Impedance Spectroscopy of Porin and Gramicidin Pores Reconstituted

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Langmuir 1998, 14, 3118-3125

Impedance Spectroscopy of Porin and Gramicidin Pores Reconstituted into Supported Lipid Bilayers on Indium-Tin-Oxide Electrodes† Stefan Gritsch,‡ Peter Nollert,§,| Fritz Ja¨hnig,§,⊥ and Erich Sackmann*,∇ Biophysics Laboratory, Physik Department E22, Technische Universita¨ t Mu¨ nchen, D-85747 Garching, Germany, Department of Membrane Biochemistry, Max Planck Institut fu¨ r Biologie, Corrensstrasse 38, D-72076 Tu¨ bingen, Germany, and Lehrstuhl fu¨ r Biophysik E22, Technische Universita¨ t Mu¨ nchen, D-85478 Garching, Germany Received September 15, 1997. In Final Form: February 23, 1998 We prepared electrolyte-membrane-electrolyte-semiconductor (EMES) interfaces by fusion and hightemperature annealing of positively charged lipid vesicles (containing 49 mol % cholesterol and 42 mol % lecithin besides 9 mol % positively charged lipid) on optical transparent indium-tin-oxide (ITO) semiconductor electrodes. Membrane resistances of up to 109 kΩ cm2 were reached. By conductivity measurements in the presence of the redox couple K3Fe(CN)6/K4Fe(CN)6 the area fraction of defects exhibited in the supported membranes was determined to be less than 0.0001. We show that by measurement of the impedance over a large frequency range (10-1-105 Hz) it is possible to discriminate between changes (1) of the capacitance of the ITO electrodes, (2) the membrane capacitance, and (3) the membrane conductivity of the sensor device. The membrane pore gramicidin and the outer membrane proteins OmpF and OmpA (from E. coli) where reconstituted into the supported membranes by transfer from vesicles. The functionality and selectivity of gramicidin and OmpF in supported membranes are demonstrated by measuring the membrane resistance in the presence of pores for various electrolyte compositions.

I. Introduction The design of biosensors based on supported membranes on semiconductor devices and signal detection through current or capacitance measurement1,2 is still an issue of great interest despite of the slow process made in this field. If one is mainly interested in sensitivity, the most promising strategy for the design of biosensors appears to be the immobilization of excitable cells or cellular networks as self-amplifying alarm systems on microelectrode arrays.3,4 Metabolic activities of cells may be sensitively recorded by light addressable semiconductor devices.5,6 A drawback of such systems is that signal formation is obscure and that it is impossible to relate the signals to distinct processes of the cells. In the present work we studied the frequency dependence of the electrical impedance of an electrolytemembrane-electrolyte-semiconductor (EMES) interface (Figure 1a) consisting of the optical transparent semiconductor indium-tin-oxide (ITO) and a supported mixed lipid bilayer containing a positively charged lipid component and a high cholesterol content. By measuring the electrical impedance over a large frequency range (10-3†

In memory of Fritz Ja¨hnig who died so early. * Corresponding author. Fax: 0049-89-2891-2469. ‡ Physik Department E22, Technische Universita ¨ t Mu¨nchen. § Max Planck Institut fu ¨ r Biologie. | Present address: Biozentrum Basel. ⊥ Deceased. ∇ Lehrstuhl fu ¨ r Biophysik E22, Technische Universita¨t Mu¨nchen. (1) Lindholm-Sethson, B. Langmuir 1996, 12, 3305-3314. (2) Plant, A. Langmuir 1993, 9, 2764. (3) Gross, G. W.; Wen, W. Y.; Lin, J. W. J. Neurosci. Methods 1985, 15, 243-252. (4) Fromherz, P.; Offenha¨usser, A.; Vetter, T.; Weis, J. Science 1991, 252, 1290-1293. (5) Hafeman, D. G.; Parce, J. W.; McConnell, H. M. Science 1988, 240, 1182-1185. (6) Parak, W. J.; Hofmann, U. G.; Gaub, H. E.; Owicki, J. C. Sens. Actuators, A, in press.

