JP3193471B2 - Integrated composite electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an integrated composite electrode having multiple electrodes, which is used in the field of electrical measurement of biological activity, particularly in the field of neuroelectrophysiology for measuring electrical activity of nerve cells.

[0002]

2. Description of the Related Art In recent years, medical examination of nerve cells and possibility of application as an electric element have been actively conducted. When a nerve cell is activated, an action potential is generated. The action potential is caused by a change in ion concentration inside and outside the cell membrane due to a change in ion permeability of a nerve cell. Detecting and examining nerve activity is performed by measuring potential changes associated with ion concentration changes (that is, ionic currents) near nerve cells using electrodes.

Conventionally, in order to measure the electrical activity of a nerve cell, a recording electrode composed of a glass electrode or the like and a stimulation electrode composed of a metal electrode or the like are inserted into cells or between cells, respectively, and the stimulation current is supplied from the stimulation electrode. It was common to measure the electrical activity of the nerve cells when a voltage (or voltage) was applied using a recording electrode.

In addition to this, for example, a cell body is pierced with a thin glass suction electrode, the inside of the cell band is refluxed with the liquid in the glass suction electrode, and an electric signal is applied from the glass suction electrode to observe the cells. There are many variations, such as the so-called intracellular perfusion method.

Further, a conductive material such as ITO (indium tin oxide) is formed on an insulating substrate with a diameter (or one side) of 1 mm.
A method of forming an electrode of 5 to 20 μm and culturing nerve cells thereon, thereby applying an electrical stimulus to the cell without inserting the electrode into the cell and recording the electrical activity of the nerve cell Have been separately proposed by the present inventors.

[0006]

In the above-mentioned prior art and its modifications, electrodes which must be considerably larger than cells, such as glass electrodes, are used. In addition, due to restrictions on operation accuracy, there is a problem that it is very difficult to perform multi-point simultaneous measurement for recording electrical activity of nerve cells by inserting two or more recording electrodes at a time in one sample.

In order to study the operation of the entire neural network, it is necessary to simultaneously record the activities of many nerve cells, and as the number of measurement points increases, the degree of difficulty increases.
There is a problem that observation between multiple cells is difficult.

Furthermore, since it is necessary to insert an electrode such as glass or metal into a cell, there is a problem that the cell is seriously damaged and it is difficult to perform measurement for a long time of several hours or more.

On the other hand, when a circular (or square) electrode having a diameter (or one side) of 15 to 20 μm is formed of an electrically conductive substance such as ITO on an insulating substrate, signal transmission between multiple cells is performed. Observation becomes possible. However, since the electrode area is small and 177μm ² ~400μm ^2, the electrode resistance is the number MΩ next culture liquid interface, usually stimulating because given by a constant current, between the electrical resistance is large electrode extremely large potential difference is generated Therefore, when electrical stimulation is applied for a long period of time at such a large voltage, ITO is destroyed, and there is a problem that observation over a long period of time is difficult.

The present invention solves such a conventional problem,
An object of the present invention is to provide an integrated composite electrode capable of easily performing simultaneous multipoint stimulation / measurement of nerve cells or the like and observing signal transmission between multiple cells for a long period of several hours or more.

[0011]

In order to solve the above-mentioned problems, an integrated composite electrode according to the present invention comprises a plurality of electrodes having the same distance between the nearest electrodes on an insulating substrate, and leads from the electrodes. a wiring portion which is disposed a line substantially radial, provided and an insulating layer covering the lead wires and electrodes, a square shape
A is greater than the length Saga 20μm its one side, and 20
It has a configuration of 0 μm or less. Also, the integration of the present invention
For composite electrodes, if the electrode is circular, its diameter
Is preferably in the range of 100 μm or more and 200 μm or less.
Good.

Further, in the integrated composite electrode of the present invention, it is preferable that the distance between the nearest electrodes is 10 to 1000 μm. Further, in the integrated composite electrode of the present invention, the insulating layer covering the lead wire has a hole on each electrode, and the insulating base is substantially formed except for the vicinity of the contact portion between the lead wire and the external circuit. The insulating layer is preferably provided on the entire surface.

Further, in the integrated composite electrode of the present invention, it is preferable that a plurality of electrode central portions are located at respective intersections on an 8 × 8 grid.

