JP6318125B2 - 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 (Patent Document 1).

Even in a measurement system using such a label, as in the case of optical immunoassay, if the unbound label is not washed sufficiently, the background becomes high and high sensitivity cannot be obtained. There are problems such as inability to obtain stability of values.
Therefore, in order to suppress non-specific adsorption of unbound label, non-specific inhibitors such as BSA, casein, and polymers for blocking are used on the working electrode in addition to the antibody (antigen) used in this reaction. Attempts have been made to immobilize so that labels other than those derived from the antigen-antibody reaction do not adsorb onto the electrode non-specifically as easily as possible.
However, when a large amount of an electrochemically inactive substance is immobilized on the electrode in this way, direct electron transfer on the electrode becomes difficult, so that the detection signal is weakened and the measurement sensitivity is reduced. This is one factor that hinders highly sensitive measurement of enzyme immunosensors.

  The method that can solve the above is, as described in Patent Document 2, a method in which immunoconjugation of an antibody or the like is performed in a concave portion, and detection is performed with a detection electrode facing the concave portion, Patent As described in Document 3 and Patent Document 4, a method of collecting and detecting on an electrode after immunoconjugation of an antigen antibody or the like with a carrier such as magnetic fine particles is described in Patent Document 5 As described above, a method is conceivable in which immunocomplexation is performed in a large area antigen-antibody reaction field, and then the complexed product is separated and concentrated in a microdetection unit for detection. However, it is difficult to stably control the reaction, and a small and highly sensitive enzyme immunosensor has not been realized.

  As an attempt to increase sensitivity, it is considered to increase the surface area of the sensing unit that detects an electrical signal. It is considered that the number of antibodies for capturing the target substance should be increased. For example, in Patent Document 1, in order to detect a substance deposited on the sensing unit and obtain a signal, it has been considered that it is necessary to generate a large amount of deposits on the sensing unit in order to increase sensitivity. . Specifically, it is described that the surface area of the sensing part should be increased and an immune complex containing a large amount of enzyme-labeled antibody formed on the sensing part.

WO2009-116534 pamphlet JP 2005-227096 A Japanese Patent No. 4102887 JP 2006-133137 A JP 2009-128233 A

As described above, in Patent Document 1, since it is necessary to reduce and deposit silver ions on the sensing part by the reducing substance generated from the enzyme-immune complex, the enzyme-immune complex is formed on the sensing part, and the sensing is performed. It was thought that it was necessary to efficiently deposit a substance produced by reduction of silver ions on the surface of the part. In addition, in order to form an enzyme-immunocomplex at the sensing unit, it was considered necessary to have a non-specific inhibitory substance present in the sensing unit so that a non-specific reaction does not occur.
In order to produce an enzyme immunosensor with higher sensitivity than ever, the present inventors further improved the silver ion deposition reaction by improving the enzyme-labeled immune complex as described in Patent Document 1. We examined the electrical measurement of sensitivity.

As a result of intensive studies by the present inventors, the sensing unit that deposits a reduced substance of silver ions and captures it as a signal is covered with a non-specific inhibitory substance, so that the signal is weakened, and sufficiently high sensitivity is achieved. It was found that could not be achieved. Furthermore, even if an antibody specific to a target substance is immobilized on a non-conductive fine particle and an immune complex is formed on the fine particle, a reduced substance of silver ions is detected on the sensing part. It was found that it was possible to measure. When using non-conductive fine particles, the antibodies on the fine particles are more distant from the surface of the sensing unit than the antibodies directly immobilized on the sensing unit. Although it was thought that the reduced substance could not be deposited and the signal became small and could be detected, it was a surprising effect that higher sensitivity could be achieved.
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,
An analytical method characterized in that a specific partner exhibiting a selective interaction is immobilized on a microparticle;
[2] The analysis method according to [1], wherein the surface of the sensing unit is not covered with a substance that hinders transmission / reception of electrical signals,
[3] The analysis method according to [1] or [2], wherein the surface of the sensing unit and the fine particles are in direct contact when performing the insolubilization reaction and electrical analysis.
[4] The analysis method according to any one of [1] to [3], wherein the fine particles are non-conductive substances.
[5] The analysis method according to any one of [1] to [4], wherein the fine particles are magnetic fine particles.
[6] The reaction between the substance to be analyzed and the specific partner solid-phased on the fine particles is performed in the complex forming unit, and the insolubilization reaction is performed on the sensing unit. [1] to [5] Any analysis method,
[7] The analysis method according to any one of [1] to [6], wherein the fine particles have a size of 3 μm or less.
[8] The analysis method according to any one of [1] to [7], wherein the soluble substance is silver ions,
[9] An apparatus used for the analysis method according to any one of [1] to [8],
An apparatus having a complex forming unit for reacting a substance to be analyzed with a specific partner solid-phased on a fine particle, and the sensing unit;
[10] The apparatus according to [9], wherein the complex forming unit and the sensing unit are separate bodies,
[11] The present invention relates to the apparatus according to [9] or [10], which includes a magnetic body capable of capturing the fine particles.

