JP7310697B2 - Test substance analysis kit and test substance analysis method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an analysis kit and an analysis method for analyzing a test substance using an antigen-antibody reaction.

Detection of biological substances is performed in fields such as medicine, health care, and the environment. There is a demand for development of an analysis method that can selectively quantify a biological substance to be measured from a plurality of biological substances with high sensitivity and simple operability.

Immunoassay is known as one of methods capable of selectively and highly sensitively measuring minute amounts of biological substances in liquids. An immunoassay method is a method of quantifying an antigen by using a reaction (antigen-antibody reaction) between a biological substance (antigen, hapten, etc.) to be measured and a substance (antibody) that binds to the antigen.

A sandwich method is known as a method for quantifying an antigen. The sandwich method is a method of sandwiching an antigen between a solid on which a primary antibody is immobilized and a label on which a secondary antibody is immobilized. That is, the sandwich method is a method of capturing an antigen with a primary antibody, binding the captured antigen with a secondary antibody, and quantifying the label immobilized on the secondary antibody bound to the antigen.
A competitive method is also known as a method for quantifying an antigen. In the competitive method, the antigen, which is the test substance contained in the solution to be analyzed, is allowed to undergo an antigen-antibody reaction with the labeled antibody, and then the unreacted labeled antibody generated at this time is quantified. It is a way to

As a method for quantifying a label, in addition to the ELISA method, a method using metal particles as a label and quantifying the amount of the metal particles using an electrochemical method is known.
US Pat. No. 6,200,000 discloses an immunoassay method comprising at least one reagent labeled with colloidal metal particles, at least one electrode, and a reagent for chemically dissolving the colloidal metal particles. In the immunoassay method disclosed in this Patent Document 1, colloidal metal particles as labels are chemically dissolved, then the solution of the metal is transferred to an electrode and reduced, and after the reduced metal is deposited on the electrode, , the amount of metal deposited on the surface of the electrode is measured by electrically redissolving the metal and analyzing the voltammetric peaks that appear after the redissolution. The colloidal metal particles are metal nanoparticles dispersed in a solvent.

On the other hand, in immunoassay methods using antigen-antibody reaction such as ELISA, non-specific adsorption of the secondary antibody to the solid phase and direct non-specific binding of the secondary antibody to the primary antibody without the intervention of the antigen are observed. In order to prevent this, proteins such as bovine serum albumin (BSA) and casein, and bio-related substances such as gelatin and skimmed milk containing proteins are used as blocking agents (for example, Patent Document 2, etc.).
However, since these proteins and substances containing proteins are easily degraded by bacteria and the like, it is difficult to store analysis kits using these substances as blocking agents for a long period of time.

Japanese Patent Publication No. 2004-512496 Japanese Patent No. 6367783

Blocking in the analysis chip of the test substance (antigen) that detects the antigen-antibody reaction by electrochemical oxidation-reduction current using the working electrode to which the primary antibody is immobilized or the metal nanoparticles to which the secondary antibody is immobilized. When proteins or substances containing proteins such as BSA, skimmed milk and gelatin are used as agents, there is a problem that the storage period of the analysis kit is short because proteins are degraded by bacteria and the like.

The present invention has been made in view of the above problems, and an object of the present invention is to provide an analysis kit capable of analyzing a test substance with high selectivity and high sensitivity with a simple operation and having a long storage period. , to provide a method for analyzing a test substance using the analysis kit.

The present inventors have found that the surface of a metal nanoparticle on which a secondary antibody that specifically binds to a test substance is immobilized or the surface of a working electrode on which a primary antibody is immobilized is treated as an unpaired electron pair or a lone electron By blocking with a heteroorganic compound having a pair or an organic compound having a triple bond, non-specific adsorption of the secondary antibody to the working electrode and direct non-specific adsorption of the secondary antibody to the primary antibody without the antigen. It is possible to analyze the test substance with high selectivity and high sensitivity with a simple operation, and it is possible to store the analysis kit for a long time. reached.
That is, the present invention provides the following means in order to solve the above problems.

(1) The analysis kit according to the first aspect comprises a sensor having a working electrode, a reference electrode, and a counter electrode, a primary antibody immobilized on the surface of the working electrode, and a test substance on the surface a secondary antibody that specifically binds to is immobilized, and the surface other than the portion where the secondary antibody is immobilized has an unpaired electron pair or a lone electron pair, or an organic compound having a triple bond characterized by containing metal nanoparticles blocked by.

(2) In the analysis kit according to the above aspect, the surface of the working electrode other than the portion where the primary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. may have been

(3) The analysis kit according to the second aspect has a working electrode, a reference electrode, and a counter electrode, a primary antibody is immobilized on the surface of the working electrode, and the primary antibody is immobilized. A sensor in which the surface of the working electrode other than the moieties is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond; It is characterized by containing metal nanoparticles to which the following antibody is immobilized.

(4) In the analysis kit according to the first aspect and the second aspect, the hetero organic compound having an unpaired electron pair or a lone electron pair is an organic sulfur compound, an organic nitrogen compound, or an organic oxygen compound, good too.

(5) In the analysis kits according to the first aspect and the second aspect, the organic compound having a triple bond may be an acetylenic organic compound.

(6) In the analysis kit according to the first aspect and the second aspect, the metal nanoparticles are at least selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel. It may contain a kind of metal.

(7) A method for analyzing a test substance according to a third aspect is a 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, in which a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode, is brought into contact with the test substance solution, and the primary antibody of the working electrode and the test substance are brought into contact with each other. a first binding step of binding a substance solution to a test substance; the working electrode; and a secondary antibody that specifically binds to the test substance is immobilized on the surface, and the secondary antibody is immobilized. The surface other than the part is contacted with a dispersion of metal nanoparticles in which metal nanoparticles blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond are dispersed. a second binding step of binding the test substance and the metal nanoparticles by binding the test substance bound to the primary antibody of the working electrode with the secondary antibody; A current amount measuring step of applying a voltage between the counter electrode to ionize the metal nanoparticles in the presence of an electrolyte solution, and measuring the amount of current until the entire amount of the metal nanoparticles is ionized; and a calculating step of obtaining the amount of metal nanoparticles from the amount and calculating the amount of the test substance from the amount of metal nanoparticles.

