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Planar patch clamp device, electrodes for said device and cell ion channel current measurement method

Foreign code F170009207
File No. AF19-03WO
Posted date Sep 12, 2017
Country EPO
Application number 12859984
Gazette No. 2801820
Date of filing Dec 5, 2012
Gazette Date Nov 12, 2014
International application number JP2012081556
International publication number WO2013094418
Date of international filing Dec 5, 2012
Date of international publication Jun 27, 2013
Priority data
  • P2011-278445 (Dec 20, 2011) JP
  • P2012-181786 (Aug 20, 2012) JP
  • 2012WO-JP81556 (Dec 5, 2012) WO
Title Planar patch clamp device, electrodes for said device and cell ion channel current measurement method
Abstract (EP2801820)
An object of the present invention is directed to reducing a noise current by suppressing variations in an applied membrane potential in the planar patch clamp device, thereby enabling accurate measurement of an ion channel current.
A planar patch clamp device comprising: an electrically insulative substrate having at least one fine through holes; a liquid reservoir and an electrode unit disposed on each of surfaces of the through-hole are configured so that the liquid reservoir retains a conductive liquid and the electrode unit is electrically communicate with the liquid reservoir, wherein the electrode comprises (a) an electrode vessel, wherein at least part of a vessel wall is made of an inorganic porous material, (b) an electrode in which a chloride (NmCl) layer is formed on the surface of a noble metal (Nm), and (c) an electrode solution containing (NmCl) and an alkali metal chloride being dissolved at a saturated concentration, is disclosed.
Scope of claims [claim1]
1. A planar patch clamp device comprising: (1) an electrically insulative substrate having at least micro through-hole interconnecting both surfaces of the electrically insulative substrate, the through-hole allowing for passage of liquid while preventing passage of a cell; and (2) a liquid reservoir and an electrode unit disposed on each of a first surface side and a second surface side of the through-hole, the liquid reservoir and the electrode unit being configured such that when a conductive liquid is conatined in the liquid reservoir, the electrode unit is in electrical contact with the conductive liquid; (3) wherein the liquid reservoir on the first surface side is configured to receive a cell, and (4) wherein the electrode unit on each of the first and second surface sides includes (a) an electrode vessel having a wall of which at least a part is made of an inorganic porous material and configured such that when a conductive liquid is conatined in the liquid reservoir, at least the part of the wall is in contact with the conductive liquid, (b) an electrode contained in the electrode vessel and made of noble metal (Nm), the electrode having a surface layer of chloride of the noble metal (NmCl), and (c) an electrode solution, filled in the electrode vessel, in which the chloride of the noble metal (NmCl) and an alkali metal chloride are dissolved at saturation concentrations.
[claim2]
2. The planar patch clamp device according to claim 1, wherein the liquid reservoir on the first surface side comprises a main liquid reservoir for cell placement, made of a light non-transmissive material, a sub liquid reservoir configured to contain the electrode unit on the first surface side, and a narrow liquid path interconnecting the main liquid reservoir and the sub liquid reservoir.
[claim3]
3. The planar patch clamp device according to claim 1 or 2, wherein the liquid reservoir on the second surface side is communicated with a liquid path to introduce and discharge a conductive liquid, and the electrode unit on the second surface side is disposed on this liquid path.
[claim4]
4. The planar patch clamp device according to any one of claims 1 to 3, wherein a plurality of liquid reservoirs are disposed on the first surface side, and a recess having a width larger than that of a cell body of a nerve cell and having a depth that can inhibit the movement of a cultured cell body is formed in these liquid reservoirs.
[claim5]
5. The planar patch clamp device according to any one of claims 1 to 4, comprising the following constitution(s) (A) and/or (B): (A) a liquid reservoir on the second surface side is communicated with a liquid suction device, which imposes negative pressure on the liquid reservoir on the second surface side, and (B) an extracellular matrix-forming substance capable of fixing cell is attached around the opening on the first surface side of the through hole on the substrate.
[claim6]
6. An electrode unit for planar patch clamp device, comprising the following constituents (a') to (c'): (a') an electrode vessel, wherein at least part of a vessel wall is composed of an inorganic porous material, (b') an electrode contained in the electrode vessel and made of noble metal (Nm), the electrode having a surface layer of chloride of the noble metal (NmCl), and (c') an electrode solution, filled in the electrode vessel, in which the chloride of the noble metal (NmCl) and an alkali metal chloride are dissolved at saturation concentrations.
[claim7]
7. The electrode unit for planar patch clamp device according to claim 6, wherein the inorganic porous material constituting at least part of the vessel wall is porous glass or porous ceramic.
[claim8]
8. The electrode unit for planar patch clamp device according to claim 6 or 7, wherein a noble metal Nm is silver (Ag) or platinum (Pt), and an alkali metal chloride is potassium chloride (KCl).
[claim9]
9. The electrode unit for planar patch clamp device according to any one of claims 6 to 8, wherein the electrode has the following characteristic (C) or (D): (C) the electrode protrudes inside the electrode vessel so as to form a stick electrode, and is composed of a core made of noble metal (Nm), having a surface layer of a chloride of the noble metal (NmCl), a noble metal chloride (NmCl) layer being formed on the surface of a core made of the noble metal Nm, or (D) the electrode is a cylindrical electrode formed on the inner periphery of an electrode vessel wall, wherein the bottom layer on the vessel wall side is a deposited layer of a noble metal (Nm) and the surface layer in contact with an electrode solution is a deposited layer of chloride of noble metal (NmCl).
[claim10]
10. A cellular ion channel current measurement method, comprising: introducing a conductive liquid into the liquid reservoir on the second surface side of the planar patch clamp device according to any one of claims 1 to 5, introducing a conductive liquid containing cells to be measured dispersed therein into the liquid reservoir on the first surface side, thereby enabling electrical communication between the liquid reservoir on the first surface side and the liquid reservoir on the second surface side; disposing the cell at a prescribed position of the liquid reservoir on the first surface side; applying a voltage between electrodes of electrode units on the first surface side and second surface side; and measuring an ion channel current of a cell to be measured.
[claim11]
11. The cellular ion channel current measurement method according to claim 10, wherein a plurality of liquid reservoirs of the constitution according to claim 4 are configured on the first surface side of the substrate in the planar patch clamp device and a cell to be measured is a nerve cell, and a conductive liquid is a cell culture medium of the nerve cell.
[claim12]
12. The cellular ion channel current measurement method according to claim 11, wherein a Ca probe for Ca imaging is introduced into the nerve cell in advance, and an ion channel current of the cell is measured by means at least including Ca imaging, which is observation of fluorescence that occurs at generation of cell action potential or at propagation of an action potential.
[claim13]
13. The cell ion channel current measurement method according to claim 12, wherein plural or many nerve cells into which a Ca probe introduced are disposed on the first surface side in the planar patch clamp device, and synapses of these nerve cells mutually linked are defined as a nerve cell network, and a current is injected or a voltage is applied to a single nerve cell of these cells to perform measurement by the Ca imaging in plural or many nerve cells.
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • URISU TSUNEO
  • WANG ZHI-HONG
  • UNO HIDETAKA
  • OBULIRAJ SENTHIL KUMAR
  • NAGAOKA YASUTAKA
IPC(International Patent Classification)
Specified countries (EP2801820)
Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Reference ( R and D project ) CREST Creation of Nanosystems with Novel Functions through Process Integration AREA
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