According to the present invention, analysis with higher detection sensitivity and accuracy than conventionally known analysis methods can be performed, and a small enzyme immunosensor can be produced.
For example, by causing an antigen-antibody reaction using magnetic fine particles, non-specific labeling can be washed by B / F by magnetic interaction, and by using fine particles, the amount of solid phase antibody 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, Facilitating direct electron exchange with the product with the electrode enables highly sensitive electrochemical enzyme immunoassay.
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. Was achieved.

It is explanatory drawing which shows typically a series of reaction utilized with the one aspect | mode of the analysis method of this invention. 3 is a plan view schematically showing an electrode part produced in Example 1. FIG. 1 is a side view schematically showing the structure of an enzyme immunoelectrode prepared in Example 1. FIG. 1 is a plan view schematically showing the structure of an enzyme immunoelectrode prepared in Example 1. FIG. In Example 2, it is a graph which shows the result of the DPV (differential pulse voltammetry) measurement which compared this invention method using an antibody fixed magnetic microparticle with the conventional method using an antibody solid-phased electrode. 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 blocking agent on the electrode in the method of this invention using the antibody fixed magnetic fine particle. In Example 4, it is a graph which shows the result of the luminescence count measurement which compared the effect by the difference in the particle diameter of the magnetic microparticle in the method of this invention using an antibody fixed magnetic microparticle. In Example 4, it is a graph which shows the result of the DPV measurement which compared the effect by the difference in the particle diameter of the magnetic fine particle in the method of this invention using an antibody fixed magnetic fine particle. 6 is a graph showing the relationship between the oxidation current peak value (Ip) of the DPV measurement value and the luminescence count (CPS) in the method of the present invention using the antibody-immobilized magnetic fine particles having different particle diameters evaluated in Example 4. 6 is a graph showing the relationship between Ip / luminescence count and particle diameter in the method of the present invention using antibody-immobilized magnetic fine particles with different particle diameters evaluated in Example 4.

  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 part and electrically analyzing (detecting or measuring) the precipitated insoluble material, wherein the selective interaction step is performed with fine particles. It is characterized by doing.

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, after describing the outline of the present invention based on the reaction schematic diagram shown in FIG. 1 showing a specific embodiment of the complex-forming analysis method of the present invention, the present invention will be described in more detail.

In the analysis system shown in FIG. 1, the antigen 103 is an analysis target substance, and an immune complex formation reaction by an antigen-antibody reaction (sandwich method) is used on the magnetic microparticle 105 as a selective interaction. 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.
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 on a magnetic 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.

  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. Further, 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, and NBT diformazan is generated by the reduction reaction, and the complex 2BCI / NBT diformazan is obtained. 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 according to 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.

  Moreover, it is preferable that the surface of the sensing unit and the fine particles are in direct contact. 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.

  In the present invention, the 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 as in the fourth embodiment, for example. 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. 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.