(8) The analysis method according to the fourth aspect has a working electrode, a reference electrode, and a counter electrode, and a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode. a first binding step of bringing the working electrode of the sensor into contact with the test substance solution to bind the primary antibody of the working electrode to the test substance of the test substance solution; A heteroorganic compound or a triplex compound having an unpaired electron pair or a lone electron pair on the surface other than the portion where the secondary antibody is immobilized that specifically binds to the test substance. By bringing into contact with a dispersion of metal nanoparticles blocked by an organic compound having a bond, the test substance bound to the primary antibody of the working electrode and the secondary antibody are bound to each other. A second binding step of binding the test substance and the metal nanoparticles, and applying a voltage between the working electrode and the counter electrode to ionize the entire amount of the metal nanoparticles by oxidation in the presence of an electrolyte solution. , a current amount measurement step of measuring the amount of current when the ionized metal nanoparticles are further reduced, and the amount of metal nanoparticles is obtained from the amount of current, and the amount of the test substance is calculated from the amount of metal nanoparticles. and a step.

(9) In the analysis methods according to the third and fourth aspects, the surface other than the portion where the primary antibody is immobilized on the working electrode is a heteroorganic compound having an unpaired electron pair or a lone electron pair, or It may be blocked by an organic compound having a triple bond.

(10) An analysis method according to a fifth aspect is a method for analyzing a test substance contained in a test substance solution, comprising a working electrode, a reference electrode, and a counter electrode, wherein the surface of the working electrode A primary antibody that specifically binds to the test substance is immobilized on the surface, and the surface other than the portion where the primary antibody is immobilized has an unpaired electron pair or a lone electron pair, or an organic compound having a triple bond a first binding step of bringing the working electrode of the sensor blocked by a compound into contact with the test substance solution to bind the primary antibody of the working electrode to the test substance of the test substance solution; , the working electrode is brought into contact with a dispersion of metal nanoparticles in which metal nanoparticles having a secondary antibody that binds to the test substance is immobilized on the surface thereof, and the primary antibody on the working electrode is a second binding step of binding the test substance and the metal nanoparticles by binding the bound test substance and the secondary antibody; and applying a voltage between the working electrode and the counter electrode. Then, the metal nanoparticles are ionized in the presence of an electrolyte solution, and a current amount measurement step of measuring the amount of current until the entire amount of the metal nanoparticles is ionized, and the amount of metal nanoparticles is obtained from the amount of current, and a calculating step of calculating the amount of the test substance from the amount of the metal nanoparticles.

(11) An analysis method according to a sixth aspect is a method for analyzing a test substance contained in a test substance solution, comprising a working electrode, a reference electrode, and a counter electrode, wherein the surface of the working electrode A primary antibody that specifically binds to the test substance is immobilized on the surface, and the surface other than the portion where the primary antibody is immobilized has an unpaired electron pair or a lone electron pair, or an organic compound having a triple bond a first binding step of bringing the working electrode of the sensor blocked by a compound into contact with the test substance solution to bind the primary antibody of the working electrode to the test substance of the test substance solution; , the working electrode is brought into contact with a dispersion of metal nanoparticles in which metal nanoparticles having a secondary antibody that binds to the test substance is immobilized on the surface thereof, and the primary antibody on the working electrode is a second binding step of binding the test substance and the metal nanoparticles by binding the bound test substance and the secondary antibody; and applying a voltage between the working electrode and the counter electrode. Then, a current amount measurement step of ionizing the entire amount of the metal nanoparticles by oxidation in the presence of an electrolyte solution and measuring the amount of current when the ionized metal nanoparticles are further reduced; and a calculating step of obtaining the amount of nanoparticles and calculating the amount of the test substance from the amount of metal nanoparticles.

(12) In the analysis methods according to the third to sixth aspects, the hetero organic compound having an unpaired electron pair or a lone electron pair may be an organic sulfur compound, an organic nitrogen compound, or an organic oxygen compound.

(13) In the analysis methods according to the third to sixth aspects, the organic compound having a triple bond may be an acetylenic organic compound.

(14) In the analysis method according to the third to sixth aspects, the metal nanoparticles are at least one metal selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel. may contain

(15) In the analysis methods according to the third to sixth aspects, the analysis method may be an immunochromatographic method.

INDUSTRIAL APPLICABILITY According to the present invention, a test substance can be analyzed with high selectivity and high sensitivity by a simple operation, and an analysis kit that can be stored for a long time, and a method for analyzing a test substance using the analysis kit. can be provided.

FIG. 2 is a plan view of a sensor used in the analysis kit according to the first embodiment of the present invention; FIG. FIG. 2 is a sectional view taken along line II-II of FIG. 1; FIG. 10 is a flow diagram illustrating analysis methods according to third to sixth embodiments of the present invention; FIG. 10 is a conceptual diagram illustrating analysis methods according to third to sixth embodiments of the present invention; A graph plotting the antigen concentration of each antigen analysis sample used in Example 1 and the amount of current required to reduce the ionized gold nanoparticles after ionizing the gold nanoparticles as antigen labels. be. The diamond plots show the results before the stability test, and the triangular plots show the results after the stability test. A graph plotting the antigen concentration of each antigen analysis sample used in Example 2 and the amount of current required to reduce the ionized gold nanoparticles after ionizing the gold nanoparticles as antigen labels. be. The diamond plots show the results before the stability test, and the triangular plots show the results after the stability test. A graph plotting the antigen concentration of each antigen analysis sample used in Example 3 and the amount of current required to reduce the ionized gold nanoparticles after ionizing the gold nanoparticles as antigen labels. be. The diamond plots show the results before the stability test, and the triangular plots show the results after the stability test. A graph plotting the antigen concentration of each antigen analysis sample used in Comparative Example 1 and the amount of current required to reduce the ionized gold nanoparticles after ionizing the gold nanoparticles as antigen labels. be. The diamond plots show the results before the stability test, and the triangular plots show the results after the stability test.

The configuration of this embodiment will be described below with reference to the drawings. In the drawings used in the following description, characteristic parts may be shown enlarged for convenience in order to make the characteristics easier to understand, and the dimensional ratio of each component may not necessarily be the same as the actual one. Also, the materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not limited to them.

"First Embodiment"
[Analysis kit]
The analysis kit of this embodiment is an analysis kit for analyzing a test substance contained in a test substance solution using an antigen-antibody reaction. Test substances are, for example, biomaterials, in particular proteins and metabolomes.
The analysis kit of this embodiment includes a sensor and a dispersion of metal nanoparticles. The sensor has a function of capturing the test substance contained in the test substance solution, and the metal nanoparticles function as labels for quantifying the captured test substance.

(sensor)
A sensor used in the analysis kit according to the first embodiment of the present invention will be described with reference to FIGS. 1 and 2. FIG. FIG. 1 is a plan view showing one embodiment of the sensor. FIG. 2 is a sectional view taken along line II--II of FIG.
A sensor 10 shown in FIG. 12, lead wires 12a, 13a and 14a formed on the first substrate 11 so as to be connected to the counter electrode 13 and the reference electrode 14 respectively and extend to the other end of the first substrate 11; 11 and a second substrate 16 formed with a window 15 through which the working electrode 12, the counter electrode 13 and the reference electrode 14 are exposed. The second substrate 16 covers portions of the lead wires 12a, 13a and 14a other than the vicinity of the other end of the first substrate 11. As shown in FIG.