105 Hz), we attempt (1) to gain better insight into the physical basis of the electrical signal transduction and (2) to explore the attainable sensitivities and potential applications of such devices. The application of impedance spectroscopy enabled the distinctions between changes (1) of the capacitance of the semiconductor electrode, (2) of the membrane conductivity, and (3) of the membrane capacitance. It is also shown that variations of the surface charge of the membrane may affect the capacitance of the ITO space charge remarkably, which is attributed to changes of the surface pH. The major thrust of the present work was to reconstitute pore-forming proteins such as the polypeptide gramicidin and the outer membrane proteins OmpF and OmpA from E. coli bacteria into preformed supported membranes of high electrical resistivity. It is shown that this is possible by transfer of reconstituted pores from vesicles. Sensitivities of 1 OmpF pore trimer per 800 µm2 of electrode surface could be achieved. II. Materials and Methods Chemicals. Dimyristoyl-L-R-phosphatidylcholine (DMPC), dimyristoyl-L-R-phosphatidylethanolamine (DMPE), and cholesterol were commercial products (Avanti Polar Lipids, Alabama, USA). The positively charged lipid dihexadecyldimethylammonium bromide (DHADAB) was purchased from Fluka (Deisenhofen, Germany). Gramicidin D and all other materials were purchased from Sigma (Deisenhofen, Germany). Several types of buffers were used: Na+-Hepes buffer consisting of 10 mM Hepes (4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid), which was adjusted to pH 7.5 by titration with NaOH; Cl--Tris buffer consisting of 5 mM Tris (Tris(hydroxymethyl)aminomethane), which was adjusted to pH 7.5 by titration with HCl. The high-frequency conductivity of these two buffers is approximately equal. The defect area fraction of supported membranes was determined by experiments with Na+-Hepes buffer containing 1 mM of the redox couple K3Fe(CN)6/K4Fe(CN)6. All aqueous solutions were prepared with Millipore water. All buffers were degassed before use.

S0743-7463(97)01038-X CCC: $15.00 © 1998 American Chemical Society Published on Web 04/24/1998

Frequency Dependence of Electrical Impedance

Figure 1. (a) Schematic view of an electrolyte-membraneelectrolyte-semiconductor (EMES) interface. The electrical properties of the stratified interface can be represented by an equivalent circuit consisting of a series-connected arrangement of capacitance-resistance pairs connected in parallel. Each RC pair represents one of the four interfaces. The circuit may be simplified by combining the RC pairs of the semiconductor and of the electrolyte layer between the semiconductor and the membrane, respectively. Similarly, the RC pairs of the membrane and the outer electrolyte layer can be combined. (b) Comparison of the impedance spectra of an electrolytesemiconductor (ES) interface (- - -) and an electrolytemembrane-electrolyte-semiconductor (EMES) interface (-). The latter was generated by deposition of a bilayer consisting of 42 mol % DMPC, 49 mol % cholesterol, and 9 mol % DHADAB. The curves (O) and (+) give the corresponding frequency dependencies of the phase shifts. Fabrication and Cleaning of Electrodes. The ITO electrodes (geometric area 8 mm2) were made out of ITO-covered glass plates (Balzers, Balzers, Switzerland) by etching with a solution of 47.5 vol % HCl (32%) and 5 vol % HNO3 (65%) in water. All electrodes were cleaned twice in 2% Hellmanex solution (Hellma, Germany) and subsequently rinsed several times with water. Immediately before use the electrodes were wiped with a tissue with 0.1 M HCl and 1 M NaOH and were finally rinsed with water. For most measurements two identical ITO electrodes on a single substrate were connected in series, making any additional reference or counter electrode unnecessary. Deposition of Bilayers by Vesicle Fusion and Annealing. A series of experiments showed that high membrane resistances

