WO2012090960A1 - Puce de détection d'échantillons, détecteur utilisant cette puce, et procédé de détection d'échantillons - Google Patents

Puce de détection d'échantillons, détecteur utilisant cette puce, et procédé de détection d'échantillons Download PDF

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WO2012090960A1
WO2012090960A1 PCT/JP2011/080121 JP2011080121W WO2012090960A1 WO 2012090960 A1 WO2012090960 A1 WO 2012090960A1 JP 2011080121 W JP2011080121 W JP 2011080121W WO 2012090960 A1 WO2012090960 A1 WO 2012090960A1
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droplet
electrode
detection
specimen
sample
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PCT/JP2011/080121
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English (en)
Japanese (ja)
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恭子 瀬尾
スンジン チョ
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シャープ株式会社
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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
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays

Definitions

  • the present invention relates to a sample detection sensor for separating and detecting only a specific trace substance contained in a liquid, and more particularly to a sample detection sensor using droplets.
  • Micro-TAS Micro-Total Analysis Systems
  • Lab-on-a-Chip Lab-on-a-Chip
  • Patent Document 1 discloses a microchannel device including a substrate portion, a photocurable resin channel, and a light-transmitting cover substrate. An uncured resin is provided between the substrate portion and the cover substrate. There has been proposed a microchannel device in which microchannels are integrally formed by forming a channel pattern by photocuring reaction after filling. Further, Patent Document 2 proposes an enzyme immunoassay chip in which an enzyme immunoassay system for measuring a change in color by coloring a substrate solution using an enzyme as a label is integrated in a microchip. .
  • a transport device using droplets that do not diffuse has advantages such as a small amount of a test reagent and a shortened reaction time. If the application is limited to a reaction or detection that is not immobilized, and the ligand is immobilized, there is a risk of impeding the transport of the droplet.
  • FIG. 13 is a diagram showing a contact angle between a droplet, a substrate, a gas, or a liquid that does not mix with the droplet.
  • the droplet 90 includes an interfacial tension 93 between the droplet 90 and the substrate 91, an interfacial tension 94 between the droplet 90 and the liquid 92 that does not mix with the gas or the droplet, a substrate 91, and the gas or the droplet.
  • the three forces of interfacial tension 95 between the liquid 92 and the unmixed liquid 92 are balanced and formed.
  • the fixing unit and the detection unit constituting the specimen detection device are not in a surface state suitable for transporting the droplet.
  • the surface state depends on each material. In the case where the contact angle of the fixed part or the detection part is lower than the other flow path surfaces, that is, in a surface state in which the liquid droplets easily get wet with respect to the substrate, the liquid droplet 90 tends to stay in place and is difficult to transport.
  • the present invention has been made in view of each of the above-mentioned problems, and its purpose is to immobilize while suppressing a decrease in sensitivity while preventing diffusion between sequentially transported solutions (droplets).
  • An object of the present invention is to provide a sample detection chip capable of detecting a sample contained in a droplet, a sensor using the same, and a sample detection method, while preventing the transport stability from being hindered by the formed ligand. .
  • a sample detection chip for detecting a sample suspended in a solvent, and a transport electrode for transporting a droplet of the solvent
  • a transfer substrate having a surface, a counter substrate facing the transfer substrate, a flow path formed between the transfer substrate and the counter substrate through which the droplets pass, and a part of the flow path And a fixing part on which a ligand is fixed, and the specimen is detected by the liquid droplets staying in the fixing part.
  • the specimen detection method of the present invention is provided in a part of the flow path, in which a droplet of the solvent in which the specimen is suspended is electrically controlled and conveyed in the flow path.
  • a sample detection method comprising: a step of causing the droplet to stay in a fixed part on which a ligand is fixed and reacting the sample with the ligand; and a step of detecting the sample,
  • the flow path is formed between a transport substrate having a transport electrode for transporting the droplets of the solvent and a counter substrate facing the transport substrate, and the droplets are transferred to the transport substrate.
  • the droplets are transported such that the area in contact with the fixed portion is larger than the area where the fixed portion faces the flow path.
  • the stability of transport is prevented from being hindered by the immobilized ligand while suppressing the decrease in sensitivity. Meanwhile, the specimen contained in the droplet can be detected.
