JP5571705B2 - Electrical analysis method - Google Patents

Description

  The present invention relates to an electrical analysis method. Note that “analysis” in the present specification includes “detection” for determining the presence or absence of an analysis target substance and “measurement” for quantitatively or semi-quantitatively determining the amount of the analysis target substance.

  An electroimmunosensor is a method that electrically measures an antigen-antibody reaction, but in order to detect the immune complex formed by the direct antigen-antibody reaction with high sensitivity, it is practical because the signal is weak. is not. Therefore, the signal is generally amplified by forming an immune complex labeled with an enzyme or the like. In this way, what is labeled with an enzyme and performing signal amplification is called an electroenzyme immunosensor, and an antibody or antigen is immobilized on the working electrode, where the enzyme-labeled immune complex formed based on the antigen-antibody reaction It takes a mechanism to electrically measure the reaction product of the enzyme in the body.

For example, in Patent Document 1, a process of converting a soluble substance into an insoluble substance by an enzyme reaction and depositing it on a sensing part, and a step of electrically analyzing the insoluble substance deposited on the sensing part are performed under fluid conditions. A method is disclosed.
In addition, as an efficient electrical analysis method for soluble substances, Patent Document 2 discloses that an electrically soluble substance is converted into an insoluble substance and deposited on a sensing unit, and the insoluble substance deposited on the sensing unit is electrically converted. A method is disclosed in which a substance is analyzed after being released.

WO2009-116534 pamphlet JP-A-6-27081

However, as a result of intensive studies by the present inventors, a plurality of reactions are carried out in the same container in the method described in Patent Document 1, so that matrix reactions other than the main reactants may be used for electrochemical measurements depending on the application conditions. It has been found that substances in the solution used for the reaction also greatly affect the electrochemical detection.
Moreover, in patent document 2, it becomes a method of achieving high sensitivity by converting the oxidation-reduction reaction to be analyzed into adhesion of an insoluble substance to the sensing part of another container that is electrically conducted. Yes. However, since a plurality of reaction vessels are required, a complicated measuring device configuration is used, and the target reaction and the detection reaction are performed in different places, it is difficult to carry out and the accuracy is not sufficient.

As a result of intensive studies by the present inventors, it has been found that sensitivity and accuracy are reduced due to by-products that affect the measured values in electrical analysis. It was also found that the sensitivity can be remarkably increased by conducting an electrical analysis in the presence of a strong electrolyte superior in current response.
Accordingly, an object of the present invention is to provide an analysis method with higher detection sensitivity and accuracy than conventional analysis methods.

That is, the present invention
[1] By performing an insolubilization reaction by reacting an analysis target substance with a specific partner exhibiting a selective interaction with the analysis target substance and correlating it with the abundance of the analysis target substance in the test sample A method for converting a soluble substance into an insoluble substance, precipitating it on a sensing part, and electrically analyzing the insoluble substance precipitated on the sensing part,
Analytical method characterized in that electrical analysis is performed in a strong electrolyte solution superior in current response,
[2] Electrical analysis
(A) removing a substance that contributes to a current response other than the insoluble substance;
(B) Next, the analysis method according to [2], including a step of replacing with a strong electrolyte solution,
[3] The analysis method according to [1] or [2], wherein the electrical analysis is performed by analyzing a single detection peak,
[4] The analysis method according to any one of [1] to [3], wherein the strong electrolyte is selected from an alkali metal halide, nitrate, sulfate, and phosphate.
[5] The analysis method according to any one of [1] to [4], wherein the strong electrolyte is potassium nitrate.
[6] The analysis method according to any one of [1] to [5], wherein the specific partner is an antibody.
[7] The analysis method according to any one of [1] to [6], wherein the soluble substance is silver ions,
[8] The analysis method according to any one of [1] to [7], wherein the specific partner is fixed to the sensing unit.
[9] The analysis method according to any one of [1] to [8], wherein the selective interaction is performed on magnetic fine particles, and the electrical measurement is an amperometric measurement method,
[10] A reagent and a kit for measuring a substance to be analyzed, characterized by being used in the analysis method according to any one of [1] to [9],
The present invention relates to a reagent and a kit including a specific partner that selectively interacts with the analyte, a soluble substance that is converted into an insoluble substance by an insolubilization reaction, a sensing unit, and a strong electrolyte.

  According to the present invention, it is possible to perform analysis with higher detection sensitivity and accuracy than conventional analysis methods. For example, the current response value obtained by the conventional analysis method is increased, and the detection sensitivity can be increased at least twice. In addition, since stable peak detection is possible, highly accurate analysis can be performed.

It is explanatory drawing which shows typically a series of reaction utilized with the one aspect | mode of the analysis method of this invention. It is explanatory drawing which shows typically a series of reaction utilized in another one aspect | mode of the analysis method of this invention. It is a block diagram which shows typically the structure of the electrochemical detection apparatus used in Example 1. FIG. In Example 1, it is a graph showing the results DPV of (differential pulse voltammetry) measurements when during electrochemical measurement using KNO ₃ as an electrolyte. In Example 2, as a solution components during the electrochemical measurements were compared with the individual components of the insolubilization reaction solution and KNO _3, it is a graph showing the results of DPV measurements. In Example 3, it is a graph which shows the result of the DPV measurement which compared the effect by the presence or absence of the electrolyte at the time of electrochemical measurement.

The present invention is an analysis method using a combination of selective interaction (for example, antigen-antibody reaction, enzyme-substrate reaction) and insolubilization reaction (preferably redox reaction), and finally produced by the insolubilization reaction. A method for precipitating (depositing, insolubilizing, precipitating) an insoluble product on the surface of a sensing unit and electrically analyzing (detecting or measuring) the precipitated insoluble material, wherein the precipitated insoluble material is electrically analyzed In this case, a substance that contributes to the current response other than the analysis target is removed and replaced with a strong electrolyte solution that is superior in the current response.
In other words, there is no substance that contributes to the current response other than the analysis target substance to such an extent that the current response derived from the analysis target substance can be detected as the main peak in the electrical analysis, and there is a strong electrolyte superior in the current response. It only has to exist.

In the analysis method of the present invention, depending on the selective interaction or insolubilization reaction used, for example,
(1) A method in which an insolubilization reaction is directly or indirectly caused by a labeling substance that can be directly or indirectly labeled on one partner involved in a selective interaction (hereinafter referred to as a complex formation type analysis method). )
(2) A method in which an insolubilization reaction is directly or indirectly caused by selective interaction itself (hereinafter referred to as an enzyme-based analysis method).
Etc. are included. In addition, the said classification | category is divided roughly based on whether a complex is formed by selective interaction, for example, the method of using an enzyme as a labeling substance is contained in a complex formation type | mold analysis method.