Langmuir, Vol. 14, No. 11, 1998 3119 of 105 Ω cm2 can be achieved by vesicle fusion using lipid mixtures containing about 40 mol % DMPC, 10 mol % positively charged lipid (DHADAB), and a high cholesterol content of 40 mol % or higher. However, parallel experiments showed that it is also possible to deposit high resistance membranes by using mixtures containing a high content of DMPE instead of cholesterol (such as 44 mol % DMPC, 46 mol % DMPE, and 10 mol % DHADAB) although the resistivities reached were smaller than those characteristic for cholesterol-containing bilayers. All experiments reported here were performed with mixtures of 42 mol % DMPC, 49 mol % cholesterol, and 9 mol % DHADAB. The vesicles were prepared by deposition of a thin lipid film on the wall of a glass flask produced by vacuum evaporation (2 h) of a chloroform solution of the lipid mixtures. Then the buffer was added while the total amount was adjusted to a concentration of 4 mg of lipid/mL of buffer. The closed flask was heated to 70 °C for 30 min and then stored for at least 2 h at 45 °C. Finally the vesicle dispersion was sonicated for 10 min and immediately afterward used for the deposition of the supported membranes. For that purpose the electrode cell was rinsed with the vesicle solution and heated for about 30 min to 60 °C. Then the residual vesicles were removed by rinsing the electrode cell with pure buffer. Protein Purification. OmpF and OmpA were obtained from OmpC-free E. coli strain P400.6 which was kindly provided by U. Henning from the Max-Planck-Institute for Biology, Tu¨bingen, Germany. The proteins were purified without the use of surfactants according to the procedure described by Surrey et al.7 In short, OmpA was extracted from preextracted cell membranes with 2-propanol at 50 °C. The OmpA-depleted membranes were digested by protease. By a subsequent extraction at 75 °C OmpF was extracted. Unfolded OmpA and OmpF were further purified by anion exchange chromatography followed by ultrafiltration and gel filtration, yielding approximately 95% pure protein solution with a concentration of approximately 30 mg/mL in 8 M urea. Reconstitution of Pores into Supported Bilayers. Both gramicidin and porins were transferred into the supported membranes by incubation of the electrode cell with dispersions of vesicles containing the polypeptides. Gramicidin was reconstituted into the vesicles by swelling of dried films of a mixture of lipid and gramicidin. It is known that some solvents used for the vesicle preparation such as chloroform can modify gramicidin to a nonconducting form. According to Cox et al.8 this problem can be overcome by heating the vesicles with the reconstituted gramicidin. For that purpose we annealed the gramicidin/DMPC/ cholesterol vesicles for 30 min at 70 °C, for 2 h at 45 °C, and finally for 30 min at 60 °C. We expect that most of the gramicidin is in the conducting state after this procedure. OmpA and OmpF were reconstituted into lipid membranes following the procedure described by Surrey7 and Eisele.9 The protein to lipid molar ratio was adjusted to 1:200. In brief, 208 µg of OmpF and 183 µg of OmpA, respectively, were dissolved in 2 mL of 10 mM Na+Hepes (pH 7.5) containing 10 mg/mL n-octyl-β-D-glucopyranoside and 0.705 mg of DMPC. The solution was extensively dialyzed at 37 °C against 10 mM Na-Hepes buffer. Before the application to preformed supported planar bilayers the proteoliposome solution was diluted by a factor of 10 and briefly sonicated. Impedance Spectroscopy. The impedance analysis was performed with a Schlumberger 1260 impedance analyzer (Schlumberger Technologies, Farnborough, GB) following Stelzle et al.10 The impedance |Ζ(υ)| and the phase angle Φ(υ) were generally measured over a frequency range of υ ) 10-1-105 Hz at 30 fixed values of the frequency which were equidistant on a logarithmic scale. However, in some cases prolonged experiments were carried out over a frequency range of υ ) 10-3-105 Hz. The measured spectra of the impedance and phase were analyzed in terms of electrical equivalent circuits using an Apple Macintosh and the analysis program IGOR (Wavemetrics, OR). (7) Surrey, T.; Schmid, A.; Ja¨hnig, F. Biochemistry 1996, 35, 2283. (8) Cox, K. J.; Ho, C.; Lombardi, J. V.; Stubbs, C. D. Biochemistry 1992, 31, 1112-1118. (9) Eisele, J. L.; Rosenbusch, J. P. J. Biol. Chem. 1990, 265, 1021710220. (10) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981.

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Gritsch et al.

Table 1. Contributions of Elements of the Equivalent Circuits Defined in Figure 1a to an ES and an EMES Interfacea

ES EMES

RE (kΩ)

RS (Ω cm2)

CS (µF/cm2)

RM (kΩ cm2)

CM (µF/cm2)

9.8 10.2

>5.7 >2.8

5.96 3.88

86.4

0.53

a

With the exception of RE all values are normalized to an area of 1 cm2. The capacitance CM correlates with that measured for similar bilayers with other electrical methods1,2,10 and, by using an electrical constant of 2.1, one can calculate a thickness for the dielectric bilayer of 35 Å, which agrees with the double thickness of the hydrocarbon chain region of DPPC measured by neutron reflection.

III. Impedance Spectroscopy of Stratified Interfaces Figure 1b shows typical impedance spectra of a bare semiconductor ITO electrode (corresponding to an electrolyte-semiconductor (ES) interface) and of a bilayercovered ITO electrode (corresponding to an electrolytemembrane-electrolyte-semiconductor (EMES) interface). The spectra are interpreted in terms of the equivalent circuit of Figure 1a. The monotone decay of the impedance of the ES interface with frequency can to a first order be well accounted for by a capacitance CS. A slight decrease of the slope at υ < 0.5 Hz and a corroborate parallel increase of the phase suggest that for the exact description the ES interface must be represented by a parallel RSCS element with the impedance:

ZS(υ) )

RS - i2πυCSRS2 (1 + (2πυ)2CS2RS2)

(1)

The element RS can be considered as a simplified Faraday impedance11 of an ES interface in the absence of a redox couple. The frequency-independent behavior of the impedance at high frequencies (υ > 103 Hz) is determined by the combined resistances of the electrolyte and the wire connections. It is represented by a resistance RE. The total impedance of the ES interface can thus be well represented as

ZES(υ) ) RE +

RS - i2πυCSRS2 (1 + (2πυ)2CS2RS2)

(2)

Fitting of the measured ZES(υ) to eq 2 yields the values of RE, RS, and CS given in Table 1. In the presence of supported membranes the Z(υ) spectrum is shifted to higher frequencies at υ < 0.4 Hz. It exhibits a pronounced shoulder at 0.4 Hz < υ < 30 Hz and reaches nearly the same value at υ > 5 × 103 Hz as the bare ITO electrode. The shoulder can be well represented by a parallel circuit composed of the membrane resistance RM and the membrane capacitance CM making up the membrane impedance ZM(υ). Considering eq 2 the total impedance of an EMES interface can be written as

ZEMES(υ) ) ZES(υ) + ZM(υ) ) RE +

RS - i2πυCS′RS2 (1 + (2πυ)2CS′2RS2)

+

RM - i2πυCMRM2 (1 + (2πυ)2CM2RM2)

(3)

Fitting of the measured ZEMES(υ) to eq 3 yields the values (11) Bard, A. Electrochemical Methods; Wiley & Sons: New York, 1980.

