WO2006070872A1

WO2006070872A1 - Method of electrically measuring and device of electrical meauring at least one of chracteristic and condition of cell membrane

Info

Publication number: WO2006070872A1
Application number: PCT/JP2005/024076
Authority: WO
Inventors: Makoto Taketani; Hiroaki Oka; Norihiro Katayama
Original assignee: Matsushita Electric Industrial Co., Ltd.; Tohoku University
Priority date: 2004-12-28
Filing date: 2005-12-28
Publication date: 2006-07-06
Also published as: JP2006184207A

Abstract

A measuring method capable of electrically measuring the conditions of cell membranes at high-speed, accurately and easily. A method of electrically measuring the conditions of cell membranes comprising the steps of providing a measuring electrode (12), a reference electrode (13) and a plurality of cells (17), electrically connecting the measuring electrode (12) with the interiors of respective cells (17) and electrically connecting the reference electrode (13) with surfaces of the respective cells (17), and, under this condition, applying a voltage to the measuring electrode (12) to measure the fluctuation of a current running between the both electrodes (12, 13). The fluctuation of a current at the plurality of cells is equal to a total of the fluctuation of a current at each cell. The condition of a cell membrane can be evaluated by analyzing the power spectrum of this current fluctuation.

Description

Method and apparatus for electrical measurement of at least one of characteristics and state of cell membrane

Technical field

The present invention relates to an electrical measurement method and an electrical measurement apparatus for at least one of characteristics and states of cell membranes.

Background art

From the early days of electrophysiology, glass electrodes were inserted into cells and the membrane potential of the cells was measured, and the presence of ion channels in the cell membrane was foreseen. The measurement of cell membrane potential has greatly progressed with the development of the patch clamp method. The patch clamp method was developed by Neher and Sakmann in 1976 (Non-Patent Document 1), which produced an epoch-making result in actually demonstrating the existence of ion channels. Furthermore, in 1981, Hamill et al. Developed a whole-cell U-clamp method, which made it possible to measure the total current of ion channels existing on the entire cell membrane surface. As the relationship between ion channel abnormalities and diseases becomes clear, the ability of whole cell patch clamps to measure the state of ion channels has become increasingly important for drug development, and to enable this to be implemented at high speed. Technology has been developed. For example, there is a flat patch device and its system for the whole cell patch clamp method (Patent Documents 1 to 4). However, this method has the following problems. That is, the patch clamp method requires the recording pipette to adhere tightly to the cells and form a tight seal that exceeds gigaohms. If this seal is loose, reliable data can be obtained. Absent. In addition, in the patch clamp method, measurement may be unsuccessful due to various factors such as hole failure, unstable baseline, and insufficient ion channel expression. Therefore, in order to obtain reliable data, a certain amount of repeated experiments are required. For this reason, for example, if the measuring device is capable of measuring 3000 cell membrane potentials per day, if it is necessary to repeat measurement four times in order to obtain reliable data, after all, 750 Only the data of can be measured. Therefore, In the whole cell clamp method, it is desired to develop a technology that can measure even faster and more accurately.

Patent Document 1: WO00159447

Patent Document 2: US 6488829

Patent Document 3: US06315940

Patent Document 4: WO00125769

Non-patent literature 1: Neher E & Sakmann B (197 Rino Single cnannel currents recorded from m embraneof denervated frog muscle fibers. Nature 260: 799—802

Non-Patent Document 2: Hamill OP, Marty A, Neher E, Sakmann B & Sigworth FJ (1981) Impro ved patch-clamp techniques for high-resolution current recording from cells and cell -free membrane patches. Pflugers Arch 391: 85-100.

Disclosure of the invention

[0003] Therefore, an object of the present invention is to provide an electrical measurement method and an electrical measurement apparatus that can measure at least one of the characteristics and state of a cell membrane faster, more accurately, and more easily than before.

[0004] In order to achieve the above object, the measurement method of the present invention is an electrical measurement method of at least one of the characteristics and state of a cell membrane, comprising preparing a measurement electrode, a reference electrode, and a plurality of cells. The measurement electrode and each cell are electrically connected, and the reference electrode and each cell membrane surface are electrically connected, and in this state, a voltage is applied to the measurement electrode to both the electrodes. This is an electrical measurement method for measuring fluctuations in the current flowing between them.