  • the decrease in sensitivity is suppressed while the diffusion of the solution at the interface between different solutions (droplets) that are sequentially fed is prevented, and the conveyance is stabilized by the immobilized ligand. Therefore, it is possible to realize a sensor that can prevent the inhibition of the sex and can detect the specimen contained in the droplet.
  • FIG. 1 is a cross-sectional view of a specimen detection sensor according to Embodiment 1.
  • FIG. 3 is a cross-sectional view of the specimen detection sensor when a droplet is sent to the specimen detection sensor according to the first embodiment.
  • FIG. 3 is a perspective view of the sample detection sensor when a droplet is sent to the sample detection sensor according to the first embodiment.
  • FIG. 3 is a plan view of the specimen detection sensor when a droplet is sent to the specimen detection sensor according to the first embodiment. It is the figure which showed the relationship between a droplet, the electrode for conveyance, a fixing
  • FIG. 10 is a cross-sectional view of a sample detection sensor when a droplet is sent to the sample detection sensor according to the second embodiment.
  • FIG. 10 is a cross-sectional view of a sample detection sensor when a droplet is sent to the sample detection sensor according to the second embodiment.
  • FIG. 9 is a cross-sectional view of a sample detection sensor when a droplet is sent to the sample detection sensor according to a third embodiment.
  • FIG. 10 is a cross-sectional view of a sample detection sensor when a droplet is sent to the sample detection sensor according to a fourth embodiment. It is a schematic plan view of IDT. It is a figure explaining the contact angle of a droplet, a board
  • the specific protein is measured by immunoassay.
  • adiponectin involved in the development of metabolic syndrome was used as the specific protein, and the concentration of the specific protein was measured by an electrochemical measurement method.
  • the present invention is not limited to these configurations.
  • FIG. 1 is a cross-sectional view of a sample detection sensor 1 according to the present embodiment.
  • electrowetting is used as a method for transporting droplets.
  • the sample detection sensor 1 includes a transport substrate 11, a transport electrode 12, a dielectric film 13, a hydrophobic film 14 a, a hydrophobic film 14 b, a substrate 15 without a transport electrode, a detection electrode 16, and a fixing unit 17. Composed.
  • “droplet” means a liquid separated by gas or liquids having different polarities.
  • the volume of each droplet is not particularly limited.
  • the volume of each droplet may be 1 to 1000 pL, 1 to 1000 nL, 1 to 1000 mL, or more.
  • a glass substrate or a Si substrate can be used as the transfer substrate 11 and the substrate 15 having no transfer electrode.
  • a plurality of transfer electrodes 12 are arranged on the transfer substrate 11, and a voltage is applied to the transfer electrodes 12 to transfer the droplets.
  • a dielectric film 13 is formed on the transfer electrode 12.
  • the dielectric film 13 is made of silicon nitride, silicon oxide, tantalum oxide, titanium oxide, barium titanate, or the like, and has dielectric properties.
  • a hydrophobic film 14a is formed on the dielectric film 13, and a hydrophobic film 14b is formed so as to face the hydrophobic film 14a.
  • a space between the hydrophobic film 14a and the hydrophobic film 14b is a space for transporting droplets.
  • the hydrophobic film 14a and the hydrophobic film 14b are made of, for example, a fluorine-based resin and have a low affinity for water, and the hydrophobic film 14a and the hydrophobic film 14b are made of the same material even if they are the same material. Nearly different materials may be used.
  • a substrate 15 having no transfer electrode is formed on the hydrophobic film 14b.
  • a fixed portion 17 and a detection electrode 16 are arranged on a substrate 15 that does not have a transfer electrode.
  • the detection electrode 16 is generally composed of two electrodes, three electrodes, or a plurality of electrodes having different shapes, and the plurality of electrodes collectively function as one detection electrode 16.
  • the detection electrode 16 can detect, for example, that a specimen is bound to a ligand described later.
  • As the electrode material gold, platinum or the like is mainly used.
  • the detection electrode 16 is directly formed on the substrate 15 that does not have a transport electrode, because it is necessary to apply a resist once before the detection electrode 16 is formed.
  • the detection electrode 16 is formed on the substrate 15 having no transfer electrode, and thereafter, only the portion of the detection electrode 16 is removed from the hydrophobic film 14b. In this way it is formed.
  • a fixing part 17 is disposed on at least a part of the detection electrode 16, and a ligand that reacts with the specimen is fixed to the fixing part 17.
