci. 1989, 30, 1-199. (10) Tien, H. T.; Salamon, Z. Bioelectrochem. Bioenerg. 1989, 22, 211-218. (11) Wardak, A.; Tien, H. T. Bioelectrochem. Bioenerg. 1990, 24, 1-11. (12) Martynski, T.; Tien, H. T. Bioelectrochem. Bioenerg. 1991, 25, 317-324. (13) Tien, H. T.; Ottova, A. L. Electrochim. Acta 1998, 43, 3587-3610. (14) Sackman, E. Science 1996, 271, 43-48. 10.1021/ac000764x CCC: $19.00
© 2000 American Chemical Society Published on Web 11/18/2000
experiments. On the surface of the GC electrode, we made layers of didodecyldimethylammonium bromide (a synthetic lipid). From electrochemical impedance experiments, it was demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We selected ClO4- as the stimulus and ruthenium(II) complex cations were useful as marker ions. The supported bilayer lipid membranes on the GC electrode, as a kind of ion-selective sensing membrane, exhibited sensitive permeability changes. The channel current is not only concentration-dependent but also timedependent. EXPERIMENTAL SECTION Reagents. Didodecyldimethylammonium bromide and tris(2,2′-bipyridine)ruthenium(II) were purchased from Acros (Belgium) and used without further purification. Analytical-grade sodium perchlorate was purchased from Beijing Chemical Reagent Factory (Beijing, China). Chloroform was analytical grade. Pure water was used throughout, obtained by means of a Millipore Q water purification set. All other chemicals were of the highest quality and used as obtained. Method for Supported Lipid Layer Formation. Didodecyldimethylammonium bromide was dissolved in chloroform to give a final concentration of 1 mg mL-1. Prior to s-BLM formation, a glassy carbon electrode was polished with 1.0-, 0.3-, and 0.05-µm alumina slurry, respectively, and then sonicated for 1 min in deionized water and acetone successively. Then the GC electrode was immersed in the 0.1 mol L-1 NaOH solution, and the potential was held at 1500 mV for 3 min in order to polarize the electrode. After the electrode was polarized, it was dried under purified nitrogen. Subsequently, a 5-µL aliquot of the lipid solution was dropped onto the surface of the electrode by a microsyringe and the electrode was immediately transferred into the 0.1 mol L-1 KCl solution, in which the supported lipid layer was formed spontaneously. Electrochemical Measurements. A computer-controlled electroanalytical system (model CS-1087, Cypress Systems, Inc., Lawrence, KS) was used to perform cyclic voltammetric (CV) measurements. The apparatus used for electrochemical impedance measurements was composed of a Solartron Co. model 1250 frequency response analyzer coupled with a model 1286 potentiostat via an IEEE interface (National Instruments, Austin, TX) to a 586 personal computer. Impedance measurement was performed in the frequency range from 0.1 to 65 535 Hz with a signal amplitude of 10 mV. All experiments were carried out with a three-electrode system consisting of a Ag/AgCl reference electrode, platinum coils as an auxiliary electrode, and a GC electrode as a working electrode. Impedance measurement was performed in the presence of a 10 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 mol L-1 KCl as a redox probe at the formal potential of the system, E° ) 266 mV. The sample solutions were purged with purified nitrogen for at least 10 min to remove oxygen prior to the beginning of a series of experiments. All experiments were carried out at the room temperature. RESULTS AND DISCUSSION Voltammetric Behavior of the Bare GC Electrode and the GC Electrode Coated with Lipid Membranes. Figure 1a shows
Figure 1. Cyclic voltammograms of GC electrode in 5 mmol L-1 K3[Fe(CN)6] solution containing 1 mol L-1 KCl: (a) bare GC electrode; (b) GC electrode coated with lipid membranes. Scan rate, 100 mV s-1.