In the present specification, “insolubilization reaction is directly caused” means that the reaction itself involving a labeling substance or selective interaction is an insolubilization reaction, and an insoluble substance is generated by the reaction. In addition, “insolubilized reaction is indirectly induced” means that a labeling substance or a substance generated by a reaction involving selective interaction is a trigger, and finally an insoluble substance is generated by the insolubilizing reaction. To do.
Hereinafter, based on the reaction schematic diagram shown in FIG. 1 showing a specific embodiment of the complex-forming analysis method of the present invention and the reaction schematic diagram shown in FIG. 2 showing a specific modification, the outline of the present invention will be described. Then, the present invention will be described in more detail.

In the analysis system shown in FIG. 1, an antigen 103 is used as an analyte, an antigen-antibody reaction (sandwich method) is used as a selective interaction, and an antibody that specifically reacts with the antigen as an enzyme [ For example, ALP-labeled antibody 4 labeled with alkaline phosphatase (ALP)] is used.
In this analysis system, the following reaction formula 1 is used as the enzyme reaction by the labeling enzyme, and the reaction formula 2 is used as the insolubilization reaction. In FIG. 1, pAPP is p-aminophenyl phosphate, pAP is p-aminophenol, pQI is p-quinoneimine, and pAPP is a substrate of ALP. Further, “(ALP)” in Reaction Scheme 1 indicates that ALP is involved as a catalyst in Reaction Scheme 1.
Further, as an electrical analysis method, an amperometric analysis method using an electrode portion having a working electrode 101, a counter electrode, and a reference electrode is used.

p-aminophenyl phosphate → p-aminophenol (ALP) (Scheme 1)
p-aminophenol + 2Ag + → p-quinoneimine + 2H ⁺ + 2Ag ↓ (Scheme 2)
Ag → Ag ⁺ + e ⁻ (Reaction Formula 3)

In the analysis system shown in FIG. 1, an antibody 102 against a substance to be analyzed (antigen) is immobilized in advance on a working electrode 101 constituting an amperometric electrode unit, and the working electrode functions as a sensing unit. When the sample to be analyzed (antigen 103) and the ALP-labeled antibody 104 are supplied from the upstream of the sensing unit along the flow direction indicated by the arrow A to the analysis system, the sample is fixed on the sensing unit. An antibody / antigen / ALP labeled antibody complex is formed. The formation amount of the complex correlates with the abundance of the analysis target substance in the test sample. When p-aminophenyl phosphate (pAPP), which is a substrate of the labeling enzyme ALP, is supplied to the analysis system from upstream of the sensing unit after the complex is formed or simultaneously with the formation, p-aminophenol (pAP At this time, if silver ions (Ag ⁺ , water-soluble) are allowed to coexist, silver (Ag, water-insoluble) is precipitated (Reaction formula 2) and is precipitated on the sensing part. To do. Since the amount of silver precipitated on the sensing unit (working electrode) is re-oxidized on the sensing unit, a current flows between the working electrode and the counter electrode (Reaction Equation 3). Therefore, by measuring the oxidation current, Can be determined. At that time, a single detection peak can be obtained by performing reoxidation on the sensing portion in the absence of a substance that contributes to the current response other than silver and in a strong electrolyte solution. Specifically, the working electrode, the counter electrode, and the reference electrode are connected to a potentiostat, the working electrode potential is swept with respect to the reference electrode potential, and the oxidation current generated by reoxidation of silver is measured.

  In the analysis system shown in FIG. 2, the antigen 103 is an analysis target substance, and an immune complex formation reaction by an antigen-antibody reaction (sandwich method) is used as a selective interaction on the magnetic microparticle 105. An ALP-labeled antibody 104 in which an antibody that specifically reacts with an antigen is labeled with an enzyme [for example, alkaline phosphatase (ALP)] is used. 1 except that the selective interaction is performed on the magnetic fine particles 105.

In the analysis system shown in FIG. 2, an antibody 102 against a substance to be analyzed (antigen) is immobilized on a magnetic fine particle 105 in advance. The working electrode 101 constituting the amperometric electrode part functions as a sensing part. First, when a test sample containing a substance to be analyzed (antigen 103), antigen 103-specific antibody 102-immobilized microparticles and ALP-labeled antibody 104 are reacted in the reaction part, immobilized antibody microparticles / antigen / ALP-labeled antibody. Is formed. The formation amount of the complex correlates with the abundance of the analysis target substance in the test sample. After the complex is formed, B / F separation is performed with an appropriate cleaning solution, and it is moved to the sensing unit, and magnetic particles are collected on the sensing unit by the magnet 106 on the back of the sensing unit. After collecting the magnetic fine particles on the sensing part, or at the same time as collecting the magnetic fine particles on the sensing part while moving to the sensing part, p-aminophenyl phosphate (pAPP), which is a substrate of the labeling enzyme ALP, is added upstream of the sensing part. When supplied to the analysis system, it is converted to p-aminophenol (pAP) (Scheme 1). At this time, if silver ions (Ag ⁺ , water-soluble) coexist, silver (Ag, water-insoluble) is converted. Precipitates (reaction formula 2) and precipitate on the sensing part. Since the amount of silver precipitated on the sensing unit (working electrode) is re-oxidized on the sensing unit, a current flows between the working electrode and the counter electrode (Reaction Equation 3). Therefore, by measuring the oxidation current, Can be determined. At that time, a single detection peak can be obtained by performing reoxidation on the sensing portion in the absence of a substance that contributes to the current response other than silver and in a strong electrolyte solution. Specifically, the working electrode, the counter electrode, and the reference electrode are connected to a potentiostat, the working electrode potential is swept with respect to the reference electrode potential, and the oxidation current generated by reoxidation of silver is measured.

  By performing antigen-antibody reaction using magnetic fine particles, non-specific labeling can be washed B / F by magnetic interaction, and the amount of solid-phase antibody can be increased by using fine particles. And the ability to catch antigens can also be increased. Furthermore, by separating the reaction part of the antigen-antibody reaction and the detection part of the product from the enzyme reaction, it is not necessary to immobilize the antibody (antigen) in the sensing part (working electrode) and immobilize the non-specific inhibitory substance, By facilitating direct electron exchange with the product with the electrode, electrochemical enzyme immunoassay with high sensitivity becomes possible. In addition, by using magnetic fine particles with a smaller particle size, it becomes possible to effectively deposit metal deposits produced by the reducing action of the products produced by the enzyme reaction on the electrode, resulting in higher sensitivity. This is preferable because it can be achieved.

