JP4510766B2 - Extracellular electrode - Google Patents

Description

  The present invention relates to an extracellular electrode that is effective in the fields of electrophysiology and medicine in order to record electrical activity of nerve cells and to apply electrical or pharmacological stimuli to nerve cells.

  The living nervous system has a signal transmission path composed of nerve cells. These nerve cells emit signals called action potentials when transmitting information. The generation of this action potential is caused by a change in potential accompanying a change in ion permeability in the cell membrane of a nerve cell. An extracellular electrode is used to measure a potential in the vicinity of a nerve cell generated by the generation of such an action potential, that is, an extracellular potential as an electrical activity of the nerve cell.

  As the extracellular electrode, for example, an electrode composed of a plurality of microelectrodes made of a conductive substance and a multipoint electrode forming a wiring can be mentioned (for example, see Non-Patent Document 1). By culturing nerve cells or tissues on the electrodes, the multipoint electrode can record an extracellular potential in a stable state for a long time or can apply electrical stimulation to the nerve cells.

In addition, in order to improve the impedance-frequency characteristics of the extracellular electrode, the surface of the extracellular electrode is subjected to platinum black plating (see, for example, Patent Document 1), or the surface of the extracellular electrode is made of an inorganic compound. For example, coating with a conductive material is performed. Such an extracellular electrode is illustrated in FIG.
The extracellular electrode 100 shown in FIG. 6 includes an insulating substrate 101, a conductive circuit 102 provided on one surface 101a thereof, and an electrode 103 electrically connected to the conductive circuit 102 and provided on the conductive circuit 102. The insulating layer 104 is provided so as to cover the conductive circuit 102.
In this extracellular electrode 100, the electrode 103 is formed by subjecting one surface 101a of the insulating base material 101 provided with the conductive circuit 102 to a platinum black porous plating process. The electrode 103 serves to suppress the impedance of the extracellular electrode 100 to 50 kΩ or less.
As a material for forming the electrode 103, a conductive porous material made of an inorganic material such as gold, titanium nitride, silver oxide, tungsten, or the like is used instead of platinum black.

In addition, nerve cells transmit information using a chemical substance secreted by nerve cells such as glutamic acid and γ-aminobutyric acid as a transmitter. These chemical substances change the chemical structure of the protein that constitutes the cell membrane of nerve cells. Changes in the chemical structure of the protein change the permeability of ions inside and outside the neuron to the cell membrane. In order to control the change in the chemical structure of the protein, an organic compound or an inorganic compound called an agonist or antagonist is used.
An agonist interacts with a receptor for a protein constituting the cell membrane of a nerve cell, and increases ion permeability. On the other hand, antagonists act antagonistically with agonists to decrease ion permeability. In the field of research relating to electrophysiology, these substances are usually administered to nerve cells via glass microelectrodes or culture medium perfusion devices.

As described above, by forming an electrode made of platinum black porous plating or an inorganic conductive porous material, the impedance of the extracellular electrode is reduced, and the extracellular electrode is resistant to electrical external noise. / N ratio is improved. As a result, by using this extracellular electrode, it becomes possible to measure a finer extracellular potential for nerve cells.
Jimbo, Y .; and Kawana, A .; , Electrical simulation of structured neural cells by planar electrode array, Annual International Conference of the IEEE IEEE in 19 and Biotech. 1741-1742 (FIG. 1). Japanese Patent No. 3193471

However, the extracellular potential of nerve cells is usually recorded at a sampling frequency of about several kHz to several tens of kHz, but the impedance-frequency characteristics in this range are non-linear. For this reason, conventionally, it has been difficult to accurately record the waveform of the extracellular potential.
In addition, when administering a drug to a nerve cell while measuring the extracellular potential of the nerve cell using such an extracellular electrode, the drug is administered from the outside to the inside of the nerve cell using a glass microelectrode, It was necessary to administer the drug to the inside of the nerve cell by exchanging the solution around the nerve cell with the perfusion device.

  The present invention has been made in view of the above circumstances, and the impedance-frequency characteristics can easily stimulate nerve cells electrically, and have characteristics sufficient for recording electrical signals, An object of the present invention is to provide an extracellular electrode mainly composed of a conductive porous polymer capable of containing an organic compound or an inorganic compound that is charged in a solution.

