KR100481743B1 - Extracellular recording electrode - Google Patents

Abstract

And a plurality of microelectrodes disposed on the substrate, and a wiring portion capable of providing an electrical signal to or extracting an electrical signal from the microelectrodes, the microelectrode having a porous conductive material on its surface. And an integrated composite electrode having an impedance of 50 mA or less. Preferably, the porous conductive material is gold and is formed by energizing for 10 to 360 seconds at a current density of 1.0 to 5.0 A / dm 2. The integrated composite electrode may include a wiring portion including a micro electrode disposed in a matrix on a substrate, a lead wire connected to the micro electrode, and an electrical contact connected to an end of the lead wire.

Description

 Integrated complex electrode, integrated cell locator, cell potential measuring device and cell potential measuring system {Extracellular recording electrode}

The present invention relates to an integrated composite electrode for extracellular recording, useful in the field of neuroelectrophysiology, for measuring potential changes, etc., in response to neuronal activity.

In recent years, nerve cells have been actively studied for their medical review and their applicability to electric devices. When a nerve cell or the like is active action potential occurs. This action potential is caused by the change in ion concentration inside and outside the cell membrane in accordance with the change in ion permeability of the nerve cell, and thus the cell membrane potential. Therefore, the activity of the nerve cell can be observed by measuring the change in potential with the change of the ion concentration in the vicinity of the nerve cell by the electrode.

Conventionally, the measurement of the potential according to the cell activity as described above was performed by placing a glass extracellular potential measurement electrode or a metal (platinum, etc.) electrode in the vicinity of the cell using a micro-manipulator or the like. Alternatively, the cell's electrical activity was measured by inserting the same electrode into the cell. Either conventional method requires a skilled technique for preparing the electrode, has a high impedance, is susceptible to external noise, and furthermore, the method of inserting the electrode into a cell may cause injury to cells or tissues. there was. For this reason, it was not suitable for stable long-term observation.

Thus, the present inventors have developed an integrated composite electrode using a conductive material on an insulating substrate to form a plurality of micro electrodes and extract patterns thereof, and to allow cell and tissue culture thereon (Japan) See Japanese Patent Application Laid-Open Nos. 6-78889 and 6-296595. This makes it possible to stably and prolong the observation of neuronal activity without causing injury to cells or tissues.

In the integrated composite electrode, the uppermost surface of the electrode close to the cell is plated with platinum black porous by electrolysis (Japanese Patent Laid-Open No. 6-78889) or forming gold by evaporation (Japan). Japanese Patent Application Laid-Open No. 6-296595). However, in the case of platinum black plating, it is easy to adjust the impedance of the electrode to about 50 kΩ or less, which is a practical level. However, since the strength is weak, there are problems such as low electrode reuse rate. On the other hand, in the case of gold formed by vapor deposition, the strength was improved, but it was difficult to adjust the impedance to about 50 Hz or less.

1A is a micrograph with an enlarged magnification of 2500 times showing the surface of a gold plating according to a comparative example, formed on a microelectrode by electrolysis at a current density of 1.0 A / dm 2. The size in the figure is 50 µm.

1B is a micrograph showing the surface of the gold plating according to the present invention formed on a microelectrode by electrolysis at a current density of 1.5 A / dm 2. The size in the figure is 50 µm.

1C is a micrograph showing the surface of the gold plating according to the present invention formed on a microelectrode by electrolysis at a current density of 2.0 A / dm 2. The size in the drawing is 50 µm.

FIG. 2A shows a print out of a computer screen displaying in 64 channels the response of a cell's potential change to a constant current stimulus on a gold plated coated microelectrode according to a comparative example, obtained by electrolysis at a current density of 1.0 A / dm 2; Drawing. Stimulus signal is applied to channel 29.

FIG. 2B shows a print out of a computer screen displaying the noise level in the absence of cells in 64 channels with respect to the gold plated coated microelectrode of FIG. 2A. FIG.

FIG. 2C shows a print out of a computer screen displaying in 64 channels the response of a cell's potential change to a constant current stimulus on a gold plated coated electrode according to the present invention obtained by electrolysis at a current density of 1.5 A / dm 2; FIG. .

FIG. 2D shows a print out of a computer screen displaying the noise level in the absence of cells in 64 channels for the gold plated coated microelectrode of FIG. 2C. FIG.

FIG. 2E shows a printout of a computer screen displaying in 64 channels the correspondence of the potential change of cells to constant current stimulation on a gold plated coated microelectrode according to the present invention obtained by electrolysis at a current density of 2.0 A / dm 2; .

FIG. 2F shows the print out of a computer screen displaying the noise level in the absence of cells in 64 channels for the gold plated coated microelectrode of FIG. 2E.

