WO2019004438A1 - Kit et procédé d'analyse - Google Patents

Kit et procédé d'analyse Download PDF

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Publication number
WO2019004438A1
WO2019004438A1 PCT/JP2018/024844 JP2018024844W WO2019004438A1 WO 2019004438 A1 WO2019004438 A1 WO 2019004438A1 JP 2018024844 W JP2018024844 W JP 2018024844W WO 2019004438 A1 WO2019004438 A1 WO 2019004438A1
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Prior art keywords
electrode
working electrode
test substance
label
secondary antibody
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PCT/JP2018/024844
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English (en)
Japanese (ja)
Inventor
坂本　健
崔　京九
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Tdk株式会社
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Priority claimed from JP2018069967A external-priority patent/JP2019012056A/ja
Application filed by Tdk株式会社 filed Critical Tdk株式会社
Publication of WO2019004438A1 publication Critical patent/WO2019004438A1/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
    • G01N27/26—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
    • 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
    • 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
    • G01N27/416—Systems
    • G01N27/42—Measuring deposition or liberation of materials from an electrolyte; Coulometry, i.e. measuring coulomb-equivalent of material in an electrolyte
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the present invention relates to an analysis kit and an analysis method for analyzing a test substance using an antigen-antibody reaction.
  • Priority is claimed on Japanese Patent Application No. 2017-129440, filed on Jun. 30, 2017, and Japanese Patent Application No. 2018-069967, filed on Mar. 30, 2018, on June 30, 2017. The contents of which are incorporated herein by reference.
  • Detection of biological substances is performed in the fields of medicine, healthcare, environment and the like. Then, development of an analysis method capable of selectively quantifying a biological substance to be measured from a plurality of biological substances with high sensitivity and easy operability is desired.
  • An immunoassay is known as one of the methods capable of selectively measuring a minute amount of biological substance in a liquid with high sensitivity.
  • the immunoassay is a method of quantifying an antigen using a reaction (antigen-antibody reaction) between a biological substance to be measured (antigen, hapten, etc.) and a substance (antibody) that binds to the antigen.
  • the sandwich method is known as a method of quantifying an antigen.
  • the sandwich method is a method of sandwiching (sandwiching) an antigen between a solid on which the primary antibody is immobilized and a label on which the secondary antibody is immobilized. That is, the sandwich method is a method of capturing an antigen with a primary antibody, binding the captured antigen to a secondary antibody, and quantifying the label immobilized on the secondary antibody bound to the antigen.
  • a method of quantifying a label a method of using metal particles as a label and quantifying the amount of the metal particles using an electrochemical method is known.
  • antigens and antibodies do not have electrical conductivity, it is difficult to quantify labels (metal particles) bound to antigens and antibodies directly using electrochemical methods.
  • Patent Document 1 discloses a diagnostic kit including at least one reagent labeled with colloidal metal particles, at least one electrode, and a reagent for chemically dissolving the colloidal metal particles.
  • a colloidal metal particle that is a label is chemically dissolved, and then a solution in which the colloidal metal particle that is a label is dissolved is transferred to an electrode for reduction. Then, after the reduced metal is deposited on the electrode, the metal deposited on the surface of the electrode is electrically redissolved. The amount of metal deposited on the surface of the electrode is then determined by analyzing the voltammetric peaks that appear after re-dissolution.
  • a method of quantifying the amount of metal particles using a metal particle as a label and using an electrochemical method is a useful method in terms of sensitivity and accuracy.
  • the operation is complicated because, for example, a step of dissolving colloidal metal particles once is required, and it takes time to obtain an analysis result. . Therefore, its introduction is difficult in clinical examinations at medical sites.
  • the present invention has been made in view of the above problems, and its object is to provide an analysis kit and an analysis method which are easy to operate, can analyze a test substance with high selectivity and high sensitivity. It is to do.
  • a sensor using a conductive diamond electrode or a conductive diamond-like carbon electrode on which a primary antibody is immobilized as a working electrode, and a secondary antibody on which a label capable of oxidation-reduction or capable of promoting an oxidation-reduction reaction is immobilized The antigen (the test substance) and the primary antibody are bound and immobilized on the working electrode. Then, the antigen and secondary antigen are bound, and the label on which the secondary antibody bound to the antigen is immobilized is brought into close contact with or in close proximity to the working electrode.