Figure 2. Effect of gramicidin pores reconstituted into in a supported bilayer on the impedance spectrum of the EMES interface: (-) bare semiconductor (ES interface); (4, solid line) supported membrane in the absence of gramicidin pores with Na+-Hepes buffer; (4, dotted line) same in the presence of Cl--Tris buffer; (O, solid line) supported membrane after transfer of gramicidin in the presence of Na+-Hepes buffer; (O, dotted line) effect of gramicidin pores in the presence of Cl--Tris buffer.

of RE, RS, CS, RM, and CM given in Table 1. The parallel shift of the impedance at low frequency υ < 0.4 Hz can be attributed either to a change in the space charge distribution of the ITO which could be caused by a variation of the pH at the ES interface as shown below or to a change of the capacitance of the electrolyte layer between the supported bilayer and the ITO surface. Formally one can simply account for the parallel shift of the impedanceversus-frequency curve to higher frequencies by reducing the capacitance CS (eq 2) to CS′ (eq 3). For the case of Figure 1b the capacitance CS is reduced from 5.96 to 3.88 µF/cm2. Below we will provide evidence for this conclusion by showing that the reduction of the resistance of the supported membrane by incorporation of pores results in a lowering of the height of the shoulder while the lowfrequency shift remains. IV. Effect of Gramicidin Channels in Supported Membranes of EMES Interfaces Figure 2 shows the modification of the impedance of an EMES interface after reconstitution of gramicidin channels in the presence of different buffers. Gramicidin (0.97 mol %) was reconstituted into DMPC/cholesterol vesicles, and then the measuring cell with the supported membrane was incubated with the vesicle dispersion for 17 min. Impedance spectra were recorded in the presence of Na+Hepes buffer and Cl--Tris buffer, respectively. In the presence of Na+ ions the height of the shoulder of the Z(υ)-vs-υ curve is drastically reduced, clearly showing that the membrane resistance RM is strongly reduced. In the presence of Cl- ions, however, the membrane resistance RM is not changed appreciably compared to the value of RM in the absence of gramicidin. This is attributed to the very low conductivity of the gramicidin channels for Clions demonstrating the ion selectivity of the gramicidin dimers in the supported membrane. Since the compositions of the buffers were adjusted in such a way that their equivalent conductivities were approximately equal, Z(υ) is not varied at υ > 5 × 103 Hz by changing the buffer. Figure 2 also shows that the membrane capacitance CM is not changed remarkably in the presence of gramicidin channels in the supported bilayer.

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Table 2. Summary of Membrane Resistances of an EMES Interface for Different Buffers after Incubation of the Measuring Cell with Vesicle Dispersions Containing 0.97 mol % Gramicidin in the Presence (3rd and 4th row) and Absence (last two rows) of Positively Charged Lipid in the Vesicles Na+/Hepes Cl-/Tris (kΩ cm2) (kΩ cm2) EMES interface incubation with DMPC/cholesterol/DHADAB vesicles incubation with DMPC/cholesterol/DHADAB vesicles + containing 0.97 mol % gramicidin incubation with DMPC/cholesterol vesicles incubation with DMPC/cholesterol vesicles containing 0.97 mol % gramicidin

Figure 3. Time dependence of the membrane resistance RM after addition of DMPC/cholesterol vesicles containing 0.97 mol % gramicidin: (O) measured membrane resistance; (-) singleexponential fit with a relaxation time of 118 s.

In separate experiments we tested the effect of DMPC/ cholesterol vesicles without gramicidin. No appreciable changes of Z(υ) were observed, demonstrating that the decrease of the membrane resistance is indeed caused by the gramicidin. In a further experiment we found that rinsing with positively charged vesicles exhibiting the same lipid composition as the supported bilayer and containing gramicidin did not result in a change of the membrane impedance ZM(υ). This is attributed to the repulsion of the equally charged membranes and shows that approach of the membranes to distances smaller than the Debye length is required for the transfer of gramicidin. The results are summarized in Table 2. The kinetics of the transfer of gramicidin has been studied by analysis of the membrane resistance as a function of time during incubation of the measuring cell with vesicles. An example is shown in Figure 3. The decay can be well represented by an exponential fit exhibiting a relaxation time of 118 s. Within the observed time range of 17 min the measured membrane resistance RM does not reach a true equilibrium. Therefore the gramicidin concentration in the supported membrane is expected to differ from that of the vesicles, which are present in excess. The density of conducting gramicidin dimers may be roughly estimated by assuming that the conductivity of a single gramicidin channel in the supported membrane is similar to that in black lipid membranes, which is approximately 0.70 pS for the buffer used in the present work.12 By assuming that this conductivity holds in positively charged supported membranes, the change of RM found in Table 2 would correspond to a lateral density of 1010 cm-2 conducting dimers in the supported membrane. Since the lateral monomer density in the vesicle (12) Hille; Bertil Ionic Channels of Excitable Membranes; Sinauer Associates: Sunderland, 1984.