In addition, the selective interaction reaction performed on the fine particles may be performed at the same place as that on the sensing unit, but a substance necessary for the selective interaction reaction is negative for the transmission and reception of an electrical signal in the sensing unit. In the case where the influence is exerted, it is preferable to be performed at a place different from that on the sensing unit.
As a specific example of such a preferred embodiment, an antigen-antibody reaction is performed in a complex forming unit using magnetic fine particles, and after completion of the antigen-antibody reaction, an electrode unit (sensing unit) which is a detection unit in another flow path Take out the magnetic particles to the sensor and collect the magnetic particles in the sensing part with a magnet. Thereafter, an enzyme reaction is performed in the sensing unit, and the product silver is electrochemically measured.

  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: Preparation of reagent and enzyme immunoelectrode
(1) Preparation of carbon electrode In order to evaluate the influence of the blocking agent on an electrode, what coated the bovine serum albumin (BSA) on the carbon electrode, and the thing which is not coated with BSA were produced.
The electrode part 12 and the lead part 13 which consist of the pattern shown in FIG. 2 were produced by the printing method using the stainless steel mask pattern. First, on an insulating substrate 11 made of polyethylene terephthalate (PET), a conductive carbon paste (FTU-20: manufactured by Asahi Chemical Research Laboratories) was printed and formed in the shape of an electrode to produce a carbon electrode. A reference electrode 16 was prepared by applying a silver / silver chloride ink (manufactured by BAS) to a part of the produced carbon electrode. Next, in order to partition the electrode portion 12 and the lead portion 13, an electrode portion including the working electrode 14, the counter electrode 15, and the reference electrode 16 was manufactured by covering a part of the lead portion with the insulating film 17. The opposite ends of the working electrode, the counter electrode, and the reference electrode function as the connection connector 18. The produced carbon electrode was used as an electrode not coated with BSA.
For comparison, an electrode coated with BSA was prepared by adding 1% BSA to a 0.1 mol / L phosphate buffer solution (pH 7.4) (hereinafter referred to as buffer solution A). The prepared carbon electrode was immersed for 1 hour.

(2) Preparation of antibody-immobilized magnetic fine particles According to a known method, mice were immunized with hepatitis B surface antigen (HBs antigen: recombinant product, subtype adw) to prepare anti-HBs antigen mouse monoclonal antibody (IgG). This was solid-phased on magnetic fine particles (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 the prepared antibody and magnetic fine particles are mixed. Thus, the antibody was chemically immobilized on the surface of the magnetic fine particle by covalent bond. Furthermore, BSA was physically and chemically immobilized to produce anti-HBs antibody-immobilized magnetic fine particles.

(3) Preparation of alkaline phosphatase (ALP) -labeled anti-HBs antigen rabbit polyclonal antibody solution (Fab ′) solution According to a known method, a rabbit is immunized with hepatitis B surface antigen (HBs antigen: recombinant product, subtype adw), An anti-HBs antigen rabbit polyclonal antibody (IgG) was generated and then prepared in the Fab ′ fraction. Using this prepared antibody, ALP (manufactured by Roche), and a crosslinking reagent (Succinimidyl 4- [N-maleimidomethyl] -cyclohexane-1-carboxylate: manufactured by PIERCE), “highly sensitive enzyme immunoassay (Eiji Ishikawa: Society Press Center) 1993) ”, ALP-labeled anti-HBs antigen rabbit polyclonal antibody (Fab ′) was prepared. A solution obtained by adjusting the enzyme-labeled antibody to a predetermined concentration with a solution obtained by adding 2% BSA to the buffer A described in (1) was used as an ALP-labeled anti-HBs antibody solution.

(4) Preparation of Washing Solution Washing solution A was prepared by adding 0.1% Tween 20 (manufactured by Wako Pure Chemical Industries) to 0.01 mol / L phosphate buffer. Further, a mixture of 10 mmol / L MES (manufactured by Dojin), 0.9% NaCl (manufactured by Wako Pure Chemical Industries) and 0.05% Tween 20 (manufactured by Wako Pure Chemical Industries) was prepared as a cleaning liquid B.