The extracellular electrode of the present invention is provided on an insulating base, a conductive circuit provided on one surface of the insulating base, and on one side of the insulating base so as to overlap at least a connection portion of the conductive circuit. An extracellular electrode comprising an electrode and an insulating layer provided so as to cover a region where the electrode of the conductive circuit does not overlap, wherein the electrode is ethylenedioxythiophene and poly (ethylenedioxythiophene) -Mainly a conductive porous polymer composed of a mixture of poly (styrene / sulfonic acid).

  The electrode may contain an organic compound or an inorganic compound that is charged in a solution.

The extracellular electrode of the present invention is provided on an insulating base, a conductive circuit provided on one surface of the insulating base, and on one side of the insulating base so as to overlap at least a connection portion of the conductive circuit. An extracellular electrode comprising a plurality of electrodes and an insulating layer provided so as to cover a region where the plurality of electrodes of the conductive circuit do not overlap, the plurality of electrodes comprising ethylenedioxythiophene, It is characterized by being mainly composed of a conductive porous polymer composed of a mixture of (ethylenedioxythiophene) -poly (styrene / sulfonic acid).

The extracellular electrode of the present invention is provided on an insulating base, a conductive circuit provided on one surface of the insulating base, and on one side of the insulating base so as to overlap at least a connection portion of the conductive circuit. An extracellular electrode comprising an electrode and an insulating layer provided so as to cover a region where the electrode of the conductive circuit does not overlap, wherein the electrode is ethylenedioxythiophene and poly (ethylenedioxythiophene) -Since it is mainly made of conductive porous polymer composed of poly (styrene sulfonic acid), it is possible to measure the electrical activity of nerve cells by bringing them into contact with nerve cells. On the other hand, a voltage can be applied as an electrical stimulus.
Further, if the electrode constituting the extracellular electrode of the present invention contains an organic compound or an inorganic compound that is charged in a solution, the organic compound or the inorganic compound is energized to the extracellular electrode, thereby providing an organic compound. Alternatively, the elution of the inorganic compound into the solution can be controlled. Therefore, the extracellular electrode of the present invention can administer a drug to nerve cells that are in contact with or close to the extracellular electrode.

  Hereinafter, an extracellular electrode embodying the present invention will be described in detail with reference to the drawings.

FIG. 1 is a schematic cross-sectional view showing an embodiment of an extracellular electrode according to the present invention.
The extracellular electrode 10 of this embodiment includes an insulating base material 11, a conductive circuit 12 provided on one surface 11a of the insulating base material 11, and a connection surface 12a of the conductive circuit 12 on the one surface 11a side of the insulating base material 11. The electrode 13 is provided so as to overlap, and the insulating layer 14 is provided so as to cover a region where the electrode 13 of the conductive circuit 12 does not overlap. In the extracellular electrode 10, the electrode 13 is electrically connected to the conductive circuit 12.
The electrode 12 is made of ethylenedioxythiophene (hereinafter abbreviated as “EDOT”) and poly (ethylenedioxythiophene) (poly (3,4-ethylenediothiophene)), hereinafter abbreviated as “PEDOT”. .)-Conducting porous polymer composed mainly of a mixture of poly (styrene sulfonic acid) (hereinafter abbreviated as “PSS”).
The chemical formula of EDOT is shown in the following formula (1), the chemical formula of PEDOT is shown in the following formula (2), and the chemical formula of PSS is shown in the following formula (3).

The electrode 13 can also contain an organic compound or an inorganic compound that is charged in a solution.
In this embodiment, examples of the solution include a general cell culture solution.

  Examples of the organic compound that is charged in the above solution include amino acids that are neurotransmitters of nerve cells, and specific examples include glutamic acid, gamma aminobutyric acid, and glycine.

  Moreover, as an inorganic compound charged in said solution, a salt etc. are mentioned as an inorganic compound, Specifically, chlorides, such as sodium and potassium, are mentioned.

  As the insulating substrate 11, a transparent material is preferably used because it is necessary to observe the nerve cell in a state where the extracellular electrode 10 is in contact with the nerve cell. For example, quartz glass, lead glass, boron Examples include inorganic materials such as glass such as silicate glass or quartz, or transparent organic materials such as polymethyl methacrylate or copolymers thereof, polystyrene, polyvinyl chloride, polyester, polypropylene, urea resin, and melamine resin. It is done. Inorganic substances are preferred because of their excellent mechanical strength and transparency.