3 is a diagram showing an impedance characteristic of the microelectrode of the present invention.

4A shows an equivalent circuit of the microelectrode of the present invention.

4B shows an equivalent circuit of the microelectrode of the present invention.

5 is a diagram showing an impedance characteristic of an equivalent circuit of the microelectrode of the present invention.

6 is a diagram showing an impedance characteristic of an equivalent circuit of the microelectrode of the present invention.

7 is a diagram showing an impedance characteristic of a microelectrode of a comparative example.

8 is a diagram showing an impedance characteristic of an equivalent circuit of the microelectrode of the comparative example.

9 is a diagram showing an impedance characteristic of a microelectrode of a comparative example.

10 is a diagram showing an impedance characteristic of an equivalent circuit of the microelectrode of the comparative example.

It is a graph which shows the result of the life test which compared the reusability of the microelectrode which carried out the electroplated gold plating by this invention with the prior art.

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems, and an object thereof is to provide an impedance-frequency characteristic having both a low impedance, a property hard to be influenced by external noise, a high intensity, and an easy electrode reuse property. An extracellular recording electrode suitable for recording an electrical signal of a cell is provided.

MEANS TO SOLVE THE PROBLEM The present inventors can obtain the porous conductive material which roughened the surface and increased the surface area by optimizing the current density at the time of forming a conductive material in the uppermost surface in preparation of an extracellular recording electrode, and It was found that this porous conductive material exhibited suitable properties as an electrode for extracellular recording. This invention was completed based on this knowledge.

The present invention is an integrated composite electrode for measuring the electrophysiological characteristics of a cell, and is capable of imparting an electrical signal to or extracting an electrical signal from a plurality of micro electrodes disposed on a substrate and the micro electrodes. An integrated composite electrode having a wiring portion, wherein the microelectrode has a porous conductive material on its surface, and the conductive material is selected from the group consisting of gold, titanium nitride, silver oxide, and tungsten, and has an impedance of 50 mA or less. To provide.

Preferably, the porous conductive material is gold and is formed by energizing for 10 to 360 seconds at a current density of 1.0 to 5.0 A / dm 2.

The present invention also provides an integrated composite electrode for measuring the electrophysiological characteristics of a cell, wherein the plurality of micro-electrodes disposed on a substrate and the micro-electrodes can be provided with electrical signals or derived from the micro-electrodes. A wiring portion having a wiring portion, and the surface area of the microelectrode calculated from the capacitance of the equivalent circuit showing substantially the same impedance characteristics as the microelectrode may be at least 10 times to less than 200 times the projected area of the microelectrode, and Provided is an integrated composite electrode having an impedance of 50 kHz or less.

Here, the term "projection area of the microelectrode" means the total area of the top surface of the microelectrode before forming the conductive material.

Preferably, the surface area measured by the gas adsorption method of the said microelectrode is a range which does not exceed 5x10 ⁵ times the projected area of this microelectrode.

In one embodiment of the present invention, the micro electrodes are arranged in a matrix on a substrate, and the wiring portion includes a lead wire connected to the micro electrode and an electrical contact connected to an end of the lead wire, At least the surface of the lead wire is covered with the insulating layer.

In one embodiment of the present invention, the porous conductive material may be formed by etching.

The present invention also provides an integrated cell mounting device having the integrated composite electrode described above and having a cell mounting region for mounting cells or tissues on a substrate of the integrated composite electrode.

The present invention also includes the integrated cell mounting device described above and an output signal processing device connected to the microelectrode in order to process an output signal by electrophysiological activity of a cell or tissue, and furthermore, Provided is a cell potential measuring device having a stimulus signal imparting device connected to the microelectrode for giving electrical stimulation.

The present invention further comprises a cell potential measuring device as described above, and furthermore, a cell potential measuring device having an optical observation device for optically observing a cell or tissue and / or a cell culture device for controlling a culture environment of the cell or tissue. Provide a system.

Hereinafter, the present invention will be described in more detail.

(Formation of porous conductive material of the micro electrode)

The integrated composite electrode for extracellular recording provided by the present invention includes a plurality of micro electrodes disposed on an insulating substrate. By arranging the cells on this microelectrode, it is possible to measure the electrical activity of the cells.

In particular, the integrated composite electrode of the present invention is characterized by having a porous conductive material on the uppermost surface of the microelectrode, and having an impedance of 50 kPa or less. The impedance of the electrode is preferably 35 kPa or less, more preferably 25 kPa or less, and still more preferably 10 kPa or less. Impedance here is defined as saying the value measured by the frequency of 1 Hz and the voltage between terminals by 50 Hz. The lower limit of the impedance in the present invention is not particularly limited and is preferably as low as possible in accordance with the teachings of the present specification.

