WO2018207937A1 - Système de mesure d'impédance, procédé de mesure d'impédance, et système de détection de substance en cours de détection - Google Patents

Système de mesure d'impédance, procédé de mesure d'impédance, et système de détection de substance en cours de détection Download PDF

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Publication number
WO2018207937A1
WO2018207937A1 PCT/JP2018/018427 JP2018018427W WO2018207937A1 WO 2018207937 A1 WO2018207937 A1 WO 2018207937A1 JP 2018018427 W JP2018018427 W JP 2018018427W WO 2018207937 A1 WO2018207937 A1 WO 2018207937A1
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Prior art keywords
electrode
electrodes
light
substance
detected
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PCT/JP2018/018427
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English (en)
Japanese (ja)
Inventor
琢也 飯田
志保 床波
山本　靖之
勇姿 西村
田村　守
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公立大学法人大阪府立大学
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Priority to JP2019517732A priority Critical patent/JP7150339B2/ja
Publication of WO2018207937A1 publication Critical patent/WO2018207937A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present disclosure relates to an impedance measurement system, an impedance measurement method, and a detection system for a substance to be detected. More specifically, the present disclosure relates to an impedance measurement system and an impedance measurement method for measuring the impedance of a minute object, and can be included in a liquid sample. The present invention relates to a detection system for a substance to be detected for detecting a substance to be detected.
  • a method for detecting a minute object having a size from the nanometer order to the micrometer order (hereinafter, also referred to as “minute object”) such as a cell, bacteria, virus, or DNA has been developed.
  • minute object such as a cell, bacteria, virus, or DNA
  • various methods such as a culture method, a PCR (Polymerase Chain Reaction) method, and an ELIZA method (Enzyme-Linked Immuno Immunosorbing Assay) method have already been put to practical use as detection methods for bacteria and the like.
  • ELIZA method Enzyme-Linked Immuno Immunosorbing Assay
  • Non-Patent Document 1 discloses a technique of inducing bacteria to the anode by induction using a cathode and measuring the impedance of the bacteria induced to the anode.
  • the electrode (cathode) functions as a concentrator that increases the density of bacteria around the electrode (anode) (see, for example, the summary of Non-Patent Document 1 and FIG. 1). .
  • Non-Patent Document 1 In order to appropriately measure the impedance of the minute object, it is necessary that a certain amount or more of the minute object exists in the vicinity of the electrode.
  • the surrounding of the electrode anode
  • Bacteria are induced in a relatively wide range. For this reason, the bacterial density in the vicinity of the electrode is unlikely to be sufficiently high.
  • the present disclosure has been made to solve the above-described problems, and an object of the present disclosure is to provide a technique capable of quickly measuring impedance in an impedance measurement system or impedance measurement method for measuring impedance of a minute object. It is.
  • another object of the present disclosure is to provide a technique capable of quickly detecting a detection target substance in a detection target substance detection system that detects a detection target substance that may be contained in a liquid sample. .
  • An impedance measurement system includes a holding member, a light source, and an impedance measurement device.
  • the holding member includes a light heating member that generates heat when irradiated with light, and first and second electrodes, and is configured to hold a dispersion liquid in which a plurality of minute objects are dispersed.
  • the light source emits light for irradiating the light heating member.
  • the impedance measuring device has a plurality of gaps between the first electrode and the second electrode due to convection in the dispersion generated by heating the dispersion due to the heat generated by the light heating member due to light irradiation from the light source. In the state where the minute objects are accumulated and the first electrode and the second electrode are bridged by the plurality of minute objects, the impedances of the plurality of minute objects between the first electrode and the second electrode are set. It is configured to measure.
  • the impedance measurement system further includes a control device configured to control the light source.
  • the control device controls the light source so that the size of the microbubbles generated in the dispersion becomes larger than the distance between the first electrode and the second electrode when the dispersion is heated by the light heating member. Control.
  • the first and second electrodes are spaced apart from each other so as to sandwich the light generating member.
  • the light heating member is included in one of the first and second electrodes.
  • the holding member includes a pair of comb-shaped electrodes arranged to face each other.
  • the first electrode is one of the pair of comb electrodes.
  • the second electrode is the other comb-shaped electrode of the pair of comb-shaped electrodes.
  • a detection system for a substance to be detected detects a substance to be detected that may be contained in a liquid sample.
  • the detection system includes a holding member, an impedance measurement device, a light source, and a detection device.
  • the holding member includes a light heating member that generates heat when irradiated with light, and first and second electrodes, and is configured to hold a liquid sample.
  • the impedance measuring device is configured to measure the impedance between the first electrode and the second electrode.
  • the light source irradiates light to the light heating member when the liquid sample contains a host molecule capable of specifically adhering the substance to be detected, and heats the liquid sample by the heat generated by the light heating member to convect the liquid sample.
  • the detection target substance is accumulated between the first electrode and the second electrode, and the first electrode and the second electrode can be cross-linked by the detection target substance. Yes.
  • a detection system for a substance to be detected detects a substance to be detected that may be contained in a liquid sample.
  • the detection system includes a holding member configured to hold a liquid sample.
  • the holding member is modified on the holding member between the light heating member that generates heat when irradiated with light, the first and second electrodes, and the first and second electrodes, and specifically detects the substance to be detected.
  • the detection system further includes an impedance measurement device, a light source, and a detection device.
  • the impedance measuring device is configured to measure the impedance between the first electrode and the second electrode.
  • the light source irradiates the light heat generating member with light, heats the liquid sample by the heat generated by the light heat generating member, and generates convection in the liquid sample, thereby detecting the substance to be detected between the first electrode and the second electrode.
  • the first electrode and the second electrode can be cross-linked by a substance to be detected.
  • the detection device is configured to detect a substance to be detected by monitoring impedance.
  • the light source heats the dispersion liquid so that the size of the microbubbles generated in the dispersion liquid is larger than the distance between the first electrode and the second electrode.
  • the detection device determines that the detected substance is detected when the amount of change in impedance exceeds a predetermined determination amount, while the amount of change in impedance until the predetermined period elapses is determined as the determination amount. If it falls below the value, it is determined that the substance to be detected was not detected.
  • the substance to be detected is a target nucleic acid that is at least one of target DNA and target RNA.
  • a host molecule is a probe nucleic acid that causes hybridization with a target nucleic acid.
  • the substance to be detected is an antigen.
  • a host molecule is an antibody that causes an antigen-antibody reaction with an antigen.
  • An impedance measurement method includes first to fourth steps.
  • the first step is a step of holding a dispersion liquid in which a plurality of minute objects are dispersed in a holding member including a light heating member that generates heat by light irradiation and the first and second electrodes.
  • the second step is a step of irradiating the light heating member with light after the holding step (first step).
  • a plurality of minute objects are integrated between the first electrode and the second electrode by using convection in the dispersion generated by heating the dispersion by the heat generated by the light generating member.
  • the first electrode and the second electrode are bridged by a plurality of minute objects.
  • the fourth step is a step of measuring impedances of a plurality of minute objects between the first electrode and the second electrode after the step of crosslinking (third step).
  • the size of the microbubbles generated in the dispersion liquid by heating the dispersion liquid is larger than the distance between the first electrode and the second electrode.
  • the present disclosure it is possible to quickly measure the impedance in the impedance measurement system or the impedance measurement method for measuring the impedance of the minute object.
  • a substance to be detected can be quickly detected in a detection system for a substance to be detected that detects a substance to be detected that may be contained in a liquid sample.
  • FIG. 1 is a diagram schematically showing a configuration of a system for measuring electrical characteristics of a minute object according to Embodiment 1.
  • FIG. It is a figure for demonstrating the structure of a measurement kit in detail.
  • 3 is a flowchart showing a method for measuring electrical characteristics of a minute object in the first embodiment. It is a figure for demonstrating the accumulation mechanism of a micro object. It is a figure for demonstrating the measurement mechanism of the electrical property between electrodes using the integration
  • FIG. 5 is a conceptual diagram for explaining the detection principle of target DNA in the second embodiment. It is a figure for demonstrating in detail the structure of a detection kit. It is a 1st figure for demonstrating the measurement mechanism of the electrical property between electrodes using hybridization. It is a 2nd figure for demonstrating the measurement mechanism of the electrical property between electrodes using hybridization. 5 is a flowchart showing a method for detecting a substance to be detected (target DNA) in Embodiment 2.
  • FIG. 5 is a flowchart showing a method for detecting a substance to be detected (complementary strand DNA) in the Example of Embodiment 2. It is a figure which shows the structure of the detection kit in Example 2 of Embodiment 2.
