{"created":"2023-06-20T13:20:13.843069+00:00","id":237,"links":{},"metadata":{"_buckets":{"deposit":"396e49f0-26b9-4627-a6ef-cd141e5b130f"},"_deposit":{"created_by":1,"id":"237","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"237"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000237","sets":["2:427:9"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"MD, MASHIUR RAHMAN"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"モシウル, ラーマン"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2005-09-30"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（学術）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　　Ion channels play key roles in functions and dysfunctions of all cells. Single channel recording methods using planar (black) lipid membranes (BLM) and patch clamp techniques are widely used at present, but they are not suited for automation and miniaturization. Therefore, in order to develop electrophysiology library arrays, several groups have recently reported on wafer-based devices that can replace the more traditional glass pipettes and Teflon partitions that are employed for investigating ion channels activities in cells and artificial membrane. The supported planar lipid bilayer (SPLB) is a lipid bilayer supported on solid surfaces. Concerning the ion-channel biosensors, single ion channel recording has been succeeded in suspended membranes made by the painting method on micro-machined supports. In the case of the suspended membrane, it is not easy to make a single bilayer with a small pore diameter (several μm), which is necessary for high speed recording and low noises. In the SPLBs made by vesicle fusion however, single channel recording have not yet been reported. The SPLB on the silicon based microelectrode are extremely attractive since small pore can be easily made, thus it has a potential of high stability, high sensitivity and high density of integration. It is considered that a major challenge in the production of tightly sealed bilayers to reduce leakage currents to the levels found in the suspended membrane, and most likely, this will require the reduction of the substrate surface roughness and the elimination of edge effects.<br /> The efforts to get high resistivity in the tethered supported membrane on Au surface have been done by several groups. Recently, the tethered lipid bilayers with a high electrical resistance of ~130 MΩ and the subsequent detection of only a few synthetic ligand-gated ion channels incorporated in the tethered lipid bilayer, have been reported. [S. Terrettaz et al. Langmuir 19 (2003) 5567] In spite of these efforts, it is clear that a much higher resistivity (GΩ seal) is required to realize a supported membrane biosensor which can be applied to the single ion channel recording. <br /><br />　　In the present thesis, based on these backgrounds, I have developed several elementary processes to realize an ideal SPLB with GΩ resistance on Si-based microelectrodes. I have developed the techniques to fabricate a hole (well) with a diameter of about 1μm for microelectrodes on a SiO<SUB>2</SUB>/CoSi<SUB>2</SUB>/Si substrate, while maintaining the SiO<SUB>2</SUB> surface roughness at less than 1 nm using a femtosecond laser microfabrication technique and synchrotron radiation etching. The SPLB membrane was formed on the surface of a microelectrode area by the fusion of giant unilamellar vesicles. I have characterized the stability, electrical resistance, capacitance, and current noise of the bilayers. <br /><br />　　After the deposition of Co on the Si(100) surface, the SiO<SUB>2</SUB> thin film consists of spin on glass (SOG) (400 nm thickness) and sputtered SiO<SUB>2</SUB> (200 nm thickness) was formed on the Co/Si. Then by annealing at 540=℃ for 10min, the Co/Si layer was changed to CoSi<SUB>2</SUB> keeping the SiO<SUB>2</SUB> surface roughness less than 1 nm. A 300 nm of Co layer was deposited on the SiO<SUB>2</SUB> surface by sputtering as an etching contact mask and circular patterns were made on the Co mask using the femto-second laser ablation. The SR etching of the SiO<SUB>2</SUB> layer to make the wells on the electrode was carried out at the beam line 4A2 of the SR facility (UVSOR) at the Institute for Molecular Science, using a mixture of SF<SUB>6</SUB> (0.05 Torr) and O<SUB>2</SUB> (0.002 Torr) as