The low impedance mentioned above is considered to be a characteristic which can be obtained derived from the porous structure of the conductive material of the electrode surface. Here, the porosity means that the surface of the conductive material has a so-called rough shape, that is, a shape including a large number of fine unevennesses. When the enlarged image of the porous conductive material surface in the present invention is observed under an optical microscope, the surface of the porous conductive material exhibits a close aggregation of fine particles having a diameter of about 0.01 to 25 µm. By making the top surface of the electrode such a porous structure, the surface area is remarkably increased, and as a result, a low impedance substantially impossible with gold of a smooth surface obtained by conventional deposition or the like can be realized.

As such, the porous structure of the electrode top surface may also be defined by the surface area of the electrode top surface. The surface area of the electrode uppermost surface can be performed using the BET method by gas adsorption well-known to those skilled in the art, for example. Alternatively, it can be calculated from the capacitance of the equivalent circuit, which represents the model of the microelectrode and solution interface as equivalent to the electrical circuit.

The porous conductive material in the present invention is typically formed by performing an electrolytic plating process under an overcurrent density. The overcurrent density preferably refers to a current density in excess of 1.2 A / dm 2, more preferably 1.0 to 5.0 A / dm 2, and most preferably 1.4 to 2.1 A / dm 2. This is in contrast to the current density used in conventional industrial electrolytically conductive material plating processes of about 1.0 A / dm 2 or less. In addition, even when a current density of more than 3.0 A / dm 2 is used, the porous electrode material plating can be obtained, but the current density is excessive and the surface is roughened to an extreme shape. For example, it becomes difficult to maintain square). In the present invention, conductive material plating can be formed by energizing for typically 10 to 360 seconds, preferably 30 to 240 seconds under an overcurrent density. If the energization time is too short, the conductive material plating may not be sufficiently formed on the microelectrode. When the energization time is too long, the growth of the conductive material is uneven on the microelectrode, resulting in a portion where the growth of the conductive material plating is rapid and a slow portion, and the shape of the electrode tends not to be square.

These electroplating conditions are exemplified, and needless to say, as long as the above-described low impedance value can be achieved, for example, they can be changed in a timely manner according to the limitation of the electroplating apparatus, an operation request, and the like.

Alternatively, the porous conductive material in the present invention can be formed by etching. For example, it is also possible to increase the surface area by performing chemical etching using an oxidizing agent and a dissolving agent, or electrochemical etching using electrolysis using direct current or alternating current in an electrolyte mainly composed of an acid.

The uppermost surface of the microelectrode in the present invention has the above-described porous structure, and the material is, for example, gold, thereby realizing high strength similarly to the low impedance characteristic. Therefore, this microelectrode has high reuse efficiency and, as a result, excellent cost performance. This is in contrast to platinum black by conventional electrode multi-use electrolytic plating, which exhibits low impedance but low strength and withstands repeated use. Specifically, when the life test was conducted under substantially the same conditions as in Example 5 below, the impedance of the electrode in the present invention was typically 30% or less after the initial increase in impedance. It is preferably within 20%, more preferably within 15%.

The porous conductive material described above is formed on the uppermost surface of the microelectrode in the present invention. The electrode material constituting the lower layer of the conductive material is not particularly limited as long as it exhibits sufficient adhesiveness with the porous conductive material. As an example of a preferable material, nickel by electroless plating or electroplating, gold by electroless plating of a conventional method, etc. are mentioned. The thickness of these lower layers is not specifically limited. For example, nickel plating may be formed to a thickness of about 3000 to 7000 angstroms, and electroless gold plating may be formed on the nickel plating to a thickness of about 300 to 700 angstroms.

In the integrated composite electrode of the present invention, typically, a plurality of micro-electrodes are arranged so as to be matrix-shaped on a substrate, that is, located at each intersection point of the grating. By this arrangement, a plurality of electrodes can be arranged at the same interval. This makes it possible for nerve cells to locate neighboring cell bodies on neighboring electrodes to detect the transmission of electrical signals between the cell bodies.

Each microelectrode is connected with a wiring section for applying an electrical signal to the electrode from the outside or for guiding the electrical signal to the outside from the electrode. Typically, the wiring portion is connected to the microelectrode and includes a lead wire extracted from the electrode in the peripheral direction of the substrate. The wiring portion may further comprise an electrical contact, usually located at the edge of the substrate, connected to the end of the lead wire. Indium oxide (ITO) is mentioned as a preferable example of a wiring part. In addition, the above-mentioned impedance is a characteristic value for the whole of the microelectrode and the wiring portion in form, but in fact, the value of the wiring portion is small enough to be negligible compared with the value defined by the material and shape of the uppermost surface of the electrode. Therefore, the choice of the material of the lower layer of the electrode and the wiring portion hardly affects the impedance.

