Supported lipid bilayer membrane arrays on micro-patterned ITO electrodes

Xuejing Wang, Ying Zhang, Hongmei Bi and Xiaojun Han*
State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, No. 92 West Da-Zhi Street, Harbin 150001, China. E-mail: hanxiaojun@hit.edu.cn

Received 21st April 2016 , Accepted 25th July 2016

First published on 26th July 2016


Abstract

Lipid bilayer arrays were formed on ITO electrodes coated with a patterned self-assembled trimethoxy(octadecyl)silane monolayer (TODS-SAM). The TODS-SAM was patterned by deep UV (254 nm) irradiation through a quartz mask to create the patterned SAM modified ITO surface. Field-emission scanning electron microscopy was applied to characterize the patterned TODS SAM modified ITO. The lipid bilayer arrays were characterized by fluorescence microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. With this bilayer array platform both the fluorescence microscopy and electrochemical detection can be realized to explore the biophysical properties of the cell membrane.


Introduction

Lipid membranes are important components of all living organisms. Apart from compartmentalizing the cell, biomembranes play a crucial role in material transport, signal transduction, energy transformation and immune recognition. As an excellent biomimetic membrane model, supported bilayer lipid membranes (sBLMs)1–10 on solid surfaces have been widely used for studying various properties of biological membranes and membrane proteins. In order to mimic more sophisticated functions that real biological membranes perform, increasing the complexity of the artificial biomembrane systems is inevitable. Since Boxer et al. pioneered patterning of lipid bilayers using lithographically fabricated corrals,11 patterned lipid membranes have received considerable attention because various components can be integrated into the membrane system with defined spatial control. Moreover, the lipid bilayer arrays have potential value in studies of the physical and biological properties of membranes, lipid mobility kinetics, diagnostics, and cellular interactions with lipid-associated biomolecules. A variety of methods have been developed to fabricate patterned sBLMs. Using pre-patterned substrates is the most common strategy to prepare lipid bilayer arrays. A number of methods to prepare pre-patterned substrates have been reported such as photolithography,11–14 micro-contact printing of self-assembled monolayers (SAMs),1,15–18 UV polymerized lipids,19–21 nanoshaving lithography.22 In addition, SAMs surfaces irradiated by patterned soft UV light (365 nm)23,24 or a deep UV light (254 nm)25–28 have been used by our group to fabricate lipid bilayer arrays.

Fluorescence microscopy and electrochemical methods are important analytical techniques for sBLMs and patterned lipid membranes. The most common substrate used for electrochemical analysis of lipid bilayer array is gold. However, gold is not suitable for fluorescence microscopy because it quenches the fluorophore. Lipid bilayer arrays on silica, glass or mica surfaces can be characterized by fluorescence, but cannot be analysed with electrochemical methods. Indium tin oxide (ITO) substrates are both transparent and have an excellent electrical conductivity. For this reason they have been widely used as substrates for lipid membranes when studying the electrical properties of biomembranes.29–33

In this work, substrates for lipid bilayer arrays were fabricated by patterned deep UV irradiation of trimethoxy(octadecyl)silane (TODS) SAM modified ITO electrodes. The intact TODS-SAM regions (nonirradiated) give a hydrophobic surface on which a lipid monolayer will form. And the irradiated domains become hydrophilic again and will support a bilayer. With this platform both fluorescence microscopy and electrochemical detection can be realized.