21.1 26 22.2 18.5 0.5

19.9 16.1 21 23.9 21

4 mM NaCl (kΩ cm2) 12.3 11.9 12.9 13.8 1.6 (4mM CsCl: 0.3)

bilayer was 4.79 × 1012 cm-2, this consideration predicts that about 0.4% of these molecules are active in the supported bilayer. However, the conductivity of gramicidin dimers in the low ionic strength used in the present work depends among other aspects on the charge of the lipid used. For negatively charged membranes the gramicidin conductivity is increased because of the higher cation concentration at the bilayer surface. Consequently the gramicidin conductivity is expected to be smaller in positively charged than in neutral membranes and the above value of the gramicidin dimer density is a lower limit. A different possibility is to estimate the lateral gramicidin dimer density from the ratio of the membrane conductivity to the gramicidin concentration which avoids the use of the unknown dimer conductivity.13 We determined the membrane conductivity to gramicidin concentration ratio by measuring the membrane resistance for various defined gramicidin concentrations in the supported membrane. Supported membranes were prepared by fusion of different vesicles consisting of 42 mol % DMPC, 49 mol % cholesterol, and 9 mol % DHADAB, which contained 0.05, 0.1, 0.5, and 1 mol % gramicidin. Within this concentration range we measured a 61-fold rise of the membrane conductivity for a 10-fold increase of the gramicidin concentration. From these measurements a lateral gramicidin dimer density of 7 × 1011 cm-2 is calculated, which is much larger than the above value of 1010 cm-2. This would correspond to a gramicidin dimer conductivity of 0.02 pS in the supported membrane. For a more exact determination of the lateral gramicidin dimer density in the supported membrane, the dimer conductivity should be measured with other techniques using the same lipid combination. In a further experiment we measured the ratio of the gramicidin conductivity for K+ and Na+ ions. The value of 2.6 measured differs by a factor of 1.45 from the value of 1.8 characterized for Black Lipid Membranes (BLM).12 V. Effect of Porin Pores on EMES Interfaces Figure 4a demonstrates that pore-forming proteins of the outer membrane of E. coli bacteria can be reconstituted into supported membranes in a functional manner by transfer from vesicles. Figure 4a shows the effect of OmpA (nonconducting) and OmpF (conducting) in the supported membrane on the impedance spectra of an EMES interface in the presence of NaCl. The vesicles were composed of DMPC and the lipid-to-protein ratio adjusted for the reconstitution of the protein into the vesicle membranes by dialysis was 200:1. By incubating the measuring cell with vesicles exhibiting reconstituted OmpA for 17 min and after exchanging the buffer with a 4 mM NaCl solution, the membrane resistance is slightly changed to 96% of its initial value (in the presence of 4 mM NaCl). The membrane resistance decreased to 52% of its initial value after the incubation of vesicles with reconstituted OmpF (13) Bamberg, E.; La¨uger, P. J. Membr. Biol. 1973, 11, 177-194.

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Figure 4. (a) Variation of impedance spectra of an EMES interface with a membrane containing nonconducting OmpA and pore-forming OmpF in the presence of 4 mM NaCl solution. (b) Variation of impedance spectra of an EMES interface with a membrane containing OmpA and OmpF, respectively in the presence of Na+-Hepes buffer. (c) Variation of impedance spectra of an EMES interface with a membrane containing OmpA and OmpF in the presence of Cl--Tris buffer.

in the same measuring cell for 17 min and replacing the original buffer again with 4 mM NaCl solution. A similar behavior of the impedance spectra as in the presence of NaCl is observed in the presence of Na+Hepes buffer as shown in Figure 4b. As expected the impedance spectrum after the incubation of vesicles with reconstituted OmpA decreases although the reduction is stronger than that in the presence of NaCl. The impedance of the EMES interface after the incubation of vesicles with reconstituted OmpF is strongly decreased.

Gritsch et al.