  The selective interaction that can be used in the present invention is an interaction in which one of the analytes can be an analyte, and is directly or indirectly correlated with the abundance of the analyte in the test sample. There is no particular limitation as long as the insolubilization reaction can be performed or a complex can be formed. Typical examples include antigen-antibody reaction, internucleic acid hybridization reaction, enzyme, and the like. -Substrate reaction, nucleic acid-protein interaction, receptor-ligand interaction, protein interaction (eg, reaction between IgG and protein A), small molecule-protein interaction (eg, reaction between biotin and avidin) ). Many of these selective interactions are selective interactions that can form complexes (eg, immune complexes), but the enzyme-substrate reaction correlates with the analyte abundance in the test sample. Reaction that triggers the insolubilization reaction or insolubilization reaction is possible.

  In addition to the above-mentioned interaction, various substances having specific partners exhibiting a selective interaction are known. Examples of the analysis target substance include proteins (enzymes, antigens / antibodies, lectins, etc.), peptides , Lipids, hormones (nitrogen-containing hormones and steroid hormones consisting of amines, amino acid derivatives, peptides, proteins, etc.), nucleic acids, sugar chains (eg, sugars, oligosaccharides, polysaccharides, etc.), drugs, dyes, low molecular weight compounds, organic Examples include substances, inorganic substances, or a fusion thereof, or molecules constituting blood or cells, blood cells, and the like.

  For example, when an antigen-antibody reaction is used as a selective interaction, a combination of an analyte and its specific partner is a combination of an antigen (analyte) and an antibody (specific partner), or an antibody (Analytical substance) and antigen (specific partner). When an enzyme-substrate reaction is used as a selective interaction, a combination of an analyte and its specific partner is a combination of a substrate (analyte) and an enzyme (specific partner), or A combination of an enzyme (analyte) and a substrate (specific partner).

  Examples of the test material containing the substance to be analyzed include blood (whole blood, plasma, serum), lymph, saliva, urine, stool, sweat, mucus, tears, fluid, nasal discharge, cervical or vaginal secretion, In addition to biological fluids such as semen, pleural fluid, amniotic fluid, ascites, middle ear fluid, joint fluid, gastric aspirate, tissue and cell extracts, and crushing fluids, food, soil, plant extracts and crushing fluids Almost all liquid samples including solutions, river water, hot spring water, drinking water, contaminated water, etc. are used.

  The reagent configuration including labeling can be appropriately selected based on the selective interaction to be used. For example, when using an antigen-antibody reaction, various known methods such as a sandwich method, two-step Methods, competition methods, inhibition methods and the like can be used. In the case of the sandwich method, as shown in FIG. 1, a combination of an immobilization partner and a labeling partner can be used. In the case of the two-step method, a combination of an immobilized partner, an unlabeled partner, and a labeled product of a substance that specifically reacts only with the unlabeled partner, specifically, a method using a primary antibody / labeled secondary antibody For example, a method using a biotinylated antibody / labeled avidin can be used. In the case of the competitive method, a combination of a labeled substance (known amount) of an analysis target substance (standard substance) and an immobilization partner can be used.

  The soluble substance used in the present invention is a substance that is soluble in a solvent used in an analysis system before undergoing an insolubilization reaction, and is converted into a substance that is insoluble in the solvent by undergoing the insolubilization reaction. The insoluble material produced by the insolubilization reaction is not particularly limited as long as it can be electrically analyzed. In the present specification, the “insolubilization reaction” includes a reaction in which a substance having low solubility is generated from a soluble substance by an “insolubilization reaction”. In this specification, “soluble” and “insoluble” are terms that can be appropriately defined depending on the solvent system used in the analysis system. For example, when an aqueous solvent is used, “water-soluble” and “water-insoluble” When an organic solvent is used, it means “organic solvent soluble” and “organic solvent insoluble”. Hereinafter, a case where an aqueous solvent is used (that is, a case where a system that converts a water-soluble substance into a water-insoluble substance by insolubilization reaction) will be mainly described as an example. However, a person skilled in the art can also use a solvent other than water. If so, the present invention can be implemented by making necessary changes as appropriate.

  The water-soluble substance used in the present invention is a substance that is soluble in an aqueous solvent used in an analysis system before undergoing an insolubilization reaction, and is converted into a substance that is insoluble in the aqueous solvent by undergoing the insolubilization reaction. As long as it is, it is not particularly limited, and examples thereof include inorganic ions (preferably metal ions), organic ions, enzyme substrates or reaction products thereof, and dyes.

  Examples of the metal ions include antimony ions, bismuth ions, copper ions, mercury ions, silver ions, palladium ions, platinum ions, and gold ions. These metal ions are water-soluble in an aqueous solvent, may be in a metal complex (preferably metal complex ion) state, and are precipitated as a metal by an insolubilization reaction.

Further, as the metal ion, a divalent cation (for example, copper ion, nickel ion, iron ion) can be used. These divalent cations are water-soluble in an aqueous solvent and are produced by oxidation of [Fe (CN) ₆ ] ³⁻ ions (eg, [Fe (CN) ₆ ] ⁴⁻ ions [Fe (CN) ₆ ]). ^When it is combined with ^3- ion), it is deposited as a metal complex MH [Fe (CN) ₆ ] (M: divalent cation).

  The reaction in which metal ions are reduced and insolubilized (deposited) depends on the redox potential of each substance. The smaller the so-called ionization tendency, the easier it is to deposit as a metal, so the reaction is not limited to the above reaction. In addition, the easiness of precipitation is greatly affected by other factors (temperature, pH, ionic strength, reaction solution composition, etc.) such as the electrochemical activity of the ions in the solution. The metal used should be interpreted in the broadest sense and should not be construed as limiting in any way. For example, the degree of insolubilization can be controlled by allowing metal ions and ions forming an insoluble salt to coexist during the insolubilization reaction. In some cases, it is preferable that the ions forming the insoluble salt are not coexistent during the insolubilization reaction but are insolubilized and precipitated as a metal. Those skilled in the art can appropriately determine suitable conditions for the amount of ions forming the insoluble salt during the insolubilization reaction. Suitable conditions can be determined in the range of 0 to 5 mmol / L or less, preferably 0 to 2 mmol / L, more preferably 0 to 1 mmol / L, and still more preferably 0 to 0.5 mmol / L. Furthermore, the substance to be deposited is not limited to metal ions, and any substance that satisfies the above conditions can be suitably used.

  Examples of the dye that can be used as the water-soluble substance include a Schiff reagent and aniline. The Schiff reagent is water-soluble in an aqueous solvent, and two aldehyde groups (for example, an aldehyde group generated by reduction of a carboxyl group) are combined with one molecule of the Schiff reagent, and are precipitated as a red-purple compound by a reduction reaction. . In addition, aniline is water-soluble in an aqueous solvent and precipitates as polyaniline by an oxidation reaction.