Experimental section

Materials

1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC) was purchased from Avanti Polar Lipids. Trimethoxy(octadecyl)silane (TODS, technical grade, 90%), chloroform, potassium ferricyanide (K3Fe(CN)6) were purchased from Sigma Aldrich (China). Texas red-labeled 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanlamine, triethylammonium salt (TR-DHPE) was obtained from Invitrogen (China). Tris(hydroxymethyl)aminomethane (≥99.0%) was purchased from Sinopharm Chemical Co., Ltd (China). The Tris buffer (pH = 7.4) is the mixture of 0.1 M Tris aqueous solution and 0.1 M hydrochloric acid solution (volume ratio = 50[thin space (1/6-em)]:[thin space (1/6-em)]42). Glass slides coated with indium tin oxide (ITO, sheet resistance ≈ 8 to 12 Ω per square, thickness ≈ 160 nm) were produced by Hangzhou Yuhong technology (China). Potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate trihydrate and toluene were supplied by Xilong Chemicals (China). The PBS (pH = 7.4) buffer was the mixture of KH2PO4 and K2HPO4·3H2O (mass ratio = 19[thin space (1/6-em)]:[thin space (1/6-em)]81). Ethanol was purchased from FuYu Chemicals (China). Detergent was obtained from LIBY Group co., Ltd. Millipore Milli-Q water with a resistivity of 18.0 MΩ cm was used for solution preparation in the experiments.

Fabrication of micro-patterned TODS SAM modified ITO substrate

The 1 × 1 cm2 ITO pieces were successively sonicated in LIBY detergent, distilled water and ethanol for 15 min each. After drying under a stream of nitrogen, the ITO slides were put into plasma cleaner (Diener electronic, Zepto) for 30 s to remove remaining trace organic compounds. Then the ITO electrodes were silanized by incubation in 1 vol% TODS toluene solution for 4 hours followed by rinsing with toluene and ethanol. Slides were dried under a nitrogen stream. TODS modified ITO surfaces were patterned by irradiation with deep UV light (20 min, 254 nm, and 6.5 mW cm−2) through a chromium/quartz mask with a 50 × 50 μm grid. The UV mercury lamp was preheated for 15 min to stabilize light intensity.

Preparation of patterned lipid bilayer arrays

Vesicles were prepared by an extrusion approach. A mixture of 1 mg DLPC and 0.005 mg TR-DHPE was dried from chloroform in glass flask under a nitrogen stream. The dried lipid film was hydrated in 1 mL distilled water by vortex, followed by extrusion through a 100 nm pore size polycarbonate membrane using a mini-extruder (Avanti Polar Lipids, Inc.). The lipid bilayer arrays were formed by incubating micro-patterned TODS SAM modified ITO surfaces in freshly prepared DLPC vesicle solution for 1 h.

Wetting angle measurements

Contact angles were measured with a contact angle analyzer (Powereach, JC2000D1). Measurements were made at least at three different points on each sample.

X-ray photoelectron spectroscopy (XPS)

Spectra were surveyed on a Thermo VG ESCALAB 250 with a base pressure maintained below 5 × 10−9 mbar during acquisition. A monochromatized Al X-ray source (15 kV 150 W) was used to irradiate the samples, with a spot diameter of approximately 0.5 mm. The spectrometer was operated in Large Area XL magnetic lens mode, using pass energies of 150 and 20 eV for survey and detailed scans, respectively. Spectra were obtained with an electron takeoff angle of 90°.

Field emission scanning electron microscopy (FE-SEM)

FE-SEM images were obtained using a Carl Zeiss Supra 55 sapphire field emission scanning electron microscope with EHT of 20 kV and working distance of 20.07 mm.

Atomic force microscopy (AFM)

AFM images were carried out with an Asylum Research Inc. CYPHER E5 and silicon nitride cantilever having nominal spring constants of 0.05 N m−1. Tapping mode images were taken in 50 mM Tris buffer, with a cantilever resonant frequency of 18 kHz. Force volume images were performed at a speed of 400 nm s−1 as 64 × 64 grids.

Electrochemical measurements

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed using Autolab electrochemical workstation (PGSTAT320N, Switzerland). A three-electrode system was used for electrochemical experiments, with the ITO substrate as working electrode, a saturated calomel electrode (SCE) as reference electrode and platinum wire as counter electrode. CV measurements were conducted in the potential range from −0.1 V to 0.8 V with a scan rate of 50 mV s−1. Impedance spectroscopy was conducted in the frequency range from 105 Hz to 0.1 Hz with AC amplitude of 5 mV, at 0 V vs. SCE in 0.1 M KCl.