[0005] The measurement apparatus of the present invention is an electrical measurement apparatus for at least one of the characteristics and state of a cell membrane, comprising a container having a plurality of holes for holding and fixing cells, a measurement electrode, a reference electrode, An application unit that applies a voltage to the measurement electrode; a detection unit that detects a current flowing between the electrodes; and a measurement unit that measures fluctuations in the current; and the measurement electrode and each cell When the reference electrode and the cell membrane surface can be electrically connected and the both electrodes are electrically connected in the state, the application means can connect the measurement electrode. A voltage is applied, a current flowing between the electrodes is detected by the detection means, and the fluctuation of the current is detected by the measurement means. Is an electrical measuring device to be measured.

[0006] As described above, the measurement method and the measurement apparatus of the present invention measure fluctuations in current caused by at least one of the characteristics and state of the cell membrane for the entirety of a plurality of cells. As will be described later, this current fluctuation reflects the characteristics and state of the cell membrane of the plurality of cells as a whole. Therefore, according to the present invention, the cell membrane state and the like of a plurality of cells are measured simultaneously at the same time. Therefore, even if the success rate of the measurement of the cell membrane state and the like of individual cells is not 100%, a single measurement is performed. Thus, highly reliable information can be obtained. In addition, when measuring the current due to the state of the cell membrane, etc. for a plurality of cells, the present invention measures the fluctuation of the force current that requires conditions such as arranging electrodes for each cell and insulating the cells. Therefore, a pair of electrodes (measurement electrode and reference electrode) that do not need to insulate each cell can measure the cell membrane state of multiple cells, and it is necessary to apply strict measurement conditions such as current measurement. There is no. Therefore, according to the present invention, it is possible to measure at least one of the characteristics and state of the cell membrane faster, more accurately and more easily than the conventional method. Brief Description of Drawings

[0007] FIG. 1 (a) is a diagram showing an example of an electric circuit presumed to be formed inside and outside a cell, and FIG. 1 (b) shows a simplified model of the electric circuit. FIG.

FIG. 2 is a diagram showing an example of an electric circuit presumed to be formed in a plurality of cells.

FIG. 3 is a cross-sectional view showing an example of a container constituting a part of the electrical measuring apparatus of the present invention.

[FIG. 4] FIG. 4 is a graph theoretically examined for the reproducibility of the number of cells per electrode and current amplitude in an example of the electrical measurement method of the present invention.

FIG. 5 is a block diagram showing an example of an electrical measuring apparatus according to the present invention.

[FIG. 6] FIG. 6 is a graph theoretically obtained dose response characteristics using the standard deviation of the current value at 20 cells in an example of the electrical measurement method of the present invention.

[FIG. 7] FIG. 7 is a diagram of a power spectrum in one embodiment of the electrical measurement method of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION [0008] In the electrical measurement method of at least one of the characteristics and state of the cell membrane of the present invention, it is further preferable to measure a power spectrum of the current fluctuation. Similarly, the electrical measurement apparatus of the present invention preferably further includes a power spectrum measurement means for measuring a power spectrum of the current fluctuation.

[0009] In the electrical measurement method of the present invention, the number of the plurality of cells is not particularly limited, and is a force determined appropriately according to various conditions. For example, the range is 2 to: LOO.

[0010] In the electrical measurement method of the present invention, the characteristics and state of the cell membrane to be measured and measured include, for example, the characteristics and state of ion channels existing in the cell membrane. Examples of the characteristics and states of the ion channel include at least one of the number of ion channels and the open / closed state of the ion channels.

[0011] In the electrical measurement method of the present invention, the cells may be, for example, individually separated cells or cells existing in a tissue.

In the electrical measurement method of the present invention, the fluctuation of the current may be measured before and after the chemical substance is administered to the cell. In this way, the influence of the chemical substance on the cell membrane can be measured, and is useful, for example, in the development of pharmaceuticals. Similarly, the electrical measuring device of the present invention is preferably used for examining the influence of chemical substances on the cell membrane.

[0013] In the electrical measurement device of the present invention, for example, the container can be filled with an electrolyte solution, and the electrolyte solution can be used between the measurement electrode and each cell and between the reference electrode and each of the cells. The cell membrane surface may be electrically connected.

