JP2020190550A - Analysis kit for test substance and method for analyzing test substance - Google Patents

Abstract

To provide an analysis kit capable of analyzing test substances with high selectivity and high sensitivity by a simple operation and enabling long-term storage, and a method for analyzing the test substances using the analysis kit.SOLUTION: An analysis kit for test substances comprises: a sensor 10 including a working electrode 12, a reference electrode 14 and a counter electrode 13, where primary antibodies are immobilized on the surface of the working electrode 12; and metallic nanoparticles in which secondary antibodies specifically binding to a test substance, are immobilized on the surface, where the surface other than the portion on which the secondary antibodies are 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 method for the test substances using the analysis kit is also provided.SELECTED DRAWING: Figure 1

Description

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 carried out in fields such as medicine, healthcare, and the environment. Then, it is desired to develop an analysis method capable of selectively quantifying a biological substance to be measured from a plurality of biological substances with high sensitivity and simple operability.

An immunoassay is known as one of the methods capable of selectively measuring a trace amount of a biological substance in a liquid with high sensitivity. The immunoassay is a method of quantifying an antigen by utilizing a reaction (antigen-antibody reaction) between a biological substance (antigen, hapten, etc.) to be measured and a substance (antibody) that binds to the antigen.

As a method for quantifying an antigen, a sandwich method is known. 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 in which an antigen is supplemented with a primary antibody, the supplemented antigen is bound to a secondary antibody, and a label immobilized on the secondary antibody bound to the antigen is quantified.
A competitive method is also known as a method for quantifying an antigen. The competitive method is an antigen-antibody reaction between an antigen, which is a test substance contained in a solution to be analyzed, and an antibody having a fixed label, and then quantifying the unreacted labeled antibody generated at this time. How to do it.

As a method for quantifying the label, in addition to the ELISA method, a method of using metal particles as the label and quantifying the amount of the metal particles by using an electrochemical method is known.
Patent Document 1 discloses an immunoassay method including 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 Patent Document 1, the colloidal metal particles as labels are chemically dissolved, then a solution of the metal is transferred to an electrode for reduction, and the reduced metal is deposited on the electrode. The amount of the metal is measured by electrically redissolving the metal deposited on the surface of the electrode and analyzing the voltammetry peak that appears after the redissolution. The colloidal metal particles are metal nanoparticles dispersed in a solvent.

On the other hand, in the immunoassay method using an antigen-antibody reaction such as ELISA, non-specific adsorption of the secondary antibody to the solid phase or direct non-specific binding of the secondary antibody to the primary antibody without an antigen is used. In order to prevent this, proteins such as bovine serum albumin (BSA) and casein, and bio-related substances such as gelatin and skim milk containing proteins are used as blocking agents (for example, Patent Document 2 and the like).
However, since these proteins and substances containing proteins are easily altered 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. 2004-512996 Japanese Patent No. 6376783

Blocking in an analysis chip of a test substance (antigen) that detects an antigen-antibody reaction by an electrochemical oxidation-reduction current using an action electrode on which a primary antibody is immobilized or metal nanoparticles on which a secondary antibody is immobilized. When a protein such as BSA, skim milk, or gelatin or a substance containing the protein is used as the agent, there is a problem that the storage period of the analysis kit is short because the protein is altered by bacteria or the like.

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

The present inventors have unpaired electron pairs or isolated electrons on the surface of metal nanoparticles on which a secondary antibody that specifically binds to a test substance is immobilized or on the surface of an action electrode on which a primary antibody is immobilized. By blocking with a hetero-organic compound having a pair or an organic compound having a triple bond, non-specific adsorption of the secondary antibody to the acting electrode or the secondary antibody is directly non-specific to the primary antibody without an antigen. The present invention is completed by finding that it is possible to analyze a test substance with high selectivity and high sensitivity by preventing binding to the antibody, and to enable long-term storage of an analysis kit with a simple operation. It came to.
That is, the present invention provides the following means for solving the above problems.

(1) The analysis kit according to the first aspect has a working electrode, a reference electrode, and a counter electrode, and a sensor in which a primary antibody is 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 to which the secondary antibody is immobilized is 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 that are blocked by.

(2) In the analysis kit according to the above embodiment, the surface other than the portion where the primary antibody of the working electrode is fixed 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 may have been done.

(3) The analysis kit according to the second aspect has a working electrode, a reference electrode, and a counter electrode, and the 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 portion 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 a sensor that specifically binds to the test substance on the surface. It is characterized by containing metal nanoparticles to which the next antibody is immobilized.

(4) In the analysis kit according to the first aspect and the second aspect, the heteroorganic 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. May be good.

(5) In the analysis kit according to the first aspect and the second aspect, the organic compound having a triple bond may be an acetylene-based organic compound.