Typically, the surface of the lead wire is covered with an insulating layer. The insulating layer may be disposed only on the lead wire, but is preferably disposed so as to cover almost the entire surface of the substrate except for the microelectrode and the vicinity of the electrical contact. Preferable examples of the material for the insulating layer include acrylic and photosensitive polyimide that are easy to process.

(Configuration of Integrated Compound Electrode)

In the detailed design of the integrated composite electrode of the present invention, any structural feature of a known integrated composite electrode can be adopted so long as it does not prevent the formation and action of the porous conductive material described above (for example, See Publication 6-78889). Below, the structural example of a typical integrated composite electrode is shown. The example described herein may be set and changed in a timely manner, reflecting various factors such as the characteristics of the nerve cell to be measured and the nature of the measurement data to be collected.

As the substrate material provided for the integrated composite electrode, a transparent insulating material is preferable for the convenience of optical observation after cell culture. As an example, glass, such as quartz glass, lead glass, borosilicate glass, or inorganic substance, such as quartz, or organic substance which has transparency, such as polymethyl methacrylate or its copolymer, polystyrene, polyethylene terephthalate, etc. are mentioned. Inorganic materials excellent in mechanical strength and transparency are preferred.

As the electrode material disposed on the substrate, for example, indium oxide (IT0), oxide, Cr, Au, Cu, Ni, Al, Pt or the like can be used. Especially, ITO and a stone oxide are preferable, and ITO which is transparency and electroconductivity is especially preferable. Said microelectrode is normally formed by performing porous conductive material plating on the uppermost surface of a part of these electrode materials arranged in desired positions and shapes.

The plurality of micro electrodes are usually arranged at the same interval so that the distance between the closest electrodes is the same. This inter-electrode distance can typically be in the range of about 10 to about 1000 μm. The shape of the electrode is typically square or circular, and the length or diameter of one side may be in the range of about 20 μm to about 200 μm. By this setting, when the neurons to be measured (i.e., the cell body and the dendritic and axonous cell bodies) are placed on one electrode, the neighboring cells are interposed through the cell protrusions extending from the cell body. The chance of other cell bodies being located on neighboring electrodes increases.

The electrode materials as described above can also be applied to lead wires connected to the microelectrodes, and ITO is also preferable. Usually, after depositing such an electrode material on a substrate, the wiring portion including the lowermost layer of the microelectrode and the lead wire is formed integrally in a desired pattern by etching using a photoresist. At this time, the thickness of the lowermost layer and the wiring portion of the microelectrode may be about 500 to 5000 angstroms.

The lead wire is typically arranged in a shape extending approximately radially from each microelectrode. In combination with this weakly radial arrangement, it is particularly preferable to position the center points of the plurality of micro-electrodes at each intersection of 8x8 lattice.

As a material of the insulating layer which coats and insulates a lead wire, transparent resins, such as a polyimide (PI) resin and an epoxy resin, are mentioned, for example. Photosensitive resins, such as negative photosensitive polyimide (NPI), are preferable. When the insulating layer material of photosensitive resin is used, it becomes possible to open a hole in the insulating layer part on a microelectrode, and to expose only this electrode, for example using pattern formation by port etching. In this way, it is preferable that the insulating layer is formed to cover almost all surfaces of the insulating substrate except for the electrical contacts on the respective electrodes and the external circuits in terms of production efficiency and the like.

(Cell potential measuring device and system)

In the design of various components in a system for effectively utilizing the integrated composite electrode of the present invention for the measurement of nerve cells or the like, a known cell potential measuring system is provided so long as it does not prevent the formation and action of the porous conductive material described above. Any characteristic in can be employ | adopted (for example, refer Unexamined-Japanese-Patent No. 8-62209).

The integrated composite electrode of the present invention can be provided as an integrated cell mounting device by adding a structure for facilitating cell culture on the composite electrode and a structure for facilitating the handling of the composite electrode itself as desired.

 For cell culture on the composite electrode, a structural part capable of holding the culture solution via the insulating layer can be added, typically on the substrate on which almost the entire surface of the substrate is coated in the insulating layer. As such a holding structure, for example, a cylindrical edge made of polystyrene can be fixed on the substrate to surround the plurality of micro electrodes. At this time, the inside of the polystyrene frame defines the cell installation region. The porous conductive material plating of the present invention may be formed on the surface of the microelectrode before or after installation of the holding structure.