  • FIG. 2 shows a state in which the droplet 2 is fed to the specimen detection sensor 1 as a sample.
  • the periphery of the droplet 2 is filled with a gas or liquid that does not mix with the droplet 2.
  • a nonpolar solvent for the liquid around the droplet 2 so as not to mix with this.
  • a hydrocarbon-based material such as dodecane, silicone oil, a fluorine-based material, or the like can be used.
  • water, alcohol (for example, ethanol, methanol), etc. can be used.
  • a nonpolar solvent as the droplet 2 and a polar solvent as the liquid around the droplet 2 that does not mix with the droplet 2.
  • a solvent may be appropriately selected according to the property of the specimen to be detected.
  • inert gas with low reactivity with a substance, such as nitrogen and argon can be used, for example. Alternatively, air may be used.
  • FIG. 3 is a perspective view of the sample detection sensor 1 of the present embodiment
  • FIG. 4 is a plan view of the sample detection sensor 1 of the present embodiment.
  • the substrate 15 having no transfer electrode is omitted.
  • the transport electrodes 12 are connected to the switches SW1, SW2, and SW3, respectively, and the voltage application state to the individual transport electrodes 12 can be controlled by turning the switches on and off. Then, the droplet 2 can be moved by controlling the voltage application state to the individual transport electrodes 12.
  • the number of switches is not particularly limited, and a necessary number can be provided.
  • the switch SW2 is in a connected state.
  • the switch SW2 is opened and the switch SW3 is connected.
  • the voltage is applied until the movement of the droplet 2 is observed, and when the threshold voltage is exceeded, the droplet 2 moves to the next transport electrode. By repeating this, the droplet 2 is moved to the target location.
  • the voltage application method is not limited to such a direct current method, and can also be performed by an alternating current method.
  • the droplet 2 can be transported not only by the above-described potential application method but also by the potential change.
  • FIG. 5A and 5B are diagrams showing the relationship between the droplet 2 and the transport electrode 12.
  • the shape of the transfer electrode 12 may be a shape as shown in FIG. 5B, or any other shape.
  • FIG. 5A the surface 2a where the droplet 2 is in contact with the hydrophobic film 14a or the hydrophobic film 14b, the detection electrode 16 and the fixing portion 17 is shown in FIG.
  • FIG. 5B When viewed from the direction of the arrow, that is, almost from the top, as shown in FIG. 5B, a plurality of transport electrodes 12 are always applied along the traveling direction of the droplet 2.
  • the surface 2a is on the transfer electrodes 12a, 12a ', 12b, 12b'.
  • the transport electrode 12 is formed of a plurality of electrodes like the transport electrodes 12a and 12a ', these are regarded as one transport electrode. Therefore, as viewed from above (b) of FIG. 5, the surface 2 a covers the two electrodes. As in this embodiment, by setting the transport electrode 12 and the droplet 2 in this way, the droplet 2 is transported.
  • a droplet control means (not shown) may be provided to control the transport amount of the droplet 2.
  • the specific configuration of the droplet control unit is not particularly limited as long as it can control the transport amount of the droplet 2.
  • the droplet control means may introduce a predetermined amount of the droplet 2 into the flow path between the hydrophobic film 14a and the hydrophobic film 14b.
  • the droplet control means may include a droplet preparation unit for forming a droplet having a predetermined volume and a pump for introducing the droplet having a predetermined volume into the flow path. It is not limited to the configuration.
  • the volume of each droplet can be set as appropriate based on the size of the transfer electrode 12 and the arrangement interval of the transfer electrodes 12.
  • the above-described droplet control means can be manufactured by combining known configurations.
  • it can be set by determining the space (height) in which the droplets 2 are transported by adjusting the arrangement of the transport substrate 11 and the substrate 15 having no transport electrodes. In this case, in order to make the contact area of the droplet 2 constant, it is preferable to keep the distance between the transfer substrate 11 and the substrate 15 having no transfer electrode in the flow path constant.
  • the detection electrode 16 and the fixing portion 17 are manufactured on the substrate 15 side having no transfer electrode, but may be manufactured on the transfer substrate 11 side. Moreover, one detection electrode 16 and the fixing
  • the detection electrode 16 and the fixed part 17 are used to smoothly transport the droplet 2. Is preferably smaller than the transfer electrode 12. Further, the detection electrode 16 and the fixing portion 17 are preferably provided smaller than the surface 2a where the droplet 2 shown in FIG. 5A is in contact with the hydrophobic film 14a or the hydrophobic film 14b. . Thereby, for example, the ligand and the specimen can be reacted efficiently.