  Examples of the material forming the conductive circuit 12 include tin-doped indium oxide (Indium Tin Oxide, hereinafter abbreviated as “ITO”), tin oxide, chromium (Cr), gold (Au), copper (Cu), nickel ( Ni), aluminum (Al), or the like is used. Among these, ITO or tin oxide is preferably used because the conductive circuit 12 becomes transparent and is suitable for observing nerve cells, and ITO is more preferably used because of its high conductivity.

  Further, the thickness of the conductive circuit 12 is not particularly limited, and is appropriately set according to the thickness and shape of the electrode 13.

  The shape and arrangement of the electrode 13 are not particularly limited, and are appropriately set according to the size and number of target nerve cells.

  As the insulating material forming the insulating layer 14, a transparent resin is used because it is necessary to observe the extracellular cells 10 in culture. For example, polyimide resin, epoxy resin, acrylate resin, polyester resin, polyamide Resin etc. are mentioned.

Next, with reference to FIG. 1 and FIG. 2, the manufacturing method of the extracellular electrode 10 of this embodiment is demonstrated.
First, the insulating base material 11 provided with the conductive circuit 12 is prepared on the one surface 11a.
Next, the insulating layer 14 made of the transparent resin is provided so as to cover the conductive circuit 12 and to have a predetermined thickness and shape. At this time, a predetermined region on the surface 12b of the conductive circuit 12 on the side in contact with the insulating layer 14 (hereinafter referred to as “one surface 12b” of the conductive circuit 12), that is, a region electrically connected to the electrode 13 is exposed. Thus, the insulating layer 14 is provided. As a result, an opening 14a for providing the electrode 13 is formed in the insulating layer 14 as shown in FIG.

Next, the one surface 12b (connecting portion 12a) of the conductive circuit 12 exposed in the opening 14a of the insulating layer 14 is cleaned by plasma treatment such as air plasma.
Next, the insulating base material 11 provided with the conductive circuit 12 and the insulating layer 14 is immersed in EDOT and PEDOT-PSS.

Next, when this mixed solution is energized with a predetermined voltage and current while the insulating base material 11 is immersed in the mixed solution, as shown in FIG. 2, one surface 12b of the conductive circuit 12 in the opening 14a. On top of this, EDOT molecules 15 and PEDOT-PSS molecules 16 are gradually deposited to form a conductive porous polymer 17.
When the conductive porous polymer 17 is energized when the predetermined thickness is reached, as shown in FIG. 1, the extracellular electrode provided with the electrode 13 mainly composed of the conductive porous polymer 17 is provided. Get 10.
In addition, the thickness of the electrode 13 can be appropriately controlled by the energization time to the mixed solution.

  The extracellular electrode 10 of this embodiment is provided on the insulating base 11, the conductive circuit 12 provided on the one surface 11a of the insulating base 11, and on the one surface 11a side of the insulating base 11 so as to overlap the conductive circuit 12. The electrode 13 and the insulating layer 14 provided so as to cover the conductive circuit 12 are roughly configured, and the electrode 13 mainly includes a conductive porous polymer composed of EDOT and PEDOT-PSS. Therefore, in the frequency range of 100 Hz to 100 kHz, the impedance hardly changes and the phase shift is small, so that the impedance is smaller than that of the ITO electrode and the impedance-frequency characteristics are excellent. .

  Further, when the electrical activity (extracellular potential) of a nerve cell is measured using the extracellular electrode 10, when an electrical signal having the same voltage is applied to the nerve cell, more nerves are used than when the ITO electrode is used. It can excite the electrical activity of cells. Furthermore, when an electrical signal having the same voltage is applied to the nerve cell, a larger current flows through the extracellular electrode 10 than when the ITO electrode is used. This is presumably because a wide range of cells are excited in the nerve cells connected to the extracellular electrode 10.

  Moreover, you may mix the organic compound or inorganic compound which is charged in said solution with the mixed solution which melt | dissolved EDOT and PEDOT-PSS. In this way, the electrode 13 mainly composed of the conductive porous polymer 17 formed by energizing the solution contains an organic compound or an inorganic compound that is charged in the solution.

  Thereby, if the electrode 13 contains an organic compound or an inorganic compound that is charged in the solution, the organic compound or the inorganic compound is eluted from the electrode 13 according to the voltage applied to the electrode 13, and the extracellular electrode The electrical activity of the nerve cell connected to 10 can be activated or suppressed. For example, the electrode 13 contains an organic compound or an inorganic compound that is negatively charged in the solution, and a voltage is applied to the extracellular electrode 10 so that the electrode 13 becomes a cathode in the solution. It is possible to administer an organic compound or an inorganic compound, that is, to apply a pharmacological stimulus, to a nerve cell in contact with the electrode and a nerve cell in the vicinity of the electrode 13.

