Lab-on-chip design to characterize pore-spanning lipid bilayers
by Theresa Kaufeld
Date of Examination:2013-10-23
Date of issue:2013-11-26
Advisor:Prof. Dr. Christoph F. Schmidt
Referee:Prof. Dr. Christoph F. Schmidt
Referee:Prof. Dr. Claudia Steinem
Persistent Address:
http://dx.doi.org/10.53846/goediss-4189
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Abstract
English
A powerful approach to study membrane proteins is the reconstitution in model membranes. Methods for arti cial bilayer formation are e.g. membranes on a solid support, or the classical BLM. In this project, the focus was on the formation of lipid bilayers on porous substrates combining the stability of solid supports and the accessibility to both sides of the bilayer of the classical BLM which is necessary for electrical recordings of membrane channels. Commercially available porous substrates, however, are typically not suitable for low-noise electrical experiments or for a combination with further manipulation techniques.Therefore, a microporous substrate was designed and fabricated meeting several demands: (i) To perform multiple experiments on one chip, the substrate was devided into four arrays of pores with separate electrolyte compartments and electrical connections. (ii) A PMMA/PDMS sample chamber was designed in a way that allows the exchange of solutions throughout the experiment. (iii) An integrated electrode facilitates switching between di erent electrical measurement techniques and allows better access for microscope objectives. The substrate was fabricated in a multi-step cleanroom technology based process. Porous arrays of 900 and 9 pores were etched into a thin silicon nitride layer, but only the small 9-pore arrays turned out to be suitable for lipid bilayer formation, because the vesicles rupturing on the pore array do not fuse completely and leave uncovered pores, as fl uorescence microscopy images showed. The surface of the substrate and the appearance of the pores was characterized using atomic force microscopy and scanning electron microscopy and showed a low surface roughness and ordered pores. In addition, the pore-diameter was determined by a pixel analysis of bright field microscopy images and matches the nominal pore diameter closely. The substrate and the in fluence of the integrated electrode as well as lipid bilayer formation was investigated using impedance spectroscopy. The circuit model for the substrate was shown to be a simple R(RC) circuit, whereas the integrated electrode contributes with an additional R/CPE element and reveals di ffusive behavior. Whether external or integrated electrodes are used does not in fluence the lipid bilayer spectrum which is dominated by the resistance of the membrane and the high capacitance of the thin silicon nitride layer of the substrate. The theoretical pore resistance for cylindrical pores was calculated and agrees well with the experimental results. As a tests for the suitability of the substrate and the newly built setup for voltage clamp recordings, alamethicin ion channels were functionally reconstituted into the lipid bilayers. Additionally, the expression of polycystin-2, a supposedly mechanosensitive membrane-spanning protein, was adapted from H. F. Canitello and modi fied to be established for future experiments. In summary, a versatile microporous substrate was developed that is suitable for solvent-free lipid bilayer formation and functional reconstitution of ion channels. The substrate can be adapted for many di fferent techniques, such as optical microscopy, impedance spectroscopy and voltage clamp recordings. Because it is custom-built, modi fications in terms of pore-size and number as well as functionalization of the surface can be adjusted easily.
Keywords: microfabrication; pore-spanning lipid bilayer; impedance spectroscopy; single ion-channel recording