JPWO2019240202A1 - measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an electrochemical measuring device capable of measuring the physical characteristics of a sample more accurately and also measuring the behavior of a sample. SOLUTION: A solution tank 5 containing a solution 3, a first electrode 7 and a second electrode 9 existing in the solution layer 5 and at least a part of the surface thereof is exposed in the solution, and a first electrode. Electricity including a voltage applying means 11 for applying a voltage between the 7 and the second electrode 9 and a current measuring means 13 for measuring the current flowing between the first electrode 7 and the second electrode 9. A measuring device which is a chemical measuring device 1 and has a surface surface S of a portion 15 exposed to the solution tank 5 of the first electrode 7 of 0.1 μm2 or more and 100 μm2 or less. [Selection diagram] Fig. 1

Description

The present invention relates to a measuring device. More specifically, the present invention relates to an electrochemical measuring device capable of measuring the physical properties and behavior of cells.

Japanese Patent No. 5617532 describes a dielectric cytometry apparatus and a cell sorting method using a dielectric cytometry.

Japanese Patent No. 5617532

With conventional dielectric cytometry, there are problems that the physical properties of the sample near the electrode cannot be measured and that the measured value fluctuates depending on the distance between the electrode and the sample.

Therefore, an object of the present specification is to provide an electrochemical measuring device capable of measuring the physical characteristics of a sample more accurately and also measuring the behavior of the sample.

The above problem is based on the finding that the physical properties of the sample existing near the electrode can also be measured by making the electrode smaller than the cross-sectional area of the sample. In addition, the physical characteristics of the sample can be measured more accurately by reducing the distance between adjacent electrodes, and the physical characteristics of the sample can be measured more accurately by providing a partition wall that allows the solution to pass but not the sample to pass between the electrodes to which the voltage is applied. Based on the knowledge that can be measured.

The invention first described herein relates to measuring apparatus 1. This measuring device is an electrochemical measuring device 1 including a solution tank 5 for accommodating the solution 3, a first electrode 7, a second electrode 9, a voltage applying means 11, and a current measuring means 13.

The first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface thereof is exposed in the solution.

The voltage applying means 11 is an element for applying a voltage between the first electrode 7 and the second electrode 9.

The current measuring means 13 is an element for measuring the current flowing between the first electrode 7 and the second electrode 9.

In this device, the surface area S of the portion 15 exposed to the solution tank 5 of the first electrode 7 is 0.1 μm ² or more and 100 μm ² or less.

This measuring device is, for example, a device for measuring the physical properties or movement of the sample 17 contained in the solution and existing in the vicinity of the first electrode. The example of sample 17 is a biological cell or a liposome, and the surface area S is preferably smaller than the area of the biological cell or the liposome.

This device preferably has an adhesion preventing means 19 for preventing the sample 17 contained in the solution from adhering to the second electrode.

It is preferable that a plurality of the first electrode 7 and the second electrode 9 are present, and the minimum distance to the adjacent first electrode 7 or the second electrode 9 is 31 mm or less.

The first electrode 7 and the second electrode 9 may be provided on the substrate 21. Further, it is preferable that the second electrode 9 is provided in the recessed portion 23 of the substrate 21, and the adhesion preventing means 19 is a net covering the recessed portion.

The first electrode 7 is provided on the substrate 21 and is provided on the substrate 21.
The second electrode 9 is provided on the side wall of the solution tank 5.
The adhesion preventing means 19 may be a net covering the second electrode.

The second electrode 9 is provided on the electrode accommodating body 23 floating in the solution, at least a part of the electrode accommodating body 23 has the adhesion preventing means 19, and the first electrode 7 is provided on the substrate 21. It may be a thing.

The invention described in this specification can provide an electrochemical measuring device capable of measuring the physical characteristics of a sample more accurately and also measuring the behavior of the sample.

FIG. 1 is a conceptual diagram showing a configuration example of a measuring device. FIG. 2 is a conceptual diagram showing an example of a measuring device in which a first electrode and a second electrode are provided on a substrate. FIG. 3 is a conceptual diagram showing the first electrode and the second electrode formed in an array. FIG. 4 is a conceptual diagram showing a measuring device in which the first electrode is provided on the side wall of the solution tank and the second electrode is provided on the bottom of the solution tank. FIG. 5 is a conceptual diagram showing an example of a measuring device in which the second electrode is immersed in a solution and is housed in an electrode container. FIG. 6 is a conceptual diagram of the electrodes manufactured in the examples. FIG. 7 is a conceptual diagram showing a dielectric measuring device for measuring a dielectric spectrum. FIG. 8 is a photograph instead of a drawing showing the state of the electrodes and cells. FIG. 9 is a graph that replaces the drawings showing the measured Cole-Cole plot (FIG. 9 (A)) and frequency-phase plot (FIG. 9 (B)). FIG. 10 is a graph that replaces the drawings showing the Cole-Cole plot (FIG. 10 (A)) and the frequency-phase plot (FIG. 10 (B)) to show the effect of the distance between the electrodes. FIG. 11 is an alternative graph to the drawing showing a frequency-phase plot for examining the effects of non-electrolytes. FIG. 12 is a conceptual diagram showing how migrating cells are tracked.

Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the forms described below, and includes those modified appropriately by those skilled in the art from the following forms.

The invention first described herein relates to measuring apparatus 1. FIG. 1 is a conceptual diagram showing a configuration example of a measuring device. As shown in FIG. 1, this measuring device includes a solution tank 5 for accommodating the solution 3, a first electrode 7 and a second electrode 9, a voltage applying means 11, and a current measuring means 13. This is an electrochemical measuring device 1. Examples of solutions may be electrolyte solutions, and depending on cells and liposomes, media or culture medium. The solution tank 5 may be any as long as it can accommodate the solution, and its size and material can be appropriately selected according to the intended use.

The first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface thereof is exposed in the solution. The entire surface of the electrode may be exposed and come into contact with the solution, or a part of the electrode may be buried in the substrate or the solution. The surface area S of the portion 15 exposed to the solution tank 5 of the first electrode 7 is 0.1 μm ² or more and 100 μm ² or less. The value of S may be in the 0.2 of the electrode [mu] m ² or more 80 [mu] m ² or less, may be a 0.5 [mu] m ² or more 70 [mu] m ² or less, may be a 1 [mu] m ² or more 50 [mu] m ² or less, 0.5 [mu] m ² or more 30 [mu] m ² or less But to good, may be a 2 [mu] m ² or more 10 [mu] m ² or less, it may be 10 [mu] m ² or more 50 [mu] m ² or less. When a voltage is applied to such a minute electrode, the shape of the diffusion layer that transports the sample to the electrode surface approaches a hemisphere, and the sample diffuses so as to converge from the hemispherical surface toward the electrode. It is thought that it can be measured accurately. In other words, if a normal electrode is used, the measured value will fluctuate depending on the sample attached to the adjacent electrode, but it is considered that such a problem can be solved by making the electrode minute. Since semiconductor chips have been miniaturized and speeded up, such microelectrodes can be manufactured by using the technology in the semiconductor manufacturing method.

The second electrode 9 may be the same as the first electrode. Further, a plurality of the first electrode 7 and the second electrode 9 may exist. FIG. 2 is a conceptual diagram showing an example of a measuring device in which a first electrode and a second electrode are provided on a substrate. In the measuring device shown in FIG. 2, the first electrode 7 and the second electrode 9 are provided on the substrate 21. Then, the second electrode 9 is provided in the recessed portion 23 of the substrate 21, and the adhesion preventing means 19 is a net covering the recessed portion. The net allows the solution to pass through the net, but the sample does not. As a result, it is possible to prevent the sample from adhering to the second electrode. As shown in FIG. 2, the first electrode 7 and the second electrode 9 may be in the form of an array provided on the substrate. Further, it is preferable that the first electrode 7 and the second electrode 9 can control the connection relationship so that the connection relationship between them can be adjusted.

FIG. 3 is a conceptual diagram showing the first electrode and the second electrode formed in an array. It is preferable that a plurality of the first electrode 7 and the second electrode 9 are present, and the minimum distance d to the adjacent first electrode 7 or the second electrode 9 is 31 mm or less. If the value of d is too small, the adjacent electrodes will be energized. Therefore, the value of d is preferably 0.1 μm or more, and may be 1 μm or more, or 5 μm or more. On the other hand, d may be 25 mm or less, 20 mm or less, 15 mm or less, 10 mm or less, 1 mm or less, 500 μm or less, 100 μm or less, or 50 μm or less. Good. In FIG. 3, a portion 15 of the electrodes exposed to the solution tank 5 is drawn. Further, in the array-shaped electrodes of FIG. 3, whichever is the first electrode 7 or the second electrode 9 may be used.

The voltage applying means 11 is an element for applying a voltage between the first electrode 7 and the second electrode 9. The voltage applying means 11 is a means for applying a voltage between the first electrode 7 and the second electrode 9. The applied voltage may be alternating current or direct current, but is usually an alternating voltage. The voltage applying means 11 is preferably one capable of controlling the frequency of the AC voltage applied between the electrodes. Since the voltage applied between the two electrodes in dielectric cytometry or the like is known, a known overvoltage voltage may be appropriately adjusted and applied between the first electrode 7 and the second electrode 9. .. Then, it is preferable that the control unit can control which electrode of the electrode array having a plurality of electrodes a voltage is applied to.