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

(7) The method for analyzing a test substance according to the third aspect is a method for analyzing a test substance contained in a solution of a test substance, which has an action electrode, a reference electrode, and a counter electrode. The action electrode of the sensor in which the primary antibody that specifically binds to the test substance is fixed on the surface of the action electrode is brought into contact with the test substance solution, and the primary antibody of the action electrode and the test are tested. The first binding step of binding the test substance of the substance solution, the action electrode, and the secondary antibody that specifically binds to the test substance are immobilized on the surface, and the secondary antibody is immobilized. The surface other than the portion is in contact with a dispersion liquid of metal nanoparticles in which metal nanoparticles blocked by a heteroorganic compound having an unpaired electron pair or an isolated electron pair or an organic compound having a triple bond are dispersed. The second binding step of binding the test substance and the metal nanoparticles by binding the test substance bound to the primary antibody of the action electrode and the secondary antibody, and the action electrode. A current amount measurement 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 the current. It is characterized by including a calculation step of obtaining the amount of metal nanoparticles from the amount and calculating the amount of a 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 the 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 and the test substance of the test substance solution, and the working electrode. A secondary antibody that specifically binds to the test substance is immobilized on the surface, and the surface other than the portion on which the secondary antibody is immobilized is a heteroorganic compound or triple having an unpaired electron pair or an isolated electron pair. The subject is brought into contact with a dispersion of metal nanoparticles blocked by a binding organic compound to bind the test substance bound to the primary antibody of the working electrode and the secondary antibody. A voltage is applied between the second bonding step of binding the test substance and the metal nanoparticles and 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 for measuring the current amount when the ionized metal nanoparticles are further reduced, and a calculation for obtaining the metal nanoparticle amount from the current amount and calculating the test substance amount from the metal nanoparticle amount. It is characterized by including a process.

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

(10) The analysis method according to the fifth aspect is a method for analyzing a test substance contained in a test substance solution, which has an action electrode, a reference electrode, and a counter electrode, and has a surface of the action electrode. A primary antibody that specifically binds to the test substance is immobilized on the substance, and the surface other than the portion on which the primary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or an isolated electron pair, or an organic having a triple bond. A first binding step in which the action electrode of the sensor blocked by the compound is brought into contact with the test substance solution to bind the primary antibody of the action electrode and the test substance of the test substance solution. The working electrode and the dispersion liquid of the metal nanoparticles in which the metal nanoparticles having the secondary antibody bound to the test substance fixed on the surface are dispersed are brought into contact with the primary antibody of the working electrode. A voltage is applied between the action electrode and the counter electrode in the second binding step of binding the test substance and the metal nanoparticles by binding the bound test substance and the secondary antibody. Then, the metal nanoparticles are ionized in the presence of an electrolyte solution, and the 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 are obtained from the current amount. It is characterized by including a calculation step of calculating the amount of a test substance from the amount of metal nanoparticles.

(11) The analysis method according to the sixth aspect is a method for analyzing a test substance contained in a test substance solution, which has an action electrode, a reference electrode, and a counter electrode, and has a surface of the action electrode. A primary antibody that specifically binds to the test substance is immobilized on the substance, and the surface other than the portion on which the primary antibody is immobilized is a heteroorganic compound having an unpaired electron pair or an isolated electron pair, or an organic having a triple bond. A first binding step in which the action electrode of the sensor blocked by the compound is brought into contact with the test substance solution to bind the primary antibody of the action electrode and the test substance of the test substance solution. The working electrode and the dispersion liquid of the metal nanoparticles in which the metal nanoparticles having the secondary antibody bound to the test substance fixed on the surface are dispersed are brought into contact with the primary antibody of the working electrode. A voltage is applied between the action electrode and the counter electrode in the second binding step of binding the test substance and the metal nanoparticles by binding the bound test substance and the secondary antibody. Then, the entire amount of the metal nanoparticles is ionized by oxidation in the presence of an electrolyte solution, and the current amount measurement step of measuring the current amount when the ionized metal nanoparticles are further reduced, and the metal from the current amount. It is characterized by including a calculation step of obtaining the amount of nanoparticles and calculating the amount of a test substance from the amount of metal nanoparticles.

(12) In the analysis method according to the third to sixth aspects, the heteroorganic 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 method according to the third to sixth aspects, the organic compound having a triple bond may be an acetylene-based 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 include.

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

According to the present invention, an analysis kit capable of analyzing a test substance with high selectivity and high sensitivity and capable of long-term storage by a simple operation, and an analysis method of the test substance using the analysis kit. Can be provided.

It is a top view of the sensor used in the analysis kit which concerns on 1st Embodiment of this invention. FIG. 2 is a sectional view taken along line II-II of FIG. It is a flow chart explaining the analysis method which concerns on 3rd to 6th Embodiment of this invention. It is a conceptual diagram explaining the analysis method which concerns on 3rd to 6th Embodiment of this 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 that are the antigen labels. is there. The diamond-shaped plot shows the results before the stability test, and the triangular plot shows 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 that are the antigen labels. is there. The diamond-shaped plot shows the results before the stability test, and the triangular plot shows 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 that are the antigen labels. is there. The diamond-shaped plot shows the results before the stability test, and the triangular plot shows 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 that are the antigen labels. is there. The diamond-shaped plot shows the results before the stability test, and the triangular plot shows the results after the stability test.

Hereinafter, the configuration of this embodiment will be described with reference to the drawings. In the drawings used in the following description, the featured portion may be enlarged for convenience in order to make the feature easy to understand, and the dimensional ratio of each component may not be the same as the actual one. Further, the materials, dimensions, etc. exemplified in the following description are examples, and the present invention is not limited thereto.