  • FIG. It is a figure which shows the observation result of a mode that a microbubble generate
  • micro object means an object having a size from nanometer order to micrometer order.
  • the shape of the minute object is not particularly limited, and may be, for example, a spherical shape, an elliptical spherical shape, a rod shape, or a coil shape.
  • the minute object is an elliptical sphere, it is sufficient that at least one of the length in the minor axis direction and the major axis direction of the ellipsoid sphere is in a range from nanometer order to micrometer order.
  • the minute object has a rod shape, it is sufficient that at least one of the width and the length of the rod is in the range from nanometer order to micrometer order.
  • the minute object has a coil shape, it is sufficient that at least one of the width and the length of the coil is in the range from nanometer order to micrometer order.
  • micro objects include metal nanoparticles, metal nanoparticle aggregates, metal nanoparticle integrated structures, semiconductor nanoparticles, organic nanoparticles, resin beads, PM (Particulate Matter), and nanocoils.
  • Metal nanoparticles are metal particles having a size on the order of nanometers.
  • the “metal nanoparticle aggregate” is an aggregate formed by aggregation of a plurality of metal nanoparticles.
  • Metal nanoparticle integrated structure is a structure in which, for example, a plurality of metal nanoparticles are fixed to the surface of a bead via an interaction site, and a gap is provided between each metal nanoparticle so as to have an interval equal to or smaller than the diameter of the metal nanoparticles. Is the body.
  • “Semiconductor nanoparticles” are semiconductor particles having a size on the order of nanometers.
  • Organic nanoparticles are particles made of an organic compound having a size on the order of nanometers.
  • Resin beads are particles made of a resin having a size ranging from the nanometer order to the micrometer order.
  • PM is a particulate material having a size on the order of micrometers. Examples of PM include PM2.5 and SPM (Suspended Particulate Matter).
  • a “nanocoil” is a coil having a size (width or length) on the order of nanometers.
  • the micro object may be a biological substance (biological substance). More specifically, the micro object may include cells, microorganisms (bacteria, fungi, etc.), biopolymers (proteins, nucleic acids, lipids, polysaccharides, etc.), antigens (allergens, etc.) and viruses. Note that the “substance to be detected” in the present disclosure may be the “micro object”.
  • the term “from nanometer order to micrometer order” refers to a range from 1 nm to 1000 ⁇ m, but typically ranges from several tens of nm to several hundreds of ⁇ m, preferably from 100 nm to 100 ⁇ m. And more preferably in the range of 1 ⁇ m to several tens of ⁇ m.
  • the impedance between the electrodes means a ratio between a voltage and a current when an electric field is applied between the electrodes. Therefore, the impedance includes a direct current resistance when a direct current (direct current voltage or direct current) is applied between the electrodes and an alternating current impedance when an alternating current (alternating voltage or alternating current) is applied between the electrodes.
  • a direct current direct current voltage or direct current
  • an alternating current impedance when an alternating current (alternating voltage or alternating current) is applied between the electrodes.
  • the “electrical characteristic” of the minute object means the responsiveness of the minute object to the electric field applied to the minute object. Examples of electrical characteristics include electrical resistivity (or electrical conductivity), capacitance, inductance, dielectric constant, carrier (electron, hole) concentration or mobility, and the like.
  • microbubble is a bubble having a size on the order of micrometers.
  • hybridization means a reassociation reaction between at least two types of single-stranded nucleic acids.
  • Hybridization is not limited to a reassociation reaction between two types of single-stranded nucleic acids as a whole, but a reassociation reaction between a part of one single-stranded nucleic acid and a part of another single-stranded nucleic acid. May be included.
  • a double strand is formed between single-stranded DNAs having complementary base sequences.
  • hybridization is not limited to this, and includes, for example, duplex formation between one single-stranded DNA and one RNA, or between two RNAs.
  • crosslinking means that the electrodes are electrically connected by linking a plurality of minute objects arranged on or between the electrodes or by forming a bond between the plurality of minute objects. It means to do.
  • connection between micro objects or the type of bond and it may be a chemical bond (covalent bond, ionic bond or metal bond), or bond by intermolecular force (hydrogen bond, attractive force acting between polar molecules) Or by van der Waals force).
  • the x direction and the y direction represent the horizontal direction.
  • the x direction and the y direction are orthogonal to each other.
  • the z direction represents the vertical direction.
  • the direction of gravity is downward in the z direction.
  • the upper direction in the z direction may be abbreviated as “upper”, and the lower direction in the z direction may be abbreviated as “lower”.
  • FIG. 1 is a diagram schematically showing the configuration of a minute object electrical characteristic measurement system (hereinafter also abbreviated as “measurement system”) 1 according to the first embodiment.
  • a measurement system 1 includes a measurement kit 2, an XYZ axis stage 10, an adjustment mechanism 20, a sample supply unit 30, a laser light source 40, an optical component 50, an objective lens 60, and illumination.
  • the light source 70, the imaging device 80, the multimeter 90, and the control apparatus 100 are provided.
  • the measurement kit 2 holds a dispersion D in which minute objects M (see FIG. 4) to be measured are dispersed.
  • the dispersion medium of the dispersion liquid D is a liquid having sufficiently high insulation (low conductivity), for example, water.
  • the detailed configuration of the measurement kit 2 will be described with reference to FIG.
  • the measurement kit 2 is placed on the XYZ axis stage 10.
  • the adjusting mechanism 20 adjusts the relative positional relationship between the XYZ axis stage 10 and the objective lens 60 in accordance with a command from the control device 100.
  • the position of the objective lens 60 is fixed. Therefore, the relative positional relationship between the XYZ axis stage 10 and the objective lens 60 is adjusted by adjusting the positions of the XYZ axis stage 10 in the x, y, and z directions.
  • a drive mechanism such as a servo motor and a focusing handle attached to the microscope can be used, but the specific configuration of the adjustment mechanism 20 is not particularly limited.
  • the adjustment mechanism 20 may be configured so that the position of the objective lens 60 can be adjusted.
  • the sample supply unit 30 supplies the dispersion D (sample) onto the measurement kit 2 in accordance with a command from the control device 100.
  • a dispenser can be used as the sample supply unit 30, for example.
  • Laser light source 40 emits, for example, near-infrared (for example, wavelength 800 nm) laser light L1 in response to a command from control device 100.
  • the wavelength of the laser beam L1 is not particularly limited as long as the wavelength is included in the absorption wavelength region of the material of the light heating member 110 (see FIG. 2) described later.
  • the wavelength of the laser light source 40 may be other wavelengths (for example, 1064 nm) included in the near infrared region, or may be wavelengths included in a wavelength region other than the near infrared region (for example, visible light region).
  • the laser light source 40 corresponds to a “light source” according to the present disclosure.
  • the optical component 50 includes, for example, a mirror, a dichroic mirror, or a prism.
  • the optical system of the measurement system 1 is adjusted so that the laser light L1 from the laser light source 40 is guided to the objective lens 60 by the optical component 50.
  • the objective lens 60 condenses the laser light L1 from the laser light source 40.
  • the light condensed by the objective lens 60 is applied to the dispersion D on the measurement kit 2.
  • “irradiate” includes the case where the laser light L1 passes through the dispersion D. That is, the present invention is not limited to the case where the beam waist of the light collected by the objective lens 60 is located in the dispersion D.
  • the optical component 50 and the objective lens 60 can be incorporated into, for example, an inverted microscope main body or an upright microscope main body (both not shown).
  • the illumination light source 70 emits white light L2 for illuminating the dispersion D on the measurement kit 2 in accordance with a command from the control device 100.
  • a halogen lamp can be employed as the illumination light source 70.
  • the objective lens 60 is also used for taking in the white light L2 irradiated to the dispersion D.
  • the white light L ⁇ b> 2 captured by the objective lens 60 is guided to the photographing device 80 by the optical component 50.
  • the photographing device 80 photographs the dispersion D irradiated with the white light L ⁇ b> 2 in accordance with a command from the control device 100 and outputs the photographed image (moving image or still image) to the control device 100.
  • a video camera including a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor is used.
  • the multimeter 90 is a measuring device (impedance measuring device) configured to measure an electrical resistance (impedance) between the electrode 111 and the electrode 112 provided in the measuring kit 2 (see FIG. 2). More specifically, the multimeter 90 measures, for example, the voltage between the electrodes 111 and 112 while passing a constant current between the electrodes 111 and 112 in accordance with a command from the control device 100, and controls the measurement result. Output to the device 100.
  • the multimeter 90 measures, for example, the voltage between the electrodes 111 and 112 while passing a constant current between the electrodes 111 and 112 in accordance with a command from the control device 100, and controls the measurement result. Output to the device 100.
