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内容記述 |
Ion channels play key roles in many cellular processes. There are many distinct<br />dysfunctions known as channelopathies caused by ion channel mutations. Therefore the<br />investigation of ion channels is of great significance for understanding how they work<br />and for drug discovery of channelopathies. Pipette patch-clamp technique has been<br />proven to be a powerful technique for the investigation of fundamental ion channel<br />biophysics and for drug discovery. This technique allows one to monitor the gating of<br />ion channels under defined conditions and enables the coupling of functional and<br />molecular studies on ion channels at the single cell level. However, this technique has <br />some weaknesses, such as the requirements of precise micromanipulation and skillful<br />experimenter, and electrode being an individual glass pipette, which are not suitable for<br />the long time measurement of the cell function and the application to high-hroughput<br />screening. Recently planar patch-clamp method has attracted great attentions. Because<br />it has some advantages compared with the pipette patch-clamp method, such as the <br />miniaturization and parallelization of the planar substrates and the availability to<br />combine with other physical probes. Many materials have been used to make the planar <br />patch-clamp substrates, such as glass, Si, quartz and PDMS etc. It has been considered<br />for Si that the background noise current is large due to the high density of free charge<br />carrier in the substrate [Fertig et al., Recept. Channels 9(2003)29]. However, they have<br />demonstrated that the noise current can be significantly reduced by using<br />silicon-on-insulator (SOI) wafer. There are several other advantages in using SOI wafer:<br />1) the structure of the micropore through the substrate can be precisely controlled by<br />using the large etching rate difference between Si and SiO2 in both plasma and wet<br />etching, and 2) it is possible to produce significantly miniaturized device by integrating<br />the biosensor and Si electronic circuits of preamplifier into the same SOI substrate. <br /> In the present study, he has fabricated the planar patch-clamp substrates using SOI<br />wafer. A device, called conventional type planar patch-clamp biosensor, was assembled<br />with the SOI-based substrate. He used this to demonstrate that SOI is a versatile<br />material for planar patch-clamp substrate fabrication used in biosensor. However, the<br />lifetime of the cell on the micropore is too short in the planar patch-clamp biosensor,<br />which is a serious problem for measuring various cell functions. To elongate the <br />cellular lifetime, the substrate in the conventional type planar patch-clamp biosensor<br />was modified with fibronectin, an extracellular matrix protein, and cells were cultured <br />under culture medium instead of buffer solution. By using this method, he has <br />developed a new generation planar patch-clamp device, called incubation type planar<br />ion-channel biosensor.<br /> In the former half of his doctoral course research, he developed several elementary<br />processes to fabricate the planar patch-clamp substrates using SOI wafer, which<br />produce low access resistance and low capacitance.<br /> Two procedures of planar patch-clamp substrate fabrication were developed. One is <br />based on electron beam lithography (EBL) combined with reactive ion etching (RIE), <br />the other is based on focused ion beam (FIB).<br /> In the fabrication method with EBL combined with RIE, firstly circular patterns<br />were made with EBL and RIE techniques. Then a SiO<sub>2</sub> layer with 1 µm thickness was <br />grown at 900ºC with thermal oxidation in which O<sub>2</sub> bubbled water vapor at 95ºC was <br />used as the reactive gas. After that large holes on the backside of the substrate were<br />made with 1-mm-diameter diamond drill polishing, followed by 8%(v/v) TMAH etching <br />at 90ºC to the buried SiO<sub>2</sub> layer. Finally the buried SiO<sub>2</sub> layer at the bottom of the<br />patterns was removed with 10% (v/v) HF solution from the topside of the substrate,<br />followed by 1-µm-thick SiO<sub>2</sub> layer formation at 900ºC with thermal oxidation in the<br />presence of O<sub>2</sub> bubbled water vapor at 95ºC. The scanning electron microscope (SEM)<br />images indicate that the initially round pore becomes faceted after the thermal<br />oxidation due to the crystallographic growth-rate dependence of single-crystal silicon, <br />and the sharp edge at the rim of the pore becomes dull after the thermal oxidation. <br />These are possibly unsuitable for the tight contact formation between the cell <br />membrane and the substrate surface. <br /> In the fabrication method using FIB, firstly a 0.2 µm thickness SiO<sub>2</sub> layer was <br />formed on the silicon surface by thermal oxidation at 900ºC with the water-saturated O<sub>2</sub><br />flow. Then large holes (～1 mm diameter and ～400 µm depth) on the backside were made by diamond drill polishing. Subsequently the pyramid-shaped holes were formed by 8% (v/v) TMAH etching at 90ºC for about 40 min, which reached the buried SiO<sub>2</sub> layer. <br />Finally micropores through the Si and SiO<sub>2</sub> layers were made by FIB milling from the<br />backside. SEM images indicate that a round pore with sharp edge can be obtained with<br />the FIB method. Therefore the planar patch-clamp substrates made with FIB were used<br />to make the device. The substrate with 1.2 µm diameter pore was used to make the<br />conventional type planar patch-clamp biosensor. The substrate was assembled into the<br />microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with<br />transient receptor potential vanilloid type 1 (TRPV1) was positioned on the micropore<br />and the whole-cell configuration was formed by suction. Capsaicin was added to the<br />extracellular solution as a ligand molecule, and the whole-cell current of HEK-293 cell<br />showing desensitization unique to TRPV1 in the extracellular solution containing Ca<sup>2+ </sup><br />[Caterina et al. Nature 389(1997)816] was measured successfully.<br /> In the latter half of his research, to overcome the cellular short-lifetime problem, <br />the incubation type planar ion-channel biosensor was developed. He modified the<br />substrate of the planar patch-clamp biosensor with fibronectin, and cultured the cell <br />positioned on the micropore under the culture medium instead of the buffer solution. <br />Atomic force microscope (AFM) and cell spreading assays of the FN-coated substrates<br />indicate that they are promising biomaterials for cell adhesion and spreading. After the<br />cell attached and spread on the pore of the FN-coated substrate, the resistance of the<br />electrolyte in the cleft between the cell membrane and the substrate surface was<br />evaluated based on a schematic model and its equivalent circuit. The obtained values of<br />seal resistance quite well agree with the experimentally measured values. Using this<br />method, the resistance of the electrolyte in the narrow space between the cell on the <br />pore and all the contacting cells was evaluated. The whole-cell configuration of the<br />HEK-293 cell spreading on the pore was obtained with nystatin perforation and the<br />whole-cell current of TRPV1 was successfully measured. It is demonstrated that<br />fibronectin modification of the substrate can make the cell live for a long time and the<br />whole-cell current of the cell spreading on the pore can be obtained during cell culture.<br />Moreover this planar ion-channel biosensor has high potential application to<br />investigate the various cell functions and the neuronal signal transductions, because the<br />cells can be cultured on this FN-coated planar ion-channel biosensor substrate.<br /> |
要旨・審査要旨 (321.9 kB)