"First embodiment"
[Analysis kit]
The analysis kit of the present embodiment is an analysis kit for analyzing a test substance contained in a test substance solution by utilizing an antigen-antibody reaction. The test substance is, for example, a biomaterial, particularly a protein or a metabolome.
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 a marker for quantifying the captured test substance.

(Sensor)
The 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. 1 is a plan view showing an embodiment of the sensor. FIG. 2 is a 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 near one end on the surface (upper surface in FIG. 2) of the first substrate 11, and a working electrode. Lead wires 12a, 13a and 14a, which are connected to 12, the counter electrode 13 and the reference electrode 14, and are formed on the first substrate 11 so as to extend to the other end of the first substrate 11, and the first substrate. It is composed of a second substrate 16 having a working electrode 12, a counter electrode 13 and a window 15 from which the reference electrode 14 is exposed, which is adhered to 11. The second substrate 16 covers a portion of the lead wires 12a, 13a, and 14a other than the vicinity of the other end of the first substrate 11.

The working electrode 12 is preferably a metal electrode, a carbon electrode, a conductive diamond electrode or a conductive diamond-like carbon electrode (DLC electrode). As the metal electrode, a copper electrode, a gold electrode, a platinum electrode, a palladium electrode, or the like can be used. The metal electrode is preferably a noble metal electrode from the viewpoint of corrosion resistance. The carbon electrode is a carbon electrode having conductivity such as graphite, and for example, a carbon printing electrode printed with a paste mainly composed of graphite can be used. As the conductive diamond electrode, a boron-doped diamond electrode doped with boron can be used. Conductive diamond electrode is a crystalline carbon electrodes having a diamond structure with sp ³ bonds. The conductive DLC electrode is an amorphous carbon electrode mainly composed of carbon and hydrogen, in which sp ³ bonds and sp ² bonds are mixed. As the conductive DLC electrode, 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 are used. Any of the doped p-type semiconductor DLC electrodes can be used.

The sp ^3- bonded carbon-based conductive diamond electrode and DLC electrode have very few processes of adsorbing chemical substances that are oxidized or reduced by an electrochemical reaction. Therefore, for example, the inner redox reaction via adsorption of hydrogen, hydroxides, and their ions caused by water to the electrode is extremely unlikely to occur. As a result, the noise current called the residual current becomes extremely small, so that the electrochemical reaction of the test substance to be detected can be detected at a high SN ratio.

The primary antibody is immobilized on the surface (upper surface in FIG. 2) of the working electrode 12. The primary antibody is appropriately selected and used according to the test substance (antigen) to be measured. The 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).

It is preferable that the surface of the working electrode 12 other than the portion where the primary antibody is fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. The heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond (blocking agent) may be used alone or in combination of two or more. Since the surface of the working electrode 12 other than the portion where the primary antibody is fixed is blocked by the blocking agent, non-specific adsorption of the secondary antibody to the working electrode is prevented, and the test substance is used. It can be analyzed with higher selectivity and sensitivity. Further, by using a heteroorganic 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 deteriorated by bacteria or the like, and the analysis kit is stored for a long period of time. Becomes possible.

Examples of the heteroorganic compound having an unpaired electron pair or a lone electron pair include an organic sulfur compound, an organic nitrogen compound, and an organic oxygen compound.

Examples of the organic sulfur compound include thiourea and its derivatives, thiazole and its derivatives, disulfides, carbamic acids and their 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 its derivatives 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 the carbamic acid and its derivatives and salts thereof include dimethyldithiocarbamic acid, diethyldithiocarbamic acid, dibutyldithiocarbamic acid, N-methyl-N-phenyldithiocarbamic acid, N-pentamethylenedithiocarbamic acid, dibenzyldithiocarbamic acid, and sodiums thereof. Examples include salt and zinc salt.

Examples of 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 compound include aliphatic amines, aromatic amines, azo compounds, imidazoles and derivatives thereof, triazoles and derivatives thereof, imines, amides, amino acids and the like.

Examples of the aliphatic amine include n-butylamine, n-hexiamine, n-octylamine, n-dodecylamine, n-hexadecylamine, n-cyclohexylamine, n-octadecylpropylenediamine, n-dodecylpropylenediamine and caproic acid. Amine, N, N'-dibutylamine, N, N'-dihexiamine, N, N'-dioctylamine, N, N'-didodecylamine, N, N'-dihexadecylamine, N, N'-dicyclohexyl Amine, N, N'-dioctadecylpropylenediamine, N, N'-didodecylpropylenediamine, N, N'-dimethylamine caproic acid, N, N'-dimethyl N'-methyloctadecylpropylenediamine, N, N' -Dimethyl N'-methyldodecylpropylene diamine, N-cyclohexyldimethylamine, morpholine, monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, diethylhydroxyamine, polyvinylamine, phosphonate amine and the like can be mentioned.

Examples of the aromatic amine include aniline, toluidine, trimethylaniline, anicidin, pyrrole, pyridine, quinoline, rhodamine, saccharin, safranin and the like.

