WO2019240202A1 - Measurement device - Google Patents

Measurement device Download PDF

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Publication number
WO2019240202A1
WO2019240202A1 PCT/JP2019/023377 JP2019023377W WO2019240202A1 WO 2019240202 A1 WO2019240202 A1 WO 2019240202A1 JP 2019023377 W JP2019023377 W JP 2019023377W WO 2019240202 A1 WO2019240202 A1 WO 2019240202A1
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electrode
solution
measuring
sample
electrodes
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PCT/JP2019/023377
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French (fr)
Japanese (ja)
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法親 緒方
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チトセ バイオ エボリューション ピーティーイー リミテッド
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Priority to JP2020525638A priority Critical patent/JP7312930B2/en
Publication of WO2019240202A1 publication Critical patent/WO2019240202A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance

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  • the present invention relates to a soot measuring device. More specifically, the present invention relates to an electrochemical measurement apparatus that can measure the physical properties and behavior of cells.
  • Japanese Patent No. 5617532 describes a dielectric cytometry apparatus and a cell sorting method using dielectric cytometry.
  • the present specification aims to provide an electrochemical measurement apparatus that can measure the physical properties of the sample with higher accuracy and can also measure the behavior of the sample.
  • the above problem is based on the knowledge that the physical properties of the sample existing near the electrode can be measured by making the electrode smaller than the cross-sectional area of the sample. In addition, if the distance between adjacent electrodes is reduced, the physical properties of the sample can be measured more accurately. By providing a partition wall that allows the solution to pass between the electrodes to which the voltage is applied but does not pass the sample, the physical properties of the sample can be measured more accurately. Based on the knowledge that can be measured.
  • This measuring apparatus is an electrochemical measuring apparatus 1 including a solution tank 5 for storing a solution 3, a first electrode 7 and a second electrode 9, a voltage applying unit 11, and a current measuring unit 13.
  • the first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface is exposed in the solution.
  • the voltage application unit 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.
  • the surface area S of the portion 15 exposed to the solution tank 5 of the first electrode 7 is not less than 0.1 ⁇ m 2 and not more than 100 ⁇ m 2 .
  • 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 near the first electrode 17a.
  • An example of the 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 apparatus preferably has an adhesion preventing means 19 for preventing the sample 17 contained in the solution from adhering to the second electrode.
  • first electrodes 7 and second electrodes 9 there are a plurality of first electrodes 7 and second electrodes 9, and the minimum distance to the adjacent first electrode 7 or second electrode 9 is 31 mm or less.
  • the first electrode 7 and the second electrode 9 may be provided on the substrate 21.
  • the second electrode 9 is preferably provided in the recessed portion 23 of the substrate 21, and the adhesion preventing means 19 is preferably a net that covers the recessed portion.
  • 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 preventing means 19 may be a net that covers the second electrode.
  • the second electrode 9 is provided in the electrode housing 23 that floats in the solution. At least a part of the electrode housing 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 measurement apparatus that can measure the physical properties of a sample with higher accuracy and can also measure the behavior of the sample.
  • FIG. 1 is a conceptual diagram illustrating a configuration example of a measuring apparatus.
  • FIG. 2 is a conceptual diagram showing an example of a measuring apparatus in which a first electrode and a second electrode are provided on a substrate.
  • FIG. 3 is a conceptual diagram showing a first electrode and a 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 apparatus in which the second electrode is accommodated in an electrode container that is in solution.
  • FIG. 6 is a conceptual diagram of the electrode manufactured in the example.
  • FIG. 7 is a conceptual diagram showing a dielectric measurement apparatus for measuring a dielectric spectrum.
  • FIG. 8 is a photograph replacing a drawing showing the state of the electrodes and cells.
  • FIG. 9 is a graph instead of a drawing showing measured Cole-Cole plots (FIG. 9A) and frequency-phase plots (FIG. 9B).
  • FIG. 10 is a graph instead of a drawing showing a Cole-Cole plot (FIG. 10A) and a frequency-phase plot (FIG. 10B) for illustrating the effect of the distance between electrodes.
  • FIG. 11 is a graph instead of a drawing showing a frequency-phase plot for examining the influence of the non-electrolyte.
  • FIG. 12 is a conceptual diagram showing a state in which moving cells are tracked.
  • FIG. 1 is a conceptual diagram illustrating a configuration example of a measuring apparatus.
  • the measuring apparatus includes a solution tank 5 that stores the solution 3, a first electrode 7 and a second electrode 9, a voltage applying unit 11, and a current measuring unit 13.
  • This is an electrochemical measurement apparatus 1.
  • the solution may be an electrolyte solution, a medium or a culture solution depending on cells and liposomes.
  • the solution tank 5 only needs to be able to store the solution, and the size and material of the solution tank 5 can be appropriately selected according to the use.
  • the first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface is exposed in the solution.
  • the entire surface of the electrode may be exposed to be in contact with the solution, or a part of the electrode may be embedded 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 not less than 0.1 ⁇ m 2 and not more than 100 ⁇ m 2 .
  • 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.
  • the second electrode 9 may be the same as the first electrode.
  • a plurality of first electrodes 7 and second electrodes 9 may exist.
  • FIG. 2 is a conceptual diagram showing an example of a measuring apparatus in which a first electrode and a second electrode are provided on a substrate.
  • the first electrode 7 and the second electrode 9 are provided on the substrate 21.
  • the 2nd electrode 9 is provided in the recessed part 23 of the board
  • the net allows the solution to pass through the net, but the sample does not pass through the net. Thereby, it is possible to prevent the sample from adhering to the second electrode.
  • 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 relation so that the connection relation 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 there are a plurality of first electrodes 7 and a plurality of second electrodes 9 and that the minimum distance d to the adjacent first electrode 7 or second electrode 9 is 31 mm or less. If the value of d is too small, the adjacent electrodes are energized, so the value of d is preferably 0.1 ⁇ m or more, 1 ⁇ m or more, or 5 ⁇ m 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 ⁇ m or less, 100 ⁇ m or less, or 50 ⁇ m or less. Good. In FIG. 3, a portion 15 of the electrode exposed to the solution tank 5 is drawn. Further, any of the arrayed electrodes in FIG. 3 may be the first electrode 7 or the second electrode 9.
  • the voltage application unit 11 is an element for applying a voltage between the first electrode 7 and the second electrode 9.