  • the catalytic function that promotes the oxidation, reduction, or redox reaction by electrochemical means that the label attached to or in close proximity to the working electrode can be quantified without chemically dissolving the label. Then, the amount of current generated when the label is exposed to a catalytic function that promotes oxidation or reduction or the redox reaction thereof by an electrochemical method has a high correlation with the amount of antigen, and the current After confirming that it is possible to determine the amount of antigen from the amount, the present invention has been completed. That is, the present invention provides the following means in order to solve the above problems.
  • the analysis kit according to the first aspect has a working electrode, a reference electrode, and a counter electrode, and the working electrode is a conductive diamond electrode or a conductive diamond-like carbon electrode, and the working electrode It is characterized in that it comprises a sensor comprising a primary antibody immobilized on the surface, and a solution comprising a secondary antibody on which a redox-enabled or a label capable of promoting a redox reaction is immobilized.
  • the label capable of the oxidation-reduction or promoting the oxidation-reduction reaction may be at least one selected from the group consisting of an enzyme, a metal complex and a metal nanoparticle.
  • the label capable of the oxidation-reduction or promoting the oxidation-reduction reaction is a metal nanoparticle, and the metal nanoparticle contains sulfur. It is also good.
  • the reference electrode may be a silver-silver chloride electrode.
  • the counter electrode may be a carbon electrode, a noble metal electrode, a conductive diamond electrode or a conductive diamond-like carbon electrode.
  • the analysis method according to the second aspect is an analysis method for analyzing a test substance contained in a test substance solution, comprising: a working electrode, a reference electrode, and a counter electrode
  • the working electrode of the sensor wherein the electrode is a conductive diamond electrode or a conductive diamond-like carbon electrode, and a primary antibody binding to the test substance is immobilized on the surface of the working electrode, and the test substance solution
  • To the primary antibody of the working electrode A second binding step of connecting the test substance with a redox-reducible label by binding the combined test substance and a secondary antibody; and washing the working electrode to thereby
  • the analysis method according to the third aspect is an analysis method for analyzing a test substance contained in a test substance solution, comprising: a working electrode, a reference electrode, and a counter electrode
  • the working electrode of the sensor wherein the electrode is a conductive diamond electrode or a conductive diamond-like carbon electrode, and a primary antibody binding to the test substance is immobilized on the surface of the working electrode, and the test substance solution
  • the primary of the working electrode A second binding step of connecting the test substance and the label promoting the redox reaction by binding the test substance bound to the body and the secondary antibody; and washing the working electrode A second washing step of removing a solution containing a secondary antibody which is not bound to the test substance and on which a label is immobilized which promotes the oxidation-reduction reaction, and between the working electrode and the counter electrode, oxidation-reduction
  • the redox-capable label required for the oxidation or reduction of the other redox-capable substance, with the possible substances present and then applying a voltage between the working electrode and the counter electrode
  • the current amount measuring step of measuring the current amount, and the calculation step of determining the labeling amount promoting oxidation-reduction reaction from the current amount and calculating the mass of the test object from the labeling amount promoting the oxidation-reduction reaction It is characterized by
  • an analysis kit and an analysis method capable of analyzing a test substance with high selectivity and sensitivity with simple operation it is possible to provide an analysis kit and an analysis method capable of analyzing a test substance with high selectivity and sensitivity with simple operation.
  • FIG. 2 is a cross-sectional view taken along line II-II of FIG. It is a flowchart explaining the analysis method concerning one embodiment of the present invention. It is a conceptual diagram explaining the analysis method concerning one embodiment of the present invention. It is the graph which plotted the antigen concentration of the sample for each antigen analysis used in Example 1, and the electric current amount required in order to ionize the cobalt nanoparticle which is a label
  • the analysis kit of the present embodiment is an analysis kit for analyzing a test substance contained in a test substance solution using an antigen-antibody reaction.
  • the test substance is, for example, a biomaterial, and in particular, a protein or metaphorome.
  • the analysis kit of the present embodiment comprises a liquid containing a sensor and a secondary antibody.
  • the sensor is immobilized with a primary antibody that supplements the test substance contained in the test substance solution, and the secondary antibody is immobilized with a label for quantifying the captured test substance.
  • FIG. 1 is a plan view showing an embodiment of a sensor.
  • FIG. 2 is a cross-sectional view taken along line II-II of FIG.