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

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

Examples of the triazole and its derivatives include benzotriazole, 1-hydroxybenzotriazole, and triltriazole.

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

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

Examples of the amino acid 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, Tween20, marialim, naphthol, ethylene glycol, polyvinylpyrrolidone, alkyrolamide, haloacetic acid, polycarboxylic acid, glyceraldehyde, dihydroxyacetone, saccharides erythrose, trehalose, and elittlerose. Ribos, liquose, xylose, arabinose, apiose, ribulose, xylulose, allose, talose, growth, glucose, altrose, mannose, galactose, idose, psicose, fructose, sorbose, tagatous, sedhepturose, collios, trehalose, isotrehalose Sorbose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, lactose, sucrose, raffinose, panose, maltotriose, meregitos, gentianose, stachiose, cyclodextrin, deoxyribose, fucose, lambnorse, glucuronic acid, galacturonic acid. , Fructoligosaccharide, palatinose, glucosamine, galactosemin, glycerin, xylitol, sorbitol, ascorbic acid, glucuronolactone, gluconolactone, amylose, starch, dextrin, amylopectin, glycogen, cellulose, pectin, glucomannose, xantagam, purulan, hyaluronic acid And so on.

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

The counter electrode 13 is composed of a conductive material for an electrode, which is usually used in a sensor for electrochemical measurement. As the counter electrode 13, for example, a noble metal electrode such as a carbon electrode, a platinum electrode and a gold electrode, a conductive diamond electrode, and 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. The reference electrode 14 is preferably a silver-silver chloride electrode.

As the second substrate 16, the same substrate as the first substrate 11 is used. The first substrate 11 and the second substrate 16 are adhered to each other by an adhesive 17.
The second substrate 16 forms a flow path with the first substrate 11. The second substrate 16 may be a smooth one in which a groove serving as a flow path is dug or a third substrate for forming a flow path is sandwiched between the first substrate and the second substrate.

The first substrate 11, the second substrate 16, and the third substrate may have physical strength that can withstand use as an analysis chip, and may not be physically or chemically damaged by a liquid containing a test substance. For example, general polymers such as polyethylene, polypropylene, polycarbonate, polystyrene, polyimide, nylon, polyethylene terephthalate, epoxy resin, polyethylene oxide, and Teflon (registered trademark) are preferable. From the viewpoint of environmental harmony, biodegradable plastics such as popolic lactic acid resin, polycaprolactone resin, polyhydroxyalkanoate resin, polyglycolic acid resin, and modified polyvinyl alcohol resin can also be used if the above conditions are satisfied.

(Metal nanoparticles)
The average particle size of the metal nanoparticles is preferably in the range of 1 nm or more and 100 nm or less, and more preferably in the range of 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, or a mixed solution thereof can be used. Examples of the aqueous solvent include water and a buffer solution. Phosphate buffered saline (PBS) can be used as the buffer solution. Examples of 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 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 type of metal may be used alone, or two or more types may be used as an alloy in combination. The metal nanoparticles are preferably gold nanoparticles and silver nanoparticles. One of these metal nanoparticles may be used alone, or two or more of these metal nanoparticles may be used in combination.

Secondary antibodies are immobilized on the surface of metal nanoparticles. The secondary antibody is appropriately selected and used according to the test substance (antigen) to be measured. The 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 fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. Examples of the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. The blocking agent may be used alone or in combination of two or more. 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 an antigen. It is possible to prevent and analyze the test substance with high selectivity and high sensitivity by a simple operation. Further, by using a heteroorganic 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 deteriorated by bacteria or the like, and the analysis kit is 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 particle nanoparticles or may be inserted 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 or more and 0.5% by mass or less.

The dispersion liquid in which the metal nanoparticles are dispersed may further contain a thickener, a surfactant, a dispersant, an antioxidant and the like.

"Second embodiment"
[Analysis kit]
In the analysis kit of the second embodiment of the present invention, the primary antibody is fixed on the surface of the working electrode, and the surface of the working electrode other than the portion where the primary antibody is fixed is an unpaired electron pair or a lone electron. The surface other than the point blocked by the heteroorganic compound having a pair or the organic compound having a triple bond and the portion where the secondary antibody of the metal nanoparticles is immobilized has an unpaired electron pair or a lone electron pair. It is configured similar to the analysis kit of the first embodiment, except that it does not have to be blocked by a heteroorganic compound or an organic compound having a triple bond.

In the analysis kit of the present embodiment, the surface of the working electrode 12 other than the portion where the primary antibody is fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. There is. Examples of the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. Since the surface of the working electrode 12 other than the portion where the primary antibody is fixed is blocked by the blocking agent, non-specific adsorption of the secondary antibody to the working electrode is prevented, and the test substance is used. It can be analyzed with high selectivity and high sensitivity. Further, by using a heteroorganic 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 deteriorated by bacteria or the like, and the analysis kit is 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 heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. It is preferable that it is. Examples of the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond (blocking agent) include those described above. Since the surface of the metal nanoparticles other than the portion where the secondary antibody is immobilized is blocked by the blocking agent, the secondary antibody directly and non-specifically binds to the primary antibody without an antigen. It is possible to analyze the test substance with higher selectivity and sensitivity with a simple operation. Further, by using a heteroorganic 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 deteriorated by bacteria or the like, and the analysis kit is stored for a long period of time. Becomes possible.