[0014] In the electrical measurement apparatus for cell membrane of the present invention, the number of holes in the container is not particularly limited, and is a force determined appropriately depending on the number of cells to be measured, etc. For example, in the range of 2 to: LOO. is there.

[0015] Next, the present invention will be described in detail.

[0016] A feature of the present invention is that, for a plurality of cells that do not measure a membrane potential, a fluctuation in current that reflects the characteristics and state of the cell membrane is measured collectively for a single cell. The fluctuation of the current of the plurality of cells is the sum of the fluctuations of the current of each cell. Then, by measuring the power spectrum of fluctuations in the membrane current of multiple cells and analyzing it, the characteristics and state of the cell membrane can be evaluated in detail. The rationale is as follows.

[0017] (1) Equivalent electrical circuit model

Figure 1 shows an equivalent circuit model when one cell is in a state of forming a seal in the device hole. The model parameters are summarized in the table described later. When the seal resistance R is sufficiently large (>> 100 MΩ) and the resistance R of the cell surface in contact with the device is small enough (<<20 MΩ), take the appropriate reference voltage. For example, the part of the circuit in Fig. 1 (a) excluding the capacitance component C can be simplified as shown in Fig. 1 (b). Die of this system

Namitas is shown by the following formula (1).

[0018] [ _Equation 1] d V _CMD

I + C _d = = 0 (1)

[0019] In the following, this simplified model (see Fig. 1 (b)) will be analyzed.

FIG. 2 shows an equivalent circuit model of an eHTS device system using multiple cells. This system has a structure in which a device hole system that forms a seal with each cell is connected in parallel. If the number of cells is N, the observed current I is given by the following equation (2).

total

[0021] [Equation 2] d V _CMD ^{d V} CMD

. _d ) + c,

dd tt

N

V ^{d V} CMD

I i + (NC _d + C _{ch ip} ) —— (2)

[0022] By correcting (cancelling) the capacitance current (second term on the right side) by the amplifier, the following formula (

3), ie, the total current is the sum of the cell currents.

[0023] [Equation 3] [0024] In the following, first, the relationship between the current flowing through one cell device hole and the channel fluctuation will be explained using linear approximation, and then the power spectral density of the fluctuation exhibited by the synthesized current will be explained. .

[0025] (2) Linear approximation

In the equivalent circuit of a single cell device pore system, the current fluctuation in the steady state recorded in the voltage-clamped mode is the conductance g that accompanies the switching fluctuation of the ion channel.

Due to the time variation of the membrane resistance caused by ch change. However, R = 1Z (R +1 m

/ g). The bandwidth of the observed signal is limited by the ch m acc filter characteristics, which consists of this resistance component, cell membrane capacitance component C, and access resistance R. The time constant of this system is given by the following equation (4).

[0026] [Equation 4]

CR _ac „R

.//R) = (4)

'' If R + R parameter is R ^ 20MΩ, R = 100MΩ, and C = 3pF, τ = 0.05ms

acc m

Calculated. The cut-off frequency is ί. = ΐΖ (2π τ) 3kHz In this case, it can be seen that the signal component below lk Hz (= 3kHz / 3) can be ignored. Since the fluctuation analysis of the membrane current often covers the band below 1kHz, for simplicity, it is approximated as C = OF. Investigating the relationship between membrane current and channel fluctuation

m

The When the membrane current is regarded as a function of the membrane conductance g, it can be expressed as the following formula (5).

[0028] [Equation 5]

However, the conductance change (δg: fluctuation component) is steady.

Assuming that it is sufficiently smaller than the value (average value), the minute change of membrane current δ I = l (g + δ g) −Kg) can be approximated as shown in the following equation (6). However, K = V / (gR +1) ^{2 and} cmd acc.

[0030] [Equation 6] d I (g)

δ I 2 δ g + o (δ g ² )

d g

VcMD

δ g = K · δ g (6)

(gRacc + 1)

[0031] The conductance change is proportional to the change in the number of open channels. For this reason, it is possible to evaluate the fluctuation of the number of aperture channels by performing spectrum analysis of the membrane current fluctuation signal.