In order to facilitate handling when the integrated composite electrode is measured in a cell, for example, a printed wiring board can be combined. A printed wiring board has a conductive pattern which electrically connects with the electrical contact of an integrated composite electrode, and serves to draw out the electrical connection which reaches an electrical contact from a microelectrode further. In order to reliably fix both while maintaining the electrical connection between the printed wiring board and the integrated composite electrode, a holder of an appropriate shape, for example, a two-split holder having a shape of sandwiching the composite electrode up and down can be used.

A cell potential measuring apparatus for giving electrical stimulation to cells installed on the composite electrode and processing the output signal as a response by combining the stimulation signal giving device and the output signal processing device in the integrated cell placing device.

The stimulus signal applying device may apply a stimulus signal between any pair of electrodes among the plurality of micro electrodes. When the cell responds to the stimulus signal, a change in the induced potential at the other electrode can be obtained, which is given to the signal processing device as an output signal. The output signal is output to, for example, a display device through appropriate signal processing. In addition, the measurement of the spontaneous potential which arises in a cell without receiving a stimulus signal can also be performed similarly.

The stimulus signal providing device and the output signal processing device are typically integrally formed by a computer having appropriate measurement software. The measurement software is detected from a parameter setting screen and a cell which can set stimulation conditions and the like on a computer screen. A recording screen capable of recording potentials and displaying in multiple channels in real time, and a data analysis screen capable of analyzing recorded data are provided. Preferably the stimulus signal from the computer is output to the integrated composite electrode via the D / A converter, and the output signal from the cell is input to the computer via the A / D converter.

A system for measuring cell potential, which makes it possible to cultivate nerve cells over a long period of time and to measure their electrophysiological activity stably and accurately by combining a cell potential measuring device and an optical observer and a cell culture device. Can be. In addition to an inverted microscope, a microscope SIT camera equipped with a high-definition display and an image pile device can be used as the optical observation device. As the cell culture device, any device or combination of devices capable of controlling temperature control of the culture atmosphere, circulation of the culture solution, supply of a mixed gas of air and carbon dioxide, and the like can be used.

Hereinafter, the present invention is more specifically illustrated. This embodiment does not limit the invention.

(Example 1)

At various current densities, the planar microelectrode (the center of each electrode having a size of 50 x 50 µm is located at each intersection of the 8 x 8 lattice. Therefore, the total surface area (projection area) of the micro electrode is 50 x 50 x 64 = 160000 μm ² ) The surface was electroplated with gold.

Specifically, electrolytic plating of gold was attempted at current densities of 1.0 A / dm 2, 1.5 A / dm 2 and 2.0 A / dm 2. The impedance of the electrode with gold plating obtained at each current density was measured at a frequency of 1 Hz and a terminal voltage of 50 Hz, and the average value of five measurements was obtained. The results are shown in Table 1. As the current density was increased, the average impedance of the microelectrode was lowered.

The gold plating surface on the microelectrode obtained here was observed with the optical microscope. Micrographs are shown in FIGS. 1A-1C. 1A is 1.0 A / dm 2, FIG. 1B is 1.5 A / dm 2, and FIG. 1C is an optical micrograph of the plating surface obtained at a current density of 2.0 A / dm 2, respectively. At a current density of 1.0 A / dm 2, an almost smooth plating surface can be obtained. As the current density is increased, the gold-plated surface shows a significantly porous shape and the area of the electrode surface is increased. .

(Example 2)

Using the microelectrode surface obtained in Example 1, the hippocampus fragment (brain) of the mouse was actually used to measure the evoked potential and the noise level. Seahorse slices were obtained from mice. Five-week-old male c57black 6 mice were anesthetized with Fluothane, followed by decapitate to extract the whole brain. The extracted brain was immediately cooled with ice Ringer's solution and a brain block containing only the hippocampus was taken out. The removed brain fragments were then sliced to a 250 micron thickness with a tissue fragmentation apparatus, and the pieces were placed on microelectrodes for testing.

Measurement of induced potential and measurement of noise level give a constant current of 10 microA as a dipolar pulse (pulse width of 100 microseconds), and the response of 64 electrodes is 5 milliseconds before stimulation and 45 milliseconds after stimulation. The measurement was performed on a computer screen displayed in 64 channels for up to a second. This result is shown to FIG. 2A-2F. 2A, 2C and 2E show the response of the cell's potential change, ie induced potential, to the constant current stimulus on a gold plated coated microelectrode. 2A shows an electrode obtained by electrolysis at a current density of 1.0 A / dm 2, FIG. 2C shows an electrode obtained by electrolysis at a current density of 1.5 A / dm 2, and FIG. 2E shows an electrode obtained by electrolysis at a current density of 2.0 A / dm 2. The induced electric potential in is shown, respectively.