  • control device 100 is a microcomputer including a CPU (Central Processing Unit), a memory, and an input / output port.
  • the control device 100 controls each device (the adjustment mechanism 20, the sample supply unit 30, the laser light source 40, the illumination light source 70, the photographing device 80, and the multimeter 90) included in the measurement system 1.
  • the control device 100 calculates an electrical characteristic (electric resistance R in the example described later) of the dispersion D by performing a predetermined calculation process on the measurement result from the multimeter 90.
  • the control device 100 is configured to perform predetermined image processing on an image photographed by the photographing device 80.
  • a configuration in which the voltage between the electrodes 111 and 112 is measured while performing constant current control by the multimeter 90 will be described as an example.
  • a galvanostat or a potentiostat may be used as the “impedance measurement device” according to the present disclosure.
  • a galvanostat the voltage between the electrodes 111 and 112 when a constant current flows between the electrodes 111 and 112 is measured.
  • a potentiostat the current flowing between the electrodes 111 and 112 when a constant voltage is applied between the electrodes 111 and 112 is measured.
  • the optical system of the measurement system 1 can irradiate the measurement kit 2 with the laser light L1 from the laser light source 40, and takes in the photographing device 80 with the white light L2 irradiated on the measurement kit 2.
  • the configuration is not limited to that shown in FIG.
  • the optical system of the measurement system 1 may be configured to include an optical fiber or the like.
  • the adjustment mechanism 20, the sample supply unit 30, the objective lens 60, the illumination light source 70, and the imaging device 80 are not essential components.
  • FIG. 2 is a diagram for explaining the configuration of the measurement kit 2 in detail.
  • measurement kit 2 includes a substrate 11, a light heating member 110, and electrodes 111 and 112.
  • the illustration of the minute object M in the dispersion D is omitted in order to prevent the drawing from becoming complicated.
  • the substrate 11 is placed on the XYZ axis stage 10 and holds the dispersion D.
  • the substrate 11 has electrical insulation and is optically transparent to the laser light L1 from the laser light source 40 and the white light L2 from the illumination light source 70.
  • a material of the substrate 11 for example, glass or quartz glass can be used.
  • the substrate 11 corresponds to an example of a “holding member” according to the present disclosure.
  • a configuration using a planar substrate as an example of the “holding member” according to the present disclosure is illustrated.
  • the “holding member” according to the present disclosure is not particularly limited as long as it can hold the dispersion D, and may be a container having a three-dimensional shape (for example, a rectangular parallelepiped or cylindrical container).
  • Each of the electrodes 111 and 112 is formed on the substrate 11.
  • the electrode 111 (first electrode) is an anode
  • the electrode 112 (second electrode) is a cathode.
  • Each of the electrodes 111 and 112 is a metal thin film having a film thickness on the order of nanometers, for example, a platinum thin film.
  • an adhesive layer 121 for enhancing the adhesiveness between the substrate 11 and the electrode 111 (for example, the adhesiveness between quartz glass and a platinum thin film) is formed.
  • an adhesive layer 122 is formed between the substrate 11 and the electrode 112.
  • Each adhesive layer 121, 122 is, for example, a titanium thin film. Note that the adhesive layers 121 and 122 are not essential as long as the adhesion of the electrodes 111 and 112 to the substrate 11 can be secured.
  • the light heating member 110 is formed on the substrate 11 between the electrode 111 and the electrode 112, for example.
  • the electrode 111 and the electrode 112 are spaced apart from each other so as to sandwich the light heating member 110.
  • the light heating member 110 is a metal thin film having a film thickness on the order of nanometers, for example, a platinum thin film.
  • an adhesive layer 120 (for example, a titanium thin film) is formed between the substrate 11 and the light heating member 110 to improve the adhesion between the substrate 11 and the light heating member 110. Not required.
  • the shape of the light heat generating member 110 is not particularly limited, and may be, for example, a perfect circle shape, a square shape, or a rectangular shape. May be.
  • the light heating member 110 absorbs the laser light L1 from the laser light source 40 and converts the light energy of the laser light L1 into heat energy. More specifically, free electrons of metal nanoparticles (for example, platinum nanoparticles) constituting the metal thin film form surface plasmons and are vibrated by the laser light L1. This causes polarization. This polarization energy is converted into lattice vibration energy by Coulomb interaction between the free electrons and the nucleus. As a result, the metal nanoparticles generate heat. Hereinafter, this effect is also referred to as a “light heating effect”.
  • metal nanoparticles for example, platinum nanoparticles
  • the material of the light heating member 110 is preferably a material having high photothermal conversion efficiency in the wavelength region of the laser light L1 (800 nm in the present embodiment).
  • the material of the light heating member 110 is not limited to platinum, but a metal element other than platinum (for example, gold or silver) or a metal nanoparticle integrated structure (for example, gold nanoparticle or silver nanoparticle) that can produce a light heat generation effect. It may be a structure using Alternatively, the material of the light heating member 110 may be a material other than a metal having high light absorption in the wavelength region of the laser light L1 and having conductivity. Examples of such a material include a material close to a black body (for example, a carbon nanotube black body).
  • the thickness of the light heating member 110 is determined by design or experiment in consideration of the output of the laser light L1 (laser output), the absorption wavelength range of the material of the light heating member 110, and the photothermal conversion efficiency.
  • FIG. 3 is a flowchart showing a method for measuring the electrical characteristics of the minute object M in the first embodiment.
  • the flowcharts shown in FIG. 3 and FIGS. 23 and 26 described later are executed when a predetermined condition is satisfied (for example, when the user operates a measurement start button (not shown)).
  • Each step (hereinafter abbreviated as “S”) included in these flowcharts is basically realized by software processing by the control device 100, but a part or all of the hardware ( Electric circuit).
  • a dispersion D in which minute objects M are dispersed is prepared.
  • the prepared dispersion D is stored in the sample supply unit 30.
  • control device 100 installs the measurement kit 2 on the XYZ axis stage 10. This process can be realized by, for example, a feed mechanism (not shown) of the substrate 11.
  • control device 100 controls the illumination light source 70 so as to emit white light L2 for irradiating the measurement kit 2, and also controls the photographing device 80 so as to start photographing of the measurement kit 2.
  • control device 100 adjusts the horizontal position of the XYZ axis stage 10 by controlling the adjustment mechanism 20 so that the laser light L1 from the laser light source 40 is irradiated onto the light heating member 110.
  • This horizontal position adjustment can be realized, for example, by extracting the outer shape pattern of the light heating member 110 from an image photographed by the photographing device 80 using an image processing technique for pattern recognition.
  • control device 100 adjusts the position of the XYZ axis stage 10 in the vertical direction by controlling the adjustment mechanism 20 so that the beam waist of the laser light L1 becomes an appropriate height.
  • the vertical position (height) of the beam waist is known from the wavelength of the laser light L1 and the specifications (magnification, etc.) of the objective lens 60. Therefore, the beam waist can be set to the target height by adjusting the position of the XYZ axis stage 10 in the vertical direction.
  • control device 100 causes the dispersion D to drop onto the measurement kit 2 by controlling the sample supply unit 30 so that an appropriate amount of the dispersion D is held.
  • the dripping amount of the dispersion liquid D may be a very small amount of, for example, about several ⁇ L to several hundred ⁇ L, or a larger amount.
  • the processing order of S103 to S105 is not limited to this.
  • the dispersion D may be dropped onto the measurement kit 2 (the process of S105) prior to the start of the irradiation of the white light L2 to the measurement kit 2 and the start of the photographing of the measurement kit 2 (the process of S103).
  • the vertical position of the XYZ axis stage 10 may be adjusted (the process of S104) after the dispersion D is dropped.
  • the control device 100 measures the electrical resistance R between the electrodes 111 and 112. More specifically, the control device 100 controls the multimeter 90 to measure the voltage between the electrodes 111 and 112 while causing a constant current to flow between the electrodes 111 and 112 of the measurement kit 2. By dividing the voltage between the electrodes 111 and 112 by the current (constant current) flowing between the electrodes 111 and 112, the electrical resistance R between the electrodes 111 and 112 can be calculated.
  • the control device 100 controls the laser light source 40 so as to start irradiation with the laser light L1 (hereinafter also referred to as “light irradiation”).
  • the laser light L1 from the laser light source 40 is condensed by the objective lens 60, and the condensed light is irradiated to the light heating member 110.
  • control device 100 ends the measurement of the electric resistance between the electrodes 111 and 112 so as to end the multimeter 90. To control.
  • control device 100 controls the laser light source 40 so as to stop the irradiation of the laser light L1 to the measurement kit 2. Moreover, the control apparatus 100 controls the illumination light source 70 so that irradiation of the white light L2 to the measurement kit 2 may be stopped.