[0032] (3) Power spectrum

Single cell membrane current I is a function of membrane conductance g. Membrane conductance is expressed as the membrane conductance _g

m, _Y a single ion channel conductance, when the opening number of units of ion channels put by P, can be expressed by the following equation (7). Where t is the time and s is the parameter governing the channel opening probability.

[0033] [Equation 7] g = g + p (t; s) γ (7)

[0034] For example, a channel having a potential dependency is included in the membrane potential V force S. Opening m

Since the number of channels is a random variable that varies with time, the membrane current is also a stochastic process. As shown above, the minute change component of the membrane current is proportional to the fluctuation of the number of open channels. Therefore, the power spectrum of the membrane current fluctuation component (difference from the average value, i (t)) is proportional to the power spectrum of channel fluctuation P. Therefore, these are equated below.

[0035] The observed current in an eHTS device in which a plurality of cell lines form a parallel circuit is the sum of the cell membrane currents. As a simple example, let us consider the current fluctuation spectrum observed in an eHTS device with two holes. If each current fluctuation is X (t) and y (t), the fluctuation z (t) of the observed current can be expressed by the following equation (8).

[0036] [Equation 8]

(t) (t) + y (t) (8)

[0037] When the Fourier transform of the membrane current fluctuation of each cell is expressed by the following equations (9) and (10), it can be expressed as the following equation (11).

[0038] [Equation 9]

[0039] [Equation 10]

[0040] [Equation 11]

(t) e— ^iwt dt

= X (ω) + Y (<(1 1) In addition, the power spectrum of the current fluctuation is given by the following equations (12) and (13). Where T is the observation time and X '(ω) is the complex conjugate of Χ (ω).

[Equation 12]

2 π

S _χ (ω) = X '(ω) X (ω) (1 2)

χ Τ

[0042] [Equation 13]

2 π

S (ω) = Υ '(ω) Υ (ω) (1 3)

y ^y Τ

From the above, the power spectrum of the total current S (ω) is calculated as shown in the following equation (14).

[0044] [Equation 14]]

,

Here, S (ω) and S (ω) are cross spectra of x and y, and are represented by the following formula (15).

[0046] [Equation 15]

2 π

S _xy (ω) = Χ '(co) Y (ω) (1 5)

Τ

[0047] Generally, it can be assumed that the membrane current fluctuation is uncorrelated and cross-correlation function: C (te) = 0) for each cell. [0048] [Equation 16]

S _xy (ω) = S _yx (ω) = 0 (1 6)

From this, the following formula (17) is obtained.

[0050] [Equation 17]

S _zz (ω) = S _XX (ω) + S _yy (ω) (1 7)

[0051] In general, the power spectrum (ω) of the total current I, which is the sum of Ν cell membrane currents Ij, can be assumed that there is no correlation between the current fluctuations. Equal to ( _ω ). That is, it can be expressed as the following formula (18).

I]

[0052] [Equation 18]

S j (ω) S j j (ω) (1 8)

j = 1

[0053] If the membrane current fluctuations of all cells follow the same stochastic process, the expected values of the respective power spectra match. This means that the power spectrum of membrane current fluctuations of N cells with the same characteristics is equal to the power spectrum of membrane current fluctuations of a single cell multiplied by N. If the cell-to-cell parameter variation is small, and as a result, the power spectrum of membrane current fluctuations is almost equal, the power spectrum of membrane current fluctuations in a parallel circuit of N cells will have a single membrane current fluctuation N The signal Z-to-noise ratio can be expected to improve.

[0054] Variable 'parameter

N: Number of cells

t: time

1 :: Cell radius (5 111)

S: Cell surface area ( ⁴ πΐ: ² )

C: membrane capacitance (= 1 F / cm ² XS 3pF)

m c: Electric capacity near the device hole

d

c: Device chip capacitance

chip

R: membrane leakage resistance (= 0.03mS X S)

m

E: resting membrane potential

m

p (t; s): Number of aperture channels (s is a parameter)

y: Single channel conductance

g: Membrane conductance by ion channel (= p (t; s) γ)

ch

g: Membrane channel conductance (g = gm + g di)

E: Equilibrium potential of ion channel current

ch

R: Membrane resistance in contact with device hole (due to estatin etc.) nys

R: Device hole resistance

hole

R: Seal resistance at cell / device contact area

seal

V: Command potential (determined by the experimenter)