the cyclic voltammetric response of the bare GC electrode in 5 mmol L-1 K3[Fe(CN)6] solution containing 1 mol L-1 KCl. After the GC electrode coated with lipid solution had been immersed in the 0.1 mol L-1 KCl solution for ∼0.5 h, it was put into 5 mmol L-1 K3[Fe(CN)6] solution and the cyclic voltammetric response was observed (Figure 1b). Comparing Figure 1b with Figure 1a, we could find a decrease in the amperometric response of the electrode and an increase in the peak-to-peak separation between the cathodic and anodic waves of K3[Fe(CN)6]. So we concluded that [Fe(CN)6]3- could be prevented from reaching the surface of GC electrode to a certain degree, which implied that the supported lipid membranes had been formed on the surface of electrode successfully. Impedance Measurements of the GC Electrode Supported Lipid Membranes. Impedance spectroscopy is an effective method for probing the features of a surface-modified electrode.15,16 The complex impedance can be presented as the sum of the real, Zre, and imaginary, Zim components that originate mainly from the resistance and capacitance of the cell, respectively. Figure 2 illustrates the results of impedance spectroscopy on a bare electrode (a) and a modified electrode (b) with supported lipid membranes in the presence of equimolar 10 mmol L-1 [Fe(CN)6]4-/3- + 0.1 mol L-1 KCl, which are measured at the formal potential of [Fe(CN)6]4-/3-. For the sake of giving more detail information about the impedance property of the membranes, a modified Randle’s equivalent circuit (inset of Figure 2b) was chosen to fit the measured results. The total impedance is determined by several parameters: (1) electrolyte resistance, Rsol; (2) the lipid membranes capacitance, Cm; (3) the lipid membranes resistance Rm (4) the double-layer capacitance, Cdl; (5) charge-transfer resistance Rct; (6) Warburg element Zw. To determine the thickness of the lipid membranes, the capacitance was chosen to show this feature. With the capacitance value of the lipid membrane and an estimate of its dielectric (15) Hall, E. A. H.; Skinner, N. G.; Jung, C.; Szunerits, S. Electroanalysis 1995, 7, 830-837. (16) Ren, X.; Pickup, P. G. J. Electroanal. Chem. 1997, 420, 251-257.
Analytical Chemistry, Vol. 72, No. 24, December 15, 2000
6031
Figure 3. Cyclic voltammeric responses of 0.5 mmol L-1 ruthenium(II) complex cation at the bare GC electrode with 5 mmol L-1 NaCl as a supporting electrolyte (a) and at the GC electrode coated with BLM in supporting electrolytes: (b) 5 mmol L-1 NaCl, (c) 5 mmol L-1 NaClO4, (d) 5 mmol L-1 NaCl. Scan rate, 50 mV s-1.
Figure 2. Complex plane impedance plots in 10 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 mol L-1 KCl at (a) a bare GC electrode; (b) a modified electrode with supported lipid membranes. Inset: modified Randle’s equivalent circuit used to model impedance data in the presence of redox couples.
constant κ, one can estimate the thickness d of the lipid membrane:17
Cm ) 0κ/d
(1)
where 0 is the permittivity of free space. From the eq 1, we show that the thickness of the lipid membranes is ∼2 nm. Because the thickness of the bilayer of didodecyldimethylammonium bromide should be ∼1.93 nm,18 it could be concluded that the membranes of didodecyldimethylammonium bromide formed on the surface of the GC electrode were bilayer membranes. Undoubtedly, we constructed an ideal model of biological membrane on the surface of GC electrode successfully. Ion Channel Behavior of the Supported Bilayer Lipid Membranes. As soon as the electrode coated with BLM was immersed in 0.5 mmol L-1 tris(2,2′-bipyridine)ruthenium(II) solution containing 5 mmol L-1 NaCl, the CV response of the electrode was recorded. Figure 3b shows the electrochemical (17) Plant, A. L. Langmuir 1999, 15, 5128-5135. (18) Einaga, Y.; Sato, O.; Iyoda, T.; Fujishima, A.; Hashimoto, K. J. Am. Chem. Soc. 1999, 121, 3745-3750.