  • Electrochemical measurement methods include a two-electrode method and a three-electrode method.
  • a three-electrode system is desirable for accurate potential measurement.
  • FIG. 6 shows the detection electrode 16 and the fixing portion 17 in the case of the three-electrode system.
  • the detection electrode 16 includes a counter electrode 161, a working electrode 162, and a reference electrode 163, which are electrochemical measurement electrodes.
  • the counter electrode 161 needs to emit (or receive) electrons at the same rate as the working electrode 162 receives (or emits) electrons.
  • the reference electrode 163 measures and controls the potential of the working electrode 162, and for example, an Ag / AgCl electrode can be used.
  • the fixing portion 17 is formed by physically adsorbing a ligand on the working electrode 162 and fixing it.
  • FIG. 6 (b) is a plan view of the three-electrode detection electrode 16 and the fixing portion 17 as viewed from the direction of the arrow in FIG. 5 (a).
  • FIG. 6C is an enlarged plan view of the detection electrode 16 and the fixing portion 17.
  • the counter electrode 161, the working electrode 162, the reference electrode 163, A region obtained by combining the surface 164 and the surface 165 can be defined as a detection electrode region 16a.
  • the detection electrode region 16a including the surface sandwiched between the respective electrodes is used as the droplet 2 in the hydrophobic film 14a or the hydrophobic film 14b. It is preferable to be provided smaller than the surface 2a in contact with the surface. By doing so, for example, the specimen almost covers the ligand, and the specimen and the ligand can be reacted efficiently. Similarly, the detection electrode region 16a is preferably provided smaller than the transfer electrode 12.
  • FIG. 7 is a flowchart showing an example of a procedure for detecting a specimen using the ELISA method. “S” represents each step in the flowchart.
  • a ligand that is a primary antibody is immobilized in advance on the immobilization unit 17 (S71).
  • a primary antibody solution MAB 10651 manufactured by R & D System
  • MAB 10651 manufactured by R & D System
  • the ligand is immobilized on the working electrode 162, but the present invention is not limited to this.
  • the fixing part 17 be close to the working electrode 162 in order to cause the electrochemical substance generated by the reaction in the fixing part 17 to undergo a redox reaction at the working electrode.
  • the detection electrode 16 a general one can be used.
  • the detection method uses electrochemical measurement here, an enzyme and a substrate that generate an electrochemical substance in an enzyme substrate reaction performed in a later step are selected, and the enzyme is pre-labeled with a secondary antibody. deep.
  • the enzyme is not particularly limited, and a desired enzyme can be appropriately used.
  • ALP Alkaline Phosphatase
  • glucose oxidase or the like
  • pAPP p-aminophenyl phosphate
  • potassium ferricyanide or the like
  • electrochemically active substance produced by the enzyme substrate reaction include pAP (paraaminophenol), potassium ferrocyanide, ferrocene, and ferrocene derivatives, or pAP, potassium ferrocyanide, ferrocene, and ferrocene derivatives.
  • ALP was used as an example of an enzyme
  • pAPP was used as a substrate.
  • a liquid 2 (a liquid droplet obtained by reacting an enzyme-labeled secondary antibody with an antigen) in which an adiponectin (R & D System 1065AP) solution and an enzyme-labeled secondary antibody are mixed in advance is used as a sample.
  • the droplet is conveyed to the fixing unit 17 and the primary antibody and the antigen are sufficiently reacted with each other by antigen-antibody reaction, and then the droplet 2 is removed (S74).
  • the specimen is detected by retaining the droplets in the fixing unit 17.
  • the residence time is not particularly limited as long as the residence time is long enough for the specimen and the ligand to react (for example, bind).
  • the antigen and the secondary antibody are mixed in advance, but these may be sequentially conveyed as separate liquids. In either case, it is possible to detect the analyte in the subsequent process, but the process can be simplified by using a droplet that has been mixed with an antigen-labeled secondary antibody in advance and then reacted. Can be
  • a Tris buffer solution is transported to the fixing part 17 as a washing solution to remove unbound proteins and the like (S75).
  • the solution used for washing is not limited to the Tris buffer, and any solution may be used as long as it does not interfere with the detection of the specimen.