  • control device 100 determines the electrical characteristics of the minute object M (for example, the electric conduction of the minute object M described in FIG. 17) based on the electric resistance R between the electrodes 111 and 112 measured in S106 to S108. Degree ⁇ ) is calculated. As a result, a series of processing ends.
  • the process of S103 is a process for observing the dispersion liquid D (taking an image), and is not an essential process for measuring the electrical characteristics of the minute object M. Therefore, even when the flowchart not including the process of S103 is executed, the electrical characteristics of the minute object M can be measured.
  • a DC voltage or DC current
  • An AC voltage may be applied between the electrodes 111 and 112, and an AC impedance measurement between the electrodes 111 and 112 may be performed.
  • DEPIM Dielectrophoretic Impedance Measurement
  • a minute object is applied according to the mechanism described below. It is possible to measure the impedance change between the electrodes 111 and 112 while accumulating in the vicinity of the electrodes 111 and 112.
  • the measurement mechanism of the electrical characteristics of the minute object M (measurement mechanism of the electrical resistance R) in the processing of S106 to S108 will be described in detail.
  • the minute objects M are integrated by the irradiation of the laser light L1 from the laser light source 40.
  • FIG. 4 is a diagram for explaining the accumulation mechanism of the minute objects M.
  • FIG. 4A when the irradiation of the laser beam L1 is started, the vicinity of the laser spot is locally heated at the irradiation position (laser spot) of the laser beam L1 due to the light heating effect of the light heating member 110. The Then, the dispersion D (dispersion medium) near the laser spot is locally boiled, and microbubbles MB are generated in the laser spot (see FIG. 4B). The microbubble MB grows with time.
  • the direction of the convection C is a direction in which the convection C once goes to the microbubble MB and then moves away from the microbubble MB.
  • the micro objects M are accumulated in the vicinity of the laser spot by being carried on the convection C toward the micro bubbles MB (see FIG. 4D). Thereafter, when the light irradiation is stopped, the convection C weakens and eventually stops.
  • the electric resistance R between the electrodes 111 and 112 is positively utilized by using the accumulation action of the minute objects M due to the light heating effect of the light heating member 110. Is measured.
  • FIG. 5 is a diagram for explaining the measurement mechanism of the electrical characteristics between the electrodes 111 and 112 using the accumulation action of the minute objects M.
  • FIG. 5A when light irradiation is started, microbubbles MB are generated on the light heating member 110 as described in FIG. 4B. At this time, the minute object M is hardly integrated between the electrode 111 and the light heating member 110 and between the electrode 112 and the light heating member 110. For this reason, the electrical resistance R of the dispersion medium (for example, water) existing between the electrode 111 and the electrode 112 is measured by the multimeter 90.
  • the dispersion medium for example, water
  • the minute objects M carried on the convection C are accumulated between the electrode 111 and the electrode 112. Then, when the accumulation amount of the minute object M between the electrode 111 and the electrode 112 increases, the electrode 111 and the electrode 112 are bridged by the minute object M at a certain time (see FIG. 5C). Then, the main component of the electric resistance R measured by the multimeter 90 changes from the electric resistance of the dispersion medium to the electric resistance of the minute object M integrated between the electrode 111 and the electrode 112.
  • FIG. 5C shows an example in which the electrode 111 and the electrode 112 are directly bridged by the minute object M.
  • the light heating member 110 is formed of a conductive material (for example, a metal such as platinum)
  • the electrode 111 and the light heating member 110 are bridged by the minute object M
  • the electrode 112 and the light The space between the heat generating member 110 and the heat generating member 110 may be bridged by the minute object M.
  • FIG. 6 is a diagram for explaining a temporal change in the electric resistance R between the electrodes 111 and 112 due to the accumulation (crosslinking) of the minute objects M.
  • the horizontal axis indicates the elapsed time.
  • the measurement start time of the electrical resistance R was set as the initial time (indicated by 0).
  • the dispersion liquid D is dropped before the measurement start time of the electric resistance R, but the measurement start time of the electric resistance R may be made coincident with the dropping time of the dispersion liquid D.
  • the vertical axis represents the electrical resistance R between the electrodes 111 and 112.
  • the electrical resistance R at the initial time is R0 (depending on the type of the dispersion medium, for example, a resistance value in the order of mega-ohms) (see FIG. 5A).
  • R0 depending on the type of the dispersion medium, for example, a resistance value in the order of mega-ohms
  • the electrode 111 and the light heating member 110 are bridged by the minute object M and the electrode 112 and the light heating member 110 are bridged by the minute object M at the time tc, that is, the electrode 111 and the electrode.
  • the electric resistance R changes from R0 to Rc.
  • the electric resistance R rapidly decreases as shown in FIG.
  • the lowered electric resistance Rc mainly represents the electric resistance R of the minute object M.
  • the light heat generating member 110 is irradiated with the laser light L1, and the convection C is generated by the light heat generating effect of the light heat generating member 110 thereby.
  • the minute objects M can be quickly accumulated near the laser spot.
  • the electrical characteristics of the minute objects M can be measured.
  • FIG. 7 is a diagram showing the configuration of the measurement kit 2A in the example of the first embodiment.
  • FIG. 7 shows the configuration of the measurement kit 2A viewed from the top to the bottom.
  • an electrode for conductivity measurement manufactured by BAS Co., Ltd. can be used for the measurement kit 2A.
  • the measurement kit 2A includes a substrate 11A made of quartz glass and eight electrodes 151 to 158 (see FIG. 8) formed on the substrate 11A.
  • eight connection pins 131 to 138 and eight wirings 141 to 148 are formed corresponding to the eight electrodes 151 to 158, respectively.
  • a clip (see FIG. 1) of the multimeter 90 is attached to any two of the connection pins 131 to 138.
  • the wirings 141 to 148 electrically connect the connection pins 131 to 138 and the electrodes 151 to 158.
  • FIG. 8 is a diagram showing the configuration of the electrodes 151 to 158 in the present embodiment in more detail.
  • each of eight electrodes 151 to 158 is a platinum electrode.
  • each electrode is bonded onto the substrate 11A via an adhesive layer that is a titanium thin film.
  • the distance between two adjacent electrodes among the eight electrodes 151 to 158 is set to various values.
  • the distance between the electrode 151 and the electrode 152 is 100 ⁇ m.
  • the distance between the electrode 152 and the electrode 155 is 10 ⁇ m.
  • the distance between the electrode 155 and the electrode 153 is 20 ⁇ m.
  • the distance between the electrode 153 and the electrode 156 is 30 ⁇ m.
  • the distance between the electrode 156 and the electrode 154 is 50 ⁇ m.
  • the distance between the electrode 154 and the electrode 157 is 100 ⁇ m.
  • the distance between the electrode 157 and the electrode 158 is 100 ⁇ m.
  • the distance between the electrodes can be set to a desired value.
  • the electrode 152 and the electrode 155 having an interelectrode distance of 10 ⁇ m were selected. 7 and 8, an example in which eight electrodes are formed will be described.
  • the number of electrodes is not particularly limited as long as it is two or more.
  • the light heating member 110 is not provided between the electrodes 151-158. This is because the electrodes 152 and 155 also serve as the “light heating member” according to the present disclosure. In other words, the light heating member is included in one of the electrodes 152 and 155 (in this embodiment, the electrode 152). Therefore, the laser light L1 from the laser light source 40 is applied to one of the electrodes 152 and 155 (electrode 152).
  • a dispersion D containing gold nanoparticles as a dispersoid and ultrapure water as a dispersion medium was used.
  • the diameter of the gold nanoparticle was 30 nm.
  • the concentration of the gold nanoparticles was 7.8 ⁇ 10 ⁇ 10 M.
  • the dripping amount of the dispersion D of gold nanoparticles was 5 ⁇ L.
  • the electrical resistance measurement in the comparative example differs from the electrical resistance measurement in the present embodiment in that the laser light L1 from the laser light source 40 is not irradiated.
  • the configuration of the measurement system according to the other comparative examples is the same as the corresponding configuration of the measurement system according to the present embodiment (see FIGS. 1, 7, and 8).
  • PBS phosphate buffered saline
  • the electrical resistance R of the PBS between the electrodes 152 and 155 was measured.
  • the electrical resistance R was in the range of 4.7 M ⁇ ⁇ 0.4 M ⁇ , it was determined that the measurement conditions were unified, and the dispersion D was further dropped onto the measurement kit 2.
  • FIG. 9 is a diagram illustrating a measurement example of a temporal change in the electrical resistance R between the electrodes 152 and 155 in the comparative example.