C D

V: Intracellular potential (membrane potential)

m

R: Access resistance (

acc = R n ^ s + R hole)

R: Membrane resistance (= R m ZZR ch = R mR ch Z (R m + R ch))

x, y: Single-cell membrane current fluctuation components

z: Fluctuation component of total current

ω: Angular frequency of signal

X, Υ, ∑: Fourier transform of each, y, z

X, Y, ゝ Z ,: complex conjugate of each X, Υ, Ζ

S, S, S: Power spectral density of each x, y, z

XX yy zz

S: Cross spectrum of x and y

Next, an example of the electrical measurement apparatus of the present invention will be described based on the cross-sectional view of FIG. 3 and the configuration diagram of FIG.

[0056] First, FIG. 3 is a cross-sectional view of the container 1 used for measurement. Examples of the material of the container 1 include single crystal substrates such as silicon, gallium arsenide, and quartz, as well as glass, quartz, and resin. Various substrate materials are used. As shown in the figure, in this container 1, the container body 11 is partitioned vertically by an internal force cutting plate 11A, and a plurality of holes 14 are formed in the partition plate 11A. Cells 17 are retained and fixed. Here, the diameter of the hole 14 is determined optimally depending on the size of the cell 17. For example, when the measurement target is HEK cells, the diameter of the hole 14 is usually 0.5 to 10 μm. Meters, preferably 1 to 5 micrometers in diameter. In addition, the optimum number of pores 14 is influenced by the activity of the cell 17 to be measured, the degree of adsorption of the cells 17 to the pore 14, etc., and is the most efficient measurement by the measurement method described later. It is decided so. Furthermore, one measurement electrode 12 is disposed on the lower surface of the partition plate 11A. In the measurement electrode 12, holes 15 are formed in portions corresponding to the holes 14 of the partition plate. Yes. The interior of the container body 11 that is partitioned up and down is filled with the electrolyte solution 16. A single reference electrode 13 is disposed on the top of the container body 11 so as to enter the electrolyte solution 16. As the material of these electrodes, for example, a conductive material such as gold, silver, copper, aluminum, stainless steel, chromium, titanium, or the like is used. Preferably, a portion of these conductive materials is provided with salty silver. It is that you are. This makes it possible to measure the potential change of the electrolyte solution 16 more accurately. In addition, the shape and size of these electrodes are not particularly limited. As shown in the measurement electrode 12 shown in the figure, the partition plate 11A can be simply immersed in the electrolyte solution 16 as in the reference electrode 13 in FIG. May be formed in close contact with the inner wall of the container 1. In order to form the measuring electrode 12 in close contact with the inner wall, the electrode material such as gold, silver, copper, aluminum, stainless steel, chromium, titanium, silver chloride, etc. is deposited, sputtered, plated, etc. An electrode may be formed on the surface. Further, in the cell 17, the cell membrane in the portion located in the hole 14 of the partition plate is partially broken. Therefore, the measurement electrode 12 and the inside of the cell 17 are electrically connected by the electrolyte solution 16. Further, the cell 17 membrane surface and the reference electrode 13 are electrically connected by the electrolyte solution 16. Although not shown, the measurement electrode 12 and the reference electrode 13 are connected to a current Z voltage conversion circuit, an AZD conversion circuit, a DZA conversion circuit, a CPU, a display device, and the like via a connector. In other words, in this container 1, the state of the whole cell clamp method can be realized for each cell 17, and it is assumed that each cell 17 Assuming that an equivalent circuit is formed, the equivalent circuit is connected in parallel as described above.