  • the substrate solution containing pAPP is conveyed to the fixing unit 17 to cause an enzyme substrate reaction (S76).
  • the generated electrochemical substance pAP (p-Aminophenol) is detected by the detection electrode 16, and the dependence of the peak current value on the adiponectin concentration is measured (S77).
  • a method for measuring the sample amount for example, a method in which a calibration curve is prepared in advance and the sample amount is calculated based on the standard curve is generally used.
  • the desired specimen can be detected from the complex substance contained in the sample by the procedure as described above.
  • the immobilization of a ligand and a ligand is not limited to those listed here, and known ones can be used.
  • the ligand can be used as long as it is a substance capable of reacting with (for example, binding to) the sample.
  • a peptide, DNA, aptamer, MIP (Molecular Imprinted Polymer), etc. can be used. is there.
  • a chemical bond for example, a covalent bond, a hydrophobic bond, an ionic bond, a hydrogen bond
  • a comprehensive method, or the like can be used to immobilize the ligand.
  • a known method such as a DNA assay using DNA hybridization can be used as a detection method for detecting the specimen, and various signals used in these methods can be used.
  • Detection means can be used. For example, electrochemical detection method, colorimetric detection method, fluorescence detection method and the like. When an optical detection method is used for detection, it is necessary to select a transparent substrate for the substrate 15 that does not have a transport electrode. Next, detection using a fluorescence method will be described.
  • the specimen detection sensor itself does not have a detection electrode, and the specimen is detected using an external detection device or the like, or the detection section is separated from the fixed section. Different.
  • FIG. 8 is a cross-sectional view when the sample droplet 2 is fed to the specimen detection sensor 1a according to the present embodiment.
  • electrowetting is used as a method of transporting the droplet 2.
  • a plurality of transfer electrodes 12 are arranged on the transfer substrate 11, a dielectric film 13 and a hydrophobic film 14 a are sequentially formed thereon, and a ligand is fixed as a fixing portion 17 on the hydrophobic film 14 a. .
  • a hydrophobic film 14b is formed on the flow path side of the substrate 15 having no transfer electrode.
  • a droplet 2 is disposed between the transfer substrate 11 and the substrate 15 having no transfer electrode. The relationship between the droplet 2 and the transport electrode 12 is the same as that described in the first embodiment.
  • the fixing portion 17 is preferably provided smaller than the transport electrode 12.
  • the fixing portion 17 is preferably provided smaller than the surface 2a where the droplet 2 shown in FIG. 5A is in contact with the hydrophobic film 14a or the hydrophobic film 14b. This is for efficiently reacting the ligand with the specimen.
  • the fixing portion 17 is manufactured on the side of the transfer substrate 11, but may be manufactured on the side of the substrate 15 that does not have the transfer electrode.
  • the detection device 26 for example, general fluorescence detection can be used. In this case, it is possible to detect only a desired substance by entering the excitation light 31 into the droplet 2 and detecting the fluorescence 32 emitted from the droplet 2. Note that the detection device 26 is not limited to the above configuration, and a configuration based on an electrochemical detection method, a colorimetric detection method, a fluorescence detection method, or the like can be used as appropriate.
  • the immobilization part 17 is formed by immobilizing a ligand (for example, an antibody) by physical adsorption. Specifically, detection can be performed as follows using the procedure shown in FIG. 7 using an antibody as a ligand and an antigen as a specimen.
  • a ligand for example, an antibody
  • a ligand that is a primary antibody is immobilized in advance on the immobilization unit 17 (S71).
  • blocking is performed to prevent non-specific adsorption (S72). This is because not all of the fixing part 17 is covered with the ligand without a gap, and there is a gap, so that it is necessary to prevent nonspecific adsorption to the gap.
  • a blocking agent BSA, casein, etc. are used, for example.
  • the detection method uses fluorescence measurement
  • an enzyme and a substrate that generate a fluorescent substance are selected in an enzyme substrate reaction performed in a later step, and the enzyme is labeled in advance with a secondary antibody.
  • HRP horseradish peroxidase
  • ADHP 10-acetyl-3,7-dihydroxyphenoxyzine
  • QuantaBlu registered trademark
  • peroxidase or the like can be used as the enzyme
  • Amplex manufactured by Invitrogen
  • Invitrogen or the like can be used as the substrate.