6032 Analytical Chemistry, Vol. 72, No. 24, December 15, 2000
characteristics of the electrode. The suppression of CV peaks of the ruthenium(II) complex cation appears to be due to the closed channels of the lipid membranes. Relatively bulky ruthenium(II) complex cations are not allowed to permeate through the closed channel toward the underlying electrode. When the electrode was immersed in 0.5 mmol L-1 tris(2,2′-bipyridine)ruthenium(II) solution containing 5 mmol L-1 NaNO3 and 5 mmol L-1 Na2SO4, respectively, the electrochemical behaviors of the electrode were almost the same as that of the electrode in solution containing 5 mmol L-1 NaCl. Compared with the electrochemical behavior of the bare GC electrode in Ru(bpy)32+ solution (Figure 3a), it can be seen that the ion channels of the membranes are almost in a closed state. The cyclic voltammetric response of the ruthenium(II) complex cation when the electrode was immersed in 0.5 mmol L-1 tris(2,2′-bipyridine)ruthenium(II) solution containing 5 mmol L-1 NaClO4 is shown in Figure 3c. A distinct CV response from the ruthenium(II) complex cation was gained as if the lipid membranes were “leaking” and the CV peaks of the marker ion appeared at almost the same potential value as those observed at the bare GC electrode. It is clear that the perchlorate anions can stimulate the ion channels of the membranes to be in an open state. In the absence of perchlorate anions, the channels were in a closed state due to the regular alignment of the lipid quaternary ammonium. The channels were open in the presence of perchlorate anions because perchlorate anions could form ion association compounds with the lipid quaternary ammonium cations in the bilayer membranes19 and change the regular alignment of the lipid; thus, the ion channels in the membranes were formed and open. When the electrode was transferred into the 0.5 mmol L-1 tris(2,2′bipyridine)ruthenium(II) solution containing 5 mmol L-1 NaCl, the channels were found in a closed state again (Figure 3d). When the electrode was put into the tris(2,2′-bipyridine)ruthenium(II) solution containing NaClO4, the channels were open a second time. The reversible open-close processes could be repeated many times. With the presence of perchlorate anions as the stimulus and ruthenium(II) complex cation as the marker ion, the cyclic (19) Sugawara, M.; Koichi, K.; Hiroyuki, S.; Umezawa, Y. Anal. Chem. 1987, 59, 2842-2846.
Figure 4. Cyclic voltammeric responses of 0.5 mmol L-1 ruthenium(II) complex cation at the GC electrode coated with BLM in different concentrations of NaClO4: (1) 0, (2) 0.4, (3) 0.8, (4) 1.2, and (5) 1.6 mmol L-1. Supporting electrolyte, 5 mmol l-1 NaCl. Scan rate, 50 mV s-1.
voltammetric responses of the supported bilayer lipid membranes constructed by didodecyldimethylammonium bromide with different concentrations of perchlorate anions were tested. The intensity of the peak current increased with the concentration of perchlorate anions (Figure 4). This can be attributed to two aspects: one is the opening gate of the channel through which the ruthenium(II) complex cation can permeate to the underlying electrode to be detected electrochemically, and the other is the increasing amount of the marker ions that can permeate through the channel toward the electrode. Furthermore, it is of interest to note that the channel behavior is not only concentrationdependent but also time-dependent. Figure 5 shows the current response of the channel as a function of time. The peak current of the ruthenium(II) complex cation increased with time and reached steady state after 80 min. As evidenced by our results described above, we can draw the conclusion that the supported bilayer lipid membranes have a function of channel switching by the following sequence: (i) In the absence of a stimulus (analyte), the channel is closed, and therefore, marker ions (monitoring ions) cannot permeate through the membrane. (ii) With the stimulus present, the channel is
Figure 5. Cathodic peak currents of 0.5 mmol L-1 ruthenium(II) complex cation through the channel as a function of time with 1.6 mmol L-1 NaClO4 as stimulator. Supporting electrolyte, 5 mmol L-1 NaCl. Scan rate, 50 mV s-1.
opened, and a large amount of marker ions are allowed to permeate through the membrane, which are immediately detected electrochemically at the underlying electrode. CONCLUSIONS In the present paper, bilayer lipid membranes on the surface of a GC electrode were made. The ion channel behavior of the membrane was discussed. The channel was formed and open in the presence of stimulus. The intensity of the channel current increases with the concentration of stimulus. In the absence of stimulus, the channel was in a closed state. The reversible openclose processes could be repeated on many times. ACKNOWLEDGMENT We thank Professor T. Kuwana (University of Kansas) for donating the CS-1087 electroanalytical system. This work was supported by the National Natural Science Foundation of China.
Received for review July 5, 2000. Accepted October 3, 2000. AC000764X
Analytical Chemistry, Vol. 72, No. 24, December 15, 2000
6033