FIG. 5 is a schematic diagram of a measuring apparatus used for measurement. The electrical measuring device 18 includes a control unit 19, a D / A conversion circuit 20 connected thereto, a current Z voltage conversion circuit 21, an A / D conversion circuit 22 and a solution driving unit 23, a mounting unit 24, and a stimulation signal. A grant unit 26 is provided. In the figure, 1 indicates the container. The measurement of the current fluctuation of the cell membrane using this apparatus is performed, for example, as follows. That is, first, the container 1 is placed on the placement unit 24. The placement unit 24 can hold the placed container 1 at a predetermined temperature, gas concentration, humidity, and atmospheric pressure. Next, the isolated cell 17 is introduced from the upper surface of the container 1. At this time, the cells 17 are held in the holes 14 by applying a negative pressure to the lower surface of the cutting plate. The partitioning plate may be coated with Ikm i poly- L-lysine ゝ gelatin ゝ polyethyienimineb or gel. The control unit 19 detects and records the current flowing between the electrodes 12 and 13 of the container 1 based on the signal input from the current Z voltage conversion circuit. The control unit 19 controls the stimulation signal applying unit 26 via the D / A conversion circuit 20 and the current Z voltage conversion circuit 21 based on the set stimulation conditions. When a current is applied to the cells on the container 1 by the stimulation signal applying unit 26 so that the voltage between the measurement electrode 12 and the reference electrode 13 becomes an arbitrary voltage, a current flows between the measurement electrode 12 and the reference electrode 13. This current reflects the cell membrane state of the plurality of cells 17 as a whole. This current is detected by the measurement electrode 12 and the reference electrode 13, and is input to the control unit 19 through the current Z voltage conversion circuit 21 and the AZD conversion circuit 22, and is measured as current fluctuation in the CPU. Power spectrum analysis. The analysis result is displayed on the display device. As described above, the current fluctuation reflects the state of the entire cell membrane of a plurality of cells, for example, the number of ion channels, opening / closing, and the like. The solution driving unit 23 discharges the electrolyte solution 16 inside the container body 11, and the tank has a function of injecting the electrolyte solution 16 into the container body 11, and is driven by the control unit 19 as necessary. The For example, use the electrolyte solution 16 having the following composition.

[0058] (Electrolyte solution composition example 1)

NaCl 137 mM, KC1 4 mM

CaCl 1.8 mM

2

MgCl 1 mM

2

glucose 10 mM

HEPES 10 mM

[0059] (Electrolyte solution composition example 2)

KC1 130 mM

MgCl 1 mM

2

EGTA 5 mM

ATP 5 mM

HEPES 10 mM

As described above, by measuring the fluctuation of the current due to the state of the cell membrane of a plurality of cells, it is possible to perform an electrical measurement of the cell membrane state at high speed and accurately. The graph in Fig. 4 shows a theoretical study of the reproducibility of the number of cells per electrode and the current amplitude. This graph shows the number of cells measured together when the success rate of measurement is not 1.0 (100%) due to factors such as hole failure, insufficient seal, unstable baseline, and insufficient ion channel expression. Increasing the value indicates that the coefficient of variation decreases, that is, the reproducibility increases. As shown in the figure, for example, even if the success rate is 0.7, if 100 cells are measured together, it can be seen that a coefficient of variation comparable to that of the success rate of 0.9 is obtained. The graph in Fig. 6 is a theoretical calculation of dose response characteristics using the standard deviation of the current value for 20 cells, and shows the simulation of noise current characteristics when a drug is applied to cells. It is a graph. Membrane current fluctuations were simulated when an open channel blocker was applied to a cell model incorporating a membrane voltage-gated ion channel, and the relationship between the block force concentration and the standard deviation (SD) of the noise current was measured in a steady state. evaluated. The dissociation constant of the block force was 10 M. SD has a maximum when the drug concentration is the dissociation constant. As shown in the figure, by utilizing this characteristic, it is possible to estimate the dissociation constant of the drug from the noise current.

Example 1 [0061] In the container 1 shown in FIG. 3, the number of the holes 14 was two, and the current fluctuation was actually measured using the electrical measuring device shown in FIG. 5 (FIG. 5). The following operation was performed at 25 ° C. The inner electrolyte solution (KC1 130 mM, MgCl 1 mM, EGTA 5 mM, ATP 5)