Working electrode 12 is preferably a metal electrode, a carbon electrode, a conductive diamond electrode or a conductive diamond-like carbon electrode (DLC electrode). A copper electrode, a gold electrode, a platinum electrode, a palladium electrode, or the like can be used as the metal electrode. A metal electrode is preferably a noble metal electrode from the viewpoint of corrosion resistance. The carbon electrode is a conductive carbon electrode such as graphite. For example, a carbon printed electrode printed with a paste mainly composed of graphite can be used. A boron-doped diamond electrode doped with boron can be used as the conductive diamond electrode. A conductive diamond electrode is a crystalline carbon electrode with a diamond structure with ^sp3 bonds. Conductive DLC electrodes are amorphous carbon electrodes composed primarily of carbon and hydrogen with a mixture of sp ³ and sp ² bonds. The conductive DLC electrode includes an n-type semiconductor DLC electrode doped with at least one element selected from the group consisting of nitrogen, phosphorus, arsenic, antimony and bismuth, and an element selected from the group consisting of boron, gallium and indium. Any DLC electrode of doped p-type semiconductor can be used.

Conductive diamond electrodes and DLC electrodes, which are mainly composed of sp ³ -bonded carbon, have very few processes of adsorption of chemical substances that are oxidized or reduced by electrochemical reactions. For this reason, for example, the inner sphere oxidation-reduction reaction through adsorption to the electrode by hydrogen or hydroxide originating from water or their ions is extremely difficult to occur. As a result, a noise current called a residual current becomes extremely small, so that the electrochemical reaction of the substance to be detected can be detected at a high SN ratio.

The working electrode 12 has a primary antibody immobilized on its surface (upper surface in FIG. 2). The primary antibody is appropriately selected and used according to the test substance (antigen) to be measured. Any primary antibody can be used without particular limitation as long as it has a high affinity for the test substance to be measured and binds to the test substance (antigen).

The surface of the working electrode 12 other than the portion where the primary antibody is immobilized is preferably blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. The hetero organic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond (blocking agent) may be used singly or in combination. The surface of the working electrode 12 other than the portion where the primary antibody is immobilized is blocked with the blocking agent, thereby preventing non-specific adsorption of the secondary antibody to the working electrode and removing the test substance. It can be analyzed with higher selectivity and sensitivity. Further, by using a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as the blocking agent, the blocking agent is not degraded by bacteria or the like, and the analysis kit can be stored for a long period of time. becomes possible.

Examples of hetero organic compounds having an unpaired electron pair or a lone electron pair include organic sulfur compounds, organic nitrogen compounds, and organic oxygen compounds.

Examples of the organic sulfur compounds include thiourea and its derivatives, thiazole and its derivatives, disulfides, carbamic acid and its derivatives, and salts thereof.

Examples of the thiourea and its derivatives include thiourea, N,N'-diethylthiourea, dibutylthiourea, dilaurylthiourea, trimethylthiourea, N,N'-diphenylthiourea, 2-mercaptoimidazoline, and the like.

Examples of the thiazole and derivatives thereof include thiazole, benzothiazole, 2-(N,N'-diethylthiocarbamoylthio)benzothiazole, 2-mercaptobenzothiazole and the like.

Examples of the disulfide include cystine, dibenzothiazyldiulfide, tetramethylthiuram disulfide, tetraethylthiuram disulfide, tetrabutylthiuram disulfide, tetramethylthiuram monosulfide, tetrakis(2-ethylhexyl)thiuram disulfide and the like.

Examples of carbamic acid and its derivatives and salts thereof include dimethyldithiocarbamate, diethyldithiocarbamate, dibutyldithiocarbamate, N-methyl-N-phenyldithiocarbamate, N-pentamethylenedithiocarbamate, dibenzyldithiocarbamate, and sodium thereof. Salts, zinc salts and the like can be mentioned.

Other organic sulfur compounds include butylxanthate, isobutylxanthate, and salts thereof, 2-di-n-butylamino-4,6-dimercapto-S-triazine, trimercapto-S-triazine, and the like. .

Examples of the organic nitrogen compounds include aliphatic amines, aromatic amines, azo compounds, imidazole and its derivatives, triazole and its derivatives, imines, amides, amino acids and the like.

Examples of the aliphatic amines include n-butylamine, n-hexamine, n-octylamine, n-dodecylamine, n-hexadecylamine, n-cyclohexylamine, n-octadecylpropylenediamine, n-dodecylpropylenediamine, and caproic acid. Amine, N,N'-dibutylamine, N,N'-dihexyamine, N,N'-dioctylamine, N,N'-didodecylamine, N,N'-dihexadecylamine, N,N'-dicyclohexyl Amines, N,N'-dioctadecylpropylenediamine, N,N'-didodecylpropylenediamine, N,N'-dimethylamine caproic acid, N,N'-dimethylN'-methyloctadecylpropylenediamine, N,N' -dimethyl N'-methyldodecylpropylenediamine, N-cyclohexyldimethylamine, morpholine, monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, diethylhydroxyamine, polyvinylamine, amine phosphonate and the like.

Examples of the aromatic amines include aniline, toluidine, trimethylaniline, anisidine, pyrrole, pyridine, quinoline, rhodamine, saccharin and safranin.

Examples of the azo compound include phenylazophenol, hydroxyazobenzene, dimethylaminoazobenzenesulfonic acid, and dimethylaminoazobenzenecarboxylic acid.

Examples of imidazole and its derivatives include imidazole, 1-methylimidazole, 2-phenylimidazole, 1,1'-thiocarbonyldiimidazole, cimetidine, emedastine difumarate, imidazole dipeptide and the like.

Examples of the triazole and derivatives thereof include benzotriazole, 1-hydroxybenzotriazole, and tolyltriazole.

Examples of the imines include aldimine, ketimine, hydroxyimine, ethyleneimine, and polyethyleneimine.

Examples of the amides include formamide, acetamide, benzamide, dimethylformamide, acetamide, acrylamide, polyacrylamide and the like.

Examples of the amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, glutamic acid and the like.

