{"created":"2023-06-20T13:20:14.395071+00:00","id":250,"links":{},"metadata":{"_buckets":{"deposit":"6eae6eb4-22f7-4a01-abe5-8a63053baef1"},"_deposit":{"created_by":1,"id":"250","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"250"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000250","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":"張, 振〓"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ツァン, ツェンロン"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2008-03-19"}]},"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（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":" Ion channels play key roles in many cellular processes. There are many distinct
dysfunctions known as channelopathies caused by ion channel mutations. Therefore the
investigation of ion channels is of great significance for understanding how they work
and for drug discovery of channelopathies. Pipette patch-clamp technique has been
proven to be a powerful technique for the investigation of fundamental ion channel
biophysics and for drug discovery. This technique allows one to monitor the gating of
ion channels under defined conditions and enables the coupling of functional and
molecular studies on ion channels at the single cell level. However, this technique has
some weaknesses, such as the requirements of precise micromanipulation and skillful
experimenter, and electrode being an individual glass pipette, which are not suitable for
the long time measurement of the cell function and the application to high-hroughput
screening. Recently planar patch-clamp method has attracted great attentions. Because
it has some advantages compared with the pipette patch-clamp method, such as the
miniaturization and parallelization of the planar substrates and the availability to
combine with other physical probes. Many materials have been used to make the planar
patch-clamp substrates, such as glass, Si, quartz and PDMS etc. It has been considered
for Si that the background noise current is large due to the high density of free charge
carrier in the substrate [Fertig et al., Recept. Channels 9(2003)29]. However, they have
demonstrated that the noise current can be significantly reduced by using
silicon-on-insulator (SOI) wafer. There are several other advantages in using SOI wafer:
1) the structure of the micropore through the substrate can be precisely controlled by
using the large etching rate difference between Si and SiO2 in both plasma and wet
etching, and 2) it is possible to produce significantly miniaturized device by integrating
the biosensor and Si electronic circuits of preamplifier into the same SOI substrate.
In the present study, he has fabricated the planar patch-clamp substrates using SOI
wafer. A device, called conventional type planar patch-clamp biosensor, was assembled
with the SOI-based substrate. He used this to demonstrate that SOI is a versatile
material for planar patch-clamp substrate fabrication used in biosensor. However, the
lifetime of the cell on the micropore is too short in the planar patch-clamp biosensor,
which is a serious problem for measuring various cell functions. To elongate the
cellular lifetime, the substrate in the conventional type planar patch-clamp biosensor
was modified with fibronectin, an extracellular matrix protein, and cells were cultured
under culture medium instead of buffer solution. By using this method, he has
developed a new generation planar patch-clamp device, called incubation type planar
ion-channel biosensor.
In the former half of his doctoral course research, he developed several elementary
processes to fabricate the planar patch-clamp substrates using SOI wafer, which
produce low access resistance and low capacitance.
Two procedures of planar patch-clamp substrate fabrication were developed. One is
based on electron beam lithography (EBL) combined with reactive ion etching (RIE),
the other is based on focused ion beam (FIB).
In the fabrication method with EBL combined with RIE, firstly circular patterns
were made with EBL and RIE techniques. Then a SiO2 layer with 1 µm thickness was
grown at 900ºC with thermal oxidation in which O2 bubbled water vapor at 95ºC was
used as the reactive gas. After that large holes on the backside of the substrate were
made with 1-mm-diameter diamond drill polishing, followed by 8%(v/v) TMAH etching
at 90ºC to the buried SiO2 layer. Finally the buried SiO2 layer at the bottom of the
patterns was removed with 10% (v/v) HF solution from the topside of the substrate,
followed by 1-µm-thick SiO2 layer formation at 900ºC with thermal oxidation in the
presence of O2 bubbled water vapor at 95ºC. The scanning electron microscope (SEM)
images indicate that the initially round pore becomes faceted after the thermal
oxidation due to the crystallographic growth-rate dependence of single-crystal silicon,
and the sharp edge at the rim of the pore becomes dull after the thermal oxidation.
These are possibly unsuitable for the tight contact formation between the cell
membrane and the substrate surface.
In the fabrication method using FIB, firstly a 0.2 µm thickness SiO2 layer was
formed on the silicon surface by thermal oxidation at 900ºC with the water-saturated O2
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 SiO2 layer.
Finally micropores through the Si and SiO2 layers were made by FIB milling from the
backside. SEM images indicate that a round pore with sharp edge can be obtained with
the FIB method. Therefore the planar patch-clamp substrates made with FIB were used
to make the device. The substrate with 1.2 µm diameter pore was used to make the
conventional type planar patch-clamp biosensor. The substrate was assembled into the
microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with
transient receptor potential vanilloid type 1 (TRPV1) was positioned on the micropore
and the whole-cell configuration was formed by suction. Capsaicin was added to the
extracellular solution as a ligand molecule, and the whole-cell current of HEK-293 cell
showing desensitization unique to TRPV1 in the extracellular solution containing Ca2+
[Caterina et al. Nature 389(1997)816] was measured successfully.
In the latter half of his research, to overcome the cellular short-lifetime problem,
the incubation type planar ion-channel biosensor was developed. He modified the
substrate of the planar patch-clamp biosensor with fibronectin, and cultured the cell
positioned on the micropore under the culture medium instead of the buffer solution.
Atomic force microscope (AFM) and cell spreading assays of the FN-coated substrates
indicate that they are promising biomaterials for cell adhesion and spreading. After the
cell attached and spread on the pore of the FN-coated substrate, the resistance of the
electrolyte in the cleft between the cell membrane and the substrate surface was
evaluated based on a schematic model and its equivalent circuit. The obtained values of
seal resistance quite well agree with the experimentally measured values. Using this
method, the resistance of the electrolyte in the narrow space between the cell on the
pore and all the contacting cells was evaluated. The whole-cell configuration of the
HEK-293 cell spreading on the pore was obtained with nystatin perforation and the
whole-cell current of TRPV1 was successfully measured. It is demonstrated that
fibronectin modification of the substrate can make the cell live for a long time and the
whole-cell current of the cell spreading on the pore can be obtained during cell culture.
Moreover this planar ion-channel biosensor has high potential application to
investigate the various cell functions and the neuronal signal transductions, because the
cells can be cultured on this FN-coated planar ion-channel biosensor substrate.
","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第1115号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"物理科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"07 構造分子科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2007"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"Zhang, Zhenlong","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲1115_要旨.pdf","filesize":[{"value":"321.9 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/250/files/甲1115_要旨.pdf"},"version_id":"a6ed28f1-6bf0-4170-a1a1-58b6694cc200"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Fabrication of incubation type planar ion-channel biosensor using silicon-on-insulator substrate","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Fabrication of incubation type planar ion-channel biosensor using silicon-on-insulator substrate"},{"subitem_title":"Fabrication of incubation type planar ion-channel biosensor using silicon-on-insulator substrate","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["9"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"250","relation_version_is_last":true,"title":["Fabrication of incubation type planar ion-channel biosensor using silicon-on-insulator substrate"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T16:02:48.253299+00:00"}