A different behavior is observed in the presence of Cl-Tris buffer (Figure 4c). OmpA leads to a pronounced shift of the membrane impedance to higher values whereas after the incubation of vesicles with incorporated OmpF the impedance is reduced again although the membrane resistance is still higher than before the addition of vesicles. Comparison of Figure 4b and Figure 4c shows that the resistance of the membranes containing OmpF is considerably more strongly reduced in the presence of Na+ ions than for Cl- ions demonstrating that OmpF is a selective pore. The experiments shown in Figure 4 clearly demonstrate that both types of outer membrane proteins are transferred into the supported membrane by incubation of the measuring cell with protein-containing vesicles. In the presence of NaCl the membrane resistance RM is only strongly reduced in the presence of OmpF whereas OmpA leads to a very small although appreciable reduction of RM. Such a behavior is expected since it is well-known that only OmpF is a pore-forming protein (as a consequence of the formation of trimers in the membrane) whereas OmpA does not form pores. The change of the membrane resistance is remarkably different for the permeable inorganic ions Na+ and Cl- as demonstrated in Figure 4b and Figure 4c where the inorganic ions are present alone together with organic ions which are not permeable. For Na+ the resistance is reduced by 73% in the presence of OmpF whereas for OmpA the reduction is only of the order of 34%. This suggests that OmpA can cause conducting defects in the supported membrane. The behavior of the membrane resistance is more complex in the presence of Cl- alone. The membrane resistance increases for both OmpA and OmpF after incubation of the measuring cell with the Cl--Tris buffer although the increase is smaller for OmpF. It is wellknown that OmpF is also permeable for Cl- although the conductivity is smaller by a factor of 7 than for Na+.7 Therefore, there must be an additional effect causing the increase of the membrane resistance in the presence of Cl-. One likely explanation is the reduction of the positive surface charge of the supported membrane caused by lipid exchange. This could lead to a reduction of the surface concentration of Cl- and an increase of that of Na+ resulting in a larger ratio (>7) of the pore conductivities of the two ions and a reduction of the conductivity of membrane defects for Cl-. A second reason may be that defects in the supported membrane heal out due to the exchange of lipid with the vesicles. Although we do not understand yet the remarkable difference between Na+ and Cl-, the present experiments show that the electrical impedance of the EMES interface depends on the type of electrolyte. It is tempting to estimate the lateral density (concentration) of functional pores reconstituted into the supported bilayer by the present transfer procedure. The trimer conductivity of active OmpF trimers has been measured for a similar ionic strength by the black lipid membrane technique to be 2.5 nS.14 From the analysis of the impedance spectra, we find a pore-induced membrane conductivity of 3.1 × 10-4 S/cm2. By assuming that the pore conductivity is the same in supported membranes as in free bilayers it follows that the lateral pore density is 1.2 × 105 OmpF trimers per cm2 corresponding to one porin trimer per 800 µm2. (14) Saint, N.; Lou, K. L.; Widmer, C.; Luckey, M.; Schirmer, T.; Rosenbusch, J. P. J. Biol. Chem. 1996, 271, 20676-20680.

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VI. Measurement of the Defect Density by a Redox Couple As shown by several groups the defect density of membrane-covered electrodes can be measured by analyzing the kinetics of the reduction-oxidation reactions of a redox couple at the electrodes. According to Amatore15 small sizes and fine distributions of membrane defects are required. Sabatini et al.16 already studied monolayers of alkanethiols on gold using impedance spectroscopy in the presence of the redox couple K4Fe(CN)6/K3Fe(CN)6. The charge transfer at the ES interface is characterized by the Faraday impedance ZF. In analogy to eq 3 the total impedance ZrEMES(υ) of an EMES interface in the presence of an active redox couple can be expressed as

Z′EMES(ω) ) ZrES + ZM ) RE +

ZF - i2πυCS′ZF2 (1 + (2πυ)2CS′2ZF2)

+

RM - i2πυCMRM2 (1 + (2πυ)2CM2RM2)

(4)

The Faraday impedance ZF can be divided into a (frequency independent) charge-transfer resistance Rct and a Warburg impedance ZW. The former accounts for the charge transfer across the semiconductor surface. The latter accounts for the diffusive transport near the surface. ZW is frequency dependent and can be described by a resistance RW and a capacitance CW in series. Following Bard11 the charge-transfer resistance can be expressed as

RCT ) RT/nFid

(5)

where F is the Faraday constant and id is the exchange current through the defects and across the semiconductor surface. In the case of an EMES interface the current for an applied alternating voltage is limited within a frequency rangeswhere the impedance ZrEMES(υ) is dominated by the charge-transfer resistancesby the fraction of the total area exhibiting conductive defects within the membrane. Therefore the charge-transfer resistance is determined by the area fraction Θ of the electrode surface covered by an ideally insulating bilayer. The defect exchange current can be expressed as

id ) FAckd

(6)

kd ) k0(1 - Θ)

(7)

where A is the electrode area and c ) cred ) cox is the concentration of the redox couple. kd is the rate constant of the electrochemical reaction at the defects of an EMES interface, k0 is the rate constant of the electrochemical reaction at a bare electrode (ES), and (1 - Θ) is the total area fraction covered by defects. Since it is assumed that k0 is constant for an ES and an EMES interface, the area fraction of the defects (1 - Θ) can be calculated from the ratio of the charge-transfer resistances Rct of the ES and the EMES interfaces. Figure 5 shows the impedance spectra of an ES interface and an EMES interface, respectively, in the presence and absence of the redox couple K4Fe(CN)6/K3Fe(CN)6. An EMES interface with a relatively low membrane resistance of 9000 Ω cm2 has been chosen in order to realize an impedance of about 107 Ω at low frequencies. Since the (15) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39-51. (16) Sabatini, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 66636669.