Examples of the organic oxygen compound include sodium lauryl sulfate, Tween 20, marialim, naphthol, ethylene glycol, polyvinylpyrrolidone, alkylolamide, haloacetic acid, polycarboxylic acid, glyceraldehyde, dihydroxyacetone, sugars erythrose, threose, erythrulose, ribose, lyxose, xylose, arabinose, apiose, ribulose, xylulose, allose, talose, gulose, glucose, altrose, mannose, galactose, idose, psicose, fructose, sorbose, tagatose, sedoheptulose, coryose, trehalose, isotrehalose, kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, lactose, sucrose, raffinose, panose, maltotriose, melezitose, gentianose, stachyose, cyclodextrin, deoxyribose, fucose, rhamnose, glucuronic acid, galacturonic acid , fructooligosaccharide, palatinose, glucosamine, galactosamine, glycerin, xylitol, sorbitol, ascorbic acid, glucuronolactone, gluconolactone, amylose, starch, dextrin, amylopectin, glycogen, cellulose, pectin, glucomannan, xanthan gum, pullulan, hyaluronic acid etc.

Examples of organic compounds having a triple bond include acetylenes such as propargyl alcohol, allyl alcohol, 2-butyne-1,4-diol, 3-hexyne-2,5-diol, and 1,4-bis(hydroxyethoxy)butyne. organic compounds, and the like.

The counter electrode 13 is made of a conductive material for electrodes normally used in sensors for electrochemical measurements. As the counter electrode 13, for example, a carbon electrode, a noble metal electrode such as a platinum electrode and a gold electrode, a conductive diamond electrode, or a conductive DLC electrode can be used.

As the reference electrode 14, for example, a silver-silver chloride electrode or a mercury-mercury chloride electrode can be used. Reference electrode 14 is preferably a silver-silver chloride electrode.

A substrate similar to the first substrate 11 is used for the second substrate 16 . The first substrate 11 and the second substrate 16 are adhered with an adhesive 17 .
The second substrate 16 forms a channel with the first substrate 11 . The second substrate 16 may be a smooth substrate in which a groove that serves as a flow path is dug, or a third substrate for forming a flow path is sandwiched between the first and second substrates.

The first substrate 11, the second substrate 16, and the third substrate should have physical strength enough to withstand use as analysis chips, and should not be physically or chemically damaged by the liquid containing the test substance. For example, common polymers such as polyethylene, polypropylene, polycarbonate, polystyrene, polyimide, nylon, polyethylene terephthalate, epoxy resin, polyethylene oxide, and Teflon (registered trademark) are preferred. From the aspect of environmental friendliness, even biodegradable plastics such as polylactic acid resin, polycaprolactone resin, polyhydroxyalkanoate resin, polyglycolic acid resin, and modified polyvinyl alcohol resin can be used if the above conditions are met.

(Metal nanoparticles)
The metal nanoparticles preferably have an average particle size in the range of 1 nm or more and 100 nm or less, more preferably 10 nm or more and 50 nm or less. The metal nanoparticles are preferably dispersed in a solvent. As the solvent, an aqueous solvent, an organic solvent, and a mixture thereof can be used. Examples of aqueous solvents include water and buffer solutions. Phosphate buffered saline (PBS) can be used as a buffer. Examples of organic solvents 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 metal nanoparticles preferably contain at least one metal selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel. One kind of metal may be used alone, or two or more kinds of metals may be used as an alloy in combination. The metal nanoparticles are preferably gold nanoparticles or silver nanoparticles. These metal nanoparticles may be used singly or in combination of two or more.

Metal nanoparticles have a secondary antibody immobilized on their surface. The secondary antibody is appropriately selected and used according to the test substance (antigen) to be measured. Any secondary antibody can be used without particular limitation as long as it has a high affinity for the test substance to be measured and binds to the test substance (antigen).

The surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. Examples of the hetero organic compound having an unpaired electron pair or lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. The blocking agents may be used singly or in combination. By blocking the surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized with the blocking agent, the secondary antibody is prevented from directly non-specifically binding to the primary antibody without an antigen. It is possible to analyze the test substance with high selectivity and high sensitivity by preventing and simple operation. Further, by using a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as the blocking agent, the blocking agent is not degraded by bacteria or the like, and the analysis kit can be stored for a long period of time. becomes possible.

The metal nanoparticles may further contain sulfur. Sulfur may adhere to the surface of the metal particles nanoparticles, or may be intercalated between metal atoms. Sulfur has the effect of suppressing oxidation of metal nanoparticles. The sulfur content of the metal nanoparticles is preferably in the range of 0.001% by mass or more and 0.5% by mass or less.

The dispersion in which the metal nanoparticles are dispersed may further contain thickeners, surfactants, dispersants, antioxidants, and the like.

"Second Embodiment"
[Analysis kit]
In the analysis kit of the second embodiment of the present invention, the working electrode has a primary antibody immobilized on its surface, and the surface of the working electrode other than the portion where the primary antibody is immobilized has an unpaired electron pair or a lone electron The surface other than the point blocked by the hetero organic compound having a pair or the organic compound having a triple bond and the portion where the secondary antibody of the metal nanoparticle is immobilized has an unpaired electron pair or a lone electron pair It is configured in the same manner as the analysis kit of the first embodiment, except that it may not be blocked by a heteroorganic compound or an organic compound having a triple bond.

In the analysis kit of this embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. there is Examples of the hetero organic compound having an unpaired electron pair or lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. The surface of the working electrode 12 other than the portion where the primary antibody is immobilized is blocked with the blocking agent, thereby preventing non-specific adsorption of the secondary antibody to the working electrode and removing the test substance. It can be analyzed with high selectivity and high sensitivity. Further, by using a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as the blocking agent, the blocking agent is not degraded by bacteria or the like, and the analysis kit can be stored for a long period of time. becomes possible.

In the analysis kit of this embodiment, the surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized is blocked by a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. It is preferable that Examples of the hetero organic compound having an unpaired electron pair or lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. The surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized is blocked by the blocking agent, so that the secondary antibody directly and non-specifically binds to the primary antibody without intervening the antigen. can be prevented, and the test substance can be analyzed with higher selectivity and sensitivity with a simple operation. Further, by using a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as the blocking agent, the blocking agent is not degraded by bacteria or the like, and the analysis kit can be stored for a long period of time. becomes possible.

"Third Embodiment"
[Analysis method]
Next, an analysis method according to a third embodiment of the present invention will be described with reference to FIGS. 3 and 4. FIG.
FIG. 3 is a flow chart explaining the analysis method according to the third embodiment of the present invention.
The analysis method of this embodiment, as shown in FIG. 3, includes a first coupling step S01, a second coupling step S02, a voltage application step S03, a current amount measurement step S04, and a calculation step S05.

(First bonding step S01)
In the first binding step S01, the working electrode 12 of the sensor 10 described above is brought into contact with the test substance solution. As shown in FIG. 4A, on the surface of the working electrode 12 is immobilized a primary antibody 31 that can selectively bind to a test substance, which is an object to be measured. 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).
In addition, when the surface of the working electrode 12 other than the portion where the primary antibody 31 is immobilized is blocked by a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond, the secondary Non-specific adsorption of antibodies to the working electrode 12 is prevented. Therefore, the test substance can be analyzed with higher selectivity and higher sensitivity.

