JP5071575B2 - Detection kit - Google Patents

Description

  The present invention relates to a detection kit.

Conventionally, various methods have been used to detect antigens or antibodies. For example, an antigen or antibody is detected using an ELISA method utilizing an antigen-antibody reaction.
In the antigen-antibody reaction detection method by this ELISA method, the antigen is detected by a dye reaction by an enzyme immobilized on the antibody during the secondary antibody reaction. However, when the abundance of the antigen is extremely small, there is a problem that the antigen detection sensitivity cannot be obtained.

On the other hand, there is an example in which a functional surface capable of detecting an antigen is produced by growing an anchorable PEG polymer having an antibody molecule at the terminal by photopolymerization or the like from a substrate surface (for example, Non-Patent Document 1).
However, antibody molecules are larger than the density and size of PEG polymer grown on a substrate. Therefore, the number of antibody molecules that can be immobilized on the substrate surface is reduced. As a result, there is a problem that the detection sensitivity of the antigen cannot be obtained.

Robert P. Serba et al, Langmuir 2005 21, 10907

  An object of the present invention is to provide a detection kit for detecting a detection object with high sensitivity.

Such an object is achieved by the present invention described below.
The detection kit of the present invention is a detection kit capable of selectively detecting a detection target,
Between the binding site that specifically binds to the detection target, a reactive first reaction site, and the binding site and the first reaction site, the binding site and the first reaction site. A first compound comprising a first linking site linking
A second reaction site that reacts with the first reaction site and a second compound comprising a labeling site;
The first linking site includes a linear polyethylene glycol chain,
The first reaction site and the second reaction site are each composed of a vinyl group,
A plurality of second reaction sites are connected to one first reaction site by reacting one first compound with a plurality of second compounds. And
As a result, a detection reagent having a binding site and a labeling site is obtained, so that the detection target can be detected with high sensitivity.
Further, the first compound has a first linking site that links the binding site and the first reaction site between the binding site and the first reaction site, thereby Since the linking site has a highly reactive group, the binding site and the first reaction site can be reliably linked.
In addition, since the first reaction site and the second reaction site are each composed of a vinyl group, the first reaction site and the second reaction site are highly reactive. A site | part and a 2nd reaction site | part can be connected easily. In addition, since a polymerization reaction occurs at a vinyl group, a plurality of second compounds can be introduced.

In the detection kit of the present invention, the binding site is preferably an antigen or an antibody.
Thereby, the detection target (antibody or antigen) that can specifically bind to the antigen or antibody can be detected with high sensitivity .

In the detection kit of the present invention, the labeling site is preferably composed of at least one of a redox substance, a fluorescent substance, and a dye compound.
As a result, the oxidation-reduction current, the fluorescence intensity, or the color tone changes, so that electrochemical detection or fluorescence detection becomes possible.
The detection kit of the present invention preferably has a plurality of the labeling sites.
As a result, a detection reagent having a plurality of labeled sites can be obtained, so that the detection sensitivity of the detection target can be further improved.

In the detection kit of the present invention, the second compound has a second linking site for linking the second reaction site and the labeling site between the second reaction site and the labeling site. It is preferable.
Thereby, since a 2nd connection site | part has a highly reactive group, a label | marker site | part and a 2nd reaction site can be connected reliably.
In the detection kit of the present invention, it is preferable that the detection kit further includes a substrate on which a captured substance capable of selectively capturing the detection target is supported.
Thereby, since a detection target object can be detected by using a base | substrate, a 1st compound, and a 2nd compound, a detection target object can be detected simply and rapidly.

In the detection kit of the present invention, the substrate comprises a well,
It is preferable that the trapped substance is supported on the inner surface of the well.
Thereby, since a detection target object can be detected by using a well, a 1st compound, and a 2nd compound, a detection target object can be detected more simply and rapidly.
In the detection kit of the present invention, the substrate is preferably formed in a granular shape.
Thereby, since the surface of a base | substrate can be made smooth, it can move in a sample solution with low resistance.

It is a figure which shows typically the 1st compound of the detection kit of this invention. It is a figure which shows typically the 1st compound of the detection kit of this invention. It is a figure which shows typically the 2nd compound of the detection kit of this invention. It is a figure (longitudinal section) for demonstrating the 1st detection method of the antigen using the detection kit of this invention. It is a perspective view of the biosensor used for the 2nd detection method of the antigen using the detection kit of this invention. It is a top view which shows typically the biosensor shown in FIG. It is the elements on larger scale of the sectional view on the AA line shown in FIG. It is a figure (longitudinal sectional view) for demonstrating the 2nd detection method of the antigen using the detection kit of this invention. It is a top view which shows typically the biosensor used for the 3rd detection method of the antigen using the detection kit of this invention. It is the elements on larger scale of the BB sectional drawing shown in FIG. It is a figure (longitudinal sectional view) for demonstrating the 4th detection method of the antigen using the detection kit of this invention. It is a cross-sectional perspective view which shows typically the well used for the 5th detection method of the antigen using the detection kit of this invention.

