Impedance measuring device and impedance measuring method

Abstract

To provide a technique that can measure the impedance of a biological specimen by using a multi-electrode array device.SOLUTION: An impedance measurement apparatus 1 comprises a measurement container 10, a plurality of first electrodes 31, a second electrode 32, a third electrode 33, a voltage application circuit 43, a current detection circuit 45, and a voltage detection circuit 47. The first electrodes 31 are arranged in array on a bottom face inside the measurement container 10. The second electrode 32 and the third electrode 33 are located inside the measurement container 10. The voltage application circuit 43 applies voltage between each of the first electrodes 31 and the second electrode 32. The current detection circuit 45 detects a current flowing in each of the first electrodes 31. The voltage detection circuit 47 detects the voltage between each of the first electrodes 31 and the third electrode 33.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The subject matter disclosed herein relates to impedance measuring devices and impedance measuring methods. [Background technology]

[0002] In the field of electrophysiology, the behavior of cellular ion channels in single cells or populations is analyzed. Examples of ion channel analyzers include intracellular action potential detection devices using the patch-clamp method and extracellular action potential detection devices using multi-electrode array (MEA) devices. Multi-electrode array devices are disclosed, for example, in Patent Document 1. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Special Publication No. 2016-529889 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] For example, impedance measurements of target biological samples (including cells and biological slices) may be performed to easily measure changes in cell morphology or cell density. However, conventionally, multi-electrode array devices have only been used to detect extracellular action potentials, and the technique for measuring the impedance of biological samples using multi-electrode array devices has not yet been established.

[0005] The objective of the present invention is to provide a technology that enables the measurement of the impedance of a biological sample using a multi-electrode array device. [Means for solving the problem]

[0006] To solve the above problems, the first embodiment is an impedance measuring device for measuring the impedance of an object, The liquid containing the aforementioned object is placed A measuring container, a plurality of first electrodes arranged in an array on the bottom surface inside the measuring container, a second electrode and a third electrode located inside the measuring container, and between each of the first electrode and the second electrode exchange A voltage application circuit for applying a voltage, a current detection circuit for detecting the current flowing through each of the first electrodes, and a voltage detection circuit for detecting the voltage between each of the first electrodes and the third electrode, A calculation unit calculates the impedance of the object based on the current detected by the current detection circuit and the voltage detected by the voltage detection circuit. It is equipped with.

[0007] The second embodiment is the impedance measuring device of the first embodiment, wherein the third electrode is located on the bottom surface.

[0008] A third embodiment is an impedance measuring device according to the first or second embodiment, further comprising an electrode selection circuit that selects one first electrode from among the plurality of first electrodes to be connected to the current detection circuit.

[0009] The fourth embodiment is an impedance measuring device according to any one of the first to third embodiments, wherein the current detection circuit is capable of outputting a voltage corresponding to the current, and the impedance measuring device further comprises a differential signal detection circuit that detects the difference between the AC voltage applied by the voltage application circuit and the voltage output by the current detection circuit.

[0011] The 5 The embodiment is an impedance measurement method, a) The liquid containing the object was placed The steps are: a) applying an AC voltage between at least one of a plurality of first electrodes arranged in an array on the bottom surface of the measuring container and a second electrode located inside the measuring container; b) detecting the current flowing through the first electrode while the AC voltage is applied by step a); c) detecting the voltage between the first electrode and a third electrode while the AC voltage is applied by step a); and d) based on the current detected by step b) and the voltage detected by step c), The aforementioned The system includes a step of calculating the impedance of the object.

Advantages of the Invention

[0012] According to the impedance measurement device of the first aspect, the impedance of the object can be measured by the three-terminal method using the first electrodes arranged in an array on the bottom surface of the measurement container.

[0013] According to the impedance measurement device of the second aspect, since the first electrode and the third electrode can be concentrated on the bottom surface, the enlargement of the device can be suppressed.

[0014] According to the impedance measurement device of the third aspect, the impedance can be measured using one first electrode selected from among the plurality of first electrodes.

[0015] According to the impedance measurement device of the fourth aspect, the impedance of the object can be calculated based on the phase difference between the AC voltage and the current.

[0016] According to the impedance measurement device of the fifth aspect, the impedance can be calculated.

Brief Description of the Drawings

[0017] [Figure 1] It is a diagram schematically showing the configuration of the impedance measurement device according to the embodiment. [Figure 2] It is a top view showing the measurement container shown in FIG. 1. [Figure 3] It is a diagram showing an equivalent circuit in the case of measuring impedance.

Modes for Carrying Out the Invention

[0018] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. Note that the components described in this embodiment are merely examples, and are not intended to limit the scope of the present invention thereto. In the drawings, for ease of understanding, the dimensions and numbers of each part may be exaggerated or simplified as necessary.