After the first bonding step S<b>01 , the working electrode 12 may be washed to remove the test substance solution adhering to the working electrode 12 . As the cleaning liquid, an aqueous solvent or an organic solvent can be used.

(Second bonding step S02)
In the second bonding step S02, the working electrode 12 is brought into contact with the metal nanoparticle dispersion. As shown in FIG. 4(c), a secondary antibody 34 capable of selectively binding to a test substance 32, which is an object to be measured, is immobilized on the surface of the metal nanoparticles 33. As shown in FIG. Therefore, in the second binding step S02, the test substance 32 and the secondary antibody 34 are bound, and the metal nanoparticles 33 are bound to the test substance 32, as shown in FIG. 4(c). Since the surface of the metal nanoparticles 33 other than the portion where the secondary antibody 34 is immobilized is blocked by a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond, the secondary antibody 34 is prevented from directly and non-specifically binding to the primary antibody 31 without antigen. Therefore, the test substance can be analyzed with high selectivity and high sensitivity.

After the second binding step, working electrode 12 may be washed to remove metal nanoparticles 33 not bound to analyte 32 . As the cleaning liquid, an aqueous solvent or an organic solvent can be used.

(Voltage application step S03)
In the voltage application step S03, the working electrode 12, the counter electrode 13, and the reference electrode 14 are immersed in the electrolyte solution, and the potential of the working electrode 12 is set to a potential that oxidizes and ionizes the metal nanoparticles 33 bound to the test substance 32. , a voltage is applied between the working electrode 12 and the counter electrode 13 .

Chlorides such as potassium chloride, sodium chloride, and lithium chloride can be used as the electrolyte of the electrolyte solution. When ionizing the metal nanoparticles 33, the chloride destroys the oxide film (passive film) formed on the surface of the metal nanoparticles 33, exposes the metal to the solution, and facilitates the progress of ionization. effective. As a solvent for the electrolyte solution, in addition to water, an organic solvent such as dimethylsulfoxide (DMSO) can be used. The chloride ion concentration of the electrolyte solution is preferably in the range of 0.05 mol/L or more and 1.0 mol/L or less.

Since the potential of the working electrode 12 is a potential at which the metal nanoparticles 33 are oxidized and ionized, the voltage applied between the working electrode 12 and the counter electrode 13 is such that the potential of the working electrode 12 can ionize the metal nanoparticles 33. There are no particular restrictions on the potential, but when the reference electrode 14 is a silver-silver chloride electrode, it is preferably 0 to 1.0 V, more preferably 0.2 to 0.8 V, with respect to the reference electrode 14 . By this voltage application step S03, the metal nanoparticles 33 bound to the test substance 32 are electrochemically oxidized and ionized.

(Current amount measurement step S04)
In the current amount measurement step S06, a voltage is applied between the working electrode 12 and the counter electrode 13, and the current amount is measured until all the metal nanoparticles 33 ionized (oxidized) in the presence of the electrolyte solution are ionized. . For example, when the metal nanoparticles 33 are gold nanoparticles, by applying a voltage between the working electrode 12 and the counter electrode 13, the amount of current when the gold nanoparticles dissolve as gold chloride complex ions is measured. . Specifically, the lead wires 12a, 13a, and 14a of the sensor 10 are connected to a potentiostat, and a voltage is applied between the working electrode 12 and the counter electrode 13 using the voltammetry method. 13 is measured.

(Calculation step S05)
In the calculation step S<b>05 , the amount of metal nanoparticles 33 is obtained from the amount of current, and the amount of the test substance is calculated from the amount of metal nanoparticles 33 . The current amount obtained in the current amount measuring step S04 correlates with the amount of the metal nanoparticles 33 (that is, the amount of the test substance). Therefore, by creating a calibration curve using a sample containing a known amount of the test substance, it is possible to accurately quantify the test substance contained in the test substance solution.

"Fourth Embodiment"
As in the third embodiment shown in FIG. 3, the analysis method of the fourth embodiment of the present invention includes a first coupling step S01, a second coupling step S02, a voltage application step S03, a current amount measurement step S04, and a calculation step. Including S05. In the analysis method of the fourth embodiment, in the voltage application step 03, instead of ionizing all the metal nanoparticles by oxidation in the presence of the electrolyte solution as in the third embodiment, all the metal nanoparticles are ionized in the presence of the electrolyte solution. In the step S04 of ionizing the ionized metal nanoparticles by oxidation and further reducing the ionized metal nanoparticles, and in the current measurement step S04, a voltage is applied between the working electrode 12 and the counter electrode 13 in the third embodiment, and the electrolyte solution Instead of measuring the amount of current until the total amount of ionized (oxidized) metal nanoparticles 33 is ionized in the presence of, a voltage is applied between the working electrode 12 and the counter electrode 13, and in the presence of the electrolyte solution After ionizing (oxidizing) all the metal nanoparticles 33, a reverse voltage is applied between the working electrode 12 and the counter electrode 13 to ionize (oxidize) the metal nanoparticles 33 in the presence of an electrolyte solution. The analysis method is configured in the same manner as the analysis method of the third embodiment, except that the amount of current until further reduction is measured.

In the voltage application step S03 of the present embodiment, first, a voltage is applied between the working electrode 12 and the counter electrode 13 in order to set the potential of the working electrode 12 to ionize the entire amount of the metal nanoparticles by oxidation in the presence of the electrolyte solution. apply. After that, a reverse voltage is applied between the working electrode 12 and the counter electrode 13 in order to set the potential of the working electrode 12 to electrochemically reduce the ionized metal nanoparticles. The potential applied to the working electrode 12 during reduction is preferably 0.0 to 1.0V, more preferably 0.1 to 0.8V.

In the current amount measurement step S04 of the present embodiment, a voltage is applied between the working electrode 12 and the counter electrode 13 to ionize the entire amount of the metal nanoparticles 33 in the presence of the electrolyte solution. By applying a reverse voltage between , the amount of current until the ionized metal nanoparticles are reduced is measured. For example, if metal nanoparticles 33 are gold nanoparticles, by applying a potential to working electrode 12 to apply a reverse voltage between working electrode 12 and counter electrode 13, gold chloride complex ions are The amount of current is measured when the gold is reduced and electrodeposited as gold. Specifically, the leads 12a, 13a, 14a of the sensor 10 are connected to a potentiostat, and the voltammetric method is used to apply a potential to the working electrode 12 while a potential is applied between the working electrode 12 and the counter electrode 13. Measure the amount of current.

