Technical Note pubs.acs.org/ac
Automated Lipid Membrane Formation Using a Polydimethylsiloxane Film for Ion Channel Measurements Hyunil Ryu,†,‡ Sangbaek Choi,†,‡ Joongjin Park,†,‡ Yeong-Eun Yoo,§ Jae Sung Yoon,§ Young Ho Seo,∥ Young-Rok Kim,⊥ Sun Min Kim,*,‡,# and Tae-Joon Jeon*,†,‡ †
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 402-751, South Korea § Nano-Mechanical Systems Research Division, Korea Institute of Machinery and Materials, Daejeon, 305-343, South Korea ∥ Department of Mechanical and Mechatronics Engineering, Kangwon National University, Chuncheon, 200-701, South Korea ⊥ Institute of Life Science and Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin, 472-864, South Korea # Department of Mechanical Engineering, Inha University, Incheon, 402-751, Republic of Korea ‡
S Supporting Information *
ABSTRACT: A black lipid membrane (BLM) is a powerful platform for studying the electrophysiology of cell membranes as well as transmembrane proteins. However, BLMs have disadvantages in terms of stability, accessibility, and transportability, which preclude their industrial applications. To resolve these issues, frozen membrane precursor (MP) was devised to improve the transportability and storability of BLMs. As described previously, MP is a storable and transportable platform that can be delivered to the pointof-use, where BLMs are automatically formed upon thawing at room temperature. However, MP has an inconsistent thinning-out time, ranging from 30 min to 24 h, as well as a low success rate of BLM formation (∼27%), which make it undesirable for practical use. In our study, polydimethylsiloxane (PDMS) was introduced as a replacement for conventionally used Teflon film to control thinning-out time. As such, we used a PDMS thin-film, a porous-structured hydrophobic polymer, and squalene, a high viscosity solvent, to facilitate membrane formation, whereas the absorption rates of solvents were controlled to achieve consistent BLM formation time. We successfully reduced thinning-out time down to 1% lipids were used. In addition, optimization experiments were carried out at four different PDMS film thicknesses (0.1−0.15 mm, 0.15−0.2 mm, 0.2−0.25 mm, 0.25− 3 mm) as well as at three different aperture sizes (750, 500, 300, 100 μm), which are typically used dimensions in conventional techniques. The highest success rate was obtained at a thickness of 0.25−0.3 mm with an aperture size of 500 μm (Table S2, Supporting Information). As a result of the optimization, the overall success rate of membrane formation from MPES notably increased to 80.8% (n > 100) while maintaining a uniform thinning-out time (40.8 min, SD: 21.3 min) as well as prolonged lifetime (39.7 min SD: 16.2) (n > 100). This constitutes a highly advanced platform in comparison to previously reported results with a 27% reconstitution success rate and wide range of thinning-out times from 30 min to 24 h. However, ∼40 min of membrane longevity may preclude applications requiring long-term ion channel measurements. Important issues with the frozen membrane precursor are readiness of membrane formation and consistent membrane formation time, which are significantly improved in our system. Although a 39.7 min lifetime of the BLMs is considered short compared to the previously reported system,14 the lifetime of our membrane is sufficient enough for ion channel studies as demonstrated as follows. Ion Channel Activities of Alpha-Hemolysin (αHL) and Gramicidin A (gA). To verify the feasibility of our platform for ion channel studies, two frequently used membrane-bound proteins, alpha-hemolysin (αHL) and gramicidin A (gA), were incorporated in our system. Conductance levels of the channels were measured to be 0.8 nS for αHL and 20 pS for gA, which are in agreement with previous reports3,24 and show the feasibility of our platform for ion channel measurement (Figure S2, Supporting Information). Of particular interest, gA was directly added to the lipid solution to make MPES. Membrane proteins are usually added to the buffer solution, which increases the time required for membrane proteins to be incorporated into the membrane. As such, gA channel activities appeared as soon as a BLM formed from the MPES. Moreover, removal of the buffer exchange process to get rid of any remaining ion channels makes the ion channel experiments readily accessible. Guanidine Screening Using gA. We were able to obtain a short and consistent membrane formation time using a PDMS film. As mentioned previously, however, continuous extraction of solvent from the PDMS film influences the longevity of the membrane. Therefore, we tested the feasibility of our platform for drug screening with membrane intact. In our experiment, guanidine was used to test the ion channel activities of gA
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RESULTS AND DISCUSSION Frozen Membrane Precursor with Expedited SelfAssembly (MPES). Frozen membrane precursor with expedited self-assembly (MPES) is a frozen lipid solution placed in a multiporous polymer film. Using a solvent mixture with a high melting temperature, lipid solution becomes freezable below 1G ohm).17 Optimization of Composition of MPES Solution and PDMS Film. To successfully make the membrane from MPES, various compositions of solvent and lipids were tested at different concentrations. The success rate of membrane formation was ∼50% using a 0.5 μL solvent mixture of hexadecane and n-decane (80% v/v) containing 1 wt % DPhPC, as described previously.14 Although the success rate of membrane formation in the PDMS film slightly increased, the times required to form the lipid bilayer were inconsistent. Moreover, membrane lifetime was only several minutes due to continuous extraction of solvents from the PDMS film. As the absorption rate is inversely proportional to viscosity based on the Carmen-Kozeny Equation,18−20 we used squalene (12 cP at 20 °C),21 a high viscosity solvent, instead of n-decane (0.92 cP at 20 °C).22 Longevity of the membrane increased when ndecane was replaced with squalene. Moreover, stability of the C
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Figure 2. (A) In the absence of guanidine, an amplitude of about 28 pS was observed (70 mV holding potential; 100 Hz Bessel low pass filter). (B) Blocking signals occurred 10 min after adding guanidine (1 M).