[0014]

The integrated composite electrode according to the present invention comprises a plurality of electrodes having the same distance between the nearest electrodes on an insulating substrate, and a wiring portion in which lead wires are arranged substantially radially from the electrodes. lead covers the insulating layer and the set Ketei Runode, gives a signal to the neural cells cultured on the integrated multiple electrode of the present invention, when measuring the transmission of signals between cells at the same time, closest electrode The inter-cell distance is adjusted to be approximately equal to the length of the nerve cell to be measured (that is, the cell body, dendrites, and axons), and by arranging these electrodes at equal intervals, one cell body is placed on the electrodes. However, the probability that the cell body extending from the cell body via the cell process is located on an adjacent electrode is increased. Therefore, transmission of a signal between adjacent cell bodies can be detected.

Further, since the lead wires extending from the electrodes are arranged substantially radially, a capacitance component (capacitance) between the lead wires is reduced as compared with, for example, a case where the lead wires are arranged in parallel, and a pulse as an electric signal is provided. Since the collapse of the signal waveform can be reduced and the time constant of the circuit is reduced, the responsiveness to a fast pulse signal is improved, and the responsiveness to a fast component of nerve cell activity is improved.

Further, by adjusting the electrode in a square shape, one side of which is larger than 20 μm and 200 μm or less (when the electrode is circular, 100 to 200 μm ).
Range), provide electrical stimulation to the cells over a long period of several hours or more, and when it is possible to measure the electrical activity of cells
In both cases, a good S / N ratio is obtained.

In the integrated composite electrode according to the present invention, the distance between the nearest electrodes is preferably 10 to 1000 μm, so that the length of a nerve cell is generally within this range. , The cell body is located on the electrode,
In addition, there is a high possibility of coupling via neurites, and the distance between electrodes is favorable for measurement of nerve cells.

In the integrated composite electrode according to the present invention, the insulating layer covering the lead has a hole on each electrode, and the insulating base except for the vicinity of a contact portion between the lead and an external circuit. By using a preferred embodiment in which the insulating layer is provided on almost the entire surface of the resin, compared with the case where the insulating layer is selectively provided only on the lead wire, the insulating material made of a photosensitive resin is used, and the resin is formed on almost the entire surface. Then, the required insulating layer can be easily formed by photo etching, such as removing the insulating layer on each electrode by photo-etching and opening a hole to expose the electrode, thereby facilitating production. This is preferable because the probability of insulation failure can be reduced.

Further, in the integrated composite electrode of the present invention, the plurality of electrode central portions are located at the respective intersections on the 8.times.8 grid, so that the lead wires are substantially separated from the electrode of the present invention. This is preferable because the maximum number of electrodes that can be radially arranged can be obtained.

[0020]

EXAMPLES As the insulating substrate material used in the present invention, a transparent substrate is preferable because it is necessary to observe the cells after culturing the cells, and a glass such as quartz glass, lead glass, borosilicate glass, or an inorganic material such as quartz is preferable. Substances, or polymethyl methacrylate or a copolymer thereof, polystyrene, polyvinyl chloride, polyester, polypropylene, urea resin, organic materials having transparency such as melamine resin, and the like, but mechanical strength and transparency In view of the above, an inorganic substance is preferred.

As the electrode material used in the present invention, for example, indium tin oxide (ITO), tin oxide, Cr, Au,
Cu, Ni, Al, etc. can be used. In particular, when ITO or tin oxide is used, the electrode becomes slightly yellowish and transparent, and the visibility of nerve cells under a microscope is good, which is advantageous in experimental operation. In particular, ITO has good conductivity. Is desirable.

A similar material can be applied to the lead wire material.
Again, ITO is preferred for the same reasons as the electrode material. Although not particularly limited, the thickness of these electrodes and lead wires is usually about 500 to 5000 Å, and these materials are usually deposited on an insulating substrate, and a desired pattern is formed by etching using a photoresist. Can be formed.

As an insulating layer material for insulating the lead wire provided in the present invention, for example, polyimide (P
I) Transparent resins such as resin, epoxy resin, acrylate resin, polyester resin and polyamide resin.

These resins are applied on the lead wires by an ordinary method to form an insulating layer. Note that it is preferable that the insulating layer material is a photosensitive resin such as light irradiation polymerizable, because a pattern can be formed by opening a hole in the insulating layer portion on the electrode to expose the electrode as described above.