  • the electrical resistance R on the vertical axis is shown on a logarithmic scale.
  • FIGS. 9A to 9C show the first to third measurement results of the electric resistance R, respectively.
  • FIG. 10 is an image near the electrodes 152 and 155 in the third measurement of the three measurements shown in FIG.
  • FIG. 9 (A) to FIG. 9 (C) a rapid decrease in electrical resistance R was observed in all three measurements.
  • the state in the vicinity of the electrodes 152 and 155 at time t1 when the rapid decrease in the electric resistance R occurs for the first time in FIG. 9C is shown in the image of FIG. From this image, it was observed that some kind of substance (actually gold nanoparticles as described later with reference to FIGS. 15 and 16) was crosslinked between the electrode 152 and the electrode 155.
  • the dispersion liquid D on the electrodes 152 and 155 disappears by evaporation as time passes from the image of FIG. 10C corresponding to the time t 3, and a small amount of gold nanoparticles and PBS crystals are formed between the electrodes 152 and 155. It can be seen that
  • the average time from the time (0) when the dispersion D was dripped to the time when the electrical resistance R changed significantly was 42 minutes. 26 seconds.
  • this time (average time until the electric resistance R rapidly decreases) is also referred to as “average measurement time”.
  • it takes a long time to measure the electrical resistance R of the gold nanoparticles.
  • the time when the change in the electric resistance R occurs is different every three measurements, and the variation is relatively large.
  • FIG. 11 is a diagram illustrating a measurement example of a temporal change in the electrical resistance R between the electrodes 152 and 155 in the present embodiment.
  • the laser beam L1 is irradiated to the electrode 152 which is an anode.
  • Each electrode 152, 155 is previously cleaned using ethanol and ultrapure water.
  • the laser output was set to 15 mW.
  • the constant current by the multimeter 90 was set to 500 nA.
  • the electrical resistance R between the electrode 152 and the electrode 155 was measured 5 times.
  • the measurement results of the electrical resistance R for the first to fifth times are shown in FIGS. 11 (A) to 11 (E), respectively.
  • the average measurement time was 69 seconds as a result of five measurements. That is, when the comparative example and this example were compared, in this example, the average measurement time could be shortened to about 1/30. Thus, according to the present Example, it turns out that an average measurement time can be shortened significantly compared with a comparative example.
  • the electric resistance R there is a large variation in time until the electric resistance R rapidly decreases.
  • the gold nanoparticles are in a thin state between the electrode 152 and the electrode 155.
  • the electrical resistance R between the electrodes 152 and 155 rapidly decreases when the density of the gold nanoparticles becomes a predetermined amount or more.
  • the electrical resistance measurement in the comparative example since the laser light L1 from the laser light source 40 is not irradiated, convection does not occur. Therefore, the probability that gold nanoparticles collide with each other is low, and it is difficult to increase the density.
  • convection is positively generated by using the light heat generation effect.
  • convection is positively generated by using the light heat generation effect.
  • the collision probability of gold nanoparticles increases compared with the case where convection does not arise, it becomes easy to make gold nanoparticles high density. Therefore, in the present embodiment, the variation required for the decrease in the electric resistance R due to percolation is smaller than that in the comparative example, so that the variation in the average measurement time can be reduced.
  • FIG. 12 is an image in the vicinity of the electrodes 152 and 155 when the electrical resistance is measured in this example.
  • 12A to 12C show images near the electrodes 152 and 155 when the laser output is set to 10 mW, 15 mW, and 20 mW, respectively.
  • the position of the laser spot is the center of each image.
  • the numerical values in the figure indicate the elapsed time from the dropping time of the dispersion D.
  • control device 100 controls the output of the laser light L1 from the laser light source 40 so that the size (diameter) of the microbubble MB is larger than the distance between the electrodes 152 and 155. The reason for this is explained as follows.
  • the direction of convection C generated by light irradiation is a direction that once heads toward the microbubble MB and then moves away from the microbubble MB. That is, since the direction of the convection C is reversed, a “stagnation region” in which the flow velocity of the convection C is zero is generated between the microbubble MB and the substrate 11.
  • the microbubbles MB function as a “fluid stopper” of the micrometer order that dams the convection C, so that most of the minute objects M stay and accumulate near the stagnation region.
  • the minute objects M are easily integrated so as to bridge between the electrodes 152 and 155.
  • the laser output was set to 20 mW
  • generation of larger microbubbles MB was confirmed and generation of more intense convection C was confirmed as compared with the case where the laser output was set to 15 mW (see FIG. 12 (C)).
  • the average measurement time of the five measurement results was 85 seconds. That is, the average measurement time when the laser output was set to 20 mW was longer than the average measurement time when the laser output was set to 15 mW. This is considered to be due to the fact that excessively intense convection C was generated by increasing the laser output, so that the gold nanoparticles were less likely to stay between the electrodes 152 and 155 and the amount of gold nanoparticles accumulated decreased.
  • the laser output has a lower limit value necessary for generating microbubbles MB and convection C and an upper limit value necessary for stable accumulation of gold nanoparticles. Therefore, it is desirable to set the laser output within a range between the upper limit value and the lower limit value based on the previous experimental result or simulation result. In this example, the laser output was set to 15 mW based on the above experimental results.
  • FIG. 13 is a diagram illustrating a measurement result of concentration dependency of gold nanoparticles in the measurement of electrical resistance of gold nanoparticles.
  • four types of dispersions D1 to D4 having different concentrations of gold nanoparticles were prepared.
  • the concentration of the gold nanoparticles is low in the order of the dispersions D1 to D4.
  • the electrical resistance R between the electrodes 152 and 155 was measured 5 times for the dispersions D1, D2 and D4, and 7 times for the dispersion D3.
  • FIG. 13A shows the average time required until the change in the electric resistance R occurs from the dripping of FIG.
  • the horizontal axis of FIG. 13B indicates the concentration of gold nanoparticles
  • the vertical axis indicates the electric resistance R.
  • FIG. 14 is an image near the laser spot in the dispersions D3 and D4.
  • FIG. 14A shows an image near the laser spot in the dispersion D3
  • FIG. 14B shows an image near the laser spot in the dispersion D4.
  • the image indicated as “0 seconds” is an image before the start of light irradiation.
  • the image indicated as “100 seconds” is an image during light irradiation after 100 seconds from the dropping of the dispersion liquid.
  • the image indicated as “300 seconds” is an image immediately after the light irradiation is stopped after 300 seconds from the dropping of the dispersion liquid.
  • the electrodes 152 and 155 are cross-linked by gold nanoparticles.
  • the dispersion D4 as shown in FIG. 14B, it was confirmed that some gold nanoparticles were accumulated in the vicinity of the electrode 152, but the electric resistance R did not change (FIG. 13). (See (A) and FIG. 13 (B)). From this measurement result, it was found that the lower limit concentration (measurement limit concentration) of the gold nanoparticles that can be measured in this example was 1.6 ⁇ 10 ⁇ 10 M, which is the concentration of the dispersion D3.
  • FIG. 15 is a diagram for explaining the identification result of the accumulation on the electrodes 152 and 155 in the comparative example.
  • FIG. 16 is a diagram for explaining the identification result of the accumulation on the electrodes 152 and 155 in the present embodiment.
  • FIG. 15A shows an optical microscope image
  • FIG. 15B shows an SEM image
  • FIG. 15C shows an EDX image. The same applies to FIGS. 16A to 16C.
  • FIG. 17 is a diagram for explaining a method of calculating the electrical conductivity of the gold nanoparticle 200 in the present example.
  • the distance d between the electrode 152 and the electrode 155 is 10 ⁇ m (see FIG. 8).
  • the width w of the accumulation range of the gold nanoparticles 200 was read from the optical microscope image and set to 78.8 ⁇ m.
  • the height h of the aggregate of the gold nanoparticles 200 is 30 ⁇ m, which is the particle diameter of the gold nanoparticles 200. did.
  • R 1.89 k ⁇ was determined from the measurement result of the electric resistance R in the dispersion D1 shown in FIG.
  • the electrical conductivity ⁇ in this example was calculated to be 2.24 ⁇ 10 ⁇ 3 [ ⁇ ⁇ 1 cm ⁇ 1 ].
  • gold nanoparticles are accumulated in the vicinity of the electrode 152 by using the photothermal effect by the irradiation of the laser beam L1 onto the electrode 152.
  • the photo exothermic effect By using the photo exothermic effect, the time required for the accumulation of gold nanoparticles can be significantly reduced.
  • the electrode 152 and the electrode 155 are cross-linked by the gold nanoparticles.