  Examples of the water-soluble dye include 5-bromo-4-chloro-3-hydroxyindole (BCI), nitro blue tetrazolium chloride (NBT) ), Indole, and 5,5′-dibromo-4,4′-dichloro-indigo (5,5′-dibromo-4,4′-dichloro-indigo), which is an insoluble substance by reduction reaction. 2BCI), BCI / nitro blue tetrazolium diformazan (NBT Diformazan), precipitated as indigo. The dye may be obtained by an enzyme reaction with a labeling enzyme (for example, alkaline phosphatase; ALP), for example, an appropriate enzyme substrate, for example, 5-bromo-4-chloro-3-indolyl phosphate (5-bromo-4-chloro-3). -indolyl phosphate) (BCIP), 3-indoxyl phosphate. Therefore, the aforementioned BCI and indole are also reaction products of enzyme substrates.

  For example, when the enzyme substrate BCIP is used, BCI is formed by an enzyme reaction involving ALP, and 2BCI is precipitated by a reduction reaction. When a mixture of the enzyme substrate BCIP and the dye NBT is used, 2BCI is generated by the ALP enzyme reaction by the ALP enzyme reaction, NBT diformazan is generated by the reduction reaction, and the complex 2BCI / NBT diformazan is Precipitate. When the enzyme substrate 3-indoxyl phosphate is used, indole is formed by the enzymatic reaction of ALP and is precipitated as indigo by the reduction reaction.

  In these examples, an enzyme reaction involving ALP used as a labeling substance causes a subsequent insolubilization reaction, and as a result, the water-soluble substance is converted into a water-insoluble substance. The present invention includes an embodiment in which the insolubilization reaction is indirectly caused by the labeling substance as a trigger and an aspect in which the insolubilization reaction is directly caused by the labeling substance.

  Enzyme substrates that can be used as water-soluble substances include, in addition to the aforementioned pAPP (or derivatives thereof), ester derivatives of thiocholine such as acetylthiocholine, propionylthiocholine, succinylbisthiocholine, and butyrylthiocholine. It is done. When an ester derivative of thiocholine is used in combination with the metal ion (for example, gold, silver, etc.), the metal ion is converted by an enzyme reaction with an appropriate labeling enzyme (for example, cholinesterase, more specifically, acetylcholinesterase, acylcholinesterase, etc.). Reduced and deposited as metal. Further, thiocholine is generated by the enzyme reaction, and a thiocholine group of thiocholine is bonded to a part of the deposited metal, so that the metal-thiocholine complex is also precipitated. Furthermore, the produced thiocholine is precipitated by bonding to a metal forming the substrate or electrode (for example, a gold substrate or a gold electrode) via a thiol group. The labeling enzyme acetylcholinesterase can be used for the enzyme substrate acetylthiocholine or propionylthiocholine, and the labeling enzyme acylcholinesterase can be used for the enzyme substrate succinylbisthiocholine or butyrylthiocholine.

As the water-soluble substance, aryl diazonium salts such as R-Ph-N ₂ BF ₄ can be used. When using an aryldiazonium salt, an active radical rich in chemical activity is generated by the reduction reaction, and it is covalently bonded to various sensing units, preferably sensing units using carbon, graphite, or carbon nanotubes. Can do.

The soluble substances (particularly water-soluble substances) that can be used in the analysis method of the present invention (including both the complex formation type analysis method and the enzyme-based analysis method) have been described above. In the analysis method, a labeling substance can be appropriately selected depending on the soluble substance and reaction system to be used. For example, in addition to the aforementioned hydrolase such as ALP or cholinesterase, transferase, lyase, ligase, isomerase, oxidoreductase and the like are used. Examples of the oxidoreductase include glucose oxidase (GOD), peroxidase, xanthine oxidase. Amino acid oxidase, ascorbate oxidase, acyl-CoA oxidase, cholesterol oxidase, galactose oxidase, oxalate oxidase, sarcosine oxidase and the like can be used. These enzymes can directly or indirectly cause an insolubilization reaction [for example, oxidation reaction, reduction reaction, hydrolysis reaction, dehydration reaction, addition polymerization, condensation polymerization (condensation polymerization), neutralization reaction] There is no particular limitation as long as it is an enzyme that converts a soluble substance into an insoluble substance by an insolubilization reaction and causes a reaction to be adsorbed and deposited by precipitation, binding, deposition, etc. on a solid surface. Alternatively, two or more kinds of enzymes can be used in combination.
In addition to these enzymes, various reducing agents or oxidizing agents can also be used.

  On the other hand, as an enzyme that can be used in the enzyme-based analysis method of the present invention, an enzyme in which either the enzyme or the enzyme substrate is an analysis target substance can be used. For example, it is used in a complex-forming analysis method. One or more of the possible enzymes can be selected.

  The electrical analysis method used in the present invention is not particularly limited as long as an insoluble substance precipitated on the surface of the sensing unit is electrically analyzed. In this specification, “electrically analyzing” means an analysis in which a change in charge on the surface of the sensing unit is captured as a change in current, an analysis in which a change in charge on the surface of the sensing unit is captured as a change in voltage (potential), and the surface of the sensing unit Analysis that is taken as a change in electrical resistance (or impedance) is included. Examples of the electrical analysis method used in the present invention include an amperometric analysis method using an electrode having at least a working electrode and a counter electrode, and a voltammetric measurement method using a transistor.

  In the amperometric analysis method, a change in charge on the surface of the sensing unit is captured as a change in current. An amperometric electrode has at least a working electrode and a counter electrode on a substrate, and includes a reference electrode as necessary. In the amperometric analysis method, an electrode active substance or a resistive substance (insulating substance) generated in the vicinity of an electrode portion is applied with a predetermined voltage between a working electrode and a counter electrode, so that the electrode active substance flowing between both electrodes or Measure the current signal corresponding to the amount of resistive material (insulating material), or apply the difference between the electrode active material or resistive material (insulating material) generated near the electrode to the applied voltage between the working electrode and the counter electrode It is a method to distinguish by.

  For example, when a metal ion is used as a water-soluble substance, the metal deposited on the sensing unit is re-oxidized to a metal ion by applying a voltage to the reference electrode to the sensing unit where the metal is precipitated, and the sensing unit. The electrochemical change in can be detected as a change in current.

  As a method for capturing a change in current, widely known methods such as cyclic voltammetry, differential pulse voltammetry, chronoamperometry, differential pulse amperometry can be used in addition to current measurement.