Further, in this embodiment, the extracellular electrode 10 in which one electrode 13 is provided on the one surface 11a of the insulating base material 11 is illustrated, but the extracellular electrode of the present invention is not limited to this. The extracellular electrode of the present invention has a plurality (two or more) of conductive porous polymers mainly composed of EDOT and PEDOT-PSS in a predetermined arrangement and a predetermined shape on one surface of an insulating substrate. ) Electrode may be provided.
Thus, the extracellular electrode provided with a plurality of electrodes can excite the electrical activity of a larger number of nerve cells at the same time.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to a following example.

Example 1
As shown in FIG. 1, an insulating base 11 (50 μm × 50 μm) provided with a conductive circuit 12 made of ITO on one surface 11a was prepared.
Next, an insulating layer 14 made of a transparent resin was provided so as to cover the conductive circuit 12, have a predetermined thickness, and have an opening 14a formed at a predetermined position.
Next, the one surface 12a of the conductive circuit 12 exposed in the opening 14a of the insulating layer 14 was cleaned by air plasma treatment.
Next, the insulating substrate 11 provided with the conductive circuit 12 and the insulating layer 14 was immersed in a mixed solution prepared by dissolving EDOT and PEDOT-PSS in a sodium chloride aqueous solution.
Next, in a state where the insulating base material 11 is immersed in the mixed solution, the mixed solution is energized at a predetermined voltage and current, and a conductive porous polymer composed of EDOT and PEDOT-PSS is obtained. The main electrode 13 was formed, and the extracellular electrode 10 was obtained.

(Electrical characteristics of extracellular electrode)
The electrical characteristics of the extracellular electrode 10 obtained in Example 1 were measured under the following conditions.
The extracellular electrode 10 is immersed in a 0.15 M sodium chloride solution, and a bipolar pulse voltage (pulse width 100 μs) of | 0.1 | V to | 0.5 | V is applied to the extracellular electrode 10 in this state. The response of impedance and phase of the extracellular electrode 10 at frequencies from 1 Hz to 100 kHz was measured. This measurement is repeated 10 times, and FIG. 3 shows an average value of these 10 measurements as a Bode diagram. 3, (a) shows the impedance of the ITO electrode, (b) shows the phase difference of the ITO electrode, (c) shows the impedance of the extracellular electrode 10, and (d) shows the phase difference of the extracellular electrode 10, respectively. . In FIG. 3, the right vertical axis represents phase, the left vertical axis represents gain, and the horizontal axis represents frequency.
From the results in FIG. 3, it was confirmed that the impedance of the extracellular electrode 10 hardly changed and the phase shift was small in the frequency range of 100 Hz to 100 kHz. Therefore, it was found that the extracellular electrode 10 has improved impedance-electric characteristics as compared with the ITO electrode.

(Measurement of extracellular potential 1)
Using the extracellular electrode 10 obtained in Example 1, the extracellular potential of the nerve cell was measured under the following conditions.
Nerve cells isolated from the cerebral cortex of rats (embryonic day 8) were cultured on the extracellular electrode 10 in the cell culture medium. For comparison, neurons isolated from the same rat-derived cerebral cortex were cultured on ITO electrodes.
Next, the spontaneous extracellular potential of nerve cells was measured. The result is shown in FIG.
4A and 4B, the vertical axis represents potential and the horizontal axis represents time.
From the results of FIGS. 4A and 4B, it was confirmed that the extracellular electrode 10 can measure the extracellular potential of the nerve cell similarly to the ITO electrode.

(Measurement of extracellular potential 2)
The number of extracellular potentials is measured by applying a | 0.5 | V bipolar pulse (pulse width 200 μs) to the extracellular electrode 10 in which the nerve cells used in the above-described extracellular potential measurement 1 are cultured. did.
Similarly, a bipolar pulse (pulse width 200 μs) of | 0.5 | V was applied to the ITO electrode used in the above-described extracellular potential measurement 1 to measure the number of extracellular potentials. .
The number of extracellular potentials is a signal derived from the same cell when the amplitude and time width distribution of each extracellular potential are within a certain range in the extracellular potential observed from 10 ms to 315 ms after stimulation. It measured using the cluster cutting method.
The result is shown in FIG.
From the results of FIG. 5, it was found that the extracellular electrode 10 excites more electrical activities of nerve cells than the ITO electrode when an electrical signal of the same voltage is applied. This is considered to be because when a voltage of the same magnitude is applied, a larger current flows through the extracellular electrode 10 and it is possible to excite neurons existing in a wide area.