2B, 2D and 2F show noise levels in the absence of cells for the gold plated coated microelectrodes of FIGS. 2A, 2C and 2, respectively.

As shown in Figs. 2A, 2C and 2E, compared with the gold plating obtained by electrolysis at a current density of 1.0 A / dm 2 (Fig. 2A), in the porous gold plating obtained at a higher current density, the electrode to which the stimulus signal is applied The response was clear and it was found that the stimulus of the constant current can be effectively applied (FIGS. 2C and 2E). In particular, the microelectrode plated at a current density of 1.5 A / dm 2 had a low impedance value and was good also in the surface form of the microelectrode.

The noise level was the lowest at 2.0 A / dm 2 (FIG. 2F), followed by the lowest at 1.5 A / dm 2 (FIG. 2D). On the other hand, plating at 1.0 A / dm 2 (FIG. 2B) was remarkably noisy, and it was difficult to accurately measure the potential change of nerve cells.

(Example 3)

The frequency characteristics of the surface of the planar microelectrode subjected to electrolytic plating with gold at various current densities were compared with the conventional products. Electrodes having a porous gold-plated surface at 2.0 A / dm 2 and 1.5 A / dm 2 exhibited frequency characteristics comparable to those of conventional electrodes having platinum black plating on the surface, respectively, by electrolysis. However, gold plating at 1.0 A / dm 2 was significantly inferior in frequency characteristics as compared with the prior art.

(Example 4)

The microelectrode surface area was measured or calculated by the following method.

1.Measurement by gas adsorption method

The surface area of the microelectrode having a porous gold plated surface at a current density of 1.5 A / dm 2 obtained in Example 1 was measured using a gas adsorption method using CO gas. As the measurement sample, 64 gold-plated microelectrodes (hereinafter referred to as gold-plated microelectrode blocks) on a glass substrate of 1.3 mm x 1.3 mm x 1.1 mm were used. It is because it is difficult to set it as the test sample by gas adsorption method too small in a micro electrode alone. Instead of gold plating, 64 platinum black plated microelectrodes (hereinafter referred to as platinum black plated microelectrode blocks) on the same glass substrate plated with platinum black by a conventional method (electrolytic plating) were used as comparative samples. In addition, either sample block and its weight were 0.004g. The measurement results are shown in Table 2.

Here, the total surface area of the microelectrode before plating (hereinafter referred to as microelectrode block) on the glass substrate of 1.3 mm x 1.3 mm x 1.1 mm is S, the surface area (projection area) of the microelectrode before plating is s, and the gold plating fine is If the total surface area of the electrode block or the platinum black plated microelectrode block is S 'and the surface area of the microelectrode is increased by α times by plating, that is, the surface area becomes αs, Ss, that is, the portion of the microelectrode block excluding the microelectrode, Since the surface area does not change, and only the surface area of the microelectrode is increased by plating, the relation of S '= (Ss) + alpha s holds. Therefore, the value of α is calculated by the formula: α = (S'-S) / s + 1.

Here, the value of S is calculated to be 7.41 mm ² in the surface area of a rectangular parallelepiped of 1.3 mm x 1.3 mm x 1.1 mm, and the value of s (projection area) is 0.16 mm ² since it is the surface area of 64 electrodes of 50 µm x 50 µm. Therefore, from the measurement result of S 'shown in Table 2, the value of (alpha) = 504955 can be obtained with respect to a platinum black plating microelectrode. In addition, the gold-plated electrode is below the detection limit of the gas adsorption method, and the increase in the surface area can be estimated as? <455.

2. Calculation of surface area of micro electrode by equivalent circuit

2.1: impedance characteristics of gold-plated electrodes

The impedance was measured by continuously changing the frequency from 1 kHz to 100 kHz for the microelectrode having a porous gold plated surface at a current density of 1.5 A / dm 2 obtained in Example 1. The measurement was performed in a 1.4 wt% NaCl aqueous solution using a 0.3 mm square platinum wire as a counter electrode. The bias voltage was 0 V and the amplitude of the measured voltage was 50 Hz. The results are shown in FIG. Fig. 3 is a board diagram well known to those skilled in the art showing the results obtained by impedance measurement, in which the logarithm of the absolute value of the measured impedance (Z) and the log angle (Z) and the phase angle (θ) are plotted against the logarithm of the frequency (f). will be. From this board diagram, the measured system was shown as an equivalent circuit to quantify the surface area of the microelectrode.