an etching gas. The SR etching results in a vertical wall and completely stops at the surface of the CoSi<SUB>2</SUB>/Si(100). SR was used because of its unique features such as high spatial resolution, extremely high material selectivity between CoSi<SUB>2</SUB> and SiO<SUB>2</SUB>, anisotropic etching, low damage, and clean etching atmosphere. Finally the Co contact mask was removed without damaging the substrate by immersion into 0.1 M HNO<SUB>3</SUB> aq. AFM images of the SiO<SUB>2</SUB> surface after the removal of the Co mask showed that the surface was very flat (R<SUB>a</SUB>=0.8 nm), which is essential for the formation of the defect-free SPLB on the surface. <br /><br />　　Ag (50 nm) was deposited by electroplating on the surface of CoSi<SUB>2</SUB> which was exposed at the bottom of the etched well. Then the surface of the Ag was changed into AgCl also by electroplating. The giant unilamellar vesicles were prepared by adding a buffer solution (10 mM KCl, pH = 6.6) to vacuum-dried films of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-3-phosphor- L-Serin (POPS) (9:1, w/w) and agitating at RT. Mixing of negatively charged lipid POPS to neutral lipid DPPC was essentially effective to form unilamellar giant vesicles without aggregation. Formation of SPLB covering the well-type electrode by the rapture of the giant vesicle was confirmed by fluorescence microscope. When substrates are immersed in an aqueous solution of lipid vesicles, the vesicles adhere to the surface, rupture, and spread to form a bilayer on hydrophilic surfaces of SiO<SUB>2</SUB>. It has been suggested that a thin water layer approximately 1-2 nm is trapped between the support and the headgroups of the lower leaflet of the bilayer. [Bayerl, T. M.; Bloom, M. Biophysical Journal, 58 (1990) 357]<br /><br />　　Fluorescence microscopy images showed that the diameter of the SPLB formed on the SiO<SUB>2</SUB>/Si(100) surface by the rapture of the giant vesicles was typically about 150 ~ 300μEm, large enough to cover the electrode area (10μEm ~ 30μEm diameter). AFM images of the bilayer showed that the thickness of the SPLB membrane was 4.5 nm, corresponding to the height of a single bilayer. The electric characteristics were measured by a patch clamp amplifier through the AgCl/Ag electrode. The resistances before and after the lipid bilayer formation were 10±3 MΩ and 1.2 GΩ, respectively. This confirmed the GΩ seal formation of SPLB on the microelectrodes. The capacitance of the bilayer measured by using a patch clamp amplifier was 10.7 pF. These values were observed with extremely good reproducibility during our experiments for more than 5 hours. <br /><br />　　Although the resistance value fulfills the condition required for the measurement of single channel measurements, even then it is much smaller than those (> 30 GΩ) realized in the planer or suspended membranes. This may be due to the edge leak current. Therefore, by depressing the edge leak current, much higher resistance of lipid bilayer is expected to be obtained. <br /><br />　　I have considered to use the hydrophobic self-assembled monolayers (SAM) as a “guard ring” to reduce the edge leak current of SPLB. I have developed a patterning method of octadecyltrichlorosilane (OTS) SAM by photo-lithography and UV ashing. OTS-SAM were formed on the sputtered SiO<SUB>2</SUB> surface by immersing the sample into a 1.0 mM solution of OTS in toluene for 10 s at 22℃. Then negative resist (7μEm height) pattern was made with lithography technique. After 30 min of UV ashing, resist pattern was removed with remover. The OTS-SAM on the open area, which was not covered with the resist, were completely removed by UV ashing, while no change was observed in the OTS-SAM on the area covered with the resist. The height of the OTS-SAM was ~2.5 nm and the roughness of the SiO<SUB>2</SUB> surface without OTS-SAM was R<SUB>a</SUB>=0.8 nm. SPLB was formed on this patterned OTS-SAM by rapture of giant unilamellar vesicles. AFM and fluorescence microscopy images have shown that SPLB forms bilayer on hydrophilic SiO<SUB>2</SUB> surfaces and a monolayer on OTS-SAM hydrophobic surfaces. This technique has been considered to use for the formation of tightly sealed bilayers to reduce leakage current of the SPLB. 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