"Third embodiment"
[Analysis method]
Next, the analysis method of the third embodiment of the present invention will be described with reference to FIGS. 3 and 4.
FIG. 3 is a flow chart illustrating an analysis method according to a third embodiment of the present invention.
As shown in FIG. 3, the analysis method of the present embodiment 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 coupling 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, a primary antibody 31 capable of selectively binding to a test substance to be measured is immobilized on the surface of the working electrode 12. Therefore, in the first binding step S01, as shown in FIG. 4B, only the test substance 32 to be measured is captured by the primary antibody 31.
Further, when the surface other than the portion where the primary antibody 31 of the working electrode 12 is fixed 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 secondary. Non-specific adsorption of the antibody 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 S01, 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 above-mentioned dispersion of metal nanoparticles. As shown in FIG. 4C, a secondary antibody 34 that can selectively bind to the test substance 32, which is the object to be measured, is immobilized on the surface of the metal nanoparticles 33. Therefore, in the second binding step S02, as shown in FIG. 4C, the test substance 32 and the secondary antibody 34 are bound, and the metal nanoparticles 33 are bound to the test substance 32. Since the surface of the metal nanoparticles 33 other than the portion where the secondary antibody 34 is fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond, the secondary antibody It is prevented that 34 directly and non-specifically binds to the primary antibody 31 without an antigen. Therefore, the test substance can be analyzed with high selectivity and high sensitivity.

After the second bonding step, the working electrode 12 may be washed to remove the metal nanoparticles 33 that are not bonded to the test substance 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 the potential for oxidizing and ionizing the metal nanoparticles 33 bonded to the test substance 32. , A voltage is applied between the working electrode 12 and the counter electrode 13.

As the electrolyte of the electrolyte solution, chlorides such as potassium chloride, sodium chloride and lithium chloride can be used. When the metal nanoparticles 33 are ionized, the chloride destroys the oxide film (passivation film) formed on the surface of the metal nanoparticles 33, exposes the metal to the solution, and facilitates the progress of ionization. effective. As the solvent of the electrolyte solution, in addition to water, an organic solvent such as dimethyl sulfoxide (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 potential of the working electrode 12 can ionize the metal nanoparticles 33 as the voltage applied between the working electrode 12 and the counter electrode 13. The potential is not particularly limited, but when the reference electrode 14 is a silver-silver chloride electrode, 0 to 1.0 V is preferable with respect to the reference electrode 14, and 0.2 to 0.8 V is more preferable. In this voltage application step S03, the metal nanoparticles 33 bonded 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 to measure the amount of current until the entire amount of the metal nanoparticles 33 ionized (oxidized) in the presence of the electrolyte solution is ionized. .. For example, when the metal nanoparticles 33 are gold nanoparticles, a voltage is applied between the working electrode 12 and the counter electrode 13 to measure the amount of current when the gold nanoparticles are dissolved as gold chloride complex ions. .. Specifically, the lead wires 12a, 13a, 14a of the sensor 10 are connected to the potentiostat, and the working electrode 12 and the counter electrode are applied while applying a voltage between the working electrode 12 and the counter electrode 13 using the voltammetry method. The amount of current flowing between the 13 and 13 is measured.

(Calculation step S05)
In the calculation step S05, the amount of the metal nanoparticles 33 is obtained from the amount of current, and the amount of the test substance is calculated from the amount of the metal nanoparticles 33. The amount of current 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"
The analysis method of the fourth embodiment of the present invention is the same as that of the third embodiment shown in FIG. 3, the first coupling step S01, the second coupling step S02, the voltage application step S03, the current amount measurement step S04, and the calculation step. Including S05. In the analysis method of the fourth embodiment, in the voltage application step 03, instead of ionizing the entire amount of the metal nanoparticles by oxidation in the presence of the electrolyte solution in the third embodiment, the total amount of the metal nanoparticles is present in the electrolyte solution. In the point of further reducing the ionized metal nanoparticles by being ionized by oxidation below, and in the current amount measurement step S04, a voltage is applied between the working electrode 12 and the counter electrode 13 of the third embodiment to apply an electrolyte solution. Instead of measuring the amount of current until the total amount of the metal nanoparticles 33 ionized (oxidized) in the presence of the working electrode 12 is ionized, a voltage is applied between the working electrode 12 and the counter electrode 13 in the presence of the electrolyte solution. After ionizing (oxidizing) the entire amount of the metal nanoparticles 33, the metal nanoparticles 33 further ionized (oxidized) in the presence of an electrolyte solution by applying a reverse voltage between the working electrode 12 and the counter electrode 13. It is configured in the same manner as the analysis method of the third embodiment except that the amount of current until reduction is measured.

In the voltage application step S03 of the present embodiment, first, in order to obtain the potential of the working electrode 12 for ionizing the entire amount of the metal nanoparticles by oxidation in the presence of the electrolyte solution, a voltage is applied between the working electrode 12 and the counter electrode 13. Apply. Then, a reverse voltage is applied between the working electrode 12 and the counter electrode 13 in order to obtain the potential of the working electrode 12 that electrochemically reduces the ionized metal nanoparticles. The potential applied to the working electrode 12 at the time of reduction is preferably 0.0 to 1.0 V, more preferably 0.1 to 0.8 V.