Figure 5. Effect of redox couple on impedance spectra of ES and EMES interfaces measured in the presence of 10 mM Na+Hepes buffer: (;) ES interface without redox couple; (4) ES interface in the presence of 1 mM K3Fe(CN)6 and 1 mM K4Fe(CN)6; (O) EMES interface without redox couple; (+) EMES interface in the presence of 1 mM K3Fe(CN)6 and 1 mM K4Fe(CN)6. The EMES interface exhibits a membrane resistance of 9000 Ω cm2.

limit of resolution of the impedance analyzer is 108 Ω, a reasonable accuracy at low frequencies was achieved in this way. Consider first the ES interface in the presence of the redox couple. The impedance is strongly reduced and shows a different frequency dependence than in the presence of pure buffer. The impedance is reduced at high frequencies (υ > 103 Hz), which is a consequence of the reduced electrolyte resistance. This is caused by the increase of the ionic strength due to the presence of the redox couple. In the frequency range 102-103 Hz a very small shoulder can be recognized, which is attributed to the charge-transfer resistance Rct exhibiting a value of 50 Ω cm2. At low frequencies the behavior of the total impedance ZrES(υ) is determined by the Warburg impedance. This leads to the observed strong reduction of the slope of the ZrES(υ)-versus-υ curve. Consider now the EMES interface. In the presence of a redox couple the impedance of the membrane leads to a clearly visible shoulder in the frequency range 101-103 Hz. A second shoulder in the low-frequency range (υ < 10-1 Hz) is induced by the charge-transfer resistance, which is increased due to the coverage of the electrode surface by the bilayer. The charge-transfer resistance Rct dominates the impedance ZrEMES(υ) at low frequencies and leads to the flattening of the slope of the impedance spectrum at υ < 10-1 Hz. From the analysis of the impedance spectra of the ES and EMES interface in the presence of the redox couple, one finds that the charge-transfer resistance increases from 50 Ω cm2 for the ES to 450 000 Ω cm2 for the EMES interface. By considering eq 5 and eq 7 it follows that the area fraction covered by defects is 1.1 × 10-4. It should be remembered that we used an EMES interface with a rather low membrane resistance. Supported membranes with defect area fractions (1 - Θ) < 10-5 can be easily prepared with the present technique. VII. Modulation of the Capacitance CS of an ES Interface The capacitance CS of ES interfaces can be sensitively modulated by changing the pH, by the application of a bias voltage, or by adsorption of a supported membrane. These effects can be understood in terms of the change of the energy levels at the semiconductor surface resulting

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Figure 6. Mott-Schottky plot of an ES interface (+) and of an EMES interface (4) produced by fusion of vesicles and hightemperature annealing. A three-electrode setup with a potentiostat was used to ensure a well-defined bias voltage across the ES interface during the impedance measurements. A flatband potential of -0.8 V (vs Ag/AgCl) was found for the ES interface (+) and of -1.23 V (vs Ag/AgCl) for the EMES interface (4).

in a change of the impedance of the semiconductor space charge layer. The space charge capacitance CS is determined by the degree of bending of the semiconductor energy levels. As is well-known from semiconductor physics17 the bending of the energy levels depends critically on external applied bias voltages and the surface potential. A convenient way to gain insight into the change of the bending of the energy levels with bias voltage is to plot the squared reciprocal capacitance of the ES interface as a function of the bias voltage. Such so-called Mott-Schottky plots are shown in Figure 6 for an ES and an EMES interface. Straight lines are found as predicted by theory.17 Extrapolating the straight lines until the squared reciprocal capacitance equals zero yields the flatband potentials. A flatband potential of about -0.8 V (vs Ag/AgCl) is obtained for the ES interface, which agrees with results of other groups.18 Experiments performed during this study showed that the impedance of the ES interface ZM(υ) shifts to higher values when a positively charged membrane is deposited. The shift (e.g., visible in the in the low-frequency regime of Figure 1b) is not correlated with the membrane impedance and is therefore attributed to a decrease of the capacitance CS. The curve on top of Figure 6 shows the Mott-Schottky plot of a freshly produced EMES interface. Extrapolation of the square of the reciprocal capacitance CS results in a flatband potential of -1.23 V (vs Ag/AgCl) so that a shift of the flatband potential by about -0.45 V is induced by the adsorption of the positive charged membrane. Similar experiments of Uchida et al. with divalent cations also show a negative shift of the flat band potential.19 To clarify the mechanism by which the deposition of the positively charged membrane causes a shift of the flatband potential of the semiconductor, we measured the impedance of the ES interface as a function of the pH. Figure 7 shows a plot of the square of the reciprocal capacitance CS versus the pH. A nearly straight line is observed for pH values between 5 and 12. This can be (17) Sze, S. M. Physics of Semiconductor Devices, 2nd ed.; John Wiley & Sons: New York, 1981; p 868. (18) Finklea, H. O. Semiconductor Electrodes; Elsevier: Amsterdam, 1988. (19) Uchida, I.; Akahoshi, H.; Toshima, S. J. Electroanal. Chem. 1978, 88, 79-84.