  • the sensor 10 shown in FIG. 1 includes a first substrate 11, a working electrode 12, a counter electrode 13 and a reference electrode 14 provided in the vicinity of one end on the surface (upper surface in FIG.
  • the second substrate 16 has a window 15 formed to expose the working electrode 12, the counter electrode 13 and the reference electrode 14 bonded thereto.
  • the second substrate 16 covers a portion of the lead wires 12 a, 13 a and 14 a other than the vicinity of the other end of the first substrate 11.
  • the working electrode 12 is a conductive diamond electrode or a conductive diamond-like carbon electrode (DLC electrode).
  • a boron-doped boron-doped diamond electrode can be used as the conductive diamond electrode.
  • the conductive diamond electrode is a crystalline carbon electrode having a diamond structure having sp 3 bonds.
  • the conductive DLC electrode is an amorphous carbon electrode mainly composed of carbon and hydrogen and in which sp 3 bonds and sp 2 bonds are mixed.
  • the doped p-type semiconductor DLC electrodes can be used.
  • the conductive diamond electrode and the DLC electrode based on sp 3 bonded carbon and the DLC electrode have very few processes in which the chemical substance to be oxidized or reduced by the electrochemical reaction is adsorbed. For this reason, for example, the inner zone oxidation-reduction reaction through adsorption on the electrode by hydrogen, hydroxide or ions thereof caused by water hardly occurs. As a result, since the noise current, which is referred to as the residual current, becomes extremely small, it is possible to detect the electrochemical reaction of the analyte to be detected with a high SN ratio.
  • the working electrode 12 has a primary antibody fixed on the surface (upper surface in FIG. 2).
  • the primary antibody is appropriately selected and used in accordance with the test substance (antigen) to be measured. Any primary antibody can be used without particular limitation as long as it has high affinity for the test substance to be measured and binds to the antigen.
  • the counter electrode 13 is composed of a conductive material for an electrode that is usually used in a sensor for electrochemical measurement.
  • a noble metal electrode such as a carbon electrode, a platinum electrode and a gold electrode, the same conductive diamond electrode or conductive DLC electrode as the working electrode 12 can be used.
  • a silver-silver chloride electrode or a mercury-mercury chloride electrode can be used as the reference electrode 14.
  • the reference electrode 14 is preferably a silver-silver chloride electrode.
  • the first substrate 11 is a support that supports the working electrode 12, the counter electrode 13, and the reference electrode 14.
  • the first substrate 11 may have physical strength that can withstand use as an electrochemical sensor.
  • the working electrode 12 is an n-type semiconductor DLC electrode
  • the first substrate 11 is preferably an n-type crystalline silicon substrate.
  • the working electrode 12 is a p-type semiconductor DLC electrode
  • the first substrate 11 is preferably a p-type crystalline silicon substrate. As a result, interface resistance such as a Schottky barrier is less likely to occur between the first substrate 11 and the working electrode 12.
  • the second substrate 16 a substrate similar to the first substrate 11 is used.
  • the first substrate 11 and the second substrate 16 are bonded by an adhesive 17.
  • the liquid containing the secondary antibody is composed of a solvent and a secondary antibody dispersed in the solvent and capable of oxidation-reduction or a label capable of promoting the oxidation-reduction reaction is immobilized.
  • the label capable of oxidation-reduction or promoting the oxidation-reduction reaction can be oxidized by releasing an electron, can be reduced by accepting an electron, or both Possible is a chemical substance that is an enzyme or catalyst that promotes the redox of another redox substance and is immobilized on a secondary antibody.
  • the label capable of redox or promoting the redox reaction is preferably at least one selected from the group consisting of an enzyme, a metal complex and a metal nanoparticle.
  • Examples of the enzyme used as a label in the present embodiment include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase and glucose amylase.
  • GDH glucose dehydrogenase
  • PQQ-GDH pyrroloquinoline quinone
  • FAD-GDH flavin adenine dinucleotide
  • NAD-GDH nicotinamide adenine dinucleotide
  • NADP nicotine adenine dinucleotide phosphate
  • Aspergillus oryzae FAD-GDH which does not use maltose other than glucose or galactose as a substrate, wild type FAD-GDH of Aspergillus oryzae or a modified form thereof is preferable.
  • wild type FAD-GDH of Aspergillus oryzae or a modified form thereof is preferable.
  • flavin adenine dinucleotide, pyrroloquinoline quinone or the like may be added.