[0019] <1. First Embodiment> Figure 1 is a schematic diagram showing the configuration of the impedance measuring device 1 according to an embodiment. Figure 2 is a top view showing the measuring container 10 shown in Figure 1. As shown in Figure 1, the impedance measuring device 1 comprises a measuring container 10 and a measuring unit 20.

[0020] The measurement container 10 is a container for measuring the impedance of the target object, the cell 9. A cell suspension containing the cell 9 is dropped into the measurement container 10. As shown in Figure 1, the measurement container 10 has a bottom 11 and side walls 13. The bottom 11 spreads out in a disc shape along the horizontal plane. The side walls 13 extend upward in a cylindrical shape from the periphery of the bottom 11. The bottom 11 is an example of a "measurement plate". The upper surface of the bottom 11 is the bottom surface inside the measurement container 10 into which the cell suspension is dropped. Note that the object to be measured in the measurement container 10 is not limited to cell 9, but may also be a slice of biological tissue, etc.

[0021] The measuring container 10 has a plurality of first electrodes 31, a second electrode 32, a third electrode 33, a plurality of first wirings 35, and a second wiring 37. The plurality of first electrodes 31, the third electrode 33, the plurality of first wirings 35, and the second wirings 37 are located on the upper surface of the bottom 11 (the bottom surface of the measuring container 10). Each first wiring 35 and second wiring 37 is covered with an insulator (for example, photosensitive polyimide). Each first electrode 31, third electrode 33, each first wiring 35, and second wiring 37 are formed, for example, by photolithography.

[0022] Multiple first electrodes 31 are arranged in an array. The measuring container 10 is a multi-electrode array (or micro-electrode array) device in which multiple minute first electrodes 31 are arranged in an array. In the examples shown in Figures 1, 2, and 3, 16 first electrodes 31 are arranged in a 4x4 matrix. The number and arrangement of the first electrodes 31 can be set arbitrarily. Preferably, 10 or more first electrodes 31 are arranged. The shape of the first electrode 31 is square when viewed from above. However, the shape of the first electrode 31 may be a polygon or a circle other than a square.

[0023] The second electrode 32 is located inside the measuring container 10. The second electrode 32 is, for example, stick-shaped, and at least a portion of it (for example, the lower end) is immersed in the liquid 91 injected into the measuring container 10.

[0024] The third electrode 33 is located radially outward from the plurality of first electrodes 31. The plurality of first electrodes 31 and the third electrode 33 are insulated from each other. In the examples shown in Figures 1 and 2, the third electrode 33 is square in top view. However, the shape of the third electrode 33 may be a polygon or a circle other than a square.

[0025] Each first wire 35 is electrically connected to a corresponding first electrode 31. In Figure 1, two first wires 35 connected to two first electrodes 31 are shown, while the first wires 35 connected to other first electrodes 31 are not shown. Each first wire 35 extends to the outside of the measuring container 10. The second wire 37 is electrically connected to the third electrode 33. The second wire 37 extends from the third electrode 33 to the outside of the measuring container 10. As shown in Figure 1, each first wire 35 and the second wire 37 are electrically connected to the measuring unit 20 located outside the measuring container 10.

[0026] As shown in Figure 1, the measurement unit 20 includes an electrode selection circuit 41, a voltage application circuit 43, a current detection circuit 45, a voltage detection circuit 47, a differential signal detection circuit 49, and a calculation unit 51.

[0027] The electrode selection circuit 41 is a circuit for selecting one first electrode 31 from among a plurality of first electrodes 31 to be electrically connected to the current detection circuit 45 and the voltage detection circuit 47. As shown in Figure 1, the electrode selection circuit 41 has a plurality of switches for opening and closing the circuits connecting each first electrode 31 to the current detection circuit 45. The switching operation of the electrode selection circuit 41 may be controlled by the calculation unit 51.

[0028] The voltage application circuit 43 is electrically connected to the first electrode 31 via the electrode selection circuit 41. The voltage application circuit 43 applies an AC voltage (AC signal) of a predetermined frequency between the first electrode 31 and the second electrode 32. In the example shown in Figure 1, the voltage application circuit 43 is connected to the non-inverting input terminal (+) of the operational amplifier 451 of the current detection circuit 45, which will be described later. The voltage application circuit 43 is also connected to the inverting input terminal (-) of the operational amplifier 491 of the differential signal detection circuit 49, which will be described later.

[0029] The current detection circuit 45 is electrically connected to the electrode selection circuit 41. The current detection circuit 45 detects the current flowing through one of the first electrodes 31 selected by the electrode selection circuit 41.