The measurement system using the micro electrode is actually generated between the IT0 circuit pattern portion and the liquid through the insulating film in addition to the resistance such as liquid resistance, ITO circuit pattern portion, capacitance by the electric double layer on the surface of the micro electrode, and the like. It can be estimated that the capacitance, resistance generated at the microelectrode interface, and the like are equivalent to those connected in series and in parallel. For example, FIG. 4A shows one equivalent circuit of a measurement system using a microelectrode. The combined impedance Z of the entire equivalent circuit shown in FIG. 4A can be represented by the following formula [I]:

Where R ₁ is the resistance of the circuit pattern portion of the ITO (the portion that is not in contact with the liquid), R ₂ is the resistance and liquid resistance of the circuit pattern portion of the ITO, R ₃ is the resistance of the microelectrode surface, and C ₁ is the microelectrode The electric double layer capacitance on the surface, C _2, is the capacitance generated between the portion of the ITO circuit pattern and the liquid through the insulating film, and ω = 2πf (ω: angular frequency, f: frequency). │ and phase θ are respectively │Z│ = (Z _Re ² + Z _Im ² ) ^1/2 , θ = tan ^-1 (-Z _Im / Z _Re ), where Z _Re : real part of Z, Z _Im Can be expressed as:

Thus, R ₁ , R ₂ , C _1, and C ₂ of the equivalent circuit shown in FIG. 4A are changed to calculate and simulate a composite impedance of the equivalent circuit, and R is given to the board diagram closest to that of FIG. 3. Combinations of ₁ , R ₂ , R ₃ , C ₁ and C ₂ were selected. 5 shows a board diagram when R ₁ = 200 Ω, R ₂ = 5 ms, R ₃ = 5 ms, C ₁ = 0.01 μF, and C ₂ = 100 pF. It can be seen that the results shown in FIG. 5 agree well with the results shown in FIG. 3, which are actual measured values.

Here, in the measurement system using the microelectrode, R ₃ >> R ₁ , R ₃ >> R _2, and C ₂ << C ₁ , and the above formula [I] is defined as R ₃ → ∞, C ₂ → 0, Can be approximated by [II].

That is, it can be seen that the measurement system using the microelectrode can be approximated by the simple circuit shown in FIG. 4B by setting R ₁ + R ₂ = R and C ₂ = C. Hereinafter, analysis was performed using the equivalent circuit of FIG. 4B.

The combined impedance Z of the entire equivalent circuit shown in FIG. 4B is represented by Z = R + (jωC) ⁻¹ . Here, R is a circuit pattern resistance, C is an electric double layer capacitance on the surface of the microelectrode, and ω = 2πf (ω: each frequency and f = frequency). And wherein the absolute value of the synthesized impedance │Z│ and the phase θ are represented by │Z│ = (R ² + (1 / ωC) ² ) ^1/2 and θ = tan ⁻¹ (−ωC / R), respectively. do.

Thus, a combination of R and C that changes the values of R and C of the equivalent circuit shown in FIG. 4B, calculates and synthesizes the composite impedance of the equivalent circuit, and gives a board diagram that most closely matches the board diagram of FIG. Selected. 6 shows board diagrams when R = 5 ms and C = 0.01 µF. It is understood that the results shown in FIG. 6 are in good agreement with the results shown in FIG. 3, which are actual measured values.

Next, a graph of the actual measured value of the impedance of the microelectrode without the gold plated surface used as the control is shown in FIG. Measurement conditions are the same as in the case where the impedance of the microelectrode having the above-described gold plated surface is measured (FIG. 3).

Next, FIG. 8 shows the results obtained by simulation of the synthesized impedance of the equivalent circuit when R = 5 kHz and C = 250 pF in the equivalent circuit shown in FIG. 4B. It is understood that the results shown in FIG. 8 are in good agreement with the results shown in FIG. 7, which are actual measured values.

Here, when the electric double layer capacitance of the microelectrode having a gold plated surface is CA and the electric double layer capacitance of the microelectrode before the gold plating treatment is CB, the electrostatic capacitance of each microelectrode is C _A = 0.01 μF. And C _B = 250PF, a relationship of C _A = 40C _B can be obtained. In general, the capacitance C _ap can be expressed as C _ap = ε ₀ ε _r S / d (ε ₀ : dielectric constant of vacuum, ε _r : dielectric constant of dielectric, S: surface area of electrode, d: thickness of dielectric) Since the value is proportional to the surface area of the electrode, the above relation indicates that the surface area of the microelectrode having a gold plated surface is increased by 40 times by gold plating.

Similar to the microelectrode having a gold plated surface, a graph of the actual measured value of the impedance of the microelectrode having a platinum black plating surface is shown in FIG. 9. Measurement conditions are the same as in the case where the impedance of the microelectrode having the above-described gold plated surface is measured (FIG. 3). Next, FIG. 10 shows the results obtained by simulation of the synthesized impedance of the equivalent circuit when R = 5 Hz and C = 0.05 µF in the equivalent circuit shown in FIG. 4B. It is understood that the result shown in FIG. 10 is in good agreement with the result shown in FIG.