"Fifth Embodiment"
In the analysis method of the fifth embodiment of the present invention, in the first binding step S01 of the third embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized has an unpaired electron pair or a lone electron pair. The configuration is the same as that of the analysis method of the third embodiment, except that the blocking is performed by a hetero organic compound having a triple bond or an organic compound having a triple bond.

In this embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. Therefore, nonspecific adsorption of the secondary antibody to the working electrode 12 is prevented. Therefore, the test substance can be analyzed with high selectivity and high sensitivity. Examples of hetero organic compounds having an unpaired electron pair or a lone electron pair or organic compounds having a triple bond are the same as those mentioned above.

In the present embodiment, in the second bonding step S02, the surface of the metal nanoparticles 33 other than the portion where the secondary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or a lone electron pair or a triple bond. may be blocked by an organic compound having In this case, direct non-specific binding of the secondary antibody 34 to the primary antibody 31 without intervening the antigen is prevented. Therefore, the test substance can be analyzed with higher selectivity and higher sensitivity.

"Sixth Embodiment"
In the analysis method of the sixth embodiment of the present invention, in the first binding step S01 of the fourth embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized has an unpaired electron pair or a lone electron pair. The configuration is the same as that of the analysis method of the fourth embodiment, except that the blocking is performed by a hetero organic compound having a triple bond or an organic compound having a triple bond.

In this embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. Therefore, non-specific adsorption of the secondary antibody to the working electrode 12 is prevented, and the test substance can be analyzed with high selectivity and high sensitivity. Examples of hetero organic compounds having an unpaired electron pair or a lone electron pair or organic compounds having a triple bond are the same as those mentioned above.

In the present embodiment, in the second bonding step S02, the surface of the metal nanoparticles 33 other than the portion where the secondary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or a lone electron pair or a triple bond. may be blocked by an organic compound having In this case, direct non-specific binding of the secondary antibody 34 to the primary antibody 31 without intervening the antigen is prevented. Therefore, the test substance can be analyzed with higher selectivity and higher sensitivity.

As described above, according to the analysis kit of the first embodiment, the surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized has an unpaired electron pair or a lone electron pair. Since the blocking is performed by an organic compound with a binding property, direct non-specific binding of the secondary antibody to the primary antibody is prevented. It can be analyzed with sensitivity.

In addition, since the analysis kit of the first embodiment uses a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as a blocking agent, the blocking agent is not altered by bacteria or the like. Therefore, long-term storage is possible.

Further, according to the analysis kit of the second embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is immobilized is a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. blocked by Therefore, nonspecific adsorption of the secondary antibody to the working electrode is prevented, and the test substance can be analyzed with high selectivity and high sensitivity.

In addition, since the analysis kit of the second embodiment uses a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond as a blocking agent, the blocking agent is not altered by bacteria or the like. Therefore, long-term storage is possible.

Further, according to the analysis methods of the third embodiment and the fifth embodiment, the surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or a lone electron pair, or Blocking by an organic compound with a triple bond prevents direct non-specific binding of the secondary antibody to the primary antibody without involving the antigen. It can be analyzed with high sensitivity.

Further, according to the analysis methods of the third embodiment and the fifth embodiment, a voltage is applied between the working electrode and the counter electrode to ionize the metal nanoparticles bound to the test substance. An electrochemical technique can be used to quantify the analyte without carrying out the step of chemically dissolving the metal.

Further, according to the analysis methods of the fourth embodiment and the sixth embodiment, the surface of the working electrode other than the portion where the primary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or a lone electron pair or a triple bond. Since the secondary antibody is blocked by the organic compound having the , non-specific adsorption of the secondary antibody to the working electrode is prevented, and the analyte can be analyzed with high selectivity and high sensitivity.

Further, according to the analysis methods of the fourth embodiment and the sixth embodiment, a voltage is applied between the working electrode and the counter electrode to ionize the total amount of metal nanoparticles bound to the test substance, and further ionized Since the metal nanoparticles are reduced, it is possible to quantify the test substance using an electrochemical method without performing the conventional step of chemically dissolving the metal. In addition, when there is a substance that is oxidized at the same time as the metal nanoparticles are oxidized, it is better to measure the current that reduces the ionized metal nanoparticles, so that the oxidation current of the substance that is oxidized at the same time as the metal nanoparticles is measured. The test substance can be analyzed more accurately than the measurement.
Therefore, according to the analysis kit and the analysis method of the present invention, it is possible to analyze a test substance with high selectivity and high sensitivity with a simple operation, and the analysis kit can be stored for a long time. . The analysis method of the present invention can also be applied to immunochromatography.

In addition, the metal nanoparticles blocked by the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond according to the present invention can also be applied in embodiments based on a competitive method. The competitive method is preferably used when the antigen is a hapten (low-molecular-weight antigen), which is a low-molecular-weight compound. For example, when the antigen to be measured is a hapten, a hapten-protein conjugate is immobilized on the working electrode. When the low-molecular-weight antigen to be measured and a limited amount of gold nanoparticle-labeled antibody are added thereto, the immobilized antigen and the released low-molecular-weight antigen to be measured competitively react with the gold nanoparticle-labeled antibody. . The more the free low-molecular-weight antigen to be measured, the less the amount of the gold nanoparticle-labeled antibody that reacts with the immobilized antigen. Conversely, the less the released low-molecular-weight antigen to be measured, the greater the amount of gold nanoparticle-labeled antibody that reacts with the immobilized antigen. After the antigen-antibody reaction, B/F separation (Bound/Free) is performed by washing to remove unreacted free low-molecular-weight antigens to be measured and gold nanoparticle-labeled antibodies. Current values are measured by electrochemical oxidation and reduction of gold nanoparticle labels on electrodes. If a calibration curve is prepared in advance using a known amount of the low-molecular-weight antigen to be measured, an unknown amount of the low-molecular-weight antigen to be measured can be measured.

EXAMPLES The present invention will be described in more detail below with specific examples, but the present invention is not limited to these examples.

[Example 1]
(1) Preparation of primary antibody-attached sensor A gold-evaporated film on a polyethylene terephthalate substrate with patterning of the counter electrode and lead wire using a mask, a conductive DLC film formed on a Si wafer as the working electrode, and Ag/AgCl pasted as the reference electrode. A sensor chip using an Ag/AgCl film produced by screen printing was prepared. An unlabeled anti-goat IgG was immobilized as a primary antibody on the working electrode (electrode area S=0.0962 cm ² ) of this sensor chip, and the working electrode other than the portion where the primary antibody was immobilized was treated with benzimidazole. By blocking the surface, an analysis chip with a primary antibody was prepared in which the primary antibody was immobilized on the surface of the working electrode.

(2) Gold nanoparticle dispersion with secondary antibody A gold nanoparticle dispersion having an average particle size of 18 nm manufactured by Tanaka Kikinzoku Co., Ltd. was used.