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, and the entire amount of the metal nanoparticles 33 is ionized in the presence of the electrolyte solution, and then the working electrode 12 and the counter electrode 13 are formed. By applying the opposite voltage between the two, the amount of current until the ionized metal nanoparticles are reduced is measured. For example, when the metal nanoparticles 33 are gold nanoparticles, the gold chloride complex ion is generated by applying an electric potential to the working electrode 12 in order to apply a reverse voltage between the working electrode 12 and the counter electrode 13. The amount of current when it is reduced and electrodeposited as gold is measured. Specifically, the lead wires 12a, 13a, 14a of the sensor 10 are connected to the potentiostat and flow between the working electrode 12 and the counter electrode 13 while applying an electric potential to the working electrode 12 using the voltammetry method. 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 other than the portion where the primary antibody of the working electrode 12 is fixed has an unpaired electron pair or an isolated electron pair. It is configured in the same manner as the analysis method of the third embodiment except that it is blocked by a heteroorganic compound having a heteroorganic compound or an organic compound having a triple bond.

In the present embodiment, the surface other than the portion where the primary antibody of the working electrode 12 is fixed is blocked by a heteroorganic 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. Therefore, the test substance can be analyzed with high selectivity and high sensitivity. Examples of the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond are the same as described above.

In the present embodiment, in the second binding step S02, the surface of the metal nanoparticles 33 other than the portion where the secondary antibody is fixed is a heteroorganic compound or a triple bond having an unpaired electron pair or a lone electron pair. It may be blocked by an organic compound having. In this case, the secondary antibody 34 is prevented from directly and non-specifically binding to the primary antibody 31 without an antigen. 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 other than the portion where the primary antibody of the working electrode 12 is fixed has an unpaired electron pair or an isolated electron pair. It is configured in the same manner as the analysis method of the fourth embodiment except that it is blocked by the heteroorganic compound having or the organic compound having a triple bond.

In the present embodiment, the surface other than the portion where the primary antibody of the working electrode 12 is fixed is blocked by a heteroorganic 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 the heteroorganic compound having an unpaired electron pair or a lone electron pair or the organic compound having a triple bond are the same as described above.

In the present embodiment, in the second binding step S02, the surface of the metal nanoparticles 33 other than the portion where the secondary antibody is fixed is a heteroorganic compound or a triple bond having an unpaired electron pair or a lone electron pair. It may be blocked by an organic compound having. In this case, the secondary antibody 34 is prevented from directly and non-specifically binding to the primary antibody 31 without an antigen. 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 other than the portion where the secondary antibody of the metal nanoparticles is fixed is a heteroorganic compound or a triple having an unpaired electron pair or a lone electron pair. Since it is blocked by an organic compound having a bond, it is prevented that the secondary antibody is directly non-specifically bound to the primary antibody without an antigen, and the test substance can be highly selective and highly selective by a simple operation. It can be analyzed by sensitivity.

Further, since the analysis kit of the first embodiment uses a heteroorganic 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 altered by bacteria or the like. Since there is no such substance, long-term storage is possible.

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

Further, since the analysis kit of the second embodiment uses a heteroorganic 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 altered by bacteria or the like. Since there is no such substance, long-term storage is possible.

Further, according to the analysis methods of the third embodiment and the fifth embodiment, the surface other than the portion where the secondary antibody of the metal nanoparticles is fixed is a heteroorganic compound having an unpaired electron pair or a lone electron pair. Since it is blocked by an organic compound having a triple bond, it is prevented that the secondary antibody directly and non-specifically binds to the primary antibody without an antigen, and the test substance can be highly selective with a simple operation. 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, which has been conventionally performed. The test substance can be quantified using an electrochemical technique without performing the step of chemically dissolving the metal.

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

Further, according to the analysis methods of the 4th embodiment and the 6th embodiment, a voltage was applied between the working electrode and the counter electrode to ionize the entire amount of the metal nanoparticles bound to the test substance, and further ionized. Since the metal nanoparticles are reduced, the test substance can be quantified by 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 oxidizing the metal nanoparticles, it is better to measure the current that reduces the ionized metal nanoparticles to reduce the oxidation current of the substance that is oxidized at the same time as the metal nanoparticles. The test substance can be analyzed more accurately than it is measured.
Therefore, according to the analysis kit and analysis method of the present invention, it is possible to analyze the test substance with high selectivity and high sensitivity by a simple operation, and the analysis kit can be stored for a long period of time. .. The analytical method of the present invention can also be applied to the immunochromatography method.