  In the voltammetric analysis method, a change in charge on the surface of the sensing unit is captured as a change in voltage (potential). A transistor used for voltammetric analysis is an element that converts a voltage signal input to a gate into a current signal output from a source electrode or a drain electrode, and a voltage is applied between the source electrode and the drain electrode. Is added, the charged particles present in the channel formed between them move along the direction of the electric field between the source electrode and the drain electrode, and are output as a current signal from the source electrode or the drain electrode. At this time, the strength of the output current signal is proportional to the density of charged particles. When a voltage is applied to the gate located above, on the side, or below the channel via an insulator, the density of charged particles in the channel changes. By using this, the gate voltage can be changed by The current signal can be changed.

  For example, when acetylthiocholine is used as the water-soluble substance, a change in electric charge in the sensing part due to thiocholine deposited on the sensing part by acetylcholinesterase can be detected as a voltage (potential) change.

As a preferable aspect of the sensing unit, when the sensing unit is conductive, it may be a conductive material, such as gold, silver, platinum, rhodium, ruthenium, iridium, mercury, palladium, or a polymer such as an osmium polymer. Carbon, nanotube-like structures (carbon nanotubes), graphite, and inorganic substances may be used alone or in combination. Moreover, the shape of the substance composed of the substance may be anything as long as it does not inhibit the reaction, and may include a planar, uneven, particle (gold colloid, etc.) substance or the like.
A preferred embodiment of the nanotube-like structure is a structure selected from the group consisting of carbon nanotubes, boron nitride nanotubes, and titania nanotubes.
Further, as long as the sensing unit has conductivity, a non-conductive material may be added to the configuration of the sensing unit together with the conductive material. Examples of non-conductive substances include insoluble carriers such as polyester resins.

  As another preferred embodiment of the sensing unit, the sensing unit is preferably a gate electrode of a field effect transistor or a single electron transistor using a nanotube-like structure (carbon nanotube), and the nanotube-like structure is a carbon nanotube or boronite. A structure selected from the group consisting of ride nanotubes and titania nanotubes is preferred.

  As another preferred embodiment of the sensing unit, in order to increase the surface area of the sensing unit, a porous carrier such as a nitrocellulose film used for immunochromatography or the like, or a polymer or latex described in International Publication No. WO2006 / 038456 pamphlet An insoluble carrier (or insoluble particles) such as a carrier may be used, and a three-dimensional body may be formed on the surface of the sensing unit using a conductive substance such as a conductive polymer or a conductive carrier together with these. . The surface of the sensing unit forms a three-dimensional body, so that the surface area of the sensing unit is remarkably increased and the detection sensitivity can be increased.

  As another preferred shape of the sensing unit, the sensing unit may be well-shaped, uneven, or convex in order to improve the efficiency of precipitation (deposition, insolubilization, deposition) of the product in the sensing unit under fluid conditions. By providing a partition or the like, it is possible to prevent the product substance deposited (insolubilized, precipitated, or deposited) under fluid conditions from flowing out in the flow direction (for example, downstream). Further, by providing these structures, an effect of increasing the reactivity of the precipitated product can be expected. The structure may be formed only in the sensing unit, or may be formed in the electrode unit or the biosensor unit, and a part thereof may be present in the sensing unit.

  Specifically, a shape having an acute three-dimensional structure is preferable, and examples include a polyhedron, a polygonal cylinder, a sphere, a cylinder, and a cone, and a cone is particularly preferable. It is sufficient that a three-dimensional structure including at least one or more acute-angled portions or protrusions is formed on the sensing unit, but it is preferable that a plurality of three-dimensional structures are formed. As the number and size of the three-dimensional structure, the number of acute-angled shapes, and the shape and arrangement thereof, a suitable one can be selected according to the measurement conditions. The acute angle may be a structure in which a part or the whole of the three-dimensional structure of the unevenness formed in the sensing part exhibits an end effect (end effect, edge effect). The edge effect is an effect widely known in the field of electroplating and the like, and is an effect in which charges are concentrated at an acute angle end. By this effect, for example, a metal such as silver which is a generated substance (insoluble substance) It is presumed that the metal ions are positively ionized again and the detection sensitivity can be increased.

  The selective interaction reaction between the analyte and the specific partner is performed at a place where the specific partner is immobilized. The place where the specific bar toner is immobilized is not limited as long as selective interaction can be performed and then electrical measurement can be performed. However, if the surface of the sensing unit or fine particles are used, the specific bar toner is not limited. A fine particle surface can be mentioned.

  The method for immobilizing the specific partner on the surface of the sensor is not limited, such as direct immobilization or indirect immobilization, and any method can be used according to the nature of the selective interaction reaction. Can be used. For example, it may be directly bonded to the substrate by physical adsorption or covalent bond, or may be indirectly bonded to the substrate in advance through a flexible spacer having an anchor portion. Further, for example, when a noble metal such as gold is used for the substrate, it may be bonded via a self-assembled film.

  In addition, after immobilizing the specific partner, the surface is treated with bovine serum albumin, polyethylene oxide or other inert molecules, or it is coated with an adhesive layer on the immobilization layer of a specific substance. It is also possible to select or control a substance that can inhibit the reaction or permeate.

  Moreover, when using microparticles | fine-particles, it is preferable that the sensing part surface and microparticles | fine-particles contact directly. The term “direct contact” means that the surface of the sensing unit that is not covered with a known substance for coating the surface of the sensing unit comes into contact with the fine particles. As a result, the insoluble material generated by the reaction on the fine particles is also directly deposited on the surface of the sensing part not covered with the coating material. Generally, since the surface of the sensing part is hydrophobic, for example, when the insoluble substance is a metal such as silver, the generated substance is also hydrophobic, so that it is considered that the sensing part is likely to be attracted. As a result, precipitation of insoluble substances on the surface of the sensing unit is promoted, and since the sensing unit surface is not covered with a substance that prevents the transmission and reception of electrical signals, electrical signals can be exchanged with high efficiency and under certain conditions. Detection with high sensitivity and high accuracy is possible.

  In addition, since the surface of the sensing unit is hydrophobic, it is considered that precipitation of the insoluble substance on the surface of the sensing unit is promoted. Therefore, the sensing unit surface is preferably not covered with a hydrophilic substance. In general, a nonspecific inhibitor used in a method utilizing an antigen-antibody reaction is often hydrophilic, and is preferably not covered with such a substance. A hydrophobic substance can be used for coating the surface of the sensing part because it may promote precipitation of the insoluble substance on the sensing part. One skilled in the art can determine the usable hydrophobic state without undue experimentation. Thereby, since precipitation of the insoluble substance on the surface of the sensing unit can be promoted, detection with higher sensitivity is possible.

Moreover, it is preferable that the surface of the sensing unit is free of substances that hinder the transmission and reception of electrical signals, such as non-specific suppressing substances such as proteins and polymers. Since the non-specific inhibitory substance is hydrophilic or hydrophobic and the surface of the sensing part is not covered with a substance that hinders the transmission and reception of electric signals, it can transfer electric signals with high efficiency and under certain conditions. Highly accurate detection is possible.
Here, “the surface of the sensing unit is not covered” only needs to reflect the state of the sensing unit in a substantial property / state.