  • a droplet 2 (a droplet obtained by reacting an enzyme-labeled secondary antibody with an antigen) in which a sample solution containing an antigen as a specimen and an enzyme-labeled secondary antibody are mixed in advance is conveyed to the fixing unit 17. To do. After the primary antibody and the antigen are sufficiently reacted with each other, the droplet is removed (S74).
  • the substrate solution is conveyed to the fixing unit 17 to cause an enzyme substrate reaction. If necessary, the enzyme substrate reaction may be stopped at this time by using an enzyme substrate reaction stop agent (S76).
  • S76 enzyme substrate reaction stop agent
  • the amount of specimen is measured by irradiating the fluorescent material generated by the enzyme substrate reaction with the excitation light 31 and detecting the resulting fluorescence 32 (S77).
  • the configuration is such that the excitation light 31 is emitted from the upper part of the fixed portion 17 and the fluorescence 32 is detected.
  • the configuration of FIG. 8 is a configuration based on fluorescence analysis, but a configuration based on general colorimetric analysis is also possible.
  • the chromogenic substrate OPD o-phenylenediamine
  • TMB 3,3 ′, 5,5′-tetramethyl-benzidine
  • the HRP enzyme can be used for the HRP enzyme to know the sample amount from the absorbance. it can.
  • a chromogenic substrate TMB, OPD, ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) can be used as an enzyme for peroxidase.
  • the sample detection sensor 1a has been described as having a configuration that does not include a detection unit.
  • the detection unit 16 ' may be provided separately from the fixing unit 17, as shown in FIG.
  • the detection means 16 ′ may be provided outside the flow path, for example, adjacent to the substrate 15 having no transfer electrode.
  • This embodiment is different from the first and second embodiments in that the transport electrodes are arranged on both upper and lower sides with the droplet 2 interposed therebetween.
  • the transport electrodes are arranged on both upper and lower sides with the droplet 2 interposed therebetween.
  • FIG. 10 is a cross-sectional view of the sample droplet 2 fed to the specimen detection sensor 1c according to the present embodiment.
  • electrowetting is used as a method of transporting the droplet 2.
  • a plurality of transfer electrodes 12 are arranged on the transfer substrate 11, a dielectric film 13 and a hydrophobic film 14 a are sequentially formed thereon, and a ligand is fixed as a fixing portion 17 on the hydrophobic film 14 a.
  • an upper electrode 120 and a hydrophobic film 14b are sequentially formed on the upper substrate 18 toward the flow path.
  • a droplet 2 is disposed between the transfer substrate 11 and the upper substrate 18.
  • the relationship between the droplet 2 and the transport electrode 12 is the same as that described in the first embodiment.
  • the fixing portion 17 is preferably provided smaller than the transfer electrode 12.
  • the fixing portion 17 is preferably provided smaller than the surface 2a where the droplet 2 shown in FIG. 5A is in contact with the hydrophobic film 14a or the hydrophobic film 14b. This is for efficiently reacting the ligand with the specimen.
  • the detection electrode 16 ′ is provided separately from the fixing portion 17 and can be provided outside the flow path, for example, along with the upper substrate 18.
  • the fixing portion 17 is manufactured on the transfer substrate 11 side, but may be manufactured on the upper substrate 18 side.
  • the voltage is applied between the transfer electrode 12 formed on the transfer substrate 11 and the upper electrode 120 formed on the upper substrate 18.
  • the upper electrode 120 is set to the ground potential, and a voltage is applied to the transfer electrode adjacent in the direction to be transferred.
  • the upper electrode 120 may be formed as one electrode or a plurality of electrodes.
  • the switch SW2 is in a connected state.
  • the switch SW2 is opened and the switch SW3 is connected.
  • the voltage is applied until the movement of the droplet 2 is observed, and when the threshold voltage is exceeded, the droplet 2 moves to the next transport electrode. By repeating this, the droplet 2 is moved to the target location.
  • the voltage application method is not limited to the direct current method, and can be performed by the alternating current method. Further, since the wettability changes according to the potential change on the substrate electrode, the droplet 2 can be transported not only by the above-described potential application method but also by applying the potential change.
  • the configuration described above includes the configuration in which the fixing portion 17 and the detection electrode 16 as shown in the first embodiment are provided at the same place, and the detection means 16 ′ described in the second embodiment is the present configuration. It is applicable to the structure detected by the detection apparatus 26, without including.
  • This embodiment is different from Embodiments 1 to 3 described above in that a droplet is transported using a surface acoustic wave (Surface Acoustic Wave).