  • ferricyanide ion, ferrocene, ruthenium compound, hexachloroiridium (IV) ion, bis (2,2'-bipyridine) dichlororuthenium, bis (2,2'-bipyridine) dichloroosmium, iodine etc. as mediator. You may.
  • metal complexes used as labels in this embodiment include iron complexes, copper complexes, iridium complexes, ruthenium complexes, and osmium complexes, such as ferricyanide ion, ferrocene, hexachloro iridium (IV) ion, bis (2,2 '-Bipyridine) dichlororuthenium, ruthenocene, bis (2,2'-bipyridine) dichlororuthenium, bis (2,2'-bipyridine) dichlororuthenium, bis (2,2'-bipyridine) dichlororuthenium, bis (2,2'-bipyridine) dichlororuthenium, bis (2,2'-bipyridine) dichloroosmium and the like.
  • the metal nanoparticles used as a label in the present embodiment preferably include at least one metal selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel.
  • the metals may be used alone or in combination of two or more.
  • gold nanoparticles and silver nanoparticles are preferred.
  • One of these metal nanoparticles may be used alone, or two or more of these metal nanoparticles may be used in combination.
  • the metal nanoparticles preferably have an average particle size in the range of 1 nm to 100 nm, and more preferably in the range of 10 nm to 50 nm.
  • the liquid containing the metal nanoparticles is preferably a dispersion of metal nanoparticles.
  • the metal nanoparticles may contain sulfur.
  • the sulfur may be attached to the surface of the metal particle nanoparticles or may be intercalated between metal atoms. Sulfur has the effect of suppressing the oxidation of metal nanoparticles.
  • the sulfur content of the metal nanoparticles is preferably in the range of 0.001% by mass to 0.5% by mass.
  • the secondary antibody is appropriately selected and used in accordance with the test substance (antigen) to be measured. Any secondary antibody may be used without particular limitation as long as it has high affinity to the analyte to be measured and binds to the antigen.
  • an aqueous solvent an organic solvent and a mixture thereof can be used.
  • aqueous solvents include water and buffers.
  • phosphate buffered saline (PBS) can be used.
  • the organic solvent include monohydric alcohols such as methanol, ethanol, 1-propanol and 2-propanol, and ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone.
  • the liquid containing the secondary antibody may further contain a thickener, a surfactant, a dispersant, an antioxidant and the like.
  • the liquid containing the secondary antibody is a liquid containing the secondary antibody to which a redox-capable label is immobilized, or a redox agent, and a secondary antibody to which a label capable of promoting the redox agent is immobilized. It is preferable that it is a liquid containing.
  • the liquid containing the secondary antibody to which the redox-provable label is fixed is preferably a liquid containing metal nanoparticles.
  • the redox agent is preferably hydrogen peroxide water
  • the redox agent is preferably an enzyme or a metal complex.
  • FIG. 3 is a flow diagram illustrating an analysis method according to an embodiment of the present invention.
  • FIG. 4 is a conceptual diagram for explaining an analysis method according to an embodiment of the present invention.
  • the analysis method of the present embodiment includes a first bonding step S01, a first washing step S02, a second bonding step S03, a second washing step S04, a magnetic field application step S05, a current amount measuring step S06, Calculation step S07 is included.
  • First bonding step S01 In the first bonding step S01, the working electrode 12 of the above-described sensor 10 is brought into contact with the test substance solution. As shown in FIG. 4A, on the surface of the working electrode 12, a primary antibody 31 capable of selectively binding to a test substance to be measured is fixed. Therefore, in the first binding step S01, only the test substance 32 to be measured is captured by the primary antibody 31, as shown in FIG. 4 (b).
  • First cleaning step S02 In the first cleaning step S02, the working electrode 12 is washed to remove the test substance solution adhering to the working electrode 12.
  • the washing solution an aqueous solvent or an organic solvent can be used.
  • the working electrode 12 is brought into contact with the aforementioned dispersion of magnetic metal nanoparticles.
  • a secondary antibody 34 capable of selectively binding to the test substance 32, which is an object to be measured, is fixed. Therefore, as shown in FIG. 4C, in the second binding step S03, the test substance 32 and the secondary antibody 34 are bound, and the magnetic metal nanoparticles 33 to be labeled are connected to the test substance 32. .
  • the working electrode 12 is washed to remove the dispersion liquid of magnetic metal nanoparticles attached to the working electrode 12.