The current measuring means 13 is an element for measuring the current flowing between the first electrode 7 and the second electrode 9. The current measuring means 13 is known. The current measuring means 13 may be capable of measuring various physical properties by measuring the current between the two electrodes.

This measuring device is, for example, a device for measuring the physical properties or movement of the sample 17 contained in the solution and existing in the vicinity of the first electrode. The example of sample 17 is a biological cell or a liposome, and the surface area S is preferably smaller than the area of the biological cell or the liposome. An example near the first electrode is a region in which the distance to the first electrode is short in the electrode adjacent to the first electrode.

This device preferably has an adhesion preventing means 19 for preventing the sample 17 contained in the solution from adhering to the second electrode. The adhesion preventing means 19 may also prevent the sample from adhering to the first electrode. Examples of the adhesion preventing means 19 are a net and a semipermeable membrane, which will be described later. When the adhesion preventing means 19 is a net, it is preferable that the mesh size of the net is smaller than that of the sample (cross-sectional area). By using such a net, it is possible to effectively prevent the sample from adhering to the electrodes while ensuring the movement of the solution.

FIG. 4 is a conceptual diagram showing a measuring device in which the first electrode is provided on the side wall of the solution tank and the second electrode is provided on the bottom of the solution tank. In the example of FIG. 4, the first electrode 7 is provided on the substrate 21, the second electrode 9 is provided on the side wall of the solution tank 5, and the adhesion preventing means 19 is a net covering the second electrode. is there. In such a positional relationship, especially when the sample is a cell, it is preferable because the situation where the sample adheres to the second electrode can be effectively prevented. In this case, the solution tank 5 has a bottom surface and a side wall extending from the bottom surface. Examples of bottom shapes are circles, ellipses, and polygons. The second electrode is preferably at a predetermined height from the bottom surface. An example of the height is 1 μm or more and 1 mm or less, and may be 10 μm or more and 100 μm or less.

The substrate may be composed of an insulator. It is preferable that the substrate is made of a transparent or translucent insulator. An example of such an insulator is transparent ceramics. The transparent ceramic can be achieved by using, for example, one or more of Al2O3, Y2O3 and YAG. If the substrate is transparent, it is easy to observe their behavior, especially when the sample is cells or liposomes.

FIG. 5 is a conceptual diagram showing an example of a measuring device in which the second electrode is housed in an electrode container in which the second electrode floats in a solution. An example of an electrode containment is a float. The second electrode 9 is provided on the electrode accommodating body 23 floating in the solution, at least a part of the electrode accommodating body 23 has the adhesion preventing means 19 (for example, a net), and the first electrode 7 is on the substrate. It may be the one provided in 21.

Next, the principle of the electrochemical measuring device will be briefly explained.
A complex resistance (complex impedance) between electrodes can be obtained by applying an AC voltage between the electrode plates and measuring the flowing current. When the frequency of the applied AC voltage is changed, the measured complex resistance also changes. Such measurement can be performed using a commercially available precision impedance analyzer (current measuring device).

The frequency-dependent complex resistance is corrected by correcting factors such as the shape of the measuring container and the transmission characteristics of the electrical wiring between the complex resistance measuring instrument and the measuring container. Can be converted to rate. The frequency dependence of the complex permittivity is called the complex permittivity dispersion (dielectric spectrum).

The complex permittivity dispersion of a cell suspension can be expressed by a single relaxation function (eg, Cole-Cole function) or by superposition of multiple relaxation functions. The variable can be optimized by performing a non-linear adaptation using the undetermined coefficient included in the relaxation function as a variable for the experimentally obtained complex permittivity variance. For example, in the case of the Cole-Cole function, the variables that characterize the dispersion curve are relaxation intensity and relaxation frequency. These dielectric variables are closely related to the structure and physical properties of cells. A method for estimating the electrical property values of the phases (cell membrane, cytoplasm, etc.) constituting the cell from the dielectric variable is described in, for example, Japanese Patent Application Laid-Open No. 2009-42141.

In manufacturing the measuring device, we developed an electrode on the substrate (chip). FIG. 6 is a conceptual diagram of the electrodes manufactured in the examples. The electrode surface is made of titanium nitride (TiN) and is the part that comes into contact with samples such as solutions and animal cells. Non-doped silicate glass (NSG, SiO2), aluminum (Al), silicon oxide (SiO2), silicon (Si) were used.