Gritsch et al.

Figure 7. Variation of the inverse of the square of the capacitance CS of an electrolyte-ITO interface as a function of pH. At acidic pH, ITO is not stable.

explained by assuming that the square of the reciprocal capacitance CS of ES interfaces depends on a surface potential or an external applied voltage in a linear way as predicted by the Mott-Schottky relation.17 The surface potential is determined by the negative surface charge of the ITO resulting from dissociable surface groups of the semiconductor ITO that are in equilibrium with the protons of the electrolyte. This behavior can be described by the Nernst equation. From the pH dependence of the capacitance CS shown in Figure 7, a shift of the flatband potential of 0.1 V per pH unit is calculated using the slope of the squared reciprocal capacitance of the ES interface obtained from Figure 6. This corresponds to a shift of the surface potential by 0.059 V per pH as predicted by the Nernst equation. According to Figure 6 a shift of the flatband potential by about -0.45 V is induced by the adsorption of the positively charged membrane. This can be interpreted as a local change of the pH at the ITO surface by about 4-5 pH units toward basic state. This shift is attributed to a change of the proton distribution at the ITO surface due to the presence of the positively charged lipid bilayer. VIII. General Discussion The sensitivity of the present setup is determined by the resolution of the impedance measurement of the impedance analyzer, which is 108 Ω. To gain insight into the minimal detectable changes of the impedance of the semiconductor or the membrane, respectively, the changes of the impedance and phase caused by variation of RM by (20% are shown in Figure 8. Judged from these effects it appears possible to detect changes of RM of (5% without difficulty. The experiments with porin showed that a density of 1.6 × 105 OmpF trimers per 1 cm2 in the presence of NaCl leads to a reduction of the membrane resistance by 45%. The minimal number of pores detectable would thus be of the order of 1500 pores for the ITO electrode used in the present work exhibiting an electrode area of 8 mm2. It must be mentioned that this is a pessimistic estimation based on a set of experiments with rather low membrane resistances. The resolution can be improved considerably by using smaller electrode areas, better electrical amplifiers, and higher membrane resistances. The present work shows that supported membranes with remarkably high electrical resistances may be deposited onto ITO semiconductor surfaces by vesicle fusion and annealing at elevated temperatures. A few mol % of positively charged lipid appears to be essential. The positive charge of the membrane favors the fusion

Frequency Dependence of Electrical Impedance

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systematic studies of physical properties of biological interfaces at semiconductors. Moreover, membranes deposited on semiconductor surfaces exhibit an increased stability compared to suspended membranes (e.g., black lipid membranes). Supported membranes can be stored for several weeks without a significant decrease of the electrical membrane impedance. Moreover, the deposition of membranes on semiconductor surfaces can be further improved and simplified by the application of the lipid bilayer spreading technique of Ra¨dler et al.20 Of particular interest is the possibility to transfer poreforming proteins from vesicles into the supported bilayer. It is hoped that this technique enables the reconstitution of more complex ion channels into supported membranes. Figure 8. Demonstration of the sensitivity of impedance spectroscopy. Variation of impedance and phase shift spectra caused by changes of the membrane resistance RM by (20%. The initial membrane resistance is RM: 1 × 106 Ω. The values for the other elements of the electrical equivalent circuit are as follows: RE, 10 kΩ; RS, 5 × 107 Ω; CS, 1 × 10-7 F; CM, 2 × 10-8 F.

onto ITO surfaces that are negatively charged at pH 7.5. Although impedance spectroscopy may be less sensitive than current measurements for the design of biosensors, it is more informative and offers several advantages for

Acknowledgment. The work was supported by the Federal Ministry for Education and Research (BMBF) under Contract Number KFA 0310851 BEO and by the Fond der Chemischen Industrie. We gratefully acknowledge helpful discussions with G. Abstreiter and M. Stutzmann concerning the problems related to semiconductors. S.G. thanks C. Thun for helpful support. LA9710381 (20) Ra¨dler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 45394545.