(5) Preparation of enzyme-labeled complexed magnetic fine particles Anti-HBs antibody-immobilized magnetic fine particles prepared in (2), hepatitis B surface antigen solution (HBs antigen: recombinant product, subtype adw), prepared in (3) The ALP-labeled anti-HBs antibody solution was mixed at a predetermined concentration, and an antigen-antibody reaction was performed at room temperature for a predetermined time. As the HBs antigen solution, a solution prepared by adjusting the HBs antigen to a predetermined concentration with a buffer A containing 1% BSA was used. Thereafter, the magnetic fine particles are separated by magnetic separation, and B / F washing is performed using the washing solution B to prepare enzyme-immunoconjugated magnetic fine particles (ALP-labeled anti-HBs antibody-HBs antigen-anti-HBs antibody-immobilized magnetic fine particles). did.

(6) Preparation of antibody-immobilized electrode Buffer A containing the anti-HBs antigen mouse monoclonal antibody (final concentration 1 mg / mL) prepared in (2) is applied to the working electrode of the non-blocking carbon electrode prepared in (1). 2 μL was dropped and incubated at 37 ° C. for 30 minutes, and then dried for 2 hours at 25 ° C. and a humidity of 40%. Then, it was immersed in buffer solution A containing 1% BSA for 1 hour to block unreacted parts, and further, the substrate was washed with demineralized water and dried to produce an antibody-immobilized electrode.

(7) Preparation of enzyme-immunoconjugate antibody-immobilized electrode Prepare the HBs antigen solution and the ALP-labeled anti-HBs antibody solution prepared in (3) in the reaction part well of the antibody-immobilized electrode prepared in (6). An antigen-antibody reaction was carried out by adding a solution mixed in a concentration and immersing the solution at room temperature for a predetermined time. Thereafter, the electrode was washed with a washing solution A to prepare an enzyme-immunoconjugated electrode (ALP-labeled anti-HBs antibody-HBs antigen-anti-HBs antibody-immobilized electrode). As the HBs antigen, a solution prepared by adjusting the HBs antigen (recombinant, subtype adw) to a predetermined concentration with buffer A containing 1% BSA was used.

(8) Preparation of substrate solution 25 mmol / L containing an aqueous solution (substrate solution A) containing 2.5 mmol / L AgNO ₃ and 2 mmol / L MgSO ₄ and 40 mmol / L p-aminophenyl phosphate (pAPP: manufactured by LKT Laboratories) A Tris aqueous solution (substrate solution B) was prepared, and immediately before the enzyme substrate reaction, the substrate solution A and the substrate solution B were diluted 10-fold and mixed one-to-one to obtain an enzyme substrate reaction solution.

(9) Silver deposition reaction conditions using enzyme-immunoconjugated magnetic microparticles The enzyme-immunoconjugated magnetic microparticles prepared in (5) are dispersed in 100 μL of an aqueous solution containing 1 mmol / L MgSO ₄ and 12.5 mmol / L Tris. As shown in FIGS. 3 and 4, with the magnet placed under the working electrode part, the enzyme-immunoconjugated magnetic fine particle dispersion solution is dropped into the reaction part well, and the enzyme-immunocomplexed substance is applied onto the working electrode. Magnetic fine particles were collected.
Thereafter, 30 μL of the substrate solution A, 30 μL of the substrate solution B, and 240 μL of an aqueous solution containing 1 mmol / L MgSO ₄ and 12.5 mmol / L Tris were added and left for a predetermined time.

(10) Silver deposition reaction conditions using enzyme-immunoconjugated antibody-immobilized electrode Electrode-immobilized antibody-immobilized electrode prepared in (7), 30 μL of substrate solution A was added to the reaction part well. 30 μL of B, 240 μL of an aqueous solution containing 1 mmol / L MgSO ₄ and 12.5 mmol / L Tris were added, and left for a predetermined time.

(11) Electrochemical measurement conditions The electrochemical measurement was performed by differential pulse voltammetry (DPV) by connecting each working electrode, reference electrode, and counter electrode connector to an electrochemical analyzer (chi1232a: manufactured by ALS). .