Fluorescence microscopy and fluorescence recovery after photobleaching (FRAP)

A Nikon 80i fluorescence microscope equipped with a Nikon DS-Fi1 digital camera was used to image the patterned lipid bilayer arrays and to carry out FRAP measurements. The FRAP data were analyzed by evaluating intensity with ImageJ (version 1.44p, USA). The lateral diffusion coefficient was calculated from D = 0.224ω2/t1/2, where ω is the radius of the bleached spot and t1/2 is the half-life of fluorescence recovery.34,35

Results and discussion

Determination of the irradiation time of TODS SAM

Deep UV (254 nm) irradiation was used to decompose the TODS SAM to form patterned TODS SAM surfaces. To determine the optimum irradiation time, the photolysis of the TODS SAM was followed by monitoring the change of contact angle as a function of time, as shown in Fig. 1a. The water contact angle decreased dramatically during the first 7 min and reached lowest point (≈5°) after 15 min. This contact angle is identical to that of ITO surface freshly after plasma cleaning. X-ray photoelectron spectroscopy was done on photo irradiated surface to characterize the remaining of TODS in a quantitative way. Fig. 1b shows the change in the XPS-spectra of the C 1s peak of a TODS SAM modified ITO substrate versus the time of photolysis. It can be seen that the C 1s peak decreased suddenly after 15 min and there was almost no change after 30 min, indicating that the organic layer was fully removed from the surface. The small peak was still evident after 30 min which can be attributed to airborne contamination. There is also a small peak for the cleaned ITO as expected for airborne contamination. The count relative to Indium after irritating 30 min is 0.229 which is close to that value of bare ITO (0.211). It illustrates that TODS SAM was removed completely after 30 min by UV irradiation.
image file: c6ra10294h-f1.tif
Fig. 1 (a) Contact angle variation during photolysis using deep UV light. (b) X-ray photoelectron spectra variation of C 1s peak region on a TODS SAM modified ITO substrate vs. time of photolysis.

We compared the lipid bilayer arrays formed on substrates with different irradiation time (15 min, 20 min, 25 min and 30 min) by fluorescence microscopy. On the substrate with 20 min of irradiation the grid was the sharpest, therefore 20 min was chosen as standard irradiation time to create the patterned substrates.

Fluorescence microscopy characterization of lipid bilayer arrays

Patterns can be created by irradiation through a mask as schematically shown in Fig. 2a and b. SAM of TODS was adsorbed onto clean ITO surfaces, from toluene solution. Irradiation with 254 nm UV light through a chromium/quartz mask led to photolysis of the exposed TODS SAM. Thus a patterned TODS SAM modified ITO substrate was formed as shown in Fig. 2b. After incubation in vesicle solution, the patterned lipid membrane arrays were formed as shown in Fig. 2c.
image file: c6ra10294h-f2.tif
Fig. 2 Schematic illustration of fabricating patterned TODS modified ITO substrate.

Image of the TODS-ITO grids with the field-emission scanning electron microscope (FE-SEM) showed that the pattern was homogeneous and very sharp, as shown in Fig. 3a. The patterned SAM was subsequently incubated with DLPC vesicles which contained 0.5% fluorescent lipid TR-DHPE. Fig. 3b is a fluorescence microscope image of the bilayer formed on such a patterned surface. The bright red rectangles correspond to regions of where lipid bilayers attached to bare ITO regions. The surrounding regions correspond to the lipid monolayers that formed on the TODS SAMs. The fluorescence intensity of lipid monolayer is almost half of lipid bilayer. And there is dark rim (the lowest intensity region) between these two regions.25,26


image file: c6ra10294h-f3.tif
Fig. 3 (a) FE-SEM image of patterned TODS modified ITO substrate. (b) Fluorescence image of lipid bilayer arrays containing 0.5% TR DHPE. White curve shows the fluorescence intensity at the position of the white line under it. Scale bar: 100 μm.