In particular, when the insulating layer material is PI and the cells to be cultured are nerve cells, it is desirable because they show good growth. Further, among PIs, negative photo-sensitive polyimide (NPI) is preferable because, similarly to the pattern formation of the wiring portion, a hole can be formed on the electrode by using a photo-etching process after applying the negative photo-sensitive polyimide over substantially the entire surface.

The thickness of the insulating layer is not particularly limited as long as the insulating property can be imparted, but is preferably 0.1 to 10 μm, more preferably about 1 to 5 μm.

The integrated composite electrode of the present invention directly cultures cells and measures and records the electrical activity of the cells. The size of the cell body or the length of cell projections such as dendrites and axons vary depending on the culture conditions or cell types, but the closest inter-electrode distance of the integrated composite electrode is preferably 10 to 1000 μm. When the distance between the electrodes is less than 10 μm, the cell bodies are too close to each other, so that the probability that the cell bodies are adjacent to each other via cell projections is reduced, and the wiring of the lead wires becomes difficult. Also, 10
When the thickness exceeds 00 μm, wiring of lead wires is easy, but since cell projections rarely extend to about 1000 μm, the probability that cell bodies are located on the electrodes is reduced. The length of cells cultured under general conditions is about 200 to 300 μm on average in the case of mammalian central nervous cells.
It is desirably about 100 to 300 μm.

The shape of the electrode is preferably circular or square because of the requirement to keep the distance between the nearest electrodes constant. The electrode area is required to have a certain size or more because it is necessary to reduce the resistance at the interface with the culture solution in order to avoid electrode destruction when applying electrical stimulation to cells for a long period of time. However, when the electrode area increases and the resistance at the interface with the culture solution decreases, the measured electrical activity of the cells decreases and the S / N ratio decreases. That is,
Assuming that the current I is constant, I = V / R. Therefore, when the resistance value R decreases, the change in the measured potential V decreases. In other words, the electrical activity of the cell to be measured decreases, and S
/ N ratio decreases. For this reason, the electrode area needs to be carefully adjusted, and in the case of a circular electrode , the diameter is 100 to 100.
200 [mu] m, 1 side when the square electrode is larger than 20 [mu] m 200 [mu] m or less, 1 00μm~200μm is preferred especially.

Further, according to the preferred embodiment of the present invention, the holes in the insulating layer of the integrated composite electrode provide electrical stimulation to the cell bodies cultured on the integrated composite electrode,
In order to detect electrical activity from adjacent cell bodies, it is formed for the purpose of exposing electrodes and is located at the center of the electrodes. The size of this hole is preferably smaller than the size of the electrode, and one side or diameter is preferably about 15 to 195 μm.

Further, when the electrode central portion of the integrated composite electrode of the present invention is located at each intersection of concentric circles or a grid of 8 × 8 or less, the lead wires can be radially wired,
In particular, from the viewpoint of arranging as many electrodes as possible and performing simultaneous stimulation and recording at multiple points, it is desirable to provide electrodes at each intersection of an 8 × 8 grid.

Hereinafter, the integrated composite electrode of the present invention will be described in more detail with reference to specific examples. Embodiment 1 FIG. 1 is a plan view showing a pattern of a wiring portion of an integrated composite electrode according to the present invention in which an electrode 1 and a lead wire 2 are formed on an insulating substrate 3 without an insulating layer. FIG. 2 is a partially cutaway view of a plan view of only the insulating layer formed on the member shown in FIG. FIG. 3 is a sectional view of a part of the integrated composite electrode of the present invention. Hereinafter, description will be made with reference to these drawings.

First, the fabrication of the composite electrode wiring section will be described. The insulating substrate 3 of the integrated composite electrode is made of a 50 × 50 × 1 mm hard glass (“IWAKI CODE 7740 GLASS”) as a transparent insulating material having high mechanical strength.
[Iwaki Glass Co., Ltd.] The same applies hereinafter).

The material of the electrode 1 and the lead wire 2 is made of ITO, and about 100
Deposited to a thickness of 0 Å and then cleaned. Next, the center of each electrode 1 is located at each intersection (the position indicated by 5 in FIG. 2) on the 8 × 8 grid, and the distance between the centers of the electrodes closest to each electrode is equal. In addition, exposure is performed using a photoresist so that the lead wire 2 is formed into a pattern of the electrode 1 and the lead wire 2 having a radially extending shape, and pure water 5 is used.
0, hydrochloric acid 50, nitric acid 1
After etching O, the photoresist was removed. A wiring portion was formed in which the diameter of the electrode 1 was 60 μm, the width of the lead wire 2 was 30 μm, and the distance between electrode centers was 300 μm.