Further, 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 of the present invention are also applicable to the embodiment by the competitive method. The competitive method is preferably used when the antigen is a hapten (small molecule antigen) which is a small molecule compound. For example, when the antigen to be measured is a hapten, the hapten-protein conjugate is fixed to the working electrode. When a small molecule antigen to be measured and a limited amount of gold nanoparticle-labeled antibody are added thereto, the immobilized antigen and the released small molecule antigen to be measured competitively react with the gold nanoparticle-labeled antibody. .. The more small molecule antigens to be measured released, the smaller the amount of gold nanoparticle-labeled antibody that reacts with the immobilized antigen. On the contrary, the smaller the amount of small molecule antigen to be measured released, the larger 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 to remove the unreacted low-molecular-weight antigen to be measured and the gold nanoparticle-labeled antibody that have not been reacted by washing. The current value is measured by electrochemical oxidation and reduction of the gold nanoparticle label on the electrode. If a calibration curve is prepared in advance using a known amount of the small molecule antigen to be measured, an unknown amount of the small molecule antigen to be measured can be measured.

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

[Example 1]
(1) Fabrication of sensor with primary antibody Gold-deposited film with counter electrode and lead wire patterned with a mask on a polyethylene terephthalate substrate, conductive DLC film formed on a Si wafer on a working electrode, and Ag / AgCl pasted on a reference electrode. A sensor chip using an Ag / AgCl film prepared by screen printing was prepared. An unlabeled anti-goat IgG as a primary antibody is fixed to 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 is fixed with benzoimidazole. By blocking the surface, an analysis chip with a primary antibody in which the primary antibody was immobilized on the surface of the working electrode was prepared.

(2) Gold Nanoparticle Dispersion with Secondary Antibody A gold nanoparticle dispersion with an average particle diameter of 18 nm manufactured by Tanaka Kikinzoku Co., Ltd. was used.

The above gold nanoparticles, benzoimidazole, anti-goat IgG, phosphate buffered saline (PBS), and polyethylene glycol sorbitan monolaurate (Tween 20, nonionic surfactant) are mixed to produce gold nanoparticles. A gold nanoparticle dispersion with a secondary antibody was prepared by immobilizing an anti-goat IgG (secondary antibody) on the surface of the sample. The concentration of gold nanoparticles in the gold nanoparticle dispersion with the secondary antibody was 0.007% by mass. As the anti-goat IgG, a polyclonal antibody commercially available from Jackson Immunoresearch Laboratories was used.

(3) Sample for antigen analysis As a sample for antigen analysis, No. 1 having different antigen concentrations as shown below. 1-No. 6 was prepared. The following No. 1-No. 6 was prepared by mixing PBS buffer containing 0.1% Tween 20 and goat IgG (antigen) so that the antigen concentration was as follows.
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-No. For each of the antigen analysis samples of No. 6, 35 μL was accurately weighed, dropped onto the working electrode of the sensor with the primary antibody prepared in (1) above, and then incubated for 40 minutes (first binding step).

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

Next, 1 μL of the gold nanoparticle dispersion with the secondary antibody prepared in (2) above was accurately weighed, and this was dropped onto the working electrode of the sensor with the primary antibody and then incubated for 3 hours (second). Bonding process).

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

Next, the lead wire of the sensor with the primary antibody was connected to the potentiostat, an electrolyte solution in which 0.1 mol / L potassium chloride was dissolved in PBS was injected, and the sensor with the primary antibody 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 and ionize the gold nanoparticles. 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 was measured during that time (current amount). Measurement process).

In FIG. 5, No. 1-No. A graph plotting the antigen concentration of each of the samples for antigen analysis of 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). From the graph of FIG. 5, it was confirmed that there is a correlation between the current value and the antigen concentration of the antigen analysis sample. 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 was possible to do so.

In addition, the sensor with the primary antibody was vacuum sterilized, wrapped in an aluminum pouch, and then subjected to a stability test at 25 ° C., 75% RH, and 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 FIG. 5, it was confirmed that there was no significant change in the calibration curve before and after the stability test, and it was possible to accurately quantify the test substance 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. 6, No. 1-No. A graph plotting the antigen concentration of each of the samples for antigen analysis of 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). From the graph of FIG. 6, it was confirmed that there is a correlation between the current value and the antigen concentration of the antigen analysis sample. 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 was possible to do so.

In addition, the sensor with the primary antibody was vacuum sterilized, wrapped in an aluminum pouch, and then subjected to a stability test at 25 ° C., 75% RH, and 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 FIG. 6, it was confirmed that there was no significant change in the calibration curve before and after the stability test, and it was possible to accurately quantify the test substance 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. 7, No. 1-No. A graph plotting the antigen concentration of each of the samples for antigen analysis of 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). From the graph of FIG. 7, it was confirmed that there is a correlation between the current value and the antigen concentration of the antigen analysis sample. 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 was possible to do so.

In addition, the sensor with the primary antibody was vacuum sterilized, wrapped in an aluminum pouch, and then subjected to a stability test at 25 ° C., 75% RH, and 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 FIG. 7, it was confirmed that there was no significant change in the calibration curve before and after the stability test, and it was possible to accurately quantify the test substance 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 skim milk was used instead of benzimidazole. The results are shown in FIG. 8 (plot before stability test in FIG. 8).

The sensor with the primary antibody was vacuum sterilized, wrapped in an aluminum pouch, and then subjected to a stability test at 25 ° C., 75% RH, and for 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, after the stability test, a decrease in the detected current was observed, and it was confirmed that the quantitativeness of the test substance decreased when stored for a long period of time.