As described above, when the electrical double layer capacity of the microelectrode having the platinum black plating surface is set to C _A , and the electrical double layer capacity of the microelectrode before performing the platinum black plating treatment is set to C _B , C _A = 0.01 μF and C _B = 0.05 Since μF, C _A = 200C _B is established. This relationship shows that the surface area of the microelectrode having the platinum black plating surface was increased by 200 times by gold plating.

In this simulation, in order to achieve 50 kΩ or less, which is the impedance limit of the microelectrode, it has been shown that gold plating requires at least 10 times the surface area compared with the projected area.

(Example 5)

The life test was performed compared with the conventional electrode (platinum black plating) using the planar microelectrode which carried out the electroplating porous by electrolysis at the current density of 1.5 A / dm <2>. The life test was repeated with the same experiment as the usual acute experiment. That is, the experiment described in Example 2 was repeated. After the end of each experiment, the cells were treated with collagenase (20 u / ml) overnight to separate the cell debris samples, washed with distilled water, and the impedance of each electrode was measured. In Fig. 11, the impedance change after each experiment is plotted. From this result, as the number of uses was repeated, the impedance increased less compared with the conventional plating method (platinum black plating), and the gap between the electrodes was smaller.

On the other hand, in the case of the conventional product having the plating of platinum black by electrolysis on the surface, as shown in Fig. 11, the impedance was significantly increased after 17 to 18 cycles. As described above, it was shown that the electrode having a porous gold plating had a higher reusability and a stable measurement of dislocation change as compared with the conventional product.

As mentioned above, although this invention was demonstrated with reference to the Example, this invention is not limited to these, The example which added various improvement, correction, and deformation on the basis of the knowledge of those skilled in the art within the range which does not deviate from the meaning of this invention. It can be carried out.

According to the present invention, there is provided an integrated composite electrode for extracellular recording which is high in intensity, can stably record regenerating activity of nerve cells and the like, and is easy to reuse. The plurality of micro-electrodes of this composite electrode use a porous gold plating to impart low impedance without compromising strength. For this reason, the application of the constant current stimulus to the electrode in the composite is easy, and is suitable for observing the response of the increasing cells due to the electrical stimulation.

Claims (9)

In the integrated composite electrode for measuring the electrophysiological characteristics of the cell, And a plurality of micro electrodes disposed on the substrate, and a wiring portion capable of providing an electrical signal to the micro electrodes or extracting an electrical signal from the micro electrodes. The microelectrode has a porous conductive material on its surface, the conductive material is selected from the group consisting of gold, titanium nitride, silver oxide, and tungsten, and the impedance is 50 kΩ or less. The integrated composite electrode according to claim 1, wherein the porous conductive material is gold and is formed by energizing for 10 to 360 seconds at a current density of 1.0 to 5.0 A / dm 2. In the integrated composite electrode for measuring the electrophysiological characteristics of the cell, A plurality of micro electrodes disposed on the substrate, and a wiring portion capable of providing an electrical signal to the micro electrodes or extracting an electrical signal from the micro electrodes; The surface area of the microelectrode calculated from the capacitance of the equivalent circuit showing substantially the same impedance characteristics as the microelectrode is at least 10 times to less than 200 times the projected area of the microelectrode, and the impedance is 50 Hz or less, Integrated composite electrode. The integrated composite electrode of Claim 3 whose surface area measured by the gas adsorption method of the said microelectrode is not more than 5x10 ⁵ times the projected area of this microelectrode. The method according to any one of claims 1 to 4, The micro electrodes are arranged in a matrix on a substrate, the wiring portion includes a lead wire connected to the micro electrode and an electrical contact connected to an end of the lead wire, and at least the surface of the lead wire is covered with an insulating layer; Integrated composite electrode. The integrated composite electrode according to any one of claims 1, 3, and 4, wherein the porous conductive material is formed by etching. An integrated cell mounting device comprising the integrated composite electrode according to any one of claims 1 to 4 and having a cell mounting region for mounting cells or tissues on a substrate of the integrated composite electrode. An integrated cell mounting device according to claim 7, and an output signal processing device connected to the microelectrode for processing an output signal by electrophysiological activity of the cell or tissue, and further electric stimulation to the cell or tissue as needed. A cell potential measuring device, comprising: a stimulus signal imparting device connected to the microelectrode for giving electrical stimulus. The cell potential measurement apparatus provided with the cell potential measuring apparatus of Claim 8, and further provided with the optical observation apparatus for optically observing a cell or tissue, and / or the cell culture apparatus for controlling the culture environment of a cell or tissue. system.