  • the voltage application unit 11 is a unit that applies 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 capable of controlling the frequency of the alternating voltage applied between the electrodes. Since a voltage applied between two electrodes in dielectric cytometry or the like is known, a voltage obtained by appropriately adjusting a known printing voltage may be applied between the first electrode 7 and the second electrode 9. . And it is preferable to be able to be controlled by the control part between which electrode among the electrode arrays which have several electrodes between which a voltage is applied.
  • 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 near the first electrode 17a.
  • An example of the 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.
  • the example in the vicinity of the first electrode is a region where the distance to the first electrode is short in the electrode adjacent to the first electrode.
  • This apparatus 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.
  • the adhesion preventing means 19 is a mesh, it is preferable that the mesh size is smaller than the sample (its cross-sectional area). By using such a net, it is possible to effectively prevent the sample from adhering to the electrode 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.
  • 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 preventing means 19 is a net that covers the second electrode. is there.
  • the sample is a cell, in particular, because it is possible to effectively prevent the sample from adhering to the second electrode.
  • the solution tank 5 has a bottom surface and a side wall extending from the bottom surface. Examples of the shape of the bottom surface are a circle, an ellipse, and a polygon.
  • the second electrode is preferably at a predetermined height from the bottom surface. Examples of the height are 1 ⁇ m or more and 1 mm or less, and may be 10 ⁇ m or more and 100 ⁇ m or less.
  • the substrate may be made of an insulator.
  • the substrate is preferably made of a transparent or translucent insulator.
  • An example of such an insulator is a transparent ceramic.
  • Transparent ceramics can be achieved, for example, by using 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 a cell or a liposome.
  • FIG. 5 is a conceptual diagram showing an example of a measuring apparatus accommodated in an electrode container in which the second electrode floats in a solution.
  • An example of the electrode container is floating.
  • the second electrode 9 is provided in an electrode container 23 floating in a solution, and at least a part of the electrode container 23 has an adhesion preventing means 19 (for example, a net), and the first electrode 7 is formed on the substrate. 21 may be provided.
  • a complex resistance (complex impedance) between the electrodes can be obtained by applying an alternating voltage between the electrode plates and measuring the flowing current. Changing the frequency of the applied AC voltage changes the measured complex resistance. Such measurement can be performed using a commercially available precision impedance analyzer (current measuring device).
  • the complex resistance depending on the frequency is corrected by correcting the factors depending on the shape of the measurement container and the transmission characteristics of the electrical wiring between the complex resistance measuring instrument and the measurement container. Can be converted to a rate.
  • the frequency dependence of the complex permittivity is called complex permittivity dispersion (dielectric spectrum).
  • the complex permittivity dispersion of the cell suspension can be expressed by a single relaxation function (for example, Cole-Cole function) or a superposition of a plurality of relaxation functions.
  • the variable can be optimized by performing nonlinear fitting with the undetermined coefficient included in the relaxation function for the experimentally obtained complex permittivity dispersion.
  • the Cole-Cole function there are relaxation intensity and relaxation frequency as variables that characterize the dispersion curve.
  • These dielectric variables are closely related to cell structure and physical properties. A method for estimating an electrical property value of a phase (cell membrane, cytoplasm, etc.) constituting a cell from a dielectric variable is described in, for example, Japanese Patent Application Laid-Open No. 2009-42141.
  • FIG. 6 is a conceptual diagram of the electrode manufactured in the example.
  • the electrode surface is made of titanium nitride (TiN) and is in contact with a sample such as a solution or animal cell.
  • Undoped silicate glass (NSG, SiO2), aluminum (Al), silicon oxide (SiO2), silicon (Si) were used.
  • Spare tip 36 electrode pads of 10 ⁇ m square with 20 ⁇ m spacing were used as a 6 ⁇ 6 grid electrode array, covering an area of 28900 ⁇ m 2 .
  • Electrop for tracking fibrillation behavior 36 electrode pads of 6.6 ⁇ m square with 3.4 ⁇ m spacing were used as a 6 ⁇ 6 grid electrode array, covering a 3200 ⁇ m 2 area.
  • FIG. 7 shows a dielectric measuring device for measuring the dielectric spectrum.
  • the dielectric spectrum was measured using an Agilent 4294A precision impedance analyzer. Z value and phase were measured.
  • Glucose solution and PBS were used as the solution.
  • 25 ⁇ m polystyrene beads were used as an example of a sample.
  • FIG. 8 is a photograph replacing a drawing showing the state of the electrodes and cells.
  • FIG. 9 is a measured Cole-Cole plot (FIG. 9A) and frequency-phase plot (FIG. 9B). The peak near 10000 Hz observed when the measurement result is illustrated on the ⁇ -Hz plane was shifted to the low frequency side in the measurement between the two electrodes on which the cell mass was placed above the electrode.
  • Two electrodes of 6.6 ⁇ m square were brought into contact with phosphate buffered saline, and impedance measurement was performed from 100 Hz to 100000000 Hz at 100 mV using an LCR meter to obtain a Z value and a ⁇ value.
  • the ⁇ value is -80 ° at 100 Hz, -60 ° at 10000 Hz, -80 ° at 10000000 Hz, and -75 ° at 100000000 Hz.
  • FIG. 10 is a graph instead of a drawing showing a Cole-Cole plot (FIG. 10A) and a frequency-phase plot (FIG. 10B) for illustrating the effect of the distance between electrodes.
  • the electrode spacing was varied from 2.5 mm to 8 mm, the ⁇ value was the same as the previous example until it reached -80 ° at 100 Hz and -60 ° at 10000 Hz.
  • the above-mentioned valley bottom Hz value is 10 ⁇ (7.1 + 0.1 (distance between electrodes (mm))). It becomes difficult to separate the electrochemical characteristics of the intermediary substance and the electrochemical characteristics of the interelectrode intermediate substance in a region away from the electrode surface as two peaks on the ⁇ -Hz plane.
  • FIG. 11 is a graph instead of a drawing showing a frequency-phase plot for examining the influence of the non-electrolyte. The peak around 10000 Hz observed when the measurement results are plotted on the ⁇ -Hz plane was shifted to the low frequency side by adding styrene beads, sugar, and eukaryotic cells to phosphate buffered saline.
  • the solution can be obtained by covering one of the electrodes with a net or the like so that the heterogeneous 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 electrode contacted with the heterogeneous structure.
  • FIG. 12 is a conceptual diagram showing a state in which moving cells are tracked. Cell positions are surrounded by dotted lines. That is, since the current value (and hence impedance) measured at the electrode changes when the cell moves, it can be measured that the cell has moved.
  • the present invention can be used in the field of analytical instruments.