[0030] As shown in Figure 1, the current detection circuit 45 includes an operational amplifier 451, a first resistor 453, and a second resistor 455. The non-inverting input terminal (+) of the operational amplifier 451 is electrically connected to the voltage application circuit 43. The inverting input terminal (-) of the operational amplifier 451 is electrically connected to the electrode selection circuit 41. The first resistor 453 is located between the electrode selection circuit 41 and the inverting input terminal (+) of the operational amplifier 451. One end of the second resistor 455 is connected between the output terminal of the operational amplifier 451 and the differential signal detection circuit 49. The other end of the second resistor 455 is connected between the inverting input terminal (-) of the operational amplifier 451 and the first resistor 453.

[0031] The voltage detection circuit 47 detects the voltage Vx between a first electrode 31 selected by the electrode selection circuit 41 and a third electrode 33. As shown in Figure 1, the voltage detection circuit 47 has an operational amplifier 471. The non-inverting input terminal (+) of the operational amplifier 471 is connected between the electrode selection circuit 41 and the first resistor 453 and is electrically connected to the first electrode 31 via the electrode selection circuit 41. The inverting input terminal (-) of the operational amplifier 471 is electrically connected to the third electrode 33.

[0032] The differential signal detection circuit 49 detects the phase difference between the AC voltage applied by the voltage application circuit 43 and the current detected by the current detection circuit 45. As shown in Figure 1, the differential signal detection circuit 49 has an operational amplifier 491. The non-inverting input terminal (+) of the operational amplifier 491 is connected to the output terminal of the operational amplifier 451 of the current detection circuit 45. The inverting input terminal (-) of the operational amplifier 491 is connected to the voltage application circuit 43.

[0033] The calculation unit 51 includes a storage device, a processing circuit, an input device, and an output device. The storage device consists of memory (storage medium) including, for example, a hard disk drive (HDD), random access memory (RAM), read-only memory (ROM), flash memory, volatile or non-volatile semiconductor memory, magnetic disk, flexible disk, optical disk, compact disk, minidisc, or DVD. The processing circuit consists of, for example, a central processing unit (CPU) that executes programs stored in the storage device. The input device consists of, for example, a device capable of inputting information, such as a mouse, keyboard, touch panel, or various switches. The output device consists of, for example, a device capable of outputting information, such as a display, liquid crystal display device, or lamp.

[0034] The calculation unit 51 calculates the impedance based on the voltage Vx output by the voltage detection circuit 47 and the voltage Vy output by the differential signal detection circuit 49. The procedure for calculating the impedance will be explained with reference to Figure 3.

[0035] <Impedance Calculation> FIG. 3 is a diagram showing an equivalent circuit when measuring impedance. When measuring the impedance of the cells 9 using the measurement container 10, the measurement container 10 is placed on a horizontal table. Then, the cell suspension is dropped onto the upper surface of the bottom 11 where a plurality of first electrodes 31 in the measurement container 10 are arranged. When the cell suspension dropped into the measurement container 10 is left for several minutes, a plurality of cells 9 sink, and a cell layer is formed on the bottom 11 of the measurement container 10. After the cell layer is formed, the measurement liquid 91 (culture medium) is injected into the measurement container 10. When the liquid 91 contacts the first electrode 31, the second electrode 32, and the third electrode 33, the first electrode 31, the second electrode 32, and the third electrode 33 become conductive. As shown in FIG. 1, the second electrode 32 is grounded during impedance measurement.

[0036] In the following description, as shown in FIG. 3, the contact impedance of the first electrode 31 is "Z1", the contact impedance of the second electrode 32 is "Z2", the contact impedance of the third electrode 33 is "Z3", and the impedance of the object (cells) (target impedance) is "Zx".

[0037] As shown in FIG. 3, when the voltage application circuit 43 applies an alternating voltage, an alternating current i flows through the first electrode 31. When the voltage application circuit 43 applies an alternating voltage, the potential differences caused by the contact impedances Z1, Z2, Z3, and the impedance Zx are respectively "V Z1 ", "V Z2 ", "V Z3 ", "V Zx ".

[0038] The voltage detection circuit 47 detects the voltage Vx between the first electrode 31 and the third electrode 33. The voltage Vx is equal to the sum of V Zx 、V Z1 、V Z3 . Also, the voltage Vx is expressed by the following formula (1) using the current i, the contact impedances Z1, Z3, and the target impedance Zx.

[0039] Vx = V Zx + V Z1 + VZ3 =(Zx+Z1+Z3)*i ···(1)

[0040] Here, by increasing the input impedance of the operational amplifier 471 in the voltage detection circuit 47 (for example, to 10 GΩ (gigaohms) or more), current is less likely to flow into the voltage detection circuit 47. As a result, the potential difference VZ3 generated at the third electrode 33 becomes almost zero (VZ3 ≈ 0). Therefore, the voltage detection circuit 47 effectively measures the sum of VZ1 and VZx (= VZ1 + VZx), and the contact impedance Z3 of the third electrode 33 can be suppressed. That is, the voltage Vx measured by the voltage detection circuit 47 is expressed by the following equation (2).