Fluorescence recovery after photobleaching (FRAP) was used to measure the lateral diffusion coefficient (D) of the lipid bilayer (Fig. 4a) and the lipid monolayer (Fig. 4b) that formed on the regions of the patterned substrate. For the lipid bilayer we observed homogeneously distributed fluorescence intensity after 8 min with the diffusion coefficient to be 0.86 ± 0.05 μm2 s−1 and a mobile fraction of 0.87. The recovery in the monolayer was slower with D value to be 0.59 ± 0.07 μm2 s−1 and a mobile fraction of 0.80. Therefore, the fluidity of the lipid bilayer is better than that of the lipid monolayer.


image file: c6ra10294h-f4.tif
Fig. 4 FRAP experiments of lipid bilayer (a) and lipid monolayer (b) formed on the patterned SAM modified ITO surfaces. Scale bars in fluorescence images are 50 μm.

Atomic force microscopy (AFM) and force curve of lipid bilayer arrays

The patterned TODS-ITO substrates before and after the incubation with 0.1 mg mL−1 DLPC vesicles solution for 1 h were imaged by AFM in air and 50 mM Tris buffer (pH = 7.4), as shown in Fig. 5a and b respectively. The 30 μm-grid can be seen clearly in the friction mode image (Fig. 5a). However, the contrast of patterned surface is not observed after the incubation with vesicle solution (Fig. 5b). This is because the outer leaflet of the lipid bilayer is in the same height as the lipid monolayer on the TODS SAM region. This result is similar to our previous work which formed lipid bilayer arrays on patterned APMES SAMs modified silica surface.24 To confirm the formation of the lipid membrane arrays, force volume imaging was carried out. Fig. 5c and d showed the typical force curve of lipid bilayer region and lipid monolayer region, respectively. Two marked steps can be seen from the force curve of Fig. 5c, and the average penetration distance of these two jumps was 3.9 nm which corresponds very well with the thickness of DLPC bilayers measured by other groups.36–38 These two jumps result from the tip passing through the outer and inner leaflet of the lipid bilayer. In Fig. 5d, there is only one obvious step with the average penetration distance of 1.9 nm which is nearly half of the lipid bilayer thickness. These two force curves illustrate the formation of lipid bilayer and monolayer on the patterned ITO surface.
image file: c6ra10294h-f5.tif
Fig. 5 (a) Friction mode AFM image of 30 μm-grid patterned TODS SAM modified ITO surface. (b) Tapping mode AFM image of patterned surface after formation of a lipid bilayer. (c) Typical force curve of lipid bilayer region. (d) Typical force curve of lipid monolayer region.

Electrochemical measurements of lipid bilayer arrays

The electrochemical analysis is well established to characterize lipid bilayers.39–41 Cyclic voltammetry was used to characterize the substrates and lipid bilayer arrays, as shown in Fig. 6a and b. The current decreased after the formation of patterned TODS SAM on the ITO electrode. The redox peaks of Fe(CN)63−/4− can still be seen as the sample has bare ITO regions where the TODS SAM was removed by the UV light irradiation through the mask. Fig. 6b is the magnified CV curve of patterned lipid bilayer. It can be seen that the current decreased to nA magnitude and the redox peaks disappeared almost after incubating the patterned SAM ITO in vesicle solution for 1 h. This result illustrates that the formation of the lipid membrane arrays hinder the electron transfer of Fe(CN)63−/4− effectively.
image file: c6ra10294h-f6.tif
Fig. 6 (a) CV curves of the bare ITO electrode, patterned TODS SAM modified ITO and patterned lipid bilayer arrays in 0.5 mM K3Fe(CN)6 solution containing 0.2 M PBS buffer and 0.1 M KCl. Scan rate is 50 mV s−1. (b) Enlarged CV curve of patterned lipid bilayer. (c) Nyquist plots of bare ITO, patterned TODS and patterned lipid bilayer modified electrodes in the frequency range from 105 Hz to 0.1 Hz with AC amplitude of 5 mV, at 0 V vs. SCE in 0.1 M KCl. (d) Enlarged Nyquist plots of bare ITO and patterned TODS-SAM modified ITO electrode.