  • a surface wave Surface Acoustic Wave
  • FIG. 11 is a cross-sectional view when the sample droplet 2 is fed to the specimen detection sensor 1d according to the present embodiment.
  • a piezoelectric film 19 and a transport electrode IDT (InterDigital Transducer) 20 are formed on the transport substrate 11 to excite surface waves.
  • the piezoelectric film 19 is made of, for example, LiNbO 3 and has a function of deforming when an electric field is applied.
  • the piezoelectric film 19 is not hydrophobic, it is desirable to provide a hydrophobic film on the piezoelectric film 19.
  • FIG. 12 is a plan view of the transfer electrode IDT20 as seen from the direction of the arrow in FIG.
  • the transfer electrode IDT 20 is a comb-shaped electrode as shown in FIG. 12, and a large electrode 202 is connected to the comb-shaped small electrode 201 so that a voltage can be applied collectively.
  • a voltage is applied from the large electrode 202 to the small electrode 201, a surface wave is excited by the small electrode 201.
  • a high frequency signal is applied to the transfer electrode IDT 20 formed on the piezoelectric film 19, an electric field is generated between the electrodes, a surface acoustic wave is excited, and the surface acoustic wave propagates on the piezoelectric film 19. .
  • the stability of the liquid feeding is prevented from being hindered by the influence of the immobilized ligand.
  • a sensor for detecting the specimen contained in the droplet could be realized.
  • the present invention can be configured as follows.
  • the transport electrode is a transport electrode that changes the wettability of the droplet, and the droplet is transported by changing the wettability of the droplet. Is preferred.
  • the transport electrode is a transport electrode for generating a surface wave, and the droplet is transported by the surface wave.
  • each area of the transport electrodes is larger than an area of the fixed portion.
  • the sample detection chip of the present invention it is preferable that at least a part of the flow path is covered with a hydrophobic film, and the fixing portion is not covered with the hydrophobic film.
  • the sample detection chip further includes droplet control means for controlling the amount of the droplets, and the droplet control means has an area where the droplets are in contact with the transport substrate.
  • the amount of the droplets is controlled to be larger than the area of the fixed portion.
  • the sample detection sensor of the present invention preferably includes the sample detection chip of the present invention and detection means for optically detecting the sample.
  • the sample detection sensor of the present invention preferably includes the sample detection chip of the present invention and at least one detection electrode for electrically detecting the sample.
  • the specimen detection sensor of the present invention it is preferable that at least a part of the flow path is covered with a hydrophobic film, and the fixing portion and the detection electrode are not covered with a hydrophobic film.
  • each area of the transport electrodes is larger than the areas of the detection electrodes and the fixing portion.
  • the sample detection sensor of the present invention includes the sample detection chip of the present invention, and the droplet control means disposed on the sample detection chip has an area where the droplet is in contact with the transfer substrate. It is preferable that the amount of the droplet is controlled so as to be larger than the area of the electrode and the fixed portion.
  • the specimen detection method of the present invention preferably includes a step of controlling the amount of the droplet.
  • the timing for performing the step of controlling the amount of droplets is not particularly limited, but can be performed, for example, before the step of transporting the inside of the flow path. More specifically, the step of controlling the amount of droplets may be a step of controlling the amount of droplets introduced into the flow path. This step can be performed by, for example, a pump for introducing a desired amount of droplets into the flow path.
  • the droplet is transported by changing the wettability of the droplet.
  • the droplet is transported by a surface wave in the transporting step.
  • the specimen is electrically detected using a detection electrode so that the area where the droplet contacts the transport substrate is larger than the areas of the fixed portion and the detection electrode.
  • the droplets are conveyed.
  • the detection electrode is preferably formed of at least a working electrode and a reference electrode.
  • the working electrode and the reference electrode are arranged so as to be in contact with the droplet simultaneously.
  • the working electrode and the reference electrode are provided at an interval that allows contact with the droplet at the same time.
  • the working electrode and the reference electrode can be provided such that there is a space between them so that they do not contact each other.
  • the width of the space and the distance between the working electrode and the reference electrode are not particularly limited as long as the working electrode and the reference electrode can be in contact with the droplet simultaneously.
  • the simultaneous contact means that there is a time for the droplets to contact the working electrode and the reference electrode at the same time in the process in which the droplets are transported in the flow path.