<< Example 2: Verification of high sensitivity by using magnetic fine particles >>
The measurement sensitivity was compared between the case of the conventional electrode in which the antibody for measurement was immobilized on the electrode and the case of the electrode in the present invention in which the antibody was immobilized on the magnetic fine particles.
As an enzyme immunoelectrode in the present invention, enzyme-labeled complexed magnetic fine particles (0.7 μm) prepared by reacting at room temperature for 30 minutes by the method of Example 1 (5) were prepared in Example 1 (1). It was dropped into the reaction part well of the carbon electrode coated with BSA, attracted with a magnet from the back of the electrode part, and magnetic fine particles were collected on the working electrode, and reacted for 30 minutes under the conditions of Example 1 (9).
On the other hand, as a conventional enzyme immunoelectrode, an enzyme-immunoconjugated antibody-immobilized electrode prepared by reacting at room temperature for 30 minutes by the method of Example 1 (7) was used, and under the conditions of Example 1 (10). The reaction was allowed for 30 minutes.
Thereafter, the enzyme substrate reaction solution was removed, 300 μL of 1 mol / L KNO ₃ solution was added, and DPV measurement was performed under the conditions of Example 1 (11).

The results (relationship between antigen concentration and silver oxidation current response value) are shown in FIG.
The enzyme immunoelectrode in the present invention showed an oxidation current peak value about 3.4 times higher than that of the conventional enzyme immunoelectrode, and it was confirmed that the silver oxidation current value could be detected with high sensitivity. This is thought to be due to the increase in the amount of solid-phase antibody by using magnetic fine particles and the fact that immunocomplexation was formed in a short time. It was found that time-sensitive measurements can be made.

<< Example 3: Verification of high sensitivity due to absence of blocking agent on electrode >>
The enzyme-immunoconjugated magnetic particles prepared in Example 1 (5) were placed in the reaction part wells of the carbon electrode with or without BSA blocking prepared in Example 1 (1), respectively, and the silver oxidation current response value was obtained. It was confirmed.
In the same manner as in Example 2, enzyme-labeled complexed magnetic fine particles (0.7 μm) prepared by reacting at room temperature for 30 minutes by the method of Example 1 (5) were used, and the conditions of Example 1 (9) were satisfied. The silver deposition reaction was carried out for 30 minutes. Thereafter, the enzyme substrate reaction solution was removed, 300 μL of 1 mol / L KNO ₃ solution was added, and DPV measurement was performed under the conditions of Example 1 (11).

The results are shown in FIG.
By using the non-blocking electrode with BSA, the silver oxidation current peak value was about 4.3 times higher than that of the electrode blocked with BSA. From this, it was found that it is possible to oxidize deposited silver more effectively by not coating the electrode surface, which is a detection part, with a blocking agent such as BSA.

<< Example 4: Verification of high sensitivity related to particle diameter of magnetic fine particles >>
Example 1 was examined except that magnetic microparticles prepared by the following preparation method and labeled antibody were used.

(1) Preparation of antibody-immobilized magnetic fine particles According to a known method, mice are immunized with influenza B virus antigen (FluB antigen: collected from the influenza strain B vaccine HA stock obtained from the Osaka University Microbial Disease Research Society). An anti-FluB mouse monoclonal antibody (IgG) was prepared. Next, the antibody-immobilized magnetic fine particles were prepared by immobilizing the antibody on magnetic fine particles (manufactured by Merck) having particle diameters of 2.6 μm, 0.7 μm, and 0.44 μm, respectively. 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, BSA was physically and chemically immobilized to produce anti-FluB antibody-immobilized magnetic fine particles.

(2) Preparation of ALP-labeled anti-FluB mouse monoclonal antibody solution (Fab ') solution In accordance with a known method, influenza B virus antigen (FluB antigen: Brisbane strain B influenza HA vaccine stock solution obtained from Osaka University Microbial Disease Research Society The mouse was immunized with an anti-FluB mouse monoclonal antibody (IgG). 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 2% BSA-containing buffer A was used as an ALP-labeled anti-FluB antibody solution.