  When fine particles are used in the present invention, a partner specific to the substance to be analyzed is immobilized on the fine particles. The fine particles are not limited as long as a specific partner can be immobilized, but magnetic fine particles capable of easily performing B / F separation are preferable. For example, magnetic fine particles capable of efficiently performing B / F separation with a magnet can be used.

  The type and size of the fine particles in the present invention are not particularly limited as long as the generated insoluble material can efficiently precipitate on the sensing unit. The type and size of the fine particles depend on various conditions such as selective interaction or insolubilization reaction to be used, labeling substance to be used, soluble substance or redox substance, flow path size, and electrical analysis method to be used. Although it varies, a person skilled in the art can appropriately determine without performing excessive trial and error by conducting a simple preliminary experiment according to the procedure described in the examples described later, for example.

  For example, regarding the type of fine particles, either conductive fine particles or non-conductive fine particles can be used. However, in the case of conductive fine particles, the particles themselves function as a sensing unit, and the conductivity of the sensing unit other than the fine particles Therefore, it is considered that the detection accuracy is not stable. On the other hand, the case of non-conductivity is more preferable because it is considered that measurement can be performed stably and accurately because an electric signal can be transmitted and received by a sensing unit under the same conditions.

  For example, regarding the size of the microparticles, it is considered preferable that a large number of immune complexes are formed near the surface of the sensing unit, so that smaller microparticles should be used to increase the surface area per volume. However, particularly in the case of non-conductive fine particles, if the fine particles cover the sensing portion, the surface of the sensing portion that can send and receive electrical signals becomes small, and as a result, electrical signals can be sent and received. Therefore, the sensitivity decreases or the detection becomes impossible, and it is necessary to determine suitable conditions in consideration of these. A person skilled in the art can easily determine suitable conditions, for example, by preparing a plurality of types of particles having different particle diameters. For example, the lower limit of the particle diameter is preferably 3 μm or less, more preferably 1 μm or less, still more preferably 0.5 μm or less, and the upper limit of the particle diameter is preferably 0.01 μm or more, more preferably 0.1 μm or more. Is mentioned.

  The method for immobilizing the specific partner to the microparticle is not limited, such as direct immobilization or indirect immobilization, and any method is used according to the nature of the selective interaction reaction. be able to. For example, the particles may be directly bonded to the fine particles by physical adsorption or covalent bond, or may be bonded to the fine particles indirectly via a flexible spacer having an anchor portion. Further, for example, when a noble metal such as gold is used for the fine particles, they may be bonded via a self-assembled film.

  In addition, after immobilizing the specific partner, the surface is treated with bovine serum albumin, polyethylene oxide or other inert molecules, or it is coated with an adhesive layer on the immobilization layer of a specific substance. It is also possible to select or control a substance that can inhibit the reaction or permeate.

  In the complex formation type analysis method of the present invention, the complex formation step (a1) based on the selective interaction is performed in the complex formation unit, and then the insolubilization reaction step based on the labeling substance contained in the complex ( a2) and the electrical analysis step (b) are performed by the sensing unit. In the present invention, the complex forming part and the sensing part may be in the same place or different places. The order in which these steps are performed is generally generally performed in this order. However, as long as an electrical signal correlated with the abundance of the analyte in the test sample can be obtained, adjacent steps ( Or a part thereof) can be performed simultaneously.

  The selective interaction reaction performed on the fine particles may be performed at the same place as the sensing unit, but a substance necessary for the selective interaction reaction has a negative effect on the transmission and reception of an electric signal in the sensing unit. In the case of giving, it is preferable to be performed at a location different from that on the sensing unit.

  The method of the present invention can be performed by any mechanism as long as an electrical analysis method can be performed. For example, a batch-type, micro-processed flow, lateral flow, capillary flow, or flow-through flow on a well such as a plate can be used. It can be performed by a flow method such as a membrane strip such as a channel or an immunochromatography method, or a combination thereof. For example, in the case of μTAS, the IEICE Transactions on CI Vol. J81-CI No. 7 pp. 385-393 July 1998 can be referred to.

  In these mechanisms, for example, when the fine particles are magnetic fine particles, a place where a magnetic force is generated is necessary to perform B / F separation, but at least the magnetic fine particles can be held on the surface of the sensing unit. Just set up your body. For example, in order to hold the magnetic fine particles on the surface of the sensing unit, it may be placed on the back side of the sensing unit, or in order to hold the magnetic fine particles on the surface of the complex forming unit, it may be placed on the back side of the complex forming unit. It ’s fine. In addition, an enzyme immunosensor having a liquid delivery system such as μTAS is preferable because the liquid exchange can be easily performed by the liquid delivery system by fixing the magnetic body.

  A solution (for example, enzyme-labeled antibody, substrate, etc.) and a washing solution used for each reaction may be sequentially sent by the liquid feeding system, or may be supplied after drying to the reaction field. For example, when a selective interaction reaction is performed on the fine particles on the sensing unit, the enzyme-labeled antibody is dried and prepared in the vicinity of the sensing unit (a place that does not affect the transmission and reception of electrical signals). When sent to the field, the enzyme-labeled antibody solution is dissolved and the selective interaction reaction can be started.

  In addition, if air is mixed during mixing or stirring during a selective interaction reaction, bubbles are likely to be generated and the stability of the reaction will be reduced. It is preferable to suppress the generation of bubbles due to a change in pressure and ultrasonic waves. Moreover, as long as this reaction is not inhibited, generally known silicone antifoaming agents and organic antifoaming agents may be used.

  Further, the reaction solution used for the insolubilization reaction can be discharged from the sensing unit, and then the solution used for electrical analysis can be supplied to the sensing unit. In order to further improve the accuracy, it is preferable to wash the sensing unit with a cleaning solution that does not contain a substance that contributes to a reaction other than the analyte before supplying the solution used for electrical analysis into the container.

The solution that can be used for the electrical analysis of the present invention is a solution that contains a strong electrolyte and does not contain any substance other than the analyte that contributes to the current response. The strong electrolyte that can be used in the present invention is not limited as long as it does not react with a solvent and does not specifically adsorb to the surface of the sensing part. For example, alkali metal halides, nitrates, sulfates, phosphates, Etc. An example is potassium nitrate. In the case of insoluble substances derived from silver ions, the use of potassium nitrate makes it possible to preferentially generate a substance that contributes to the current response (for example, silver nitrate) and to detect it as a single peak. Therefore, it is particularly preferable because sensitivity and accuracy are increased.
Moreover, it is only necessary that a single detection peak can be obtained more stably by not including a substance that contributes to the current response other than the analyte.
The electrolyte that can be used in the present invention may be one type or a combination of two or more types, but a combination that generates a substance that causes a plurality of electrical signals to be detected is not desirable.