Next, a negative photo-sensitive polyimide (hereinafter abbreviated as NPI) as the insulating layer 4 is spin-coated so that the thickness after drying becomes 1 μm, and one side of the electrode is placed at the center of each electrode of the wiring section as shown in FIG. The insulating layer pattern was exposed and formed so that a square hole 5 of 50 μm was formed.

The contact of the lead wire 2 with the external circuit near the end in the direction opposite to the electrode 1 was coated with gold 7 and nickel 8 to improve the durability. Further, the electrode 1 at the hole 5 portion of the insulating layer 4 was immersed in a mixed aqueous solution of 1% chloroplatinic acid hexahydrate and 0.01% lead acetate,
A current of mA / cm ² was supplied for 30 seconds to deposit platinum black 6 on the electrode surface, thereby lowering the impedance.

In this embodiment, the electrode 1 and the lead 2
Although ITO was used for the portion and NPI was used for the insulating layer, it was already described that the material to be used is not limited to these. Further, the process for forming the integrated composite electrode of the present invention is not limited to the method of this embodiment.

Example 2 Next, the culture of nerve cells on the integrated composite electrode will be described. The rat cerebral visual cortex was cultured as neurons on the integrated composite electrode configured as in Example 1.

Hereinafter, the culturing method will be described in detail. (A) The brain of the fetal rat of SD rat 16-18 days after pregnancy was excised and ice-cooled Hanks balanced salt solution (hereinafter HBB)
(Abbreviated as S).

(B) The visual cortex is cut out from the brain in ice-cold HBBS, and Eagle's minimum essential medium (hereinafter abbreviated as MEM)
Transfer into solution. (C) In the MEM solution, the visual cortex is cut as finely as possible and cut into a maximum of 0.2 mm square.

(D) The finely cut visual cortex is placed in a centrifuge tube (test tube for centrifugation), washed three times with HBBS containing no calcium and magnesium (hereinafter abbreviated as CMF-HBBS), and then an appropriate amount of the same solution is used. Disperse in.

(E) In the centrifuge tube of (d), a trypsin CMF-HBBS solution (0.25% by weight) was added.
Double the total amount. 15 minutes at 37 ° C with gentle stirring
The mixture was kept at a constant temperature for 20 minutes to 20 minutes to carry out the enzyme reaction.

(F) Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and HamF-
DMEM / F-1 in which 12 mediums were mixed at a volume ratio of 1: 1
(2) The mixed medium is added to the centrifuge tube passed through the above (e) to further double the total amount. Using a pasteur pipette whose tip is burned with a burner and whose diameter is reduced, gently repeat pipetting (up to about 20 times) to loosen the cells.

(G) Centrifuge at 9806.65 m / sec ² (ie, 1000 g) for about 5 minutes. After the centrifugation, the supernatant was discarded, and the precipitate was washed with DM containing 5% FCS.
Suspend in EM / F-12 mixed medium.

(H) The above (g) and (h) are repeated two more times (a total of three times). (Nu) The finally obtained precipitate was washed with DM containing 5% FCS.
The cells are suspended in an EM / F-12 mixed medium, and the cell concentration in the suspension is measured using a red blood cell counter. Using the same medium, the cell concentration is adjusted to 2 to 4 × 10 ⁶ cells / ml.

(L) 25 mm in diameter on the integrated composite electrode
A plastic cylinder with a height of 6 mm is attached to the center of the composite electrode.
And the center of the plastic cylinder
5% F in the cell culture well
Add 500 μl of DMEM / F-12 mixed medium containing CS
Huh, CO^TwoIn the incubator (O_Two95% concentration, CO _Two
(5% concentration, 97% humidity, 37 ° C temperature).

(Iii) 100 μl of the suspension whose cell concentration has been adjusted is gently added to the well of the above (l), and the mixture is left again in the CO ₂ incubator. (G) Three days after the above operation (I), half of the medium is replaced with a new medium. The exchange medium was DM without FCS.
An EM / F-12 mixed medium is used.