(Example 2)
In the EDOT and PEDOT-PSS mixed solution, the insulating base material 11 provided with the conductive circuit 12 and the insulating layer 14 is immersed, and the insulating base material 11 is immersed in the mixed solution. Then, the mixture solution was energized, and the cell 13 was formed in the same manner as in Example 1 except that the electrode 13 mainly composed of a conductive porous polymer containing glutamic acid and composed of EDOT and PEDOT-PSS was formed. An outer electrode 10 was obtained.

(Administration of organic compounds to nerve cells)
Using the extracellular electrode 10 obtained in Example 2, glutamic acid was administered to nerve cells in direct contact with the electrode 13 and nerve cells in the vicinity of the electrode 13 under the conditions shown below.
Nerve cells isolated from the rat cerebral cortex (embryonic day 8) were cultured on the extracellular electrode 10.
Next, the spontaneous activity of nerve cells cultured on the extracellular electrode 10 was measured.
As a result, it was confirmed that the extracellular potential of the above-described nerve cell could be measured.
Next, for 1 minute before and after applying a negative pulse voltage (pulse width 500 ms) that does not excite the electrical activity of the nerve cell to the extracellular electrode 10 brought into contact with the nerve cell 10 times every second. Changes in the spontaneous electrical activity of neurons were measured as the number of extracellular potentials.
As a result, no change was observed in the number of extracellular potentials of nerve cells after applying the pulse voltage. Therefore, it is considered that glutamic acid contained in the electrode 13 was eluted into the aqueous sodium chloride solution and excited the electrical activity of the nerve cells.

From the above results, according to the extracellular electrode of this example, when the same voltage is applied with a low impedance, it is possible to excite the electrical activity of more nerve cells than when the ITO electrode is used. It was confirmed that it was possible.
Further, according to the extracellular electrode of this example, the extracellular electrode brought into contact with the nerve cell is immersed in the solution, and a voltage is applied to the extracellular electrode, thereby directly contacting the electrode. It was confirmed that the drug can be locally administered to nerve cells and nerve cells in the vicinity of the electrode without using a special instrument.

  The extracellular electrode of the present invention can also be applied to drug administration in the medical field.

It is a schematic sectional drawing which shows one Embodiment of the extracellular electrode which concerns on this invention. It is a schematic sectional drawing which shows the manufacturing method of the extracellular electrode which concerns on this invention. In the Example of this invention, it is a graph which shows the result of having measured the response of the impedance and phase of an extracellular electrode. In the Example of this invention, it is the graph which shows having measured the extracellular potential of the nerve cell. In the Example of this invention, it is a graph which shows the result of having measured the number of the extracellular potential. It is a schematic sectional drawing which shows the conventional extracellular electrode.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Extracellular electrode, 11 ... Insulating base material, 12 ... Conductive circuit, 13 ... Electrode, 14 ... Insulating layer.

Claims (3)

An insulating base, a conductive circuit provided on one surface of the insulating base, an electrode provided on one side of the insulating base so as to overlap at least a connection portion of the conductive circuit, and the conductive circuit An extracellular electrode provided with an insulating layer provided so as to cover a region where the electrodes do not overlap,
The electrode is mainly composed of a conductive porous polymer composed of a mixture of ethylenedioxythiophene and poly (ethylenedioxythiophene) -poly (styrene / sulfonic acid). .  The extracellular electrode according to claim 1, wherein the electrode contains an organic compound or an inorganic compound that is charged in a solution. An insulating base, a conductive circuit provided on one surface of the insulating base, a plurality of electrodes provided on one side of the insulating base so as to overlap at least a connection portion of the conductive circuit, and the conductive circuit An extracellular electrode provided with an insulating layer provided so as to cover a region where the plurality of electrodes are not overlapped,
The plurality of electrodes are mainly composed of a conductive porous polymer composed of a mixture of ethylenedioxythiophene and poly (ethylenedioxythiophene) -poly (styrene / sulfonic acid). Outer electrode.

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