Figure 3. Optical and electrical properties of membranes formed in the multipore array system. When each bilayer started to form, the width of the square wave increased rapidly. Image on the left shows frozen membrane precursor. The black arrows mean that the bilayer had formed. Bilayers formed in the order of left-bottom, right-top, left-top, and right-bottom. The bottom five figures show the electrical properties when each membrane forms. The resistance of the membranes was 5.05 G ohm.
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against guanidine. gA shows flicker phenomena in the presence of specific target molecules. When a flicker phenomenon occurs, ion currents are first reduced and then revert back to their original state. Several biosensing applications have been introduced using the properties of gA.25,26 For instance, iminium ions, including tetrodotoxin, amiloride, and nonylguanidinium, undergo characteristic events such as blocking of gA channels.27 Similarly, guanidine HCl shows specific flicker events on gA channels.28−30 As shown in Figure 2, current drops of over 1.5 pA were observed in the presence of 1 M guanidine upon reconstitution of BLM and gA channels from MPES. Blocking frequency increased to 8.41 Hz (p < 0.01) in the presence of guanidine while the blocking frequency of gA in the control experiments was 0.013 Hz (p < 0.01). Multipore Array System. The statistical probability of detecting target materials should correspond to the surface area of a membrane. Target molecules at low concentration are more readily measurable when a membrane with a large surface area is achieved, resulting in higher sensitivity. In other words, the surface area of a membrane is proportional to the sensitivity and high throughput of the system.31 However, enlarged aperture size yields a membrane with lower stability and reproducibility.32 Correspondingly, an array system was suggested to maintain the original aperture size as well as increase membrane surface area. To demonstrate an array system using MPES, we made a 2 × 2 array system by creating four apertures (300 μm) in PDMS thin-film (0.2−0.25 mm) using a CNC machine. To successfully perform membrane formation using the array system, optical and electrical measurements were simultaneously carried out, resulting in four different current jumps as soon as each membrane formed (Figure 3). With an increased number of membranes, an array system provides a more sensitive drug screening platform compared to a system with a single membrane.31
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Technical Note
ASSOCIATED CONTENT
S Supporting Information *
Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Authors
*Phone:+82-32-860-7328. Fax: +82-32-867-7328. E-mail:
[email protected]. *Phone:+82-32-860-7511. Fax: +82-32-872-4046. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a National Research Foundation Grant (NRF-2012R1A1B4002413) and the Pioneer Research Center Program (NRF-2012-0009575), National Research Foundation of Korea.