Electrochemical impedance spectroscopy (EIS) offers a more quantitatively analysis of the lipid bilayer arrays. EIS was done in a frequency regime of 10−1 to 105 Hz. The resulting Nyquist plot is shown in Fig. 6c and d, the impedance increased markedly after the formation of lipid bilayer arrays on the ITO electrode. This agrees with the CV results. When fitting the data with equivalent circuit in Fig. 6c, a constant phase element (CPE) Q was introduced in the form of ZCPE(ω) = 1/j(ωQ)α where ZCPE is the angular frequency (ω)-dependent impedance of CPE, α is the CPE exponent which represents the deviation from a pure capacitor. When α value is close to 1, Q can be regarded as a pure capacitor. When the lipid membrane system is simulated, CPE may represent the roughness of the underlying solid surface or defects in the membranes.42,43 In the equivalent circuit in Fig. 6c, Rs represents solution resistance, R and Q represent membrane resistance and membrane capacitance of lipid bilayer array (when α is close to 1), respectively. In this case the α value was obtained to be 0.92, therefore the Q value was regarded as membrane capacitance. The overall membrane capacitance of 0.96 ± 0.05 μF cm−2 is on the upper end of the range of characteristic capacitance of lipid bilayers (0.4–1.0 μF cm−2).44,45

The lipid array membrane system can be regarded as two capacitors in parallel, a hybrid bilayer lipid membrane (HBM) and a bilayer lipid membrane (BLM) on hydrophilic ITO surfaces. The pattern on the mask defines the coverage of the two lipid membranes. Lipid bilayer regions cover 25% and monolayer regions cover 75% of the surface area, respectively. Thus the total capacitance of patterned lipid bilayer arrays is given by eqn (1) as below.

 
Ctotal = 0.25CBLM + 0.75CHBM (1)

This equation contains two unknowns CBLM and CHBM. To measure the individual contributions, EIS measurements were carried out on pure hybrid bilayer membranes and pure BLMs. Incubating DLPC vesicles with non-irradiated hydrophobic TODS SAM modified ITO surface, led to the formation of hybrid bilayer membranes (HBMs). A capacitance of 0.78 ± 0.04 μF cm−2 was measured for the hybrid bilayer membrane. Incubation of vesicles on bare hydrophilic ITO electrodes yielded pure BLMs. However, the capacitance of lipid bilayer membrane on hydrophilic ITO surface was 2.2 ± 0.3 μF cm−2 which is larger than the range of the characteristic membrane capacitance (0.4–1.0 μF cm−2). A possible reason for this is there were still some defects visible (Fig. S1b) after the formation of lipid bilayers which led to the current leakage in electrochemical measurements. According to eqn (1), the total capacitance of patterned lipid bilayer is given by 0.75 × 0.78 μF cm−2 + 0.25 × 2.2 μF cm−2 = 1.135 μF cm−2. This value is comparable with the fitting capacitance (0.96 ± 0.05 μF cm−2) of patterned lipid membranes.

Conclusions

Lipid bilayer arrays have many advantages over plain sBLM, but so far no method was reported to create bilayer arrays that allowed both the use of fluorescence microscopy and electrochemical analysis, which are both important characterizations for biological samples. Supported lipid bilayer arrays formed on micro-patterned TODS SAM modified ITO electrodes overcome this limitation and allow characterization with both fluorescence microscopy and electrochemical methods. This may find great potential on simultaneously study the electric property and fluid property of cell membranes.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 21273059, 21511130060, 21528501), National Key Research and Development Programme (2016YFC0401104), the State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (Grant No. 2014DX09), and Harbin Science and Technology Research Council (grant No. 2014RFXXJ063).