The above gold nanoparticles, benzimidazole, anti-goat IgG, phosphate-buffered saline (PBS), and polyethylene glycol sorbitan monolaurate (Tween 20, nonionic surfactant) were mixed to form gold nanoparticles. A secondary antibody-attached gold nanoparticle dispersion liquid in which anti-goat IgG (secondary antibody) was immobilized on the surface of was prepared. The concentration of gold nanoparticles in the secondary antibody-attached gold nanoparticle dispersion was set to 0.007% by mass. As anti-goat IgG, a polyclonal antibody commercially available from Jackson Immunoresearch Laboratories was used.

(3) Samples for Antigen Analysis As samples for antigen analysis, No. 2 samples with different antigen concentrations were used as shown below. 1 to No. 6 was prepared. No. below. 1 to No. 6 was prepared by mixing PBS buffer containing 0.1% Tween 20 and goat IgG (antigen) at the following antigen concentrations.
No. 1: Antigen concentration = 0.01 ng/mL
No. 2: Antigen concentration = 0.1 ng/mL
No. 3: Antigen concentration = 1.0 ng/mL
No. 4: Antigen concentration = 10 ng/mL
No. 5: Antigen concentration = 100 ng/mL
No. 6: Antigen concentration = 1000 ng/mL

(4) Antigen analysis No. prepared in (3). 1 to No. 35 μL of each sample for antigen analysis in 6 was accurately weighed, dropped onto the working electrode of the primary antibody-attached sensor prepared in (1) above, and then incubated for 40 minutes (first binding step).

Next, the working electrode of the primary antibody-attached sensor was washed with a washing liquid to wash away the sample for antigen analysis. PBS was used as a washing solution.

Next, 1 μL of the secondary antibody-attached gold nanoparticle dispersion prepared in (2) above was accurately weighed, dropped onto the working electrode of the primary antibody-attached sensor, and incubated for 3 hours (second bonding step).

Next, the working electrode of the primary antibody-attached sensor was washed with a washing liquid to wash away the secondary antibody-attached gold nanoparticle dispersion. PBS was used as a washing solution.

Next, the lead wire of the primary antibody-attached sensor was connected to the potentiostat, an electrolyte solution in which 0.1 mol/L potassium chloride was dissolved in PBS was injected, and the primary antibody-attached sensor was immersed in the electrolyte solution.

Next, using a potentiostat, a voltage was applied between the working electrode and the counter electrode to once electrochemically oxidize the gold nanoparticles and ionize them. After that, a reverse voltage was applied between the working electrode and the counter electrode to electrochemically reduce the ionized gold nanoparticles, and the amount of current flowing between the working electrode and the counter electrode during this time was measured (current amount measurement process).

In FIG. 1 to No. A graph plotting the antigen concentration of each antigen analysis sample in 6 and the amount of current flowing between the working electrode and the counter electrode (that is, the amount of current required to electrochemically reduce the ionized gold nanoparticles) (plot before stability test in FIG. 5). It was confirmed from the graph of FIG. 5 that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis. Therefore, by creating a calibration curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance), the antigen (test substance) contained in the test substance solution can be accurately quantified. It was confirmed that it is possible to

Further, the sensor with the primary antibody was vacuum sterilized, packed in an aluminum pouch, and then subjected to a stability test at 25° C., 75% RH for 3 months. After 3 months, the package was opened and the antigen concentration was measured. The results are shown in FIG. 5 (plot after stability test in FIG. 5). As shown in Figure 5, there was no significant change in the calibration curve before and after the stability test, confirming that the test substance can be accurately quantified even after long-term storage. was done.

[Example 2]
A sensor with a primary antibody, a gold nanoparticle dispersion with a secondary antibody, and a sample for antigen analysis were prepared in the same manner as in Example 1 except that benzotriazole was changed to cystine, and the antigen was analyzed.
In FIG. 1 to No. A graph plotting the antigen concentration of each antigen analysis sample in 6 and the amount of current flowing between the working electrode and the counter electrode (that is, the amount of current required to electrochemically reduce the ionized gold nanoparticles) (plot before stability test in FIG. 6). It was confirmed from the graph of FIG. 6 that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis. Therefore, by creating a calibration curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance), the antigen (test substance) contained in the test substance solution can be accurately quantified. It was confirmed that it is possible to

Further, the sensor with the primary antibody was vacuum sterilized, packed in an aluminum pouch, and then subjected to a stability test at 25° C., 75% RH for 3 months. After 3 months, the package was opened and the antigen concentration was measured. The results are shown in FIG. 6 (plot after stability test in FIG. 6). As shown in Figure 6, there was no significant change in the calibration curve before and after the stability test, confirming that the test substance can be accurately quantified even after long-term storage. was done.

[Example 3]
A sensor with a primary antibody, a gold nanoparticle dispersion with a secondary antibody, and a sample for antigen analysis were prepared in the same manner as in Example 1 except that benzotriazole was changed to sucrose, and the antigen was analyzed.
In FIG. 1 to No. A graph plotting the antigen concentration of each antigen analysis sample in 6 and the amount of current flowing between the working electrode and the counter electrode (that is, the amount of current required to electrochemically reduce the ionized gold nanoparticles) (plot before stability test in FIG. 7). It was confirmed from the graph of FIG. 7 that there is a correlation between the current value and the antigen concentration of the sample for antigen analysis. Therefore, by creating a calibration curve (current value-antigen concentration curve) using a sample containing a known amount of antigen (test substance), the antigen (test substance) contained in the test substance solution can be accurately quantified. It was confirmed that it is possible to

Further, the sensor with the primary antibody was vacuum sterilized, packed in an aluminum pouch, and then subjected to a stability test at 25° C., 75% RH for 3 months. After 3 months, the package was opened and the antigen concentration was measured. The results are shown in FIG. 7 (plot after stability test in FIG. 7). As shown in Figure 7, there was no significant change in the calibration curve before and after the stability test, confirming that the test substance can be accurately quantified even after long-term storage. was done.

[Comparative Example 1]
A sensor was prepared and the antigen concentration was measured in the same manner as in Example 1, except that skimmed milk was used instead of benzimidazole. The results are shown in FIG. 8 (plot before stability test in FIG. 8).

After vacuum-sterilizing the sensor with the primary antibody and packaging it in an aluminum pouch, a stability test was conducted at 25° C., 75% RH, and 3 months. After 3 months, the package was opened and the antigen concentration was measured. The results are shown in FIG. 8 (plot after stability test in FIG. 8). As shown in FIG. 8, the detection current decreased after the stability test, confirming that long-term storage reduces the quantification of the test substance.