Spare tip
Thirty-six electrode pads of 10 μm square at 20 μm intervals were used as a grid electrode array of 6 rows and 6 columns, covering a region of ^{28900 μm 2.}

Tip for tracking fibrillation behavior
36 electrode pads of 6.6 μm square were used as a grid electrode array of 6 rows and 6 columns with an interval of 3.4 μm, and the area of ^{3200 μm 2 was covered.}

Dielectric measuring device FIG. 7 shows a dielectric measuring device for measuring a dielectric spectrum. In this example, the dielectric spectrum was measured using an Agilent 4294A Precision Impedance Analyzer. The Z value and phase were measured.

Glucose solution and PBS were used as the solutions. As an example of the sample, beads made of polystyrene of 25 μm were used here.

Two 10 μm square electrodes were brought into contact with the MGM-450 insect medium at intervals of 20 μm, and an insect cell mass was added to the medium. The electrodes were observed using an optical microscope, and impedance measurements from 100 Hz to 1000000 Hz were performed at 100 mV using an LCR meter between two electrodes with no cell mass on top of the electrodes to obtain Z and θ values. Similarly, two 10 μm square electrodes were brought into contact with the MGM-450 insect medium at intervals of 20 μm, and an insect cell mass was added to the medium. The electrodes were observed using an optical microscope, and impedance measurements from 100 Hz to 1000000 Hz were performed at 100 mV using an LCR meter between the two electrodes with cell clusters on top of the electrodes to obtain Z and θ values. FIG. 8 is a photograph instead of a drawing showing the state of the electrodes and cells. FIG. 9 is a measured Cole-Cole plot (FIG. 9 (A)) and a frequency-phase plot (FIG. 9 (B)). The peak near 10000 Hz observed when the measurement result was illustrated on the θ-Hz plane was shifted to the low frequency side in the measurement between the two electrodes with the cell mass on the upper part of the electrode.

Examination of the distance between the electrodes As the electrodes, a 10 μm square electrode, a 6.6 μm square electrode, and a probe needle of a manual prober were used. The 10 μm square electrode and the 6.6 μm square electrode are both connected by independent conductors, and the opposite end of the conductors is probed with a probe needle of a manual prober so that conduction can be obtained.

Two 6.6 μm square electrodes were brought into contact with phosphate buffered saline, and impedance measurements from 100 Hz to 100000000 Hz were performed at 100 mV using an LCR meter to obtain Z and θ values. The θ value is -80 ° at 100Hz, -60 ° at 10000Hz, -80 ° at 10000000Hz, and -75 ° at 100000000, which is between the electrodes in the region away from the electrode surface that appears in the high frequency region measured by the prior art. The electrochemical properties of the intermediate material and the electrochemical properties of the intermediate material between the electrodes in the electrode surface region appearing near 10000 Hz, which was not measured by the conventional technique, were confirmed. The measurement results did not change when the electrode spacing was in the range of 3 μm to 60 μm.

One 6.6 μm square electrode and a probe needle of a manual prober were brought into contact with phosphate buffered saline, and impedance measurements were performed at 100 mV from 100 Hz to 100 000000 Hz using an LCR meter to obtain Z and θ values. FIG. 10 is a graph that replaces the drawings showing the Cole-Cole plot (FIG. 10 (A)) and the frequency-phase plot (FIG. 10 (B)) to show the effect of the distance between the electrodes. When the electrode spacing was changed in the range of 2.5 mm to 8 mm, the θ value was -80 ° at 100 Hz and -60 ° at 10000 Hz, which was the same as in the above embodiment, but the value of θ was around 10000000 Hz. Increased from -80 ° to -70 ° depending on the distance between the electrodes, and the value of θ increased from -75 ° to -45 ° depending on the distance between the electrodes near 100000000 Hz. The electrochemical properties of the inter-electrode intermediate material in the electrode surface region appeared around 10000 Hz and did not change by changing the distance between the electrodes. On the other hand, the electrochemical properties of the inter-electrode intermediate material in the region away from the electrode surface appeared in the high-frequency region and changed by changing the distance between the electrodes.