(T) Thereafter, the same medium exchange as above is performed every 4 to 5 days. Through a series of these operations, the neurons of the rat cerebral cortex could be cultured on the integrated composite electrode.

The cells grew well both on the insulating layer (NPI) and on the electrode on which platinum black was deposited. Therefore, simultaneous multi-point measurement of nerve cell electrical activity was possible by using an electrode at an appropriate position as a stimulation electrode or a recording electrode.

In a state where the wells were filled with the DMEM / F-12 culture solution, a constant current stimulus of 100 μA at 0.2 Hz was applied through two electrodes, one of which was a positive electrode and the other was a negative electrode.
Even after the application for 8 hours or more, no destruction of the electrode was observed.

Therefore, simultaneous multipoint measurement of the nerve cell electrical activity could be continuously performed over a long period of 48 hours or more. The method for culturing nerve cells has many variations other than the present embodiment, and is not limited to the present embodiment.

[0051]

Industrial Applicability The present invention is capable of culturing nerve cells and simultaneously measuring multi-points of nerve cell electrical activity and long-term observation of signal transmission over multiple cells for several hours or more, which was impossible or extremely difficult in the past. And an integrated composite electrode having excellent responsiveness can be provided.

The distance between the nearest electrodes is 10 to 10
By adopting a preferred embodiment of the present invention, which is 00 μm, each cell body is located on each electrode, and the possibility of binding via neurites can be increased, and an integrated composite electrode convenient for measurement of nerve cells can be obtained. Can be provided.

Further, the insulating layer covering the lead wire has a hole on each electrode, and is provided on almost the entire surface of the insulating base except for the vicinity of the contact portion between the lead wire and the external circuit. According to a preferred embodiment of the present invention, a desired insulating layer pattern can be easily formed by a photo-etching method by applying this resin to almost the entire surface using an insulating material made of a photosensitive resin. An integrated composite electrode that is easy and has a low probability of insulation failure can be provided.

In a preferred aspect of the present invention, in which a plurality of electrode central portions are located at respective intersections on an 8 × 8 grid, the maximum number of electrodes from which the lead wires can be arranged substantially radially from the electrodes. Can be provided.

[0055]

[Brief description of the drawings]

[0056]

FIG. 1 is a plan view showing a pattern of a wiring portion without an insulating layer of an integrated electrode of the present invention in which an electrode and a lead wire are formed on an insulating substrate according to an embodiment of the present invention.

[0057]

FIG. 2 is a partially cutaway view of a plan view of only an insulating layer of one embodiment of the integrated composite electrode of the present invention.

[0058]

FIG. 3 is a partial cross-sectional view of one embodiment of the integrated composite electrode of the present invention.

[0059]

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Electrode 2 Lead wire 3 Insulating base 4 Insulating layer 5 Hole 6 Platinum black 7 Gold 8 Nickel

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁷ Identification symbol FI G01N 27/30 A61B 5/04 300Z 27/416 300J 33/48 G01N 27/46 341M H01B 5/14 (56) References JP Hei 4-204244 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/483 A61B 5/0408 A61B 5/0492 G01N 27/26 G01N 27/30 G01N 27/416 G01N 33/48 H01B 5/14

Claims (5)

(57) [Claims] 1. A wiring section comprising a plurality of electrodes having the same distance between the nearest electrodes on an insulating base, and a lead portion disposed substantially radially from the electrodes, and an insulating layer covering the lead wires. preparative provided, and electrodes, a square shape, its 1
Greater than the length Saga 20μm sides and integrated multiple electrode is 200μm or less. 2. An electrode having a circular shape and a diameter of 100
The integrated composite electrode according to claim 1, wherein the integrated composite electrode is in a range of not less than 200 m and not more than 200 m . 3. The closest distance between electrodes is 10 to 1000.
3. The integrated composite electrode according to claim 1 , which has a range of μm . 4. The method according to claim 1 , wherein the insulating layer covering the lead wire comprises an electrode.
With a hole on the top and near the contact point of the lead wire with the external circuit
Excluding the insulating layer provided on almost the entire surface of the insulating base.
Integrated composite electrode according to claim 1 that. 5. A plurality of electrode central parts are arranged on an 8 × 8 grid.
The method according to any one of claims 1 to 4, which is located at each intersection of
Integrated composite electrode.