Abstract

[Problem] To provide an electrochemical measurement device with which the physical properties of a sample can be more accurately measured, and with which the behavior of the sample can also be measured. [Solution] An electrochemical measurement device 1 comprises: a solution tank 5 that accommodates a solution 3; a first electrode 7 and a second electrode 9 that are located within the solution tank 5, at least a portion of the surface of the electrodes being exposed in the solution; a voltage application means 11 that applies a voltage between the first electrode 7 and the second electrode 9; and a current measurement means 13 that measures a current which flows between the first electrode 7 and the second electrode 9. The surface area S of a portion 15 of the first electrode 7 which is exposed in the solution tank 5 is from 0.1 μm2 to 100 μm2.

Description

測定装置measuring device
　本発明は， 測定装置に関する。より詳しく説明すると，本発明は，細胞の物性や挙動を測定できる電気化学的測定装置に関する。 The present invention relates to a soot measuring device. More specifically, the present invention relates to an electrochemical measurement apparatus that can measure the physical properties and behavior of cells.
　特許５６１７５３２号公報には，誘電サイトメトリ装置及び誘電サイトメトリによる細胞分取方法が記載されている。 Japanese Patent No. 5617532 describes a dielectric cytometry apparatus and a cell sorting method using dielectric cytometry.
特許５６１７５３２号公報Japanese Patent No. 5617532
　従来の誘電サイトメトリでは，電極付近にある試料の物性を測定できないという問題や，電極と試料との距離により測定値が変動するという問題があった。 Conventional dielectric cytometry has a problem that the physical properties of the sample in the vicinity of the electrode cannot be measured, and a measurement value varies depending on the distance between the electrode and the sample.
　そこで，本明細書は，試料の物性をより精度よく測定でき，試料の挙動をも測定できる電気化学的測定装置を提供することを目的とする。 Therefore, the present specification aims to provide an electrochemical measurement apparatus that can measure the physical properties of the sample with higher accuracy and can also measure the behavior of the sample.
　上記の課題は，電極を試料の断面積よりも小さくすることで，電極付近に存在する試料の物性をも測定できるという知見に基づく。また，隣接する電極の間の距離を小さくするとより精度よく試料の物性を測定でき，電圧を印可する電極の間を溶液は通るものの試料は通さない隔壁を設けることで，さらに精度よく試料の物性を測定できるという知見に基づく。 The above problem is based on the knowledge that the physical properties of the sample existing near the electrode can be measured by making the electrode smaller than the cross-sectional area of the sample. In addition, if the distance between adjacent electrodes is reduced, the physical properties of the sample can be measured more accurately. By providing a partition wall that allows the solution to pass between the electrodes to which the voltage is applied but does not pass the sample, the physical properties of the sample can be measured more accurately. Based on the knowledge that can be measured.
　本明細書において最初に記載される発明は測定装置１に関する。この測定装置は，溶液３を収容する溶液槽５と，第１の電極７及び第２の電極９と，電圧印加手段１１と，電流測定手段１３と，を備える電気化学測定装置１である。 The invention first described in this specification relates to the measuring apparatus 1. This measuring apparatus is an electrochemical measuring apparatus 1 including a solution tank 5 for storing a solution 3, a first electrode 7 and a second electrode 9, a voltage applying unit 11, and a current measuring unit 13.
　第１の電極７及び第２の電極９は，溶液槽５内に存在し，少なくとも表面の一部が溶液中に露出する。 The first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface is exposed in the solution.
電圧印加手段１１は，第１の電極７及び第２の電極９との間に電圧を印可するための要素である。 The voltage application unit 11 is an element for applying a voltage between the first electrode 7 and the second electrode 9.
電流測定手段１３は，第１の電極７及び第２の電極９との間に流れる電流を測定するための要素である。 The current measuring means 13 is an element for measuring the current flowing between the first electrode 7 and the second electrode 9.
そして，この装置は，第１の電極７の溶液槽５に露出した部分１５の表面積Ｓが０．１μｍ２以上１００μｍ２以下である。 In this apparatus, the surface area S of the portion 15 exposed to the solution tank 5 of the first electrode 7 is not less than 0.1 μm 2 and not more than 100 μm 2 .
　この測定装置は，例えば，溶液中に含まれる試料１７であって第１の電極付近に存在するもの１７ａの物性又は動きを測定するための装置である。試料１７の例は，生物細胞又はリポソームであり，表面積Ｓが生物細胞又はリポソームの面積より小さいことが好ましい。 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 near the first electrode 17a. An example of the 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.
　この装置は，溶液中に含まれる試料１７が第２の電極に付着することを防止する付着防止手段１９を有することが好ましい。 This apparatus preferably has an adhesion preventing means 19 for preventing the sample 17 contained in the solution from adhering to the second electrode.
　第１の電極７及び第２の電極９はそれぞれ複数存在し，隣接する第１の電極７又は第２の電極９までの最小距離が３１ｍｍ以下であるものが好ましい。 It is preferable that there are a plurality of first electrodes 7 and second electrodes 9, and the minimum distance to the adjacent first electrode 7 or second electrode 9 is 31 mm or less.
　第１の電極７及び第２の電極９は，基板２１上に設けられてもよい。また，第２の電極９は，基板２１の凹み部分２３に設けられ，付着防止手段１９は，凹み部を覆う網であるものが好ましい。 The first electrode 7 and the second electrode 9 may be provided on the substrate 21. The second electrode 9 is preferably provided in the recessed portion 23 of the substrate 21, and the adhesion preventing means 19 is preferably a net that covers the recessed portion.
　第１の電極７は，基板上２１に設けられ，
　第２の電極９は，溶液槽５の側壁に設けられ，
　付着防止手段１９は，第２の電極を覆う網であってもよい。 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 preventing means 19 may be a net that covers the second electrode.
　第２の電極９は，溶液に浮遊する電極収容体２３に設けられ，電極収容体２３の少なくとも一部が，付着防止手段１９を有し，第１の電極７は，基板上２１に設けられるものであってもよい。 The second electrode 9 is provided in the electrode housing 23 that floats in the solution. At least a part of the electrode housing 23 has the adhesion preventing means 19, and the first electrode 7 is provided on the substrate 21. It may be a thing.
　この明細書に記載した発明は，，試料の物性をより精度よく測定でき，試料の挙動をも測定できる電気化学的測定装置を提供できる。  The invention described in this specification can provide an electrochemical measurement apparatus that can measure the physical properties of a sample with higher accuracy and can also measure the behavior of the sample.