References

  1. A. T. A. Jenkins, N. Boden, R. J. Bushby, S. D. Evans, P. F. Knowles, R. E. Miles, S. D. Ogier, H. Schonherr and G. J. Vancso, J. Am. Chem. Soc., 1999, 121, 5274–5280 CrossRef CAS .
  2. L. J. C. Jeuken, R. J. Bushby and S. D. Evans, Electrochem. Commun., 2007, 9, 610–614 CrossRef CAS .
  3. L. J. C. Jeuken, S. D. Connell, P. J. F. Henderson, R. B. Gennis, S. D. Evans and R. J. Bushby, J. Am. Chem. Soc., 2006, 128, 1711–1716 CrossRef CAS PubMed .
  4. L. J. C. Jeuken, N. N. Daskalakis, X. J. Han, K. Sheikh, A. Erbe, R. J. Bushby and S. D. Evans, Sens. Actuators, B, 2007, 124, 501–509 CrossRef CAS .
  5. A. L. Plant, M. Gueguetchkeri and W. Yap, Biophys. J., 1994, 67, 1126–1133 CrossRef CAS PubMed .
  6. A. Erbe, R. J. Bushby, S. D. Evans and L. J. C. Jeuken, J. Phys. Chem. B, 2007, 111, 3515–3524 CrossRef CAS PubMed .
  7. E. Sackmann, Science, 1996, 271, 43–48 CAS .
  8. K. H. Sheikh, H. K. Christenson, R. J. Bushby and S. D. Evans, J. Phys. Chem. B, 2007, 111, 379–386 CrossRef CAS PubMed .
  9. J. T. Groves and S. G. Boxer, Biophys. J., 1995, 69, 1972–1975 CrossRef CAS PubMed .
  10. J. T. Groves and S. G. Boxer, Acc. Chem. Res., 2002, 35, 149–157 CrossRef CAS PubMed .
  11. J. T. Groves, N. Ulman and S. G. Boxer, Science, 1997, 275, 651–653 CrossRef CAS PubMed .
  12. P. S. Cremer and T. L. Yang, J. Am. Chem. Soc., 1999, 121, 8130–8131 CrossRef CAS .
  13. J. T. Groves, L. K. Mahal and C. R. Bertozzi, Langmuir, 2001, 17, 5129–5133 CrossRef CAS .
  14. D. Stroumpoulis, H. N. Zhang, L. Rubalcava, J. Gliem and M. Tirrell, Langmuir, 2007, 23, 3849–3856 CrossRef CAS PubMed .
  15. A. Kumar and G. M. Whitesides, Appl. Phys. Lett., 1993, 63, 2002–2004 CrossRef CAS .
  16. A. Kumar and G. M. Whitesides, Science, 1994, 263, 60–62 CAS .
  17. M. Mrksich and G. M. Whitesides, Trends Biotechnol., 1995, 13, 228–235 CrossRef CAS .
  18. J. L. Wilbur, A. Kumar, E. Kim and G. M. Whitesides, Adv. Mater., 1994, 6, 600–604 CrossRef CAS .
  19. K. Morigaki, T. Baumgart, U. Jonas, A. Offenhausser and W. Knoll, Langmuir, 2002, 18, 4082–4089 CrossRef CAS .
  20. K. Morigaki, T. Baumgart, A. Offenhausser and W. Knoll, Angew. Chem., Int. Ed., 2001, 40, 172–174 CrossRef CAS .
  21. T. Okazaki, Y. Tatsu and K. Morigaki, Langmuir, 2010, 26, 4126–4129 CrossRef CAS PubMed .
  22. J. J. Shi, J. X. Chen and P. S. Cremer, J. Am. Chem. Soc., 2008, 130, 2718–2719 CrossRef CAS PubMed .
  23. X. J. Han, K. Critchley, L. X. Zhang, S. N. D. Pradeep, R. J. Bushby and S. D. Evans, Langmuir, 2007, 23, 1354–1358 CrossRef CAS PubMed .