  • the magnetic metal nanoparticles 33 not connected to the test substance 32 are removed by the second cleaning step S04.
  • As the washing solution an aqueous solvent or an organic solvent can be used.
  • Magnetic field application step S05 In the magnetic field application step S05, a magnetic field is applied to the magnetic metal nanoparticles 33 connected to the test substance 32 in the presence of a solvent.
  • the magnetic field is preferably applied from the back side of the working electrode 12 in the direction in which the magnetic metal nanoparticles 33 are attracted.
  • the magnetic field can be applied, for example, by disposing the magnet 35 on the side of the back surface (the lower surface in FIG. 4D) of the working electrode 12 as shown in FIG. 4D.
  • a permanent magnet or an electromagnet can be used.
  • the magnetic metal nanoparticles 33 connected to the test substance 32 come into contact with the working electrode 12.
  • the solvent is not particularly limited as long as it can move the magnetic metal nanoparticles 33 into contact with the working electrode 12 by the applied magnetic field, but the conductivity can be used in the next current amount measurement step S06. It is preferred to use an organic solvent.
  • a voltage is applied between the working electrode 12 and the counter electrode 13 to ionize (oxidize) the magnetic metal nanoparticles 33 in the presence of the conductive solvent, and the total amount of the magnetic metal nanoparticles 33 is Measure the amount of current until ionization.
  • the magnetic metal nanoparticles 33 are cobalt nanoparticles, as shown in FIG. 4 (d)
  • the cobalt nanoparticles are divalent ions by applying a voltage between the working electrode 12 and the counter electrode 13. Measure the amount of current when dissolving.
  • the lead wires 12a, 13a and 14a of the sensor 10 are connected to a potentiostat, and while applying a voltage between the working electrode 12 and the counter electrode 13 using the voltammetry method, the working electrode 12 and the counter electrode Measure the amount of current flowing between it and 13.
  • an electrolyte solution as the conductive solvent.
  • chlorides such as potassium chloride, sodium chloride and lithium chloride can be used.
  • the chloride destroys the oxide film (passive film) formed on the surface of the magnetic metal nanoparticles 33 when the magnetic metal nanoparticles 33 are ionized, exposing the magnetic metal to a solution, and advancing the ionization. It has the effect of making it easy to do.
  • An aqueous solvent can be used as a solvent of the electrolyte solution. Examples of aqueous solvents include water and buffers.
  • the chloride ion concentration of the electrolyte solution is preferably in the range of 0.05 mol / L to 1.0 mol / L.
  • the amount (the labeled amount) of the magnetic metal nanoparticles 33 is obtained from the current amount, and the mass of the test object is calculated from the amount of the magnetic metal nanoparticles 33.
  • the amount of current obtained in the step of current amount measurement step S06 correlates with the amount of the magnetic metal nanoparticles 33 (that is, the mass of the test object). Therefore, by preparing a calibration curve using a sample containing a known amount of a test substance, it becomes possible to accurately quantify the test substance contained in the test substance solution.
  • the analysis method of the present embodiment has been described with an example where magnetic metal nanoparticles are used as a label capable of redox or promoting a redox reaction.
  • an enzyme, a metal complex, or a metal nanoparticle having no magnetism is used as a label capable of redox or promoting a redox reaction, it is not necessary to perform the magnetic field application step S05.
  • the magnetic field application step S05 may be omitted even when magnetic metal nanoparticles are used as a label capable of oxidation reduction or promoting oxidation reduction reaction.
  • the conductive diamond electrode and the conductive diamond-like carbon electrode (DLC electrode) used as the working electrode of the sensor in this embodiment have high sensitivity, and the minute redox current flowing between the working electrode and the label is detected at a high SN ratio can do. For this reason, even if the label and the working electrode are not in close contact with each other, the amount of label can be measured with high accuracy.
  • the labeling amount is calculated by ionizing (oxidizing) the magnetic metal nanoparticles 33 in the current amount measurement step S06, but the present invention is not limited to this.
  • a substance capable of redox reaction is present between the working electrode and the counter electrode, and then a voltage is applied between the working electrode and the counter electrode to promote the redox reaction.
  • the amount of labeling may be calculated by measuring the amount of current required for oxidation or reduction.
  • a mediator may be used for this.
  • a method of using hydrogen peroxide as a substance capable of oxidation and reduction and a ferrocyanide ion as a mediator it can be performed as follows.