図１は，測定装置の構成例を示す概念図である。FIG. 1 is a conceptual diagram illustrating a configuration example of a measuring apparatus. 図２は，第１の電極及び第２の電極が基板上に設けられた測定装置の例を示す概念図である。FIG. 2 is a conceptual diagram showing an example of a measuring apparatus in which a first electrode and a second electrode are provided on a substrate. 図３は，アレイ状に形成された第１の電極及び第２の電極を示す概念図である。FIG. 3 is a conceptual diagram showing a first electrode and a second electrode formed in an array. 図４は，第１の電極が溶液槽の側壁に設けられ，第２の電極は溶液槽の底に設けられる測定装置を示す概念図である。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. 図５は，第２の電極が溶液にうく電極収容体に収容された測定装置の例を示す概念図である。FIG. 5 is a conceptual diagram showing an example of a measuring apparatus in which the second electrode is accommodated in an electrode container that is in solution. 図６は，実施例において製造された電極の概念図である。FIG. 6 is a conceptual diagram of the electrode manufactured in the example. 図７は，誘電スペクトルを測定するための誘電測定装置を示す概念図である。FIG. 7 is a conceptual diagram showing a dielectric measurement apparatus for measuring a dielectric spectrum. 図８は，電極と細胞の様子を示す図面に代わる写真である。FIG. 8 is a photograph replacing a drawing showing the state of the electrodes and cells. 図９は，測定されたCole-Coleプロット（図９（Ａ））及び周波数－位相プロット（図９（Ｂ））を示す図面に代わるグラフである。FIG. 9 is a graph instead of a drawing showing measured Cole-Cole plots (FIG. 9A) and frequency-phase plots (FIG. 9B). 図１０は，電極間の距離の効果を示すためのCole-Coleプロット（図１０（Ａ））及び周波数－位相プロット（図１０（Ｂ））を示す図面に代わるグラフである。FIG. 10 is a graph instead of a drawing showing a Cole-Cole plot (FIG. 10A) and a frequency-phase plot (FIG. 10B) for illustrating the effect of the distance between electrodes. 図１１は，非電解質の影響を検討するための周波数－位相プロットを示す図面に代わるグラフである。FIG. 11 is a graph instead of a drawing showing a frequency-phase plot for examining the influence of the non-electrolyte. 図１２は，移動する細胞を追跡した様子を示す概念図である。FIG. 12 is a conceptual diagram showing a state in which moving cells are tracked.
　以下，図面を用いて本発明を実施するための形態について説明する。本発明は，以下に説明する形態に限定されるものではなく，以下の形態から当業者が自明な範囲で適宜修正したものも含む。 Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, but includes those appropriately modified by those skilled in the art from the following embodiments.
　本明細書において最初に記載される発明は測定装置１に関する。図１は，測定装置の構成例を示す概念図である。図１に示されるように，この測定装置は，溶液３を収容する溶液槽５と，第１の電極７及び第２の電極９と，電圧印加手段１１と，電流測定手段１３と，を備える電気化学測定装置１である。溶液の例は電解質溶液や，細胞やリポソームによっては，培地や培養液であってもよい。溶液槽５は溶液を収容できるものであればよく，用途に合わせてその大きさや素材を適宜選択できる。 The invention first described in this specification relates to the measuring apparatus 1. FIG. 1 is a conceptual diagram illustrating a configuration example of a measuring apparatus. As shown in FIG. 1, the measuring apparatus includes a solution tank 5 that stores the solution 3, a first electrode 7 and a second electrode 9, a voltage applying unit 11, and a current measuring unit 13. This is an electrochemical measurement apparatus 1. Examples of the solution may be an electrolyte solution, a medium or a culture solution depending on cells and liposomes. The solution tank 5 only needs to be able to store the solution, and the size and material of the solution tank 5 can be appropriately selected according to the use.
　第１の電極７及び第２の電極９は，溶液槽５内に存在し，少なくとも表面の一部が溶液中に露出する。電極の表面全体が露出して溶液と接触するものであってもよし，電極の一部が基板や溶液に埋没していてもよい。第１の電極７の溶液槽５に露出した部分１５の表面積Ｓが０．１μｍ２以上１００μｍ２以下である。Sの値は０．２の電極はμｍ２以上８０μｍ２以下でもよいし，０．５μｍ２以上７０μｍ２以下でもよいし，１μｍ２以上５０μｍ２以下でもよいし，０．５μｍ２以上３０μｍ２以下でもよいし，２μｍ２以上１０μｍ２以下でもよいし，１０μｍ２以上５０μｍ２以下でもよい。このように微小な電極に電圧を印加した場合，電極表面へ試料を輸送する拡散層の形が半球に近づき，試料が半球表面から電極に向けて収束するように拡散するので，試料の物性を精度よく測定できると考えられる。つまり，通常の電極を用いると隣接する電極に付着等した試料により測定値が変動するものの，電極を微小とすることでそのような問題を解消できると考えられる。半導体チップは微小化及び迅速化が達成できているので，このような微小電極を，半導体製造方法における技術を用いることで製造できる。 The first electrode 7 and the second electrode 9 exist in the solution tank 5, and at least a part of the surface is exposed in the solution. The entire surface of the electrode may be exposed to be in contact with the solution, or a part of the electrode may be embedded 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 not less than 0.1 μm 2 and not more than 100 μm 2 . 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 small electrode, the shape of the diffusion layer that transports the sample to the electrode surface approaches the hemisphere, and the sample diffuses so that it converges from the hemispheric surface toward the electrode. It is thought that it can measure with high accuracy. In other words, when a normal electrode is used, the measured value varies depending on the sample attached to the adjacent electrode, but such a problem can be solved by making the electrode minute. Since the semiconductor chip can be miniaturized and speeded up, such a microelectrode can be manufactured by using a technique in a semiconductor manufacturing method.
　第２の電極９も第１の電極と同様のものであってもよい。また，第１の電極７及び第２の電極９は，複数存在してもよい。図２は，第１の電極及び第２の電極が基板上に設けられた測定装置の例を示す概念図である。図２に示される測定装置は，第１の電極７及び第２の電極９が，基板２１上に設けられている。そして，第２の電極９が，基板２１の凹み部分２３に設けられ，付着防止手段１９が，凹み部を覆う網である。網により，溶液は網を通過するものの，試料は網を通過しない。これにより，第２の電極に試料が付着する事態を防止できる。図２に示されるように第１の電極７及び第２の電極９は，基板に設けられたアレイ状のものであってもよい。また，第１の電極７及び第２の電極９は，それぞれの接続関係を調整できるように接続関係を制御できるものが好ましい。 The second electrode 9 may be the same as the first electrode. A plurality of first electrodes 7 and second electrodes 9 may exist. FIG. 2 is a conceptual diagram showing an example of a measuring apparatus in which a first electrode and a second electrode are provided on a substrate. In the measuring apparatus shown in FIG. 2, the first electrode 7 and the second electrode 9 are provided on the substrate 21. And the 2nd electrode 9 is provided in the recessed part 23 of the board | substrate 21, and the adhesion prevention means 19 is the net | network which covers a recessed part. The net allows the solution to pass through the net, but the sample does not pass through the net. Thereby, 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 relation so that the connection relation can be adjusted.