  • the detection electrode is preferably formed of a working electrode, a reference electrode, and a counter electrode.
  • the working electrode, the reference electrode, and the counter electrode are arranged so as to be in contact with the droplet simultaneously.
  • the working electrode, the reference electrode, and the counter electrode are provided at intervals such that they can be contacted simultaneously with the droplet. That is, the working electrode, the reference electrode, and the counter electrode can be provided such that a space is provided between them so that they do not contact each other.
  • the width of the space and the distance between the electrodes are not particularly limited as long as the working electrode, the reference electrode, and the counter electrode can be in contact with the droplet at the same time.
  • the simultaneous contact means that there is a time during which the droplet contacts the working electrode, the reference electrode, and the counter electrode at the same time in the process of transporting the droplet in the flow path. To do.
  • the present invention can be used in an analyzer for detecting a desired specimen.

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  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Spectroscopy & Molecular Physics (AREA)
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  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

Cette invention concerne un détecteur capable de détecter un échantillon contenu dans une gouttelette de liquide, sans perte de sensibilité, et sans diffusion entre des solutions (gouttelettes de liquide) acheminées les unes après les autres. A cette fin, on utilise une puce de détection d'échantillons qui détecte un échantillon au moyen d'une gouttelette retenue dans une partie d'immobilisation, et qui comprend : un substrat de transport comportant une électrode de transport ; un substrat faisant face au substrat de transport ; un conduit formé entre le substrat de transport et le substrat lui faisant face, et une partie d'immobilisation comprenant la partie du conduit où se trouve immobilisé un ligand.
PCT/JP2011/080121 2010-12-28 2011-12-26 Puce de détection d'échantillons, détecteur utilisant cette puce, et procédé de détection d'échantillons WO2012090960A1 (fr)

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JP2010-291433 2010-12-28
JP2010291433 2010-12-28
JP2011-264288 2011-12-02
JP2011264288A JP2012150098A (ja) 2010-12-28 2011-12-02 検体検出用チップ、それを用いたセンサ、及び検体検出方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112444547A (zh) * 2019-08-30 2021-03-05 希森美康株式会社 预处理方法及生物体内成分测定装置

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9458543B2 (en) * 2012-12-04 2016-10-04 Sharp Kabushiki Kaisha Active matrix electrowetting-on-dielectric device
GB2533951A (en) 2015-01-08 2016-07-13 Sharp Kk Active matrix device and method of driving
JP6654951B2 (ja) * 2016-03-31 2020-02-26 株式会社エンプラス 流体取扱装置
TW202024627A (zh) * 2018-11-09 2020-07-01 中國商深圳華大智造科技有限公司 基板上的數位微流體的多層電氣連接

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005199231A (ja) * 2004-01-19 2005-07-28 Tsukuba Technology Seed Kk 送液装置及びその駆動方法
JP2005257407A (ja) * 2004-03-10 2005-09-22 Olympus Corp 微量液体制御装置
JP2006317299A (ja) * 2005-05-13 2006-11-24 Hitachi High-Technologies Corp 液体搬送デバイス及び分析システム
JP2007132749A (ja) * 2005-11-09 2007-05-31 Hitachi Ltd 液体搬送装置
JP2008502882A (ja) * 2004-06-04 2008-01-31 ユニベールシテ・デ・スジャンス・エ・テクノロジー・ドゥ・リル 生化学的分析のために液滴を扱う装置、前記装置を製造する方法及びマイクロ流体分析

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005199231A (ja) * 2004-01-19 2005-07-28 Tsukuba Technology Seed Kk 送液装置及びその駆動方法
JP2005257407A (ja) * 2004-03-10 2005-09-22 Olympus Corp 微量液体制御装置
JP2008502882A (ja) * 2004-06-04 2008-01-31 ユニベールシテ・デ・スジャンス・エ・テクノロジー・ドゥ・リル 生化学的分析のために液滴を扱う装置、前記装置を製造する方法及びマイクロ流体分析
JP2006317299A (ja) * 2005-05-13 2006-11-24 Hitachi High-Technologies Corp 液体搬送デバイス及び分析システム
JP2007132749A (ja) * 2005-11-09 2007-05-31 Hitachi Ltd 液体搬送装置

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112444547A (zh) * 2019-08-30 2021-03-05 希森美康株式会社 预处理方法及生物体内成分测定装置

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