  • the measurement target of the measurement system 1 changes from the electrical resistance of the dispersion medium between the electrode 152 and the electrode 155 to the electrical resistance of the gold nanoparticle that is bridged between the electrode 152 and the electrode 155.
  • the electrical conductivity ⁇ can be calculated with high accuracy from the measured electrical resistance R.
  • a detection system for detecting a detection target substance that is a minute object that may be contained in a liquid sample will be described.
  • the detection system according to the second embodiment is different from the measurement system 1 according to the first embodiment (see FIGS. 1 and 2) in that a detection kit 2B is provided instead of the measurement kit 2.
  • the configuration of the detection kit 2B will be described with reference to FIG.
  • the control device 100 corresponds to a “detection device” according to the present disclosure. Since the other configuration (overall configuration) of the detection system according to Embodiment 2 is basically the same as the configuration of measurement system 1, description thereof will not be repeated.
  • the substance to be detected is DNA.
  • This DNA is also referred to as “target DNA”.
  • metal nanoparticles are used for detection of the target DNA.
  • the metal nanoparticles are, for example, gold nanoparticles.
  • Each of the gold nanoparticles is modified with DNA capable of specifically attaching the target DNA.
  • DNA capable of specifically attaching the target DNA is also referred to as “probe DNA”.
  • the probe DNA corresponds to an example of a “host molecule” according to the present disclosure.
  • the metal nanoparticles are not limited to gold nanoparticles, and may be, for example, silver nanoparticles or copper nanoparticles.
  • target RNA may be used instead of target DNA, or probe RNA may be used instead of probe DNA.
  • Target DNA and target RNA are collectively referred to as “target nucleic acid”, and probe DNA and probe RNA are collectively referred to as “probe nucleic acid”.
  • FIG. 18 is a conceptual diagram for explaining the detection principle of the target DNA in the second embodiment.
  • An example of the base sequences of the target DNA and probe DNA is shown in FIG.
  • the target DNA 210 is a single-stranded DNA having a base sequence of 24 adenines (represented by A), for example.
  • A 24 adenines
  • two types of probe DNAs 211 and 212 for specifically attaching the target DNA 210 are prepared.
  • Probe DNA 211 is a single-stranded DNA having, for example, a thiol group (represented by SH) at the 3 'end.
  • the probe DNA 211 has a base sequence complementary to the base sequence on the 3 'end side of the target DNA between the thiol group and the 5' end. This complementary base sequence is 12 thymines (denoted by T).
  • the probe DNA 212 is a single-stranded DNA having, for example, a thiol group at the 5 'end.
  • the probe DNA 212 has a base sequence complementary to the base sequence on the 5 'end side of the target DNA between the 3' end and the thiol group. This complementary base sequence is 12 thymines.
  • the gold nanoparticle 201 is modified with the probe DNA 211.
  • the gold nanoparticle 202 is modified with the probe DNA 212.
  • known methods can be used as a method for modifying the gold nanoparticles 201 and 202 with the probe DNAs 211 and 212.
  • the gold nanoparticles 201 and 202 When the gold nanoparticles 201 and 202 are introduced into the liquid containing the target DNA 210, hybridization occurs between the target DNA 210 and the probe DNA 211 and between the target DNA 210 and the probe DNA 212. Thereby, when the gold nanoparticles 201 and 202 are dispersed in the liquid, the gold nanoparticles 201 and 202 aggregate.
  • FIG. 19 is a diagram for explaining the configuration of the detection kit 2B in detail.
  • detection kit 2B includes a substrate 11B and electrodes 111B and 112B.
  • the electrode 111B is an anode, and the electrode 112B is a cathode.
  • Each of the electrodes 111B and 112B is a metal thin film (for example, a platinum thin film or a gold thin film) having a film thickness on the order of nanometers.
  • One of the electrodes 111B and 112B also serves as the “light heating member” according to the present disclosure as described in the example of the first embodiment.
  • the liquid sample SP that may contain the target DNA 210 is dropped so as to cover the electrodes 111B and 112B. Since substrate 11B and adhesive layers 121B and 122B are basically the same as the corresponding configuration of measurement kit 2 in Embodiment 1 (see FIG. 2), description thereof will not be repeated.
  • electrical resistance measurement is realized by adopting at least one of the following two configurations (first and second configurations) described below.
  • FIG. 20 is a first diagram for explaining a measurement mechanism of electrical characteristics (for example, electrical resistance R) between the electrodes 111 and 112 using hybridization.
  • electrical characteristics for example, electrical resistance R
  • FIG. 20A in the first configuration, gold nanoparticles 201 and 202 are dispersed in liquid sample SP. Although the gold nanoparticles 201 and 202 are very small, they are schematically enlarged in FIG. 20 and FIG.
  • a dispersion medium for the liquid sample SP exists between the electrode 111B and the electrode 112B. Since the dispersion medium is an insulating liquid (for example, water), the electrode 111B and the electrode 112B are electrically insulated.
  • the target DNA 210 when the target DNA 210 is included in the liquid sample SP, as shown in FIG. 20B, hybridization occurs between the target DNA 210 and the probe DNA 211 and between the target DNA 210 and the probe DNA 212. Occurs, and the gold nanoparticles 201 and 202 aggregate. As a result, the electrode 111B and the electrode 112B are cross-linked by an aggregate of the gold nanoparticles 201 and 202. Hybridization occurs, and the complementary bases of the single-stranded DNA are bonded to increase conductivity. As a result, the electrical resistance between the electrode 111B and the electrode 112B decreases via the gold nanoparticles 201 and 202 and the double-stranded DNA (the probe DNAs 211 and 212 and the target DNA 210 after hybridization).
  • FIG. 21 is a second diagram for explaining a measurement mechanism of electrical characteristics (for example, electrical resistance R) between the electrodes 111 and 112 using hybridization.
  • electrical characteristics for example, electrical resistance R
  • gold nanoparticles 201 and 202 are fixed in advance on substrate 11B between electrodes 111B and 112B.
  • the distance between the gold nanoparticle 201 and the gold nanoparticle 202 is close enough to allow DNA hybridization between the gold nanoparticle 201 and the gold nanoparticle 202 (for example, the length of the target DNA 210 or less).
  • the gold nanoparticles 201 and 202 can be fixed on the substrate 11B by a known method such as a method using a thiol group.
  • the electrode 111B and the electrode 112B are electrically insulated.
  • the target DNA 210 when the target DNA 210 is contained in the liquid sample SP, hybridization occurs between the probe DNA 211 modified to the gold nanoparticle 201 and the target DNA 210 as shown in FIG. At the same time, hybridization occurs between the probe DNA 212 modified to the gold nanoparticle 202 and the target DNA 210. As a result, the electrode 111B and the electrode 112B are bridged and become electrically conductive.
  • the electric resistance R is determined based on the electric resistance value of the dispersion medium of the liquid sample SP.
  • the electrical resistance value of the aggregate of the particles 201 and 202 and the double-stranded DNA (see FIG. 20B) or the electrical resistance of the cross-linked structure between the electrode 111B and the electrode 112B (see FIG. 21B) Decreases to value.
  • the target DNA 210 can be detected by monitoring the electrical resistance R between the electrode 111B and the electrode 112B.
  • the two configurations shown in FIGS. 20 and 21 can be combined. Further, instead of fixing the gold nanoparticles 201 and 202 on the substrate 11B, only the thiol group for fixing the gold nanoparticles 201 and 202 is modified on the substrate 11B, and the gold nanoparticles 201 and 202 are liquidated. It may be dispersed in the sample SP.
  • FIG. 22 is a flowchart showing a method for detecting a substance to be detected (target DNA 210) in the second embodiment.
  • the processing of S201 to S205 is the same as the processing of S101 to S105 (see FIG. 3) in the first embodiment, and therefore detailed description will not be repeated.
  • control device 100 controls the multimeter 90 so as to measure the electric resistance R between the electrodes 111B and 112B (start or continue the measurement).
  • control device 100 controls the laser light source 40 so as to start (continue) light irradiation.
  • the control device 100 determines whether or not the electrical resistance R between the electrodes 111 and 112 has changed. More specifically, the decrease amount of the electrical resistance R from the initial value (R0 in FIG. 21) is larger than the reference amount, or the decrease rate of the electrical resistance R (decrease amount per unit time) is faster than the reference rate. In such a case, the control device 100 determines that the electrical resistance R has changed.
