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Electrode having NANO structure at tip

An electrode and recording electrode technology, which is applied in the determination/inspection of microorganisms, methods of stress-stimulated microbial growth, and bioreactors/fermenters for specific purposes, etc. The effect of cell damage

Pending Publication Date: 2021-06-15
ION CHAT RES CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] As a report of an attempt at intracellular recording by an MEA-based method, although there is an example of successfully destroying a cell membrane by seeding cells on a mushroom-shaped electrode and applying a high voltage thereto (Non-Patent Document 1), the recording of intracellular potential is maintained The time of the condition was extremely short, within 3 minutes, and it did not show effectiveness as a recording method

Method used

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  • Electrode having NANO structure at tip
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  • Electrode having NANO structure at tip

Examples

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Embodiment

[0210] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0211] Other terms and concepts in the present invention are based on the meanings of the terms commonly used in this field; especially in addition to the technology that expresses its source, as far as the various technologies used to implement the present invention are concerned, as long as those skilled in the art , it can be implemented easily and reliably based on known documents and the like. In addition, various analyzes and the like are carried out using the methods described in the manuals of the analytical instruments, reagents, and kits used, catalogs, and the like.

[0212]In addition, the description contents in the technical documents, patent publications, and patent application specifications cited in this specification are referred to as the description contents of the present invention.

reference example 1

[0213] (Reference Example 1) Determination of intracellular potential in HEK cells stably expressing Nav1.5 / Kir2.1

[0214] This experiment shows that HEK cells stably expressing Nav1.5 / Kir2.1 that spontaneously generate action potentials can be used to measure intracellular potentials with conductive nanoparticles that penetrate the cell membrane.

[0215] (Refer to 1-1) Production of HEK cells stably expressing Nav1.5 / Kir2.1

[0216] HEK cells (JCRB cell bank) were transformed with the Nav1.5 / Kir2.1 gene using the vector to produce HEK cells stably expressing Nav1.5 / Kir2.1.

[0217] Specifically, first, the Nav1.5 gene was excised from the α subunit (SCNA5, BC140813: Source Bioscience) of the human Na+ channel (Nav1.5), and inserted into pcDNA3.1(-) hygromycin (Invitrogen) .

[0218] Since BC140813 is an embryonic Nav1.5 gene, it was replaced with the Nav1.5 gene expressed in human adult myocardium by PCR or the like using human heart cDNA (Zymogen).

[0219] For the Kir2...

reference example 2

[0242] (Reference example 2) Measurement method using the charge amplifier principle

[0243] (Refer to 2-1) Preparatory experiments for the application of the charge amplifier principle

[0244] This experiment is an experiment to demonstrate that, when measuring biopotentials, the measurement system is effective by combining a charge amplifier with nanoparticles introduced into cells ( FIG. 12 ).

[0245] Circuit A is a circuit in which a model cell (cell equivalent circuit: 500MΩ resistor and 33pF capacitor) is connected in parallel in a patch clamp amplifier Axopatch 200A in current-clamp mode (Current-clamp mode) for recording. 20ms, 120pA), measure the potential change through the equivalent circuit ( Figure 8 Circuit A).

[0246] Next, connect the conductive glass of the sensor that acts as a charge amplifier between the equivalent circuit and the negative input of the amplifier. In order to make this conductive glass function as a capacitor, an aluminum foil ( Fi...

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Abstract

The purpose of the present invention is to provide a method which is designed to form an intracellular recording electrode for a cell by a simple operation which is less invasive to the cell and does not need a magnetic force, and with which the short-term or long-term intracellular potential can be accurately measured. Specifically, provided is a method comprising: securing, to a manipulator or the like, a holder provided on a conductor having a conductive nano structure at a tip; making the tip nano structure part penetrate a cell membrane while adjusting the amount of pressure applied to the target cell, thereby forming an intracellular recording electrode independently secured above the cell; and measuring the intracellular potential. The conductive nano structure at the tip and the conductor main body do not have to be magnetic but may be stuck together by magnetic force or may be formed as one body. When the cell membrane potential of a target cell cultured in a typical culture vessel is recorded, by forming the conductor main body of a magnetic electrode (Magele) and independently securing same using a ring-shaped magnet that is provided on the lower surface of the culture vessel and that secures a light projection path or a light observation path through the center thereof, measurement of the intracellular potential of the target cell and fluorescent observation of changes in intracellular potential due to a light stimulus or intracellular calcium dynamics can be performed simultaneously.

Description

technical field [0001] The present invention relates to a method for measuring and / or controlling intracellular potential or potential change by using an electrode with a nanostructure at the front end and the electrode used in the method. Background technique [0002] All cells have different ion compositions inside and outside the cell, and the intracellular potential (membrane potential) is maintained by transporters (such as sodium pumps) that maintain differences in their ion distribution and differences in their ion composition. In the resting state, although the membrane potential is stable (resting membrane potential), due to the activation and opening of ion channels on the membrane surface, due to the difference in ion concentration inside and outside the membrane, ions are released or flowed in at once, and the potential in the entire cell occurs. changes (depolarization or hyperpolarization), resulting in changes in the intracellular potential. As a result, acti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34C12Q1/02
CPCC12M35/02C12M1/34C12M1/42C12Q1/02G01N33/5061G01N33/5088
Inventor 斋藤光义
Owner ION CHAT RES CORP
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