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REFERENCES
(1) Hanke, W.; Schulue, W. Planar lipid bilayers: Methods and applications; Academic Press: New York, 1993. (2) Mirzabekov, T. A.; Silberstein, A. Y.; Kagan, B. L. Methods Enzymol. 1999, 294, 661−674. (3) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226−230. (4) Fang, Y.; Lahiri, J.; Picard, L. Drug Discovery Today 2003, 8, 755− 761. (5) Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M. Curr. Opin. Biotechnol. 2010, 21, 439−476. (6) Wood, C.; Williams, C.; Waldron, G. J. Drug Discovery Today 2004, 9, 434−441. (7) Mueller, P.; Rudin, D. O.; Ti Tien, H.; Wescott, W. C. Nature 1962, 194, 979−980. (8) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U. S. A. 1972, 69, 3561−3566. (9) Baaken, G.; Sondermann, M.; Schlemmer, C.; Rühe, J.; Behrends, J. C. Lab Chip 2008, 8, 938−944. (10) Costello, R. F.; Peterson, I. P.; Heptinstall, J.; Byrne, N. G.; Miller, L. S. Adv. Mater. Opt. Electron. 1998, 8, 47−52. (11) Ide, T.; Yanagida, T. Biochem. Biophys. Res. Commun. 1999, 265, 595−599. (12) Jeon, T.-J.; Malmstadt, N.; Schmidt, J. J. J. Am. Chem. Soc. 2006, 128, 42−43. (13) Malmstadt, N.; Jeon, T. J.; Schmidt, J. J. Adv. Mater. 2008, 20, 84−89. (14) Jeon, T.-J.; Poulos, J. L.; Schmidt, J. J. Lab Chip 2008, 8, 1742. (15) Malmstadt, N.; Nash, M. A.; Purnell, R. F.; Schmidt, J. J. Nano Lett. 2006, 6, 1961−1965. (16) Miller, C. Ion channel reconstitution; Plenum Press: New York, 1986. (17) Priel, A.; Gil, Z.; Moy, V. T.; Magleby, K. L.; Silberberg, S. D. Biophys. J. 2007, 92, 3893−3900. (18) Kozeny, J. Sitzungsber. Akad. Wiss. Wien 1927, 136, 271−306. (19) Carman, P. Trans. Inst. Chem. Eng. 1937, 15, 150−166. (20) Carman, P. C. Flow of gases through porous media; Butterworth’s Scientific Publications: London, 1956. (21) Yaws, C. L. Chemical properties handbook : Physical, thermodynamic, environmental, transport, safety, and health related properties for organic and inorganic chemicals; McGraw-Hill: New York, 1999. (22) Windholz, M., Budavari, S., Fertig, M. N., Eds. The Merck Index: An Encyclopedia of Chemicals and Drugs; Merck: Rahway, NJ, 1976. (23) Jeon, T.-J.; Malmstadt, N.; Poulos, J. L.; Schmidt, J. J. Biointerphases 2008, 3, FA96−FA100.
CONCLUSION
The BLM system is a powerful tool for studying ion channels and electrophysiology, as well as for biosensing applications. However, its practical applicability has been slow to date, as the stability and longevity of BLMs do not meet industrial standards. In our studies, we devised a frozen MPES to achieve a consistent membrane formation time as well as a higher success rate of membrane formation with transportability and storability. Membranes formed upon thawing of MPES after addition of buffer at ambient temperature, resulting in a uniform BLM reconstitution time of ∼40 min and a success rate of over ∼80%. Moreover, we incorporated gA into lipid solution for reconstituting ion channels, thereby enabling immediate channel incorporation upon membrane formation as well as eliminating two important processes: protein incorporation and buffer exchange. To prove the feasibility of our platform for drug screening applications, ion channel activities were successfully measured in the presence of guanidine. As our platform requires no expertise for membrane formation and frozen MPES is transportable and indefinitely storable, MPES can be widely implemented for both industrial and academic uses. Moreover, a disposable PDMS thin-film should cost no more than $0.20, which widens its usability by significantly reducing costs for ion channel experiments. E
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(24) Hladky, S.; Haydon, D. Biochim. Biophys. Acta, Biomembr. 1972, 274, 294−312. (25) Yellen, G. J. Gen. Physiol. 1984, 84, 157−186. (26) Heinemann, S. H.; Sigworth, F. J. Biophys. J. 1988, 54, 757−764. (27) Hemsley, G.; Busath, D. Biophys. J. 1991, 59, 901−907. (28) Ritchie, J. M.; Rogart, R. B. Rev. Physiol., Biochem. Pharmacol. 1977, 79, 1−50. (29) Bentley, P. J. J. Physiol. 1968, 195, 317−330. (30) Farley, J. M.; Yeh, J. Z.; Watanabe, S.; Narahashi, T. J. Gen. Physiol. 1981, 77, 273−293. (31) Le Pioufle, B.; Suzuki, H.; Tabata, K. V.; Noji, H.; Takeuchi, S. Anal. Chem. 2008, 80, 328−332. (32) Mach, T.; Chimerel, C.; Fritz, J.; Fertig, N.; Winterhalter, M.; Fütterer, C. Anal. Bioanal. Chem. 2008, 390, 841−846.
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