  • the control device 100 returns the process to S206 and continues the electrical resistance measurement until the electrical resistance R between the electrodes 111B and 112B changes (NO in S208 and NO in S210). If electric resistance R has changed before a predetermined period (eg, several tens of seconds to several minutes) has elapsed (YES in S208), control device 100 determines that target DNA 210 has been detected in liquid sample SP (S209). . On the other hand, when the predetermined period has passed without the electrical resistance R changing (NO in S208 and YES in S210), control device 100 determines that target DNA 210 has not been detected in liquid sample SP (S211). Instead of using the elapsed time (predetermined period) for determining whether or not the electric resistance R has changed, the determination of whether or not the electric resistance R has changed may be performed a predetermined number of times.
  • a predetermined period e.g, several tens of seconds to several minutes
  • control device 100 controls the multimeter 90 so as to end the electrical resistance measurement (S212). Further, the control device 100 controls the laser light source 40 and the illumination light source 70 so as to stop the irradiation of the laser light L1 and the white light L2 (S213). As a result, a series of processing ends.
  • the laser light L1 may be irradiated for a predetermined time in S207, and then the presence or absence of a change in the electrical resistance R may be determined after the irradiation of the laser light L1 is stopped (S208).
  • the irradiation of the laser beam L1, the stop of the irradiation of the laser beam L1, and the measurement of the electric resistance R are repeatedly executed until the predetermined period of S210 elapses. Become.
  • the target DNA 210 which is a substance to be detected that may be contained in the liquid sample SP, is detected by monitoring the change in the electrical resistance R between the electrodes 111B and 112B. can do. Further, by irradiating the laser light L1 from the laser light source 40, the period required for detecting the target DNA 210 (predetermined period in S210) can be shortened as compared with the case where the laser light L1 is not irradiated.
  • Example 1 of Embodiment 2 a measurement result of the electric resistance R in the first configuration illustrated in FIG. 20 will be described.
  • the base sequences of the probe DNAs 211 and 212 in this example are 12 thymines (T) as in the example described with reference to FIG.
  • A 24 adenines
  • T 24 thymines
  • mismatch DNA The concentrations of complementary strand DNA, mismatch DNA, and probe DNAs 212 and 212 in the dispersion were all 1 ⁇ M. The amount of each dispersion dropped was 5 ⁇ L.
  • FIG. 23 is a diagram for explaining the change over time of the electrical resistance R between the electrodes 111B and 112B in the dispersion liquid containing complementary strand DNA or mismatched DNA.
  • FIG. 23 (A) shows the measurement results with a dispersion containing complementary strand DNA
  • FIG. 23 (B) shows the measurement results with a dispersion containing mismatched DNA.
  • FIG. 23A and FIG. 23B shows four measurement results.
  • FIG. 23 (A) it can be seen that in the dispersion containing the complementary strand DNA, a rapid decrease in the electric resistance R is detected within a few minutes (about 2 to 6 minutes). As explained in FIG. 20, this is considered to be due to the cross-linking between the electrodes 111B and 112 due to the hybridization between the complementary strand DNA and the probe DNAs 211 and 212. Further, from the result shown in FIG. 23A, it can be seen that the variation in time until the electric resistance R decreases is somewhat large (about 2 to 3 minutes).
  • the electrical resistance R sharply decreases even in the dispersion containing the mismatched DNA.
  • the time until the electric resistance R decreases is long, and the time variation until the electric resistance R decreases is large.
  • the reason why the electric resistance R decreases even in the dispersion containing mismatched DNA is considered to be that the gold nanoparticles 201 and 202 having conductivity are deposited between the electrodes 111B and 112 over time.
  • the measurement results shown in FIG. 23 are for the case where the DNA concentration (complementary strand DNA concentration or mismatched DNA concentration) is 1 ⁇ M. Such a decrease in electrical resistance R occurs when the DNA concentration is 500 nM (ie, half). Also confirmed.
  • the electric resistance R is rapidly reduced.
  • the time variation until the electric resistance R decreases is relatively large. Therefore, in order to detect a difference in the base sequence of DNA, it is desirable to measure the time change of the electric resistance R a plurality of times and average the measurement results as described below.
  • FIG. 24 is a diagram obtained by averaging the measurement results of the temporal change in the electrical resistance R between the electrodes 111B and 112B.
  • FIG. 24 shows the average value of the measurement results when the electrical resistance measurement is performed nine times in the dispersion containing the complementary strand DNA. The same applies to a dispersion containing mismatched DNA.
  • the average value of electrical resistance R in a dispersion containing complementary strand DNA is the average value of electrical resistance R in a dispersion containing mismatched DNA (“ It can be seen that the time to reach the lower limit is shorter than that of “mismatched average resistance”. Therefore, the electrical resistance R may be measured in a time range in which the complementary strand average resistance reaches the lower limit while the mismatch average resistance does not reach the lower limit. More specifically, in the example shown in FIG. 24, the difference between the complementary strand average resistance and the mismatch average resistance is large in the time range from about 280 seconds to about 450 seconds.
  • the complementary strand average resistance is 0.15 M ⁇ and the mismatch average resistance is 0.75 M ⁇ . Therefore, the electric resistance R in the time range from about 280 seconds to about 450 seconds is measured a plurality of times, and the average value of the measurement results, the complementary chain average resistance (0.15 M ⁇ ), and the mismatch average resistance (0.75 M ⁇ ) are obtained. By comparing, the difference in the base sequence of DNA can be detected.
  • FIG. 25 is a flowchart showing a method for detecting a substance to be detected (complementary strand DNA) in the example of the second embodiment.
  • the processing with the measurement preparation in S301 is a comprehensive description of the processing in S201 to S205 (see FIG. 22) in the second embodiment because of space limitations.
  • control device 100 controls the laser light source 40 so as to start the irradiation with the laser light L1. Then, control device 100 continues the light irradiation until the predetermined period elapses (NO in S303), and when the predetermined period elapses (YES in S303), control device 100 causes laser light source 40 to stop irradiation with laser light L1. Control is performed (S304).
  • the predetermined period is a period (for example, 300 seconds) within the time range from about 280 seconds to about 450 seconds in the example shown in FIG.
  • control device 100 controls the multimeter 90 so as to measure the electric resistance R between the electrodes 111B and 112B.
  • control device 100 determines whether or not the number of measurements of the electrical resistance R has reached a specified number of measurements (a specified number).
  • a specified number of measurements a specified number
  • control device 100 returns the process to S301. Thereby, the measurement of the electrical resistance R is repeated until the specified number of times is reached. For example, the sequential measurement of the electric resistance R can be repeated by arranging the detection kits 2B shown in FIG. 19 in an array.
  • the control device 100 advances the process to S307 and calculates the average value of the electrical resistance R for the specified number of times. And the control apparatus 100 determines whether the average value of the electrical resistance R is below a predetermined reference value.
  • the reference value is a resistance value (for example, 0.30 M ⁇ ) between 0.15 M ⁇ and 0.75 M ⁇ .
  • control device 100 determines that the complementary strand DNA that is the detection target is detected (S309). On the other hand, when the average value of electrical resistance R is higher than the reference value (NO in S308), control device 100 did not detect the substance to be detected (or mismatch DNA was detected in the example of FIG. 24). ) (S310). As a result, a series of processing ends.
  • the electrical resistance R between the electrodes 111B and 112B is measured a plurality of times within a predetermined time range assuming that the difference between the complementary strand average resistance and the mismatch average resistance is large. . And it is determined whether the average value of the measurement result of the electrical resistance R is below the reference value. As a result, even when a single-stranded DNA (mismatch DNA) having a base sequence different from that of the substance to be detected (complementary strand DNA) is contained in the liquid sample, the difference in the base sequence is determined, and the substance to be detected is It can be detected accurately.
  • mismatch DNA complementary strand DNA
  • Example 2 of Embodiment 2 a configuration in which a detection kit including a comb electrode is employed will be described.
  • the detection target (substance to be detected) in this example is the target DNA 210 (see FIG. 8), as in Example 1 described above.
  • the measurement kit including the comb-shaped electrode can be applied to the electrical characteristic measurement system according to Embodiment 1 instead of the measurement kit 2A shown in FIG. 7, for example.
  • FIG. 26 is a diagram showing a configuration of the detection kit 3 in Example 2 of the second embodiment.
  • FIG. 26A shows a schematic diagram of the detection kit 3.
  • substrate 31 is provided at the tip (one end) of cable 32, and comb-shaped electrodes 301 and 302 are formed on the top surface of substrate 31.
  • Fig. 26 (B) shows an enlarged view of the comb-type electrode.
  • the comb electrodes 301 and 302 are a pair of electrodes arranged so as to face each other. More specifically, each of the comb-shaped electrodes 301 and 302 includes a plurality of electrodes arranged in a stripe shape. The comb-shaped electrode 301 and the comb-shaped electrode 302 are arranged so that the plurality of electrodes of the comb-shaped electrode 301 and the plurality of electrodes of the comb-shaped electrode 302 are engaged with each other.