  • the conductive diamond electrode or the conductive diamond-like carbon electrode (DLC electrode) is used as the working electrode 12 of the sensor 10, the electrochemistry of the magnetic metal nanoparticles It is possible to detect the reaction at a high SN ratio.
  • the primary antibody is immobilized on the working electrode 12 of the sensor 10, the test substance contained in the test substance solution can be complemented with high selectivity.
  • the dispersion liquid of magnetic metal nanoparticles to which the secondary antibody is immobilized is provided, magnetic metal nanoparticles are used as a label, and the amount of the magnetic metal nanoparticles is determined by By quantifying using a chemical method, it is possible to analyze with high sensitivity the analyte captured by the primary antibody.
  • the magnetic metal nanoparticles connected to the test substance contain a solvent
  • the test is carried out using an electrochemical method without carrying out the conventional chemical dissolution step of the metal.
  • the substance can be quantified. Therefore, according to the analysis kit and the analysis method of the present invention, it is possible to simplify the operation and analyze the test substance with high selectivity and high sensitivity.
  • Cobalt nanoparticles described above ovalbumin (OA, grade III), anti-goat IgG, phosphate buffered saline (PBS), and polyethylene glycol sorbitan monolaurate (Tween 20, non-ionic surfactant) It mixed and the cobalt nanoparticle dispersion liquid with a secondary antibody which fixed anti-goat IgG (secondary antibody) on the surface of cobalt nanoparticles was produced.
  • the concentration of cobalt nanoparticles in the cobalt nanoparticle dispersion liquid with secondary antibody was 0.007% by mass.
  • anti-goat IgG a polyclonal antibody commercially available from Jackson Immunoresearch Laboratories was used.
  • the working electrode of the sensor with a primary antibody was washed using a washing solution to wash away the sample for antigen analysis (first washing step).
  • PBS was used as the washing solution.
  • the working electrode of the sensor with a primary antibody was washed using a washing solution to wash out the cobalt nanoparticle dispersion with a secondary antibody (second washing step).
  • PBS was used as the washing solution.
  • a neodymium magnet was disposed closely to the outside of the bottom of the plastic rectangular container, and a magnetic field was applied to the working electrode of the sensor with a primary antibody in the direction to attract cobalt nanoparticles from the back side of the sensor (magnetic field application step).
  • FIG. 1 to No. The graph which plotted the antigen concentration of the sample for each antigen analysis of 6, and the electric current amount (namely, electric current amount required in order to ionize a cobalt nanoparticle) which flowed between the working electrode and the counter electrode is shown. From the graph of FIG. 5, it was confirmed that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis. Therefore, by preparing a standard curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance), it is possible to accurately determine the antigen (test substance) contained in the test substance solution. It was confirmed that it would be possible.
  • a standard curve current value-antigen concentration curve
  • Example 2 In the magnetic field application step, that is, a neodymium magnet is disposed closely to the outside of the bottom of a plastic rectangular container, and a magnetic field is not applied to the working electrode of the sensor with primary antibody from the back side of the sensor Except for the above, in the same manner as in Example 1, a graph was created in which the antigen concentration of each antigen analysis sample was plotted with the amount of current required to ionize the cobalt nanoparticle that is the label of the antigen. The results are shown in FIG. From the graph of FIG. 6, it was confirmed that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis.
  • the antigen contained in the test substance solution is prepared by preparing a calibration curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance). It was confirmed that it was possible to quantify (the test substance).
  • Gold Nanoparticles-Dispersed Liquid with Secondary Antibody Gold Colloid Solution (20 nm particle size) manufactured by Tanaka Kikinzoku Co., Ltd., ovalbumin (OA, grade III), anti-goat IgG, phosphate buffered saline (PBS) And polyethylene glycol sorbitan monolaurate (Tween 20, non-ionic surfactant) mixed, and a gold nanoparticle dispersion liquid with a secondary antibody on which anti-goat IgG (secondary antibody) is immobilized on the surface of the gold nanoparticles Made.
  • the concentration of gold nanoparticles in the secondary antibody-loaded gold nanoparticle dispersion was 0.007% by mass.
  • anti-goat IgG a polyclonal antibody commercially available from Jackson Immunoresearch Laboratories was used.
  • the working electrode of the sensor with a primary antibody was washed using a washing solution to wash away the sample for antigen analysis (first washing step).
  • PBS was used as the washing solution.