(3) Examination of relationship between particle diameter of magnetic fine particles and silver oxidation current value Anti-FluB antibody-immobilized magnetic fine particles (particle diameters: 0.4 μm, 0.7 μm, 2.6 μm) prepared in (1), FluB antigen Solution (FluB antigen was diluted with buffer A containing 1% BSA to a predetermined concentration (0%, 0.001%, 0.01%)), ALP-labeled anti-antigen prepared in (2) The FluB antibody solution was mixed at a concentration of 1 mAbs, and the original antibody reaction was performed at room temperature for 5 minutes. Thereafter, the magnetic fine particles were separated by magnetic separation, and B / F washing was performed using the washing liquid B to prepare enzyme-immunoconjugated magnetic fine particles.

First, the enzyme-immunoconjugated magnetic fine particles of each particle size were allowed to react with CDP-Star, which is a luminescent substrate, for 3 minutes. Was confirmed. The result is shown in FIG.
As a result, it was confirmed that the light emission amount was small and the reactivity was low as the particle size was reduced. That is, a smaller particle size indicates that a smaller amount of enzyme-labeled antibody could be conjugated at the time of enzyme-immunoconjugation.

On the other hand, next, using the same enzyme-immunoconjugated magnetic fine particles, a silver deposition reaction was performed at room temperature for 3 minutes according to Example 1 (9), and then the silver oxidation current response value was confirmed by DPV measurement. . The confirmed result is shown in FIG.
The silver oxidation current peak value increased as the particle size decreased. This indicates that the smaller the particle diameter, the more silver is deposited on the sensing part electrode (working electrode) that is the sensing part.

  FIG. 9 shows the relationship between the oxidation current peak value (Ip) of the DPV measurement value and the relationship between the Ip / luminescence count and the particle diameter in FIG. Show. From FIG. 9, it can be seen that the smaller the particle size, the larger the inclination. Further, as shown in FIG. 10, the smaller the particle diameter, the larger the value of Ip / luminescence count. These indicate that silver can be more effectively deposited on the sensing portion electrode (working electrode) with a smaller amount of enzyme-labeled antibody. In other words, by reducing the size of the magnetic fine particles to be used, the electrode surface and the distance at which silver deposition occurs will be closer, and it is possible to deposit more silver that can directly transfer and receive electrons on the electrode. Highly sensitive measurement is possible.

  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 (7)

An insolubilization reaction is carried out by reacting an analyte with a specific partner exhibiting a selective interaction with the analyte and performing an insolubilization reaction by correlating with the abundance of the analyte in the test sample. A soluble substance added as a reagent before the step is converted into an insoluble substance, precipitated in a sensing part, and the insoluble substance precipitated in the sensing part is electrically analyzed,
A specific partner exhibiting a selective interaction is immobilized on the microparticle, and the microparticle is a non-conductive substance,
The method of analysis characterized in that the surface of the sensing part is not covered with a substance that hinders the transmission and reception of electrical signals.   The analysis method according to claim 1, wherein the sensing unit surface is configured such that the sensing unit surface and the fine particles are in direct contact with each other when performing insolubilization reaction and electrical analysis.   The analysis method according to claim 1, wherein the fine particles are magnetic fine particles.   The reaction between the substance to be analyzed and the specific partner solid-phased on the fine particle is performed in the complex forming unit, the formed complex is sent to the sensing unit, and the insolubilization reaction is performed on the sensing unit. The analysis method according to any one of claims 1 to 3, which is performed in step (1).   The analysis method according to any one of claims 1 to 4, wherein the fine particles have a size of 3 µm or less.   The analysis method according to claim 1, wherein the soluble substance is silver ions. A combination of a sensing part , a fine particle, a soluble substance, and a labeling substance for use in the analysis method according to any one of claims 1 to 6,
Specific partner showing the selective interaction are immobilized on the particles, the fine particles are electrically non-conductive material,
The surface of the sensing unit is not covered with a substance that hinders the transmission and reception of electrical signals,
The surface of the sensing unit is configured so that the sensing unit surface and fine particles are in direct contact when performing an insolubilization reaction and electrical analysis ,
The combination, wherein the labeling substance is an enzyme, a reducing agent, or an oxidizing agent that can cause an insolubilization reaction directly or indirectly and can convert the soluble substance into an insoluble substance by the insolubilization reaction .

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