Effect of non-electrode: Increase in θ due to the electrochemical properties of the inter-electrode intermediate material (sample) in the electrode surface region and θ due to the electrochemical properties of the inter-electrode intermediate material in the region away from the electrode surface. The increase means that two peaks are generated on the θ-Hz plane, and the value of Hz at the valley bottom generated between these two peaks is around 10000 Hz where the electrochemical characteristics of the inter-electrode intermediate material in the electrode surface region appear. When approaching, the electrochemical characteristics of the inter-electrode intermediate material in the electrode surface region and the electrochemical characteristics of the inter-electrode intermediate material in the region away from the electrode surface are separated as two peaks on the θ-Hz plane. Is expected to be difficult. When the measurement results so far are extrapolated, the Hz value at the bottom of the valley is 10 ^ (7.1 + 0.1 (distance between electrodes (mm))), and when the distance between electrodes is 31 mm or more, the electrodes in the electrode surface region It becomes difficult to separate the electrochemical characteristics of the intermediate material and the electrochemical characteristics of the intermediate material between the electrodes in a region away from the electrode surface as two peaks on the θ-Hz plane.

One 6.6 μm square electrode and a probe needle of a manual prober were brought into contact with phosphate buffered saline, and impedance measurements were performed at 100 mV from 100 Hz to 1000000 Hz using an LCR meter to obtain Z and θ values. FIG. 11 is an alternative graph to the drawing showing a frequency-phase plot for examining the effects of non-electrolytes. The peak around 10000 Hz observed when the measurement results were illustrated on the θ-Hz plane was shifted to the low frequency side by adding styrene beads, sugar, and eukaryotic cells to phosphate buffered saline.

In the impedance measurement using an LCR meter in which two electrodes are brought into contact with a dispersed solution having an inhomogeneous structure such as micelles and particles, the electrochemical characteristics of the intermediate material between the electrodes in the electrode surface region and the region away from the electrode surface Under the limited conditions of separating and measuring the electrochemical properties of an intermediate substance between electrodes, one electrode is covered with a net so that inhomogeneous structures such as micelles, particles, and cells do not come into contact with each other. It was possible to know which of the two electrodes in contact with the inhomogeneous structure was in contact with.

Sample behavior analysis The durability of the chips was verified by continuously measuring the culture medium for 72 hours without cells. As a result, the impedance did not change. Next, 60 adjacent electrode pairs were repeatedly measured at 1 hour and 15 minute intervals. As a result, we were able to track the migrating cells. The situation is shown in FIG. FIG. 12 is a conceptual diagram showing how migrating cells are tracked. The location of the cells is surrounded by a dotted line. In other words, when the cell moves, the current value (and therefore the impedance) measured at the electrode changes, so it is possible to measure that the cell has moved.

The present invention can be used in the field of analytical instruments.

1 Electrochemical measuring device 3 Solution 5 Solution tank 7 First electrode 9 Second electrode 11 Voltage applying means 13 Current measuring means 15 Part exposed in the solution tank of the first electrode

Claims (8)

A solution tank (5) for accommodating the solution (3) and
A first electrode (7) and a second electrode (9) existing in the solution layer (5) and having at least a part of the surface exposed in the solution.
A voltage applying means (11) for applying a voltage between the first electrode (7) and the second electrode (9), and
An electrochemical measuring device (1) including a current measuring means (13) for measuring a current flowing between a first electrode (7) and a second electrode (9).
The surface area S of the portion (15) of the first electrode (7) exposed to the solution tank (5) is 0.1 μm ² or more and 100 μm ² or less.
measuring device. The measuring device according to claim 1.
The measuring device is a measuring device for measuring the physical properties or movement of a sample (17) contained in the solution and existing in the vicinity of the first electrode (17a). The measuring device according to claim 1.
A measuring device in which the sample (17) is a biological cell or a liposome, and the surface area S is smaller than the area of the biological cell or the liposome. The measuring device according to claim 1.
A measuring device having an adhesion prevention means (19) for preventing the sample (17) contained in the solution from adhering to the second electrode. The measuring device according to claim 1, wherein a plurality of the first electrode (7) and the second electrode (9) are present, and the adjacent first electrode (7) or second electrode (9) is adjacent to each other. A measuring device with a minimum distance of 31 mm or less. The electrochemical measuring apparatus according to claim 4.
The first electrode (7) and the second electrode (9) are provided on the substrate (21).
The second electrode (9) is provided in the recessed portion (23) of the substrate (21), and the adhesion preventing means (19) is a net covering the recessed portion, which is an electrochemical measuring device. The electrochemical measuring apparatus according to claim 4.
The first electrode (7) is provided on the substrate (21).
The second electrode (9) is provided on the side wall of the solution tank (5).
The adhesion prevention means (19) is a net covering the second electrode.
measuring device. The electrochemical measuring apparatus according to claim 4.
The second electrode (9) is provided on the electrode accommodating body (23) suspended in the solution, and at least a part of the electrode accommodating body (23) has the adhesion preventing means (19).
The first electrode (7) is an electrochemical measuring device provided on the substrate (21).

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