DESCRIPTION OF SYMBOLS 10... Sensor, 11... First substrate, 12... Working electrode, 13... Counter electrode, 14... Reference electrode, 12a, 13a, 14a... Lead wire, 15... Window, 16... Second substrate, 17... Adhesive, 31... Primary antibody 32 Test substance 33 Metal nanoparticles 34 Secondary antibody

Claims (15)

A sensor having a working electrode, a reference electrode, and a counter electrode, wherein a primary antibody is immobilized on the surface of the working electrode, and a secondary antibody that specifically binds to a test substance is immobilized on the surface and the surface other than the portion where the secondary antibody is immobilized is blocked by a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. An analysis kit for a test substance. The analysis according to claim 1, wherein the surface of the working electrode other than the portion where the primary antibody is immobilized is blocked with a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. kit. A working electrode, a reference electrode, and a counter electrode, wherein a primary antibody is immobilized on the surface of the working electrode, and the surface of the working electrode other than the portion where the primary antibody is immobilized is an unpaired electron pair Alternatively, a sensor blocked by a heteroorganic compound having a lone electron pair or an organic compound having a triple bond, and a metal nanoparticle on the surface of which a secondary antibody that specifically binds to a test substance is immobilized. A test substance analysis kit characterized by: The analysis kit according to any one of claims 1 to 3, wherein the hetero organic compound having an unpaired electron pair or a lone electron pair is an organic sulfur compound, an organic nitrogen compound, or an organic oxygen compound. The analysis kit according to any one of claims 1 to 3, wherein the organic compound having a triple bond is an acetylenic organic compound. 6. Any one of claims 1 to 5, wherein the metal nanoparticles contain at least one metal selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel. analysis kit as described in . A method for analyzing a test substance contained in a test substance solution, comprising:
The working electrode of a sensor having a working electrode, a reference electrode, and a counter electrode, wherein a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode; and the test substance solution. a first binding step of bringing the primary antibody of the working electrode into contact with the test substance of the test substance solution;
The working electrode and a secondary antibody that specifically binds to a test substance are immobilized on the surface, and the surface other than the portion where the secondary antibody is immobilized has an unpaired electron pair or a lone electron pair. The test substance bound to the primary antibody of the working electrode is brought into contact with a dispersion of metal nanoparticles in which metal nanoparticles blocked by a heteroorganic compound or an organic compound having a triple bond are dispersed. a second binding step of binding the test substance and the metal nanoparticles by binding the secondary antibody;
A current measurement step of applying a voltage between the working electrode and the counter electrode to ionize the metal nanoparticles in the presence of an electrolyte solution, and measuring the amount of current until the entire amount of the metal nanoparticles is ionized. and,
A calculation step of obtaining the amount of metal nanoparticles from the amount of current and calculating the amount of a test substance from the amount of metal nanoparticles;
A method for analyzing a test substance, comprising: A method for analyzing a test substance contained in a test substance solution, comprising:
The working electrode of a sensor having a working electrode, a reference electrode, and a counter electrode, wherein a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode; and the test substance solution. a first binding step of bringing the primary antibody of the working electrode into contact with the test substance of the test substance solution;
The working electrode and a secondary antibody that specifically binds to a test substance are immobilized on the surface, and the surface other than the portion where the secondary antibody is immobilized has an unpaired electron pair or a lone electron pair. A dispersion of metal nanoparticles blocked by a heteroorganic compound or an organic compound having a triple bond is contacted to bind the test substance bound to the primary antibody of the working electrode to the secondary antibody. a second binding step of binding the test substance and the metal nanoparticles;
A voltage is applied between the working electrode and the counter electrode to ionize the entire amount of the metal nanoparticles by oxidation in the presence of an electrolyte solution, and the amount of current when the ionized metal nanoparticles are further reduced is a current amount measurement step to be measured;
A calculation step of obtaining the amount of metal nanoparticles from the amount of current and calculating the amount of a test substance from the amount of metal nanoparticles;
A method for analyzing a test substance, comprising: 9. The method according to claim 7, wherein the surface of the working electrode other than the portion where the primary antibody is immobilized is blocked with a hetero organic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. analysis method. A method for analyzing a test substance contained in a test substance solution, comprising:
A working electrode, a reference electrode, and a counter electrode, wherein a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode, and the surface other than the portion where the primary antibody is immobilized is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond, and the working electrode of the sensor is brought into contact with the analyte solution to obtain the primary a first binding step of binding the antibody and the test substance of the test substance solution;
The working electrode is brought into contact with a dispersion liquid of metal nanoparticles in which metal nanoparticles having a secondary antibody that binds to the test substance is immobilized on the surface is dispersed, so that the working electrode binds to the primary antibody. a second binding step of binding the test substance and the metal nanoparticles by binding the test substance and the secondary antibody;
A current measurement step of applying a voltage between the working electrode and the counter electrode to ionize the metal nanoparticles in the presence of an electrolyte solution, and measuring the amount of current until the entire amount of the metal nanoparticles is ionized. and,
A calculation step of obtaining the amount of metal nanoparticles from the amount of current and calculating the amount of a test substance from the amount of metal nanoparticles;
A method for analyzing a test substance, comprising: A method for analyzing a test substance contained in a test substance solution, comprising:
A working electrode, a reference electrode, and a counter electrode, wherein a primary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode, and the surface other than the portion where the primary antibody is immobilized is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond, and the working electrode of the sensor is brought into contact with the analyte solution to obtain the primary a first binding step of binding the antibody and the test substance of the test substance solution;
The working electrode is brought into contact with a dispersion liquid of metal nanoparticles in which metal nanoparticles having a secondary antibody that binds to the test substance is immobilized on the surface is dispersed, so that the working electrode binds to the primary antibody. a second binding step of binding the test substance and the metal nanoparticles by binding the test substance and the secondary antibody;
A voltage is applied between the working electrode and the counter electrode to ionize the entire amount of the metal nanoparticles by oxidation in the presence of an electrolyte solution, and the amount of current when the ionized metal nanoparticles are further reduced is a current amount measurement step to be measured;
A calculation step of obtaining the amount of metal nanoparticles from the amount of current and calculating the amount of a test substance from the amount of metal nanoparticles;
A method for analyzing a test substance, comprising: The analysis method according to any one of claims 7 to 11, wherein the hetero organic compound having an unpaired electron pair or a lone electron pair is an organic sulfur compound, an organic nitrogen compound, or an organic oxygen compound. The analysis method according to any one of claims 7 to 11, wherein the organic compound having a triple bond is an acetylenic organic compound. 14. Any one of claims 7 to 13, wherein the metal nanoparticles contain at least one metal selected from the group consisting of gold, platinum, silver, copper, rhodium, palladium, iron, cobalt and nickel. Analysis method described in. The analysis method according to any one of claims 7 to 14, wherein the analysis method is an immunochromatographic method.

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