  • the working electrode of the sensor with a primary antibody was washed using a washing solution to wash out the secondary antibody-bearing gold nanoparticle dispersion (second washing step).
  • PBS was used as the washing solution.
  • FIG. 1 to No. The graph which plotted the antigen concentration of the sample for each antigen analysis of 6, and the electric current amount (namely, electric current amount required in order to ionize a gold nanoparticle) which flowed between the working electrode and the counter electrode is shown. From the graph of FIG. 5, it was confirmed that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis. Therefore, by preparing a standard curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance), it is possible to accurately determine the antigen (test substance) contained in the test substance solution. It was confirmed that it would be possible.
  • a standard curve current value-antigen concentration curve
  • bioscience anti-8-hydroxydeoxyguanosine (anti-8-OHdG) antibody SMC-155D
  • Enzyme-labeled secondary antibody solution Enzyme-labeled secondary antibody includes horseradish peroxidase (HRP) -labeled 8-hydroxydeoxyguanosine (anti-8-OHdG) antibody (SMC-155D-HRP) manufactured by Stressmark Biosciences Inc. Using this, it was diluted to 10 ⁇ g / mL with phosphate buffered saline (PBS) to prepare an enzyme-labeled secondary antibody solution.
  • HRP horseradish peroxidase
  • SMC-155D-HRP 8-hydroxydeoxyguanosine
  • No. 1 with different antigen concentrations. 1 to No. I prepared six.
  • the following No. 1 to No. 6 was prepared by mixing PBS buffer containing 0.1% Tween 20 with 8-OH dG (antigen) so that the antigen concentration became the following concentration.
  • No. 1: Antigen concentration 0.01 ng / mL
  • No. 2: Antigen concentration 0.1 ng / mL
  • No. 3: Antigen concentration 1 ng / mL
  • No. 4: Antigen concentration 10 ng / mL
  • Antigen concentration 100 ng / mL
  • the working electrode of the sensor with a primary antibody was washed using a washing solution to wash away the sample for antigen analysis (first washing step).
  • PBS was used as the washing solution.
  • the present invention can provide an analysis kit and an analysis method capable of analyzing a test substance with high sensitivity and sensitivity with simple scanning.

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Abstract

L'invention concerne un kit d'analyse qui est caractérisé en ce qu'il comprend : un capteur ayant une électrode active, une électrode de référence et une contre-électrode, l'électrode active étant une électrode en diamant électroconducteur ou une électrode en carbone de type diamant électroconducteur, des anticorps primaires étant fixés à une surface de l'électrode active ; et un liquide comprenant des anticorps secondaires auxquels est fixée un marqueur qui est apte à mettre en œuvre une oxydoréduction ou de favoriser une réaction d'oxydoréduction.
PCT/JP2018/024844 2017-06-30 2018-06-29 Kit et procédé d'analyse WO2019004438A1 (fr)

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JP2017129440 2017-06-30
JP2018-069967 2018-03-30
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021205454A1 (fr) * 2020-04-08 2021-10-14 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Systèmes et procédés de détermination de la prévalence du sras-cov-2 dans une population

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62501171A (ja) * 1984-12-19 1987-05-07 アイキュ−・（バイオ）・リミテッド 生化学的アッセイのための方法と装置
JP2008157730A (ja) * 2006-12-22 2008-07-10 Rohm Co Ltd 生体分子または生体関連物質の測定方法
JP2009025217A (ja) * 2007-07-20 2009-02-05 Japan Advanced Institute Of Science & Technology Hokuriku 銀イオンの測定方法及び被検物質の測定方法
JP2009276343A (ja) * 2008-04-17 2009-11-26 Canon Inc 免疫測定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62501171A (ja) * 1984-12-19 1987-05-07 アイキュ−・（バイオ）・リミテッド 生化学的アッセイのための方法と装置
JP2008157730A (ja) * 2006-12-22 2008-07-10 Rohm Co Ltd 生体分子または生体関連物質の測定方法
JP2009025217A (ja) * 2007-07-20 2009-02-05 Japan Advanced Institute Of Science & Technology Hokuriku 銀イオンの測定方法及び被検物質の測定方法
JP2009276343A (ja) * 2008-04-17 2009-11-26 Canon Inc 免疫測定方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021205454A1 (fr) * 2020-04-08 2021-10-14 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Systèmes et procédés de détermination de la prévalence du sras-cov-2 dans une population

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