　図３は，アレイ状に形成された第１の電極及び第２の電極を示す概念図である。第１の電極７及び第２の電極９はそれぞれ複数存在し，隣接する第１の電極７又は第２の電極９までの最小距離ｄが３１ｍｍ以下であるものが好ましい。ｄの値があまりに小さいと隣接する電極において通電してしまうので，ｄの値は０．１μｍ以上であることが好ましく，１μｍ以上でもよいし，５μｍ以上でもよい。一方，ｄは，２５ｍｍ以下でもよいし，２０ｍｍ以下でもよいし，１５ｍｍ以下でもよいし，１０ｍｍ以下でもよいし，１ｍｍ以下でもよいし，５００μｍ以下でもよいし，１００μｍ以下でもよいし，５０μｍ以下でもよい。図３において，電極のうち溶液槽５に露出した部分１５が描画されている。また，図３のアレイ状の電極において，いずれが第１の電極７又は第２の電極９であってもよい。 FIG. 3 is a conceptual diagram showing the first electrode and the second electrode formed in an array. It is preferable that there are a plurality of first electrodes 7 and a plurality of second electrodes 9 and that the minimum distance d to the adjacent first electrode 7 or second electrode 9 is 31 mm or less. If the value of d is too small, the adjacent electrodes are energized, so the value of d is preferably 0.1 μm or more, 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 electrode exposed to the solution tank 5 is drawn. Further, any of the arrayed electrodes in FIG. 3 may be the first electrode 7 or the second electrode 9.
電圧印加手段１１は，第１の電極７及び第２の電極９との間に電圧を印可するための要素である。電圧印加手段１１は，第１の電極７及び第２の電極９の間に電圧を印可する手段である。印加される電圧は交流であっても，直流であってもよいものの，通常は交流電圧である。電圧印加手段１１は，電極間に印加する交流電圧の周波数を制御できるものが好ましい。誘電サイトメトリ等において２つの電極の間に印加される電圧は公知であるから，公知の印過電圧を適宜調整したものを第１の電極７及び第２の電極９との間に印加すればよい。そして，複数の電極を有する電極アレイのうち，いずれの電極間に電圧を印可するかは，制御部により制御できるようにされていることが好ましい。 The voltage application unit 11 is an element for applying a voltage between the first electrode 7 and the second electrode 9. The voltage application unit 11 is a unit that applies 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 capable of controlling the frequency of the alternating voltage applied between the electrodes. Since a voltage applied between two electrodes in dielectric cytometry or the like is known, a voltage obtained by appropriately adjusting a known printing voltage may be applied between the first electrode 7 and the second electrode 9. . And it is preferable to be able to be controlled by the control part between which electrode among the electrode arrays which have several electrodes between which a voltage is applied.
電流測定手段１３は，第１の電極７及び第２の電極９との間に流れる電流を測定するための要素である。電流測定手段１３は，公知である。電流測定手段１３は，２つの電極間の電流を測定することで，各種物性を測定できるものであってもよい。 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.
　この測定装置は，例えば，溶液中に含まれる試料１７であって第１の電極付近に存在するもの１７ａの物性又は動きを測定するための装置である。試料１７の例は，生物細胞又はリポソームであり，表面積Ｓが生物細胞又はリポソームの面積より小さいことが好ましい。第１の電極付近の例は，第１の電極と隣接する電極において，第１の電極への距離が近い領域である。 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 near the first electrode 17a. An example of the 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. The example in the vicinity of the first electrode is a region where the distance to the first electrode is short in the electrode adjacent to the first electrode.
　この装置は，溶液中に含まれる試料１７が第２の電極に付着することを防止する付着防止手段１９を有することが好ましい。　付着防止手段１９は，第１の電極に試料が付着することをも防止してもよい。付着防止手段１９の例は，後述する網や半透膜である。付着防止手段１９が網の場合，網の目の大きさが試料（の断面積）よりも小さいものが好ましい。そのような網を用いることで溶液の移動を確保しつつ，試料が電極に付着する事態を効果的に防止できる。 This apparatus 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 mesh, it is preferable that the mesh size is smaller than the sample (its cross-sectional area). By using such a net, it is possible to effectively prevent the sample from adhering to the electrode while ensuring the movement of the solution.
　図４は，第１の電極が溶液槽の側壁に設けられ，第２の電極は溶液槽の底に設けられる測定装置を示す概念図である。図４の例では，第１の電極７が，基板上２１に設けられ，第２の電極９が，溶液槽５の側壁に設けられ，付着防止手段１９が，第２の電極を覆う網である。このような位置関係にある場合，特に試料が細胞であると，試料が第２の電極に付着する事態を効果的に防止できたため，好ましい。この場合の，溶液槽５は，底面と底面から伸びる側壁とを有するものである。底面の形状の例は，円形，楕円形，及び多角形である。第２の電極は，底面から所定の高さにあることが好ましい。高さの例は，１μｍ以上１ｍｍ以下であり，１０μｍ以上１００μｍ以下でもよい。 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 that covers the second electrode. is there. In such a positional relationship, it is preferable that the sample is a cell, in particular, because it is possible to effectively prevent the sample from adhering to the second electrode. In this case, the solution tank 5 has a bottom surface and a side wall extending from the bottom surface. Examples of the shape of the bottom surface are a circle, an ellipse, and a polygon. The second electrode is preferably at a predetermined height from the bottom surface. Examples of the height are 1 μm or more and 1 mm or less, and may be 10 μm or more and 100 μm or less.
　基板は，絶縁体により構成されていてもよい。基板が透明又は半透明な絶縁体により構成されていることが好ましい。そのような絶縁体の例は，透明セラミックスである。透明セラミックスは，例えばＡｌ２Ｏ３，Ｙ２Ｏ３及び ＹＡＧのいずれか又は複数を用いることで達成できる。基板が透明であれば，特に試料が細胞やリポソームの場合に，それらの挙動を観測しやすい。 The substrate may be made of an insulator. The substrate is preferably made of a transparent or translucent insulator. An example of such an insulator is a transparent ceramic. Transparent ceramics can be achieved, for example, by using 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 a cell or a liposome.
　図５は，第２の電極が溶液に浮く電極収容体に収容された測定装置の例を示す概念図である。電極収容体の例は，浮きである。第２の電極９は，溶液に浮遊する電極収容体２３に設けられ，電極収容体２３の少なくとも一部が，付着防止手段１９（例えば網）を有し，第１の電極７は，基板上２１に設けられるものであってもよい。 FIG. 5 is a conceptual diagram showing an example of a measuring apparatus accommodated in an electrode container in which the second electrode floats in a solution. An example of the electrode container is floating. The second electrode 9 is provided in an electrode container 23 floating in a solution, and at least a part of the electrode container 23 has an adhesion preventing means 19 (for example, a net), and the first electrode 7 is formed on the substrate. 21 may be provided.
　次に，電気化学測定装置の原理を簡単に説明する。
　電極板間に交流電圧を印加して，流れる電流を測定することで，電極間の複素抵抗（複素インピーダンス）が得られる。印加される交流電圧の周波数を変化させると，測定される複素抵抗も変化する。このような測定は，市販されている精密インピーダンスアナライザ（電流測定装置）を用いて行うことができる。 Next, the principle of the electrochemical measurement device will be briefly described.
A complex resistance (complex impedance) between the electrodes can be obtained by applying an alternating voltage between the electrode plates and measuring the flowing current. Changing the frequency of the applied AC voltage changes the measured complex resistance. Such measurement can be performed using a commercially available precision impedance analyzer (current measuring device).