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 and having a primary antibody immobilized on the surface of the working electrode, and a secondary antibody that specifically binds to a test substance are immobilized on the surface. It is characterized in that the surface other than the portion where the secondary antibody is immobilized contains 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. Analysis kit for the test substance. The analysis according to claim 1, wherein the surface other than the portion where the primary antibody of the working electrode is fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. kit. It has a working electrode, a reference electrode, and a counter electrode, and the primary antibody is fixed on the surface of the working electrode, and the surface of the working electrode other than the portion where the primary antibody is fixed is an unpaired electron pair. Alternatively, it includes a sensor blocked by a heteroorganic compound having a lone electron pair or an organic compound having a triple bond, and metal nanoparticles having a secondary antibody immobilized on the surface that specifically binds to the test substance. An analysis kit for the test substance. The analysis kit according to any one of claims 1 to 3, wherein the heteroorganic 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 acetylene-based organic compound. 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. The analysis kit described in. This is a method for analyzing the test substance contained in the test substance solution.
The working electrode of a sensor having a working electrode, a reference electrode, and a counter electrode, and a primary antibody that specifically binds to the test substance is immobilized on the surface of the working electrode, and the test substance solution. In the first binding step of binding the primary antibody of the working electrode and the test substance of the test substance solution.
A secondary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode, and the surface other than the portion on which the secondary antibody is immobilized has an unpaired electron pair or a lone electron pair. With the test substance bound to the primary antibody of the working electrode by contacting 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, and
A current amount measuring 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. When,
A calculation step of obtaining the amount of metal nanoparticles from the amount of the current and calculating the amount of the test substance from the amount of the metal nanoparticles.
A method for analyzing a test substance, which comprises. This is a method for analyzing the test substance contained in the test substance solution.
The working electrode of a sensor having a working electrode, a reference electrode, and a counter electrode, and a primary antibody that specifically binds to the test substance is immobilized on the surface of the working electrode, and the test substance solution. In the first binding step of binding the primary antibody of the working electrode and the test substance of the test substance solution.
A secondary antibody that specifically binds to a test substance is immobilized on the surface of the working electrode, and the surface other than the portion on which 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 brought into contact with the test substance bound to the primary antibody of the working electrode to bind the secondary antibody. Thereby, the second bonding step of bonding the test substance and the metal nanoparticles, and
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. The current amount measurement process to be measured and
A calculation step of obtaining the amount of metal nanoparticles from the amount of the current and calculating the amount of the test substance from the amount of the metal nanoparticles.
A method for analyzing a test substance, which comprises. The 7th or 8th claim, wherein the surface other than the portion where the primary antibody of the working electrode is fixed is blocked by a heteroorganic compound having an unpaired electron pair or a lone electron pair or an organic compound having a triple bond. Analysis method. This is a method for analyzing the test substance contained in the test substance solution.
It has a working electrode, a reference electrode, and a counter electrode, and a primary antibody that specifically binds to the test substance is immobilized on the surface of the working electrode, and the surface other than the portion to which the primary antibody is immobilized is immobilized. The working electrode of the sensor, which is blocked by a heteroorganic compound having an unpaired electron pair or a lone pair of electrons, or an organic compound having a triple bond, is brought into contact with the test substance solution to bring the primary of the working electrode into contact with the test substance solution. The first binding step of binding the antibody and the test substance of the test substance solution, and
The working electrode and the dispersion liquid of the metal nanoparticles in which the metal nanoparticles having the secondary antibody bound to the test substance fixed on the surface are dispersed are brought into contact with each other and bound to the primary antibody of the working electrode. A second binding step of binding the test substance and the metal nanoparticles by binding the test substance and the secondary antibody.
A current amount measuring 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. When,
A calculation step of obtaining the amount of metal nanoparticles from the amount of the current and calculating the amount of the test substance from the amount of the metal nanoparticles.
A method for analyzing a test substance, which comprises. This is a method for analyzing the test substance contained in the test substance solution.
It has a working electrode, a reference electrode, and a counter electrode, and a primary antibody that specifically binds to the test substance is immobilized on the surface of the working electrode, and the surface other than the portion to which the primary antibody is immobilized is immobilized. The working electrode of the sensor, which is blocked by a heteroorganic compound having an unpaired electron pair or a lone pair of electrons, or an organic compound having a triple bond, is brought into contact with the test substance solution to bring the primary of the working electrode into contact with the test substance solution. The first binding step of binding the antibody and the test substance of the test substance solution, and
The working electrode and the dispersion liquid of the metal nanoparticles in which the metal nanoparticles having the secondary antibody bound to the test substance fixed on the surface are dispersed are brought into contact with each other and bound to the primary antibody of the working electrode. 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. The current amount measurement process to be measured and
A calculation step of obtaining the amount of metal nanoparticles from the amount of the current and calculating the amount of the test substance from the amount of the metal nanoparticles.
A method for analyzing a test substance, which comprises. The analysis method according to any one of claims 7 to 11, wherein the heteroorganic 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 acetylene-based organic compound. 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. The analysis method described in. The analysis method according to any one of claims 7 to 14, wherein the analysis method is an immunochromatography method.

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