[0041] Vx = V Zx +V Z1 =(Zx+Z1)*i ···(2)

[0042] Here, the alternating current i is expressed by the following equation (3), using the applied voltage Vs of the voltage application circuit 43 and the resistance value R1 of the first resistor 453.

[0043] i = Vs / R1 ... (3)

[0044] The voltage Vy output by the differential signal detection circuit 49 is the difference between the voltage output by the current detection circuit 45 (=R2*i) and the applied voltage Vs. That is, the voltage Vy is expressed by the following equation (4).

[0045] Vy = (R² * i) - Vs ... (4)

[0046] By measuring voltages Vx and Vy, the phase difference between their signals can be detected, allowing the combined impedance Zx+Z1 to be measured. Additionally, contact impedance Z1 is measured by performing impedance measurements in a state with only liquid 91 (i.e., without cells 9; Zx=0). The target impedance Zx is calculated by subtracting the measured contact impedance Z1 from the combined impedance Zx+Z1.

[0047] As described above, the impedance measuring device 1 can measure the impedance of the target cell 9 by measuring the current flowing through multiple first electrodes 31, which are a multi-electrode array. This makes it possible to measure the impedance of the cell 9, as well as the conventional extracellular action potential, using the measuring container 10, which is a multi-electrode array device.

[0048] Furthermore, the impedance measuring device 1 measures impedance using a three-terminal method with three electrodes. Therefore, it is possible to measure the impedance of cells 9 with higher accuracy than with the two-terminal method.

[0049] <2. Variant Example> Although embodiments have been described above, the present invention is not limited to the embodiments described above.

[0050] For example, the second electrode 32 may be formed on the surface of the side wall portion 13, for example, by photolithography.

[0051] Furthermore, it is not essential that the third electrode 33 is located on the upper surface of the bottom 11 of the measuring container 10. For example, similar to the second electrode 32, the third electrode 33 may be in the shape of a stick, so that it is immersed in the liquid injected into the measuring container 10. Alternatively, multiple third electrodes 33 may be arranged. In this case, a selection circuit (not shown) may be used to select one of the multiple third electrodes 33 that is connected to the voltage detection circuit 47.

[0052] Although this invention has been described in detail, the above description is illustrative in all respects, and the invention is not limited thereto. It is understood that countless variations not illustrated can be conceived without falling outside the scope of this invention. The components described in each of the above embodiments and variations can be combined or omitted as appropriate, as long as they do not contradict each other. [Explanation of symbols]

[0053] 1. Impedance measuring device 10 Measuring containers 11 Bottom 20 Measurement section 31 1st electrode 32 2nd electrode 33 Third electrode 41 Electrode Selection Circuit 43 Voltage Application Circuit 45 Current detection circuit 47 Voltage detection circuit 49 Differential signal detection circuit 51 Calculation Section

Claims

1. An impedance measuring device for measuring the impedance of an object, A measuring container in which the liquid containing the aforementioned object is placed, Multiple first electrodes are arranged in an array on the bottom surface inside the measuring container, The second electrode and the third electrode located inside the measuring container, A voltage application circuit that applies an AC voltage between each of the first electrode and the second electrode, A current detection circuit for detecting the current flowing through each of the first electrodes, A voltage detection circuit for detecting the voltage between each of the first electrodes and the third electrode, A calculation unit that calculates the impedance of the object based on the current detected by the current detection circuit and the voltage detected by the voltage detection circuit, An impedance measuring device equipped with the following features.

2. An impedance measuring device according to claim 1, An impedance measuring device in which the third electrode is located on the bottom surface.

3. An impedance measuring device according to claim 1 or claim 2, An electrode selection circuit that selects one first electrode from among the plurality of first electrodes to be connected to the current detection circuit. An impedance measuring device that also features [the following].

4. An impedance measuring device according to claim 1 or claim 2, The current detection circuit is capable of outputting a voltage corresponding to the current, The impedance measuring device is A differential signal detection circuit that detects the difference between the AC voltage applied by the voltage application circuit and the voltage output by the current detection circuit. An impedance measuring device that also features [the following].

5. An impedance measurement method, a) A step of applying an AC voltage between at least one of a plurality of first electrodes arranged in an array on the bottom surface of a measuring container in which a liquid containing the object is placed, and a second electrode located inside the measuring container, b) A step of detecting the current flowing through the first electrode while an AC voltage is applied by step a), c) A step of detecting the voltage between the first electrode and the third electrode while an AC voltage is applied by step a), d) A step of calculating the impedance of the object based on the current detected in step b) and the voltage detected in step c), An impedance measurement method comprising the following features.

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