　周波数に依存した複素抵抗は，測定容器の形状に依存した因子，複素抵抗測定器と測定容器の間の電気配線の伝送特性に依存した因子などを補正することにより，細胞懸濁液の複素誘電率に変換できる。複素誘電率の周波数依存性を，複素誘電率分散（誘電スペクトル）という。 The complex resistance depending on the frequency is corrected by correcting the factors depending on the shape of the measurement container and the transmission characteristics of the electrical wiring between the complex resistance measuring instrument and the measurement container. Can be converted to a rate. The frequency dependence of the complex permittivity is called complex permittivity dispersion (dielectric spectrum).
　 細胞懸濁液の複素誘電率分散は，単一の緩和関数（例えば，Cole-Cole関数），あるいは複数の緩和関数の重ね合わせにより表現できる。実験的に得られた複素誘電率分散に対して，緩和関数が含む未定係数を変数とした非線形適合を行うことにより，その変数を最適化できる。例えばCole-Cole関数の場合，分散曲線を特徴づける変数として，緩和強度及び緩和周波数がある。これらの誘電変数は，細胞の構造や物性と密接に関連している。誘電変数から細胞を構成する相（細胞膜，細胞質など）の電気的物性値を推定する方法は，例えば特開２００９－４２１４１号公報に記載されている。 複 素 The complex permittivity dispersion of the cell suspension can be expressed by a single relaxation function (for example, Cole-Cole function) or a superposition of a plurality of relaxation functions. The variable can be optimized by performing nonlinear fitting with the undetermined coefficient included in the relaxation function for the experimentally obtained complex permittivity dispersion. For example, in the case of the Cole-Cole function, there are relaxation intensity and relaxation frequency as variables that characterize the dispersion curve. These dielectric variables are closely related to cell structure and physical properties. A method for estimating an electrical property value of a phase (cell membrane, cytoplasm, etc.) constituting a cell from a dielectric variable is described in, for example, Japanese Patent Application Laid-Open No. 2009-42141.
測定装置を製造するにあたり，基板（チップ）上に電極を開発した。図６は，実施例において製造された電極の概念図である。電極表面は，窒化チタン（TiN）で形成され，溶液や動物細胞などの試料と接することとなる部分である。非ドープケイ酸塩ガラス（NSG，SiO2），アルミニウム（Al），酸化ケイ素（SiO2），ケイ素（Si）が用いられた。 In manufacturing the measuring device, an electrode was developed on the substrate (chip). FIG. 6 is a conceptual diagram of the electrode manufactured in the example. The electrode surface is made of titanium nitride (TiN) and is in contact with a sample such as a solution or animal cell. Undoped silicate glass (NSG, SiO2), aluminum (Al), silicon oxide (SiO2), silicon (Si) were used.
予備チップ
20μm間隔の10μm四方の36個の電極パッドを6行6列のグリッド電極アレイとし，28900μm2の領域を覆った。 Spare tip
36 electrode pads of 10 μm square with 20 μm spacing were used as a 6 × 6 grid electrode array, covering an area of 28900 μm 2 .
細動の挙動追跡用チップ
3.4μmの間隔をおいて6.6μm角の36個の電極パッドを6行6列のグリッド電極アレイとし，3200μm2の領域を覆った。  Tip for tracking fibrillation behavior
36 electrode pads of 6.6 μm square with 3.4 μm spacing were used as a 6 × 6 grid electrode array, covering a 3200 μm 2 area.
　誘電測定装置
　図７に誘電スペクトルを測定するための誘電測定装置を示す。この例では，アジレント４２９４Ａプレシジョンインピーダンスアナライザーを用いて，誘電スペクトルを測定した。Ｚ値と位相を測定した。 Dielectric Measuring Device FIG. 7 shows a dielectric measuring device for measuring the dielectric spectrum. In this example, the dielectric spectrum was measured using an Agilent 4294A precision impedance analyzer. Z value and phase were measured.
　溶液としてグルコース溶液及びＰＢＳを用いた。試料の例として，ここでは，２５μｍのポリスチレン製のビーズを用いた。 Glucose solution and PBS were used as the solution. Here, as an example of a sample, 25 μm polystyrene beads were used.
　MGM-450昆虫培地に10μm四方の電極２個を20μm間隔で接触させ，培地に昆虫細胞塊を加えた。光学顕微鏡を用いて電極を観察し，電極上部に細胞塊が乗っていない２つの電極間でLCRメーターを用いて100mVで100Hzから1000000Hzまでのインピーダンス測定を行ってZ値とθ値を得た。同様に，MGM-450昆虫培地に10μm四方の電極２個を20μm間隔で接触させ，培地に昆虫細胞塊を加えた。光学顕微鏡を用いて電極を観察し，電極上部に細胞塊が乗っている２つの電極間でLCRメーターを用いて100mVで100Hzから1000000Hzまでのインピーダンス測定を行ってZ値とθ値を得た。図８は，電極と細胞の様子を示す図面に代わる写真である。図９は，測定されたCole-Coleプロット（図９（Ａ））及び周波数－位相プロット（図９（Ｂ））である。測定結果をθ-Hz平面上に図示したときに観察される10000Hz付近のピークは電極上部に細胞塊が乗っている２つの電極間の測定において低周波側にシフトした。 2 pieces of 10 μm square electrodes were brought into contact with the MGM-450 insect medium at 20 μm intervals, and the insect cell mass was added to the medium. The electrodes were observed using an optical microscope, and the impedance was measured from 100 Hz to 1000000 Hz 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 20 μm intervals, and the insect cell mass was added to the medium. The electrodes were observed using an optical microscope, and the impedance was measured from 100 Hz to 1000000 Hz at 100 mV using an LCR meter between two electrodes with cell clumps on top of the electrodes to obtain Z and θ values. FIG. 8 is a photograph replacing a drawing showing the state of the electrodes and cells. FIG. 9 is a measured Cole-Cole plot (FIG. 9A) and frequency-phase plot (FIG. 9B). The peak near 10000 Hz observed when the measurement result is illustrated on the θ-Hz plane was shifted to the low frequency side in the measurement between the two electrodes on which the cell mass was placed above the electrode.
電極間の距離についての検討
電極として，10μm四方の電極と6.6μm四方の電極とマニュアルプローバーのプローブ針を用いた。10μm四方の電極と6.6μm四方の電極は，いずれも独立の導線で繋がっており，導線の反対側の端はマニュアルプローバーのプローブ針を用いてプロービングすることで導通が得られるようにした。 Examination of distance between electrodes We used a 10μm square electrode, a 6.6μm square electrode, and a probe needle of a manual prober. The 10 μm square electrode and the 6.6 μm square electrode are both connected by independent conductors, and the opposite end of the conductor is probed with a probe needle of a manual prober so that continuity can be obtained.
リン酸緩衝生理食塩水に6.6μm四方の電極２個を接触させ，LCRメーターを用いて100mVで100Hzから100000000Hzまでのインピーダンス測定を行ってZ値とθ値を得た。θ値は100Hzで-80°，10000Hzで-60°，10000000Hzで-80°となり，100000000で-75°となり，従来技術で測定されてきた高周波領域に現れる電極表面から離れた領域にある電極間中間物質の電気化学的特性と，従来技術では測定されなかった10000Hz付近に現れる電極表面領域にある電極間中間物質の電気化学的特性が確認された。電極の間隔が3μmから60μmの範囲では測定結果が変化しなかった。 Two electrodes of 6.6 μm square were brought into contact with phosphate buffered saline, and impedance measurement was performed from 100 Hz to 100000000 Hz at 100 mV using an LCR meter to obtain a Z value and a θ value. The θ value is -80 ° at 100 Hz, -60 ° at 10000 Hz, -80 ° at 10000000 Hz, and -75 ° at 100000000 Hz. Between the electrodes in the region away from the electrode surface appearing in the high-frequency region measured by the prior art The electrochemical properties of the intermediate material and the inter-electrode intermediate material in the electrode surface region appearing near 10000 Hz, which was not measured by the prior art, were confirmed. The measurement results did not change when the electrode spacing was in the range of 3 μm to 60 μm.