2

mM, HEPES lOmM, pH 7.4), the upper surface of the device (vessel 1 upper side) is the outer electrolyte solution (NaCl 137 mM, KC1 4 mM, CaCl 1.8 mM, MgCl 1 mM, glucose 10 mM, HEPES

twenty two

10 mM, pH 7.4). HEK cells in which hERG channels were steadily expressed were isolated by pipetting in an external solution in a separate container and then placed on the upper surface of the device. By applying negative pressure (up to 530mmHg) to the lower surface of the device by suction, the sealing resistance between the device and cells was increased. When the sealing resistance was sufficiently increased, the inner electrolyte solution on the lower surface of the device was replaced with a solution containing -statin (nystatin concentration 250 g / ml) to construct perforations with nystatin in the cell membrane. After perforation of the cell membrane by nystatin, hERG-expressing HEK cells were first fixed at −80 mV using an EPC-10 amplifier (HEKA), then the fixed voltage was raised to OmV for 6 seconds. Fluctuations were measured. The measured current fluctuation was digitally sampled at 10 kHz (PATCHMASTER software, manufactured by HEKA), and the power spectrum was obtained by fast Fourier transform of the data for the last 2 seconds (Origin 7.0). Next, the hERG channel inhibitor E-4031 (manufactured by Sigma) is placed in the outer electrolyte solution on the upper surface of the device to a final concentration of 1M, and the voltage fluctuation is similarly fixed to measure the current fluctuation. The power spectrum was determined. These power spectra are shown in the graph of Fig. 7.

[0062] As shown in Fig. 7, the power decreased below lOOHz after adding E-4031 (dotted line) as compared to before E-4031 was covered (solid line). This clearly shows that E-4031 is a hERG channel inhibitor, and that the change in the behavior of the ion channel could be measured by obtaining the power spectrum of the current fluctuation with the method of the present invention. .

[0063] As described above, the electrical measurement method and the electrical measurement device for at least one of the characteristics and states of the cell membrane of the present invention summarize the fluctuations in current caused by the characteristics and states of the cell membranes of a plurality of cells. Therefore, electrical measurement of cell membrane state and the like is possible at high speed, accurately and easily. Therefore, the electrical measurement method and electrical measurement apparatus of the present invention are applicable to all fields for analyzing characteristics and states of cell membranes such as ion channel states. For example, it is useful in fields such as biology, medicine, pharmacy, and agriculture, and is particularly suitable for the development of pharmaceuticals.

Claims

The scope of the claims

[1] An electrical measurement method for at least one of the characteristics and state of a cell membrane, comprising preparing a measurement electrode, a reference electrode, and a plurality of cells, and electrically connecting the measurement electrode and the inside of each cell And the electrical measurement method which electrically connects the said reference electrode and each said cell membrane surface, applies a voltage to the said measurement electrode, and measures the fluctuation | variation of the electric current which flows between the said both electrodes.

[2] The electrical measurement method according to claim 1, further comprising measuring a power spectrum of the fluctuation of the current.

[3] The electrical measurement method according to claim 1, wherein the number of the plurality of cells is in the range of 2 to: LOO.

[4] The electrical measurement method according to claim 1, wherein the characteristics and state of the cell membrane to be measured are those of the ion channel existing in the cell membrane.

5. The electrical measurement method according to claim 4, wherein the ion channel has a characteristic and a state force that are at least one of the number of the ion channels and the open / closed state of the ion channels.

6. The electrical measurement method according to claim 1, wherein the cell is a cell present in a tissue.

7. The electrical measurement method according to claim 1, wherein the fluctuation of the current is measured before and after the chemical substance is administered to the cell.

[8] An electrical measurement device for at least one of the characteristics and state of a cell membrane, a container having a plurality of holes for holding and fixing cells, a measurement electrode, a reference electrode, and an application for applying a voltage to the measurement electrode Means, a detecting means for detecting a current flowing between the electrodes, and a measuring means for measuring fluctuations in the current, and the measuring electrode and each cell can be electrically connected, In addition, when the reference electrode and the cell membrane surface are electrically connectable and the electrodes are electrically connected in the state, a voltage is applied to the measurement electrode by the applying means, and the detection is performed. Means for detecting the current flowing between the electrodes and measuring the fluctuation of the current by the measuring means.

9. The electrical measurement device according to claim 8, further comprising power spectrum measurement means for measuring a power spectrum of the fluctuation of the current.

[10] The container can be filled with an electrolyte solution, and the electrolyte solution electrically connects between the measurement electrode and each cell and between the reference electrode and each cell membrane surface. The electrical measuring device according to claim 8 to be connected.

[11] The electrical measuring device according to claim 8, wherein the number of holes in the container is 2 to: LOO.

[12] The measuring device according to claim 8, which is used for examining the influence of a chemical substance on a cell membrane.