The concentration of the electrolyte that can be used in the present invention can be used at about 0.05 mol / L or more, but it is preferable that the electrolyte concentration is as high as possible because the detection peak in the present invention becomes large. For example, when potassium nitrate is used as the electrolyte, it is 0.05 to 1.0 mol / L, preferably 0.1 to 1.0 mol / L, more preferably 0.5 to 1.0 mol / L, and particularly preferably 0. .8 to 1.0 mol / L.
The solvent of the solution used for electrical measurement is not limited as long as the potential window is wider than the redox potential of the insoluble substance and the insoluble substance does not dissolve, but is preferably water.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Effect of using KNO ₃ as electrolyte during electrochemical measurement >>
In the following examples, the effects of the present invention were verified by measuring influenza virus type B (hereinafter abbreviated as FluB) antigen.
FIG. 3 is a block diagram showing the configuration of the electrochemical detection device used in Example 1. The effect of using or not using KNO ₃ as an electrolyte during electrochemical measurement was examined.
The electrode was produced with reference to the international publication WO2009-116534 pamphlet. On an insulating substrate 1 made of polyethylene terephthalate (PET), an electrode part composed of a working electrode 2, a reference electrode 3, and a counter electrode 4 was formed with carbon paste (FTU-20: manufactured by Asahi Chemical Research Laboratory). The working electrode 2 functions as a sensing unit. A part of the reference electrode 3 was coated with silver / silver chloride ink (manufactured by BAS).

  Next, anti-FluB monoclonal antibody-immobilized magnetic microparticles (0.055%, particle diameter 0.7 μm), 1/10 diluted FluB antigen solution (BD), alkaline phosphatase (hereinafter referred to as “prepared”) prepared in advance by each production method described below. , ALP) labeled anti-FluB monoclonal antibody (Fab ′) (1 μg / mL), 50 μL each, and after washing, magnesium sulfate 1 mmol / L, trishydroxymethylaminomethane (hereinafter, Tris) 12.5 mmol / L The solution was dispersed in 100 μL to obtain an ALP-labeled antibody-antigen-antibody-immobilized magnetic fine particle solution, and 100 μL of this solution was added dropwise to the working electrode 2.

  The anti-FluB monoclonal antibody-immobilized magnetic fine particles were prepared as follows. In accordance with a known method, mice were immunized with influenza B virus antigen (FluB antigen: collected from the influenza strain B vaccine HA stock obtained from Osaka University Microbial Disease Research Society), and anti-FluB mouse monoclonal antibody (IgG) Was made. Next, the antibody was immobilized on magnetic fine particles (manufactured by Merck) having a particle diameter of 0.7 μm to produce antibody-immobilized magnetic fine particles. Specifically, according to a known method, the carboxyl group (—COOH) on the surface of the magnetic fine particles is activated with water-soluble carbodiimide (WSC: manufactured by Dojin), and then mixed with the antibody and the magnetic fine particles. The antibody was chemically immobilized on the surface of the microparticles by covalent bonding. Furthermore, bovine serum albumin (BSA) was physically and chemically immobilized to produce anti-FluB antibody-immobilized magnetic microparticles.

  Next, an ALP-labeled anti-FluB monoclonal antibody (Fab ') was prepared as follows. In accordance with a known method, mice were immunized with influenza B virus antigen (FluB antigen: Brisbane strain B influenza HA vaccine stock obtained from Osaka University Microbial Disease Research Society), and anti-FluB mouse monoclonal antibody (IgG) Was made. This was prepared in the Fab 'fraction and used as an anti-FluB mouse monoclonal antibody for ALP labeling. Next, using this antibody, ALP (Roche) and a cross-linking reagent (Succinimidyl 4- [N-maleimidomethyl] -cyclohexane-1-carboxylate: PIERCE), a “highly sensitive enzyme immunoassay (Eiji Ishikawa: Academic Publishing Center) 1993) ”, an ALP-labeled anti-FluB mouse monoclonal antibody (Fab ′) was prepared. A solution in which the prepared enzyme-labeled antibody was adjusted to a 1 mAbs concentration with a 2% BSA-containing buffer was used as an ALP-labeled anti-FluB antibody solution.

The ALP-labeled antibody-antigen-antibody-immobilized magnetic fine particle solution dropped on the working electrode 2 was fixed to the sensing portion (working electrode 2) on the substrate 1 by magnetic force by placing a permanent magnet 5 on the back surface of the working electrode 2.
Using a substrate 1 on which ALP-labeled antibody-antigen-antibody-immobilized magnetic microparticles are fixed to the sensing part and an acrylic plate 8 having a liquid inlet 6 and a liquid outlet 7 open, the double-sided tape is cut into the shape of a flow path. A sensor unit having an electrode portion held in a hollow state was produced by sandwiching the gasket 9 produced by the above and below.

A solution containing p-aminophenyl phosphate (hereinafter referred to as pAPP) 2 mmol / L, silver nitrate 0.125 mmol / L, magnesium sulfate 1 mmol / L, Tris 12.5 mmol / L was prepared, and the insolubilized reaction solution was used as the silver ion-containing substrate solution. Prepared in reservoir 10.
A solution containing 1 mol / L of potassium nitrate (hereinafter referred to as KNO ₃ ) was prepared and prepared in the electrical measurement solution reservoir 11.
In the reservoir 12, air was prepared for an air gap that enters between the solutions.

  Each reservoir was connected to the pump 15 via the manifold 13 and to the liquid inlet 6 via the manifold 14. Liquid feeding control was performed by opening and closing the solenoid valve in the manifold. The liquid discharge port 7 was connected to the waste liquid server 16. Thereby, each solution can be flowed in a fixed direction at a predetermined flow rate.

First, was solution during the electrochemical measurements under the conditions substituted 1 mol / L KNO ₃ solution. Specifically, first, in order to perform the insolubilization reaction, the silver ion-containing substrate solution was sent from the insolubilization reaction solution reservoir 10 to the sensing unit at a flow rate of 150 μL / min for 10 minutes. After 10 minutes, the reservoir 12 was switched to send air for 2 minutes, and the solution in the sensing unit was removed. After 2 minutes, the electrical measurement reservoir 11 was switched over, and a 1 mol / L KNO ₃ solution was fed at a flow rate of 250 μL / min for 3 minutes. After 3 minutes, the air was switched to the reservoir 12 again and air was sent for 2 minutes, and then the electrical measurement reservoir 11 was switched to feed 1 mol / L KNO ₃ solution at a flow rate of 250 μL / min for 3 minutes. After 3 minutes, the liquid feeding was stopped and electrochemical measurement was performed.