　リン酸緩衝生理食塩水に6.6μm四方の電極１個とマニュアルプローバーのプローブ針を接触させ，LCRメーターを用いて100mVで100Hzから100000000Hzまでのインピーダンス測定を行ってZ値とθ値を得た。図１０は，電極間の距離の効果を示すためのCole-Coleプロット（図１０（Ａ））及び周波数－位相プロット（図１０（Ｂ））を示す図面に代わるグラフである。電極の間隔を2.5mmから8mmの範囲で変化させると，θ値は100Hzで-80°，10000Hzで-60°となるまでは前述の実施例と同様であったが，10000000Hz付近でθの値が電極間距離依存的に-80°から-70°まで増大し，100000000Hz付近でθの値が電極間距離依存的に-75°から-45°まで増大した。電極表面領域にある電極間中間物質の電気化学的特性は10000Hz付近に現れ，電極間の距離を変化させることによって変化しなかった。一方で電極表面から離れた領域にある電極間中間物質の電気化学的特性は高周波領域に現れ，電極間の距離を変化させることによって変化した。 1) A 6.6 μm square electrode and a probe needle of a manual prober were brought into contact with phosphate buffered saline, and impedance measurement was performed from 100 Hz to 100000000 Hz at 100 mV using an LCR meter to obtain a Z value and a θ value. FIG. 10 is a graph instead of a drawing showing a Cole-Cole plot (FIG. 10A) and a frequency-phase plot (FIG. 10B) for illustrating the effect of the distance between electrodes. When the electrode spacing was varied from 2.5 mm to 8 mm, the θ value was the same as the previous example until it reached -80 ° at 100 Hz and -60 ° at 10000 Hz. Increased from -80 ° to -70 ° depending on the interelectrode distance, and the value of θ increased from -75 ° to -45 ° in the vicinity of 100000000Hz depending on the interelectrode distance. The electrochemical properties of the interelectrode intermediate material in the electrode surface region appeared around 10000Hz, and did not change by changing the distance between the electrodes. On the other hand, the electrochemical characteristics of the interelectrode 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.
　非電解質の効果
　電極表面領域にある電極間中間物質（試料）の電気化学的特性に由来するθの増大と電極表面から離れた領域にある電極間中間物質の電気化学的特性に由来するθの増大とは，θ-Hz平面上に２つのピークを生じるが，この２つのピークの間に生じる谷底のHzの値が，電極表面領域にある電極間中間物質の電気化学的特性の現れる10000Hz付近に近づくと，電極表面領域にある電極間中間物質の電気化学的特性と電極表面から離れた領域にある電極間中間物質の電気化学的特性をθ-Hz平面上の２つのピークとして分離することが困難になると予測される。これまでの測定結果を外挿すると，前述の谷底のHzの値は10^(7.1+0.1(電極間距離(mm)))であり，電極間距離が31mm以上になると電極表面領域にある電極間中間物質の電気化学的特性と電極表面から離れた領域にある電極間中間物質の電気化学的特性をθ-Hz平面上の２つのピークとして分離することが困難になる。 Effect of non-electrolyte Increase in θ derived from the electrochemical properties of the interelectrode intermediate material (sample) in the electrode surface region and θ derived from the electrochemical properties of the interelectrode intermediate material in the region away from the electrode surface The increase means two peaks on the θ-Hz plane. The value of Hz at the bottom of the valley between these two peaks is around 10000 Hz where the electrochemical properties of the interelectrode intermediate material in the electrode surface region appear. When approaching, separate the electrochemical properties of the interelectrode intermediate material in the electrode surface region and the electrochemical properties of the interelectrode intermediate material in the region away from the electrode surface into two peaks on the θ-Hz plane. Is expected to be difficult. Extrapolating the measurement results so far, the above-mentioned valley bottom Hz value is 10 ^ (7.1 + 0.1 (distance between electrodes (mm))). It becomes difficult to separate the electrochemical characteristics of the intermediary substance and the electrochemical characteristics of the interelectrode intermediate substance in a region away from the electrode surface as two peaks on the θ-Hz plane.
　リン酸緩衝生理食塩水に6.6μm四方の電極１個とマニュアルプローバーのプローブ針を接触させ，LCRメーターを用いて100mVで100Hzから1000000Hzまでのインピーダンス測定を行ってZ値とθ値を得た。図１１は，非電解質の影響を検討するための周波数－位相プロットを示す図面に代わるグラフである。測定結果をθ-Hz平面上に図示したときに観察される10000Hz付近のピークは，リン酸緩衝生理食塩水にスチレンビーズ，砂糖，真核細胞を加えることによって低周波側にシフトした。 1) A 6.6 μm square electrode and a manual prober probe needle were brought into contact with phosphate buffered saline, and impedance measurement was performed from 100 Hz to 1000000 Hz at 100 mV using an LCR meter to obtain a Z value and a θ value. FIG. 11 is a graph instead of a drawing showing a frequency-phase plot for examining the influence of the non-electrolyte. The peak around 10000 Hz observed when the measurement results are plotted on the θ-Hz plane was shifted to the low frequency side by adding styrene beads, sugar, and eukaryotic cells to phosphate buffered saline.
　電極２個をミセルや粒子などの不均質構造の分散した溶液に接触させるLCRメーターを用いたインピーダンス測定において，電極表面領域にある電極間中間物質の電気化学的特性と電極表面から離れた領域にある電極間中間物質の電気化学的特性を分離してそれぞれ測定する限定された条件では，片方の電極をミセルや粒子，細胞などの不均質構造が接触しないように網などで覆うことで，溶液に接触した２個の電極のうちどちらに不均質構造が接触したのか知ることができた。 In impedance measurement using an LCR meter that contacts two electrodes with a solution with a heterogeneous structure such as micelles or particles, the electrochemical characteristics of the interelectrode intermediate material 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 a certain inter-electrode intermediate material, the solution can be obtained by covering one of the electrodes with a net or the like so that the heterogeneous 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 electrode contacted with the heterogeneous structure.
　試料の挙動分析
細胞なしで72時間培養培地を連続的に測定することにより，チップの耐久性を検証した。その結果，インピーダンスは変化しなかった。次に，隣り合っている60個の電極対を1時間15分間隔で繰り返し測定した。その結果，移動する細胞を追跡することができた。その様子を図１２に示す。図１２は，移動する細胞を追跡した様子を示す概念図である。細胞の位置は点線で囲ってある。つまり，細胞が移動すると電極において測定される電流値（したがってインピーダンス）が変化するので，細胞が移動したことを測定できる。 Analysis of sample behavior The durability of the chip 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 measured repeatedly at 1 hour and 15 minute intervals. As a result, we were able to track the moving cells. This is shown in FIG. FIG. 12 is a conceptual diagram showing a state in which moving cells are tracked. Cell positions are surrounded by dotted lines. That is, since the current value (and hence impedance) measured at the electrode changes when the cell moves, it can be measured that the cell has moved.
　本発明は分析機器の分野で利用されうる。 The present invention can be used in the field of analytical instruments.
１　電気化学測定装置
３　溶液
５　溶液槽
７　第１の電極
９　第２の電極
１１　電圧印加手段
１３　電流測定手段
１５　第１の電極の溶液槽に露出した部分