  24. X. J. Han, S. N. D. Pradeep, K. Critchley, K. Sheikh, R. J. Bushby and S. D. Evans, Chem.–Eur. J., 2007, 13, 7957–7964 CrossRef CAS PubMed .
  25. X. J. Han, A. S. Achalkumar, R. J. Bushby and S. D. Evans, Chem.–Eur. J., 2009, 15, 6363–6370 CrossRef CAS PubMed .
  26. X. J. Han, M. R. Cheetham, K. Sheikh, P. D. Olmsted, R. J. Bushby and S. D. Evans, Integr. Biol., 2009, 1, 205–211 RSC .
  27. X. J. Han, G. D. Qi, X. T. Xu and L. Wang, Chem.–Eur. J., 2011, 17, 14741–14744 CrossRef CAS PubMed .
  28. Y. Zhang, L. Wang, X. J. Wang, G. D. Qi and X. J. Han, Chem.–Eur. J., 2013, 19, 9059–9063 CrossRef CAS PubMed .
  29. J. Feng, Y. X. Ci, C. Y. Zhang, A. L. Ottova and H. T. Tien, Electrochem. Commun., 1999, 1, 145–147 CrossRef CAS .
  30. S. Gritsch, P. Nollert, F. Jahnig and E. Sackmann, Langmuir, 1998, 14, 3118–3125 CrossRef CAS .
  31. M. Nagata, Y. Yoshimura, J. Inagaki, Y. Suemori, K. Iida, T. Ohtsuka and M. Nango, Chem. Lett., 2003, 32, 852–853 CrossRef CAS .
  32. H. T. Tien, S. H. Wurster and A. L. Ottova, Bioelectrochem. Bioenerg., 1997, 42, 77–94 CrossRef .
  33. G. Wiegand, N. Arribas-Layton, H. Hillebrandt, E. Sackmann and P. Wagner, J. Phys. Chem. B, 2002, 106, 4245–4254 CrossRef CAS .
  34. D. M. Soumpasis, Biophys. J., 1983, 41, 95–97 CrossRef CAS PubMed .
  35. L. Q. Zhang, M. L. Longo and P. Stroeve, Abstr. Pap. Am. Chem. S., 2000, 220, U257–U258 Search PubMed .
  36. L. M. Grant and F. Tiberg, Biophys. J., 2002, 82, 1373–1385 CrossRef CAS PubMed .
  37. L. J. Lis, M. McAlister, N. Fuller, R. P. Rand and V. A. Parsegian, Biophys. J., 1982, 37, 667–672 CAS .
  38. H. Mueller, H. J. Butt and E. Bamberg, J. Phys. Chem. B, 2000, 104, 4552–4559 CrossRef CAS .
  39. W. L. Cheng, X. J. Han, E. Wang and S. J. Dong, Electroanalysis, 2004, 16, 127–131 CrossRef CAS .
  40. X. J. Han, A. Studer, H. Sehr, I. Geissbuhler, M. Di Berardino, F. K. Winkler and L. X. Tiefenauer, Adv. Mater., 2007, 19, 4466–4470 CrossRef CAS .
  41. C. Hennesthal and C. Steinem, J. Am. Chem. Soc., 2000, 122, 8085–8086 CrossRef CAS .
  42. M. Bart, E. C. A. Stigter, H. R. Stapert, G. J. de Jong and W. P. van Bennekom, Biosens. Bioelectron., 2005, 21, 49–59 CrossRef CAS PubMed .
  43. E. D. Bidoia, L. O. S. Bulhoes and R. C. Rocha, Electrochim. Acta, 1994, 39, 763–769 CrossRef CAS .
  44. M. Montal and P. Mueller, Proc. Natl. Acad. Sci., 1972, 69, 3561–3566 CrossRef CAS .
  45. W. Romer and C. Steinem, Biophys. J., 2004, 86, 955–965 CrossRef PubMed .

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10294h

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