  As Comparative Experimental Example A, a conventional condition, that is, a solution at the time of electrochemical measurement was performed under the condition of a silver ion-containing substrate solution. Specifically, first, in order to perform the insolubilization reaction, the silver ion-containing substrate solution was fed from the insolubilization reaction solution reservoir 10 to the sensing unit at a flow rate of 150 μL / min for 10 minutes. After 10 minutes, the liquid feeding was stopped and electrochemical measurement was performed.

  As Comparative Experimental Example B, the solution at the time of electrochemical measurement was performed under the condition of a silver ion-free substrate solution. For Comparative Experiment B, a solution containing pAPP 2 mmol / L, magnesium sulfate 1 mmol / L, Tris 12.5 mmol / L was prepared and prepared in the insolubilized reaction solution reservoir 10 as a silver ion-free substrate solution. From the insolubilized reaction solution reservoir 10 for Comparative Experiment B, a silver ion-free substrate solution was fed to the sensing unit at a flow rate of 150 μL / min for 10 minutes. After 10 minutes, the liquid feeding was stopped and electrochemical measurement was performed.

  In the electrochemical measurement, the working electrode 2, the reference electrode 3, and the counter electrode 4 are connected to the electrochemical analyzer 17, and the electric potential is changed between -0.1V and 0.4V by differential pulse voltammetry (DPV). The chemical response was measured.

The results are shown in Table 1 and FIG.
From the comparison with Comparative Experimental Example A, the current response peak value derived from silver seen between 0V to 0.25V is increased about twice by replacing the solution at the time of electrochemical measurement with 1 mol / L KNO ₃ solution. There was found. In Comparative Experimental Example B, no silver ion insolubilization reaction occurs, so that no current response peak is observed between 0 V and 0.25 V, but the pAPP-derived current response peak that is commonly present in the substrate solution is 0. It was observed after 25 V, and from this, it was inferred that, in the measurement of the silver ion-containing substrate solution of Comparative Experimental Example A, two peaks overlap, so that a large peak top cannot be obtained.
On the other hand, by substituting with a 1 mol / L KNO ₃ solution, it becomes possible to control the product so that by-products with different reactivities are not generated, and a single peak is formed, and the sensitivity and It was considered possible to increase the accuracy.

<< Example 2: Comparison of each component of insolubilized reaction solution in solution at the time of electrochemical measurement and KNO ₃ >>
Next, after carrying out the insolubilization reaction using the silver ion-containing substrate solution, the current response peak value when each component of the insolubilization reaction solution other than pAPP was used as a solution at the time of electrochemical measurement was examined.
As a solution at the time of electrochemical measurement, instead of 1 mol / L KNO ₃ solution, (1) 12.5 mmol / L Tris solution, (2) 1 mmol / L magnesium sulfate solution, (3) 12.5 mmol / L Tris and 1 mmol Measurement was performed according to the procedure described in Example 1 except that a solution containing / L magnesium sulfate was used.

The results are shown in Table 2 and FIG.
When the 1 mol / L KNO ₃ solution is replaced with a solution containing each component of the insolubilized reaction solution other than pAPP, the current response peak value derived from silver seen between 0 V and 0.25 V is larger, and a single peak is obtained. Indicated. Further, since Tris is a weak electrolyte, the current response peak value is small, and magnesium sulfate is an electrolyte. However, since the concentration in the solution is small, the current response peak value is considered to be small. In addition, no single peak was formed. It was confirmed that the effect obtained in Example 1 was not an influence of the electrolyte contained in the insolubilized reaction solution.

<< Example 3: Comparison in the presence and absence of electrolyte >>
The current response peak values in the presence and absence of the electrolyte that affect the current response peak were compared.
The measurement was carried out according to the procedure described in Example 1 except that H ₂ O was used instead of the 1 mol / L KNO ₃ solution as a replacement solution during electrochemical measurement.

The results are shown in Table 3 and FIG. It was found that H ₂ O in the absence of electrolyte has a small current response peak value. From the above, it was shown that the reaction product increases due to the presence of the electrolyte.

<< Example 4: Change in current response peak depending on electrolyte concentration >>
Instead of 1 mol / L KNO ₃ solution, 0.5 mol / L, 0.1 mol / L, 0.05 mol / L, 0.01 mol / L, 0.001 mol / L of KNO was used as a replacement solution for electrochemical measurement. Measurements were performed according to the procedure described in Example 1 except that a solution containing ₃ was used.

The results are shown in Table 4. As the concentration increases, the current response peak value derived from silver seen between 0 V and 0.25 V increases, but no significant change is observed after 0.5 mol / L. Compared to Example 1, it was found that the current response peak value was increased by replacing with 0.05 mol / L or more of KNO ₃ solution. Furthermore, an increase of about 1.5 times was observed at 0.1 mol / L, and a current response peak value of about 2 times or more was increased at 0.5 mol / L or more.

  The present invention, for example, can analyze a sample with high sensitivity and can be applied to clinical tests, diagnosis, food fields, and environmental analysis.

Claims (8)

Soluble substances are obtained by reacting an analyte and a specific partner that exhibits a selective interaction with the analyte, and performing an insolubilization reaction by correlating with the abundance of the analyte in the test sample. Is converted into an insoluble substance, precipitated in a sensing part, and the insoluble substance precipitated in the sensing part is electrically analyzed,
Electrical analysis, are performed by the dominant strong electrolyte solution in the current response,
The strong electrolyte is selected from alkali metal halides, nitrates, sulfates and phosphates;
The electrical analysis is
(A) removing a substance that contributes to a current response other than the insoluble substance;
(B) Next, the step of replacing with the strong electrolyte solution
The analysis method characterized by including . The analysis method according to claim 1, wherein the electrical analysis is performed by analyzing a single detection peak. The analysis method according to claim 1 or 2 , wherein the strong electrolyte is potassium nitrate. The analysis method according to any one of claims 1 to 3 , wherein the specific partner is an antibody. The analysis method according to any one of claims 1 to 4 , wherein the soluble substance is silver ions. Wherein the specific partner in the sensing section is fixed analysis method according to any one of claims 1-5. The selective interaction performed on magnetic microparticles, is an electrical measurement amperometric measurement analysis method according to any one of claims 1-6. A reagent and a kit for measuring a substance to be analyzed, characterized by being used in the analysis method according to any one of claims 1 to 7 ,
A strong partner selected from a specific partner that selectively interacts with the analyte, a soluble substance that is converted to an insoluble substance by an insolubilization reaction, a sensor, an alkali metal halide, a nitrate, a sulfate, and a phosphate. Including reagents and kits.

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