  DESCRIPTION OF SYMBOLS 1 Electrochemical measuring device 3 Solution 5 Solution tank 7 1st electrode 9 2nd electrode 11 Voltage application means 13 Current measurement means 15 The part exposed to the solution tank of 1st electrode

Claims (8)

  1. 　溶液（３）を収容する溶液槽（５）と，
    　前記溶液層（５）内に存在し，少なくとも表面の一部が前記溶液中に露出する第１の電極（７）及び第２の電極（９）と，
    　第１の電極（７）及び第２の電極（９）との間に電圧を印可する電圧印加手段（１１）と，
    　第１の電極（７）及び第２の電極（９）との間に流れる電流を測定する電流測定手段（１３）と，を備える電気化学測定装置（１）であって，
    　第１の電極（７）の前記溶液槽（５）に露出した部分（１５）の表面積Ｓが０．１μｍ２以上１００μｍ２以下である，
    　測定装置。     A solution tank (5) containing the solution (3);
    A first electrode (7) and a second electrode (9) which are present in the solution layer (5) and at least a part of the surface of which is exposed in the solution;
    Voltage applying means (11) for applying a voltage between the first electrode (7) and the second electrode (9);
    An electrochemical measurement device (1) comprising a current measurement means (13) for measuring a current flowing between the first electrode (7) and the second electrode (9),
    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.
    measuring device.
  2. 　請求項１に記載の測定装置であって，
    　前記測定装置は，前記溶液中に含まれる試料（１７）であって第１の電極付近に存在するもの（１７ａ）の物性又は動きを測定するための装置である，測定装置。     The measuring device according to claim 1,
    The measuring device is a device for measuring physical properties or movements of a sample (17) contained in the solution and present near the first electrode (17a).
  3. 　請求項１に記載の測定装置であって，
    　前記試料（１７）が生物細胞又はリポソームであり，前記表面積Ｓが前記生物細胞又はリポソームの面積より小さい，測定装置。     The measuring device according to claim 1,
    The measurement apparatus, wherein 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.
  4. 　請求項１に記載の測定装置であって，
    　前記溶液中に含まれる試料（１７）が第２の電極に付着することを防止する付着防止手段（１９）を有する，測定装置。     The measuring device according to claim 1,
    A measuring apparatus comprising adhesion preventing means (19) for preventing the sample (17) contained in the solution from adhering to the second electrode.
  5. 　請求項１に記載の測定装置であって，第１の電極（７）及び第２の電極（９）はそれぞれ複数存在し，隣接する第１の電極（７）又は第２の電極（９）までの最小距離が３１ｍｍ以下である，測定装置。 2. The measuring apparatus according to claim 1, wherein a plurality of first electrodes (7) and a plurality of second electrodes (9) exist, and the adjacent first electrode (7) or second electrode (9). Measuring device whose minimum distance is 31mm or less.
  6. 　請求項４に記載の電気化学測定装置であって，
    　第１の電極（７）及び第２の電極（９）は，基板（２１）上に設けられ，
    　第２の電極（９）は，前記基板（２１）の凹み部分（２３）に設けられ，前記付着防止手段（１９）は，前記凹み部を覆う網である，電気化学測定装置。     The electrochemical measurement device according to claim 4,
    The first electrode (7) and the second electrode (9) are provided on the substrate (21),
    The electrochemical measurement apparatus, wherein the second electrode (9) is provided in a recessed portion (23) of the substrate (21), and the adhesion preventing means (19) is a net covering the recessed portion.
  7. 　請求項４に記載の電気化学測定装置であって，
    　第１の電極（７）は，基板上（２１）に設けられ，
    　第２の電極（９）は，前記溶液槽（５）の側壁に設けられ，
    　前記付着防止手段（１９）は，第２の電極を覆う網である，
    　測定装置。     The electrochemical measurement device 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 preventing means (19) is a net covering the second electrode.
    measuring device.
  8. 　請求項４に記載の電気化学測定装置であって，
    　第２の電極（９）は，前記溶液に浮遊する電極収容体（２３）に設けられ，前記電極収容体（２３）の少なくとも一部が，前記付着防止手段（１９）を有し， 
    　第１の電極（７）は，基板上（２１）に設けられる，電気化学測定装置。
     
                                                                                                               
     
          The electrochemical measurement device according to claim 4,
    The second electrode (9) is provided on the electrode container (23) floating in the solution, and at least a part of the electrode container (23) has the adhesion preventing means (19),
    The first electrode (7) is an electrochemical measurement device provided on a substrate (21).



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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981268A (en) * 1997-05-30 1999-11-09 Board Of Trustees, Leland Stanford, Jr. University Hybrid biosensors
WO2002055653A1 (en) * 2001-01-09 2002-07-18 Matsushita Electric Industrial Co., Ltd. Device for measuring extracellular potential, method of measuring extracellular potential by using the same and apparatus for quickly screening drug provided therewith
US20080293997A1 (en) * 2005-05-26 2008-11-27 Philippe Buhlmann Chemical Sensor
WO2010070538A1 (en) * 2008-12-17 2010-06-24 Koninklijke Philips Electronics N.V. Microelectronic device for measuring cell adhesion

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981268A (en) * 1997-05-30 1999-11-09 Board Of Trustees, Leland Stanford, Jr. University Hybrid biosensors
WO2002055653A1 (en) * 2001-01-09 2002-07-18 Matsushita Electric Industrial Co., Ltd. Device for measuring extracellular potential, method of measuring extracellular potential by using the same and apparatus for quickly screening drug provided therewith
US20080293997A1 (en) * 2005-05-26 2008-11-27 Philippe Buhlmann Chemical Sensor
WO2010070538A1 (en) * 2008-12-17 2010-06-24 Koninklijke Philips Electronics N.V. Microelectronic device for measuring cell adhesion

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