  • any one electrode (may be two or more) of the plurality of electrodes included in the comb-shaped electrode 301 corresponds to the “first electrode” according to the present disclosure
  • Any one of the electrodes (may be two or more) corresponds to the “second electrode” according to the present disclosure.
  • at least one of the comb electrodes 301 and 302 also serves as the “light heating member” according to the present disclosure.
  • a light heating member may be provided separately.
  • the substrate 31 is made of an optically transparent material having electrical insulation, and is a glass substrate such as a slide glass.
  • the other end of the cable 32 (the end where the board 31 is not provided) is electrically connected to the control device 100 (see FIG. 1).
  • the other configuration is basically the same as the configuration of measurement system 1, and therefore the description will not be repeated.
  • the sodium chloride concentration in dispersion D was 0.2M, and the phosphoric acid concentration was 10 mM.
  • the dripping amount of the dispersion D was 15 ⁇ L.
  • the wavelength of the laser light from the laser light source 40 was 800 nm, and the laser output after passing through the substrate 31 was 17 mW.
  • the light irradiation time was 10 minutes.
  • FIG. 27 is a diagram showing an observation result of a state in which microbubbles MB are generated in the comb electrodes 301 and 302.
  • the optical microscope image near the comb-shaped electrodes 301 and 302 is shown on the left side of the drawing, and the fluorescence observation image near the comb-shaped electrodes 301 and 302 (the same part) is shown on the right side.
  • microbubbles MB were generated in the vicinity of the laser spot by irradiation with laser beam L1. Moreover, it was confirmed that the diameter of the microbubble MB is larger than the distance between the electrode 301 and the electrode 302.
  • a fluorescent dye is inserted between the base pairs of the target DNA 210, and when the detection kit 3 is irradiated with light having an excitation wavelength of the fluorescent dye, a location where the target DNA 210 exists (that is, the target DNA and the probe DNA). Fluorescence is emitted from the point where hybridization with the
  • FIG. 28 is a diagram illustrating an example of measurement of an accumulation between the comb-shaped electrodes 301 and 302.
  • FIG. 28 shows a state in the vicinity of the comb electrodes 301 and 302 in a state where the dispersion D is dried after the irradiation of the laser beam L1 is stopped.
  • the optical microscope image of the location irradiated with the laser beam L1 is shown on the upper side in the figure, and the fluorescence observation image of the same location as the optical microscope image is shown on the lower side.
  • the target DNA was fluorescently labeled (probe DNA was not fluorescently labeled).
  • the target DNA used was labeled with a fluorescent dye ALEXA488 that emits green light at the 5 'end.
  • the fluorescence emitted from the accumulation between the comb electrodes 301 and 302 was observed in the fluorescence observation image. From this, it can be said that it was confirmed that hybridization occurred between the target DNA of the fluorescently labeled complementary strand and the probe DNA to cause crosslinking.
  • FIG. 29 is an image in the vicinity of the comb-shaped electrodes 301 and 302 after the irradiation of the laser beam L1 is stopped.
  • an optical microscope image when complementary strand DNA is contained in the dispersion D is shown on the upper side
  • an optical microscope image when mismatched DNA is contained in the dispersion D instead of the complementary strand DNA is shown on the lower side. From FIG. 29, it can be seen that an accumulation was formed in the vicinity of the comb-shaped electrodes 301 and 302 regardless of whether the DNA in the dispersion D was complementary DNA or mismatched DNA.
  • FIG. 30 is a diagram showing a measurement example of the electric resistance R in the comb-shaped electrodes 301 and 302.
  • the vertical axis in FIG. 30A indicates the electric resistance R between the comb electrodes 301 and 302 on a linear scale.
  • the vertical axis in FIG. 30B indicates the electrical resistance R between the comb electrodes 301 and 302 on a logarithmic scale.
  • the magnitude of the electric resistance R after the decrease was several times different between the mismatch DNA and the complementary strand DNA. From this, it is possible to distinguish whether the DNA in the dispersion D is a complementary strand DNA or a mismatched DNA by comparing the magnitude of the electric resistance R after the decrease with a predetermined reference value. That is, when the magnitude of the electrical resistance R after a sufficient time has elapsed from the start of light irradiation is greater than or equal to the reference value, it is determined that mismatched DNA is included, and the magnitude of the electrical resistance R after the decrease is determined. Is less than the reference value, it can be determined that complementary strand DNA is contained.
  • the number of combinations of the positive electrode and the negative electrode is larger than that of the pair of microelectrodes, the number of locations where cross-linking by hybridization can occur is large. If any one of these locations is cross-linked, the electrical resistance R decreases, and therefore the electrical resistance R is likely to decrease.
  • a plurality of electrodes included in each are alternately arranged.
  • two electrodes of opposite electrodes (the other of the comb electrodes 301 and 302) are disposed opposite to each side of each electrode of one of the comb electrodes 301 and 302. Therefore, if one of the plurality of electrodes arranged alternately is irradiated with laser light, at least one of the electrode irradiated with the laser light and two electrodes of opposite poles disposed opposite to both sides of the electrode. Cross-linking can occur between As a result, the electrical resistance R between the comb-shaped electrodes 301 and 302 can be reduced.
  • the distance between the comb electrodes (more specifically, one electrode among a plurality of electrodes included in one comb electrode and a plurality of adjacent electrodes included in the other comb electrode).
  • the distance between one of the electrodes) is narrower than the distance between the pair of microelectrodes.
  • the distance between the comb electrodes 301 and 302 was 5 ⁇ m.
  • the size of the microbubbles MB grows larger than the distance between the electrodes.
  • the shorter the distance between the electrodes the shorter the time required for the microbubbles MB to grow to such a size.
  • the detection time of the target DNA 210 can be shortened by using the comb-shaped electrodes 301 and 302.
  • the detection method of the detection target substance is not limited to this.
  • the substance to be detected is an antigen
  • the substance to be detected can be detected by modifying an antibody that causes an antigen-antibody reaction with the antigen into nanoparticles.
  • 1 measurement system 2, 2A measurement kit, 2B, 3 detection kit, 10 XYZ axis stage, 11, 11A, 11B, 31 substrate, 111, 111B, 112, 112B, 151-158 electrode, 301, 302 comb electrode, 20 adjustment mechanism, 30 sample supply unit, 32 cable, 40 laser light source, 50 optical components, 60 objective lens, 70 illumination light source, 80 imaging device, 90 multimeter, 100 control device, 110 light heating member, 120, 121, 121B , 122, 122B adhesive layer, 131-138 connecting pins, 141-148 wiring, 200-202 gold nanoparticles, 210 target DNA, 211, 212 probe DNA, D, D1-D4 dispersion, SP liquid sample, M micro object .

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Abstract

L'invention concerne un système de mesure (1) qui comporte une trousse de mesure (2), une source de lumière laser (40), et un multimètre (90). La trousse de mesure (2) inclut un organe générateur de chaleur optique (110) qui génère de la chaleur lorsqu'il est irradié par de la lumière, et des électrodes (111, 112), et est configurée pour pouvoir contenir un liquide de dispersion (D) dans lequel est dispersée une pluralité de micro-objets (M). Le multimètre (90) est configuré pour mesurer une impédance entre l'électrode (111) et l'électrode (112) dans un état dans lequel une pluralité des micro-objets (M) s'est collectée entre l'électrode (111) et l'électrode (112) en résultat d'une convection dans le liquide de dispersion (D) causée par le chauffage du liquide de dispersion (D) grâce à la chaleur générée par l'organe générateur de chaleur optique (110) par projection de lumière par la source de lumière laser (40), et dans lequel un espace entre l'électrode (111) et l'électrode (112) a été relié en pont par la collecte de la pluralité de micro-objets (M).
PCT/JP2018/018427 2017-05-12 2018-05-11 Système de mesure d'impédance, procédé de mesure d'impédance, et système de détection de substance en cours de détection WO2018207937A1 (fr)

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WO2022234849A1 (fr) * 2021-05-07 2022-11-10 公立大学法人大阪 Procédé de détection d'une substance à détecter, kit de détection et système de détection, et procédé de fabrication d'un kit de détection
WO2023163096A1 (fr) * 2022-02-24 2023-08-31 公立大学法人大阪 Procédé de concentration de micro-objets, kit de concentration de micro-objets et système de concentration de micro-objets

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WO2023163096A1 (fr) * 2022-02-24 2023-08-31 公立大学法人大阪 Procédé de concentration de micro-objets, kit de concentration de micro-objets et système de concentration de micro-objets

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