Hereinafter, the detection kit of the present invention will be described in detail.
<Detection kit>
The detection kit of the present embodiment includes a first compound having a binding site that specifically binds to the detection target and a second compound having a labeling site that increases the detection sensitivity of the detection target.

First, the first compound and the second compound of the detection kit of the present invention will be described.
1 and 2 are diagrams schematically showing the first compound, and FIG. 3 is a diagram schematically showing the second compound.
The first compound 1 shown in FIG. 1 includes a binding site 11, a reactive first reaction site 12, and a first connection site 13 that connects the binding site 11 and the first reaction site 12. Have.

The binding site 11 is not particularly limited as long as it can specifically recognize the detection target, and examples thereof include antibodies, antigens, microorganisms, enzymes, sugars, and proteins. Among these, in the present embodiment, the binding site 11 is preferably an antigen or an antibody.
The kind of such an antibody is not specifically limited, For example, IgG, IgM, IgA, IgE etc. are mentioned. Among these, IgG is preferable. IgG is easy to produce and increases the types of antigens that can be detected.

The first reaction site 12 is a site that reacts with and binds to a second reaction site 21 of the second compound 2 described later. That is, the first reaction site 12 has a function of linking the first compound 1 and the second compound 2.
The first reaction site 12 is not particularly limited, and examples thereof include a polymerizable group such as vinyl group, allyl group, acrylic group, methacryl group, and styryl group, maleimide group, pyridyl disulfide group, and N-hydroxysuccinimide. Group, NHS group and the like. Of these, a polymerizable group is preferable, and a vinyl group is more preferable. Since the polymerizable group, particularly the vinyl group, is a highly reactive group, the first reaction site 12 and the second reaction site 21 can be reacted efficiently.

The first linking site 13 has a linking group (functional group) that can link the linking site 11 and the first reaction site 12. Examples of such a linking group include an amide bond, an ether bond, an ester bond, a sulfide bond, and a carbonyl bond. Two or more of these may be included.
For example, when the binding site 11 is an antibody, a free amino group is present on the surface of the binding site 11, so that the binding group preferably contains an amide bond. Thereby, since an antibody and a binding group react efficiently, an antibody and a binding group can be bound reliably.

In addition, for example, when the binding site 11 is a protein having cysteine, it is preferable that the cysteine has an SH group and therefore includes a sulfide bond.
Moreover, it is preferable that the 1st connection site | part 13 contains the polyethyleneglycol (PEG) chain | strand (spacer). Thereby, since the binding site 11 and the first reaction site 12 are separated from each other by a certain distance, the first reaction site 12 and the second reaction site 21 can be reacted efficiently.

Compared to alkyl chains, PEG chains contain an ether bond on the chain, so it is easy to form interactions with metal ions and polar functional groups due to the influence of electrons in the non-bonded pair of ether oxygen atoms, and also associate with water molecules. Easy to do. Moreover, the dipole moment is relatively large due to the influence of the dipole moment between the ether oxygen atom and the adjacent carbon atom, which also affects the ease of hydration.
Thus, since the PEG chain generally has a chain structure that tends to be water-soluble, it is easy to take a relaxed extension structure in water.

On the other hand, since the alkyl chain is hydrophobic, the chain shrinks in water due to the hydrophobic aggregation effect, and the distance between the reaction sites cannot be maintained.
Accordingly, since the first linking site 13 contains a PEG chain, it becomes easy to take an elongated structure in water, so that the detection target in the liquid sample 151 can be detected with high sensitivity.
Moreover, it is preferable that the 1st connection site | part 13 contains the particle | grains 13a. Thereby, since the particle | grain 13a has a large surface area, the some coupling | bond part 11 and the 1st reaction site | part 12 can be couple | bonded with the surface of the particle | grain 13a.
The first compound 1 having the binding site 11, the first reaction site 12, and the first linking site 13 as described above includes, for example, the following compound (1), compound (2), and the structure shown in FIG. Can be mentioned. In both compound (1) and compound (2), the binding site (site where the PEG chain and amide bond) 11 is an antibody.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The first compound 1 shown in FIG. 2 has a binding site 11, a first reaction site 12, and a first coupling part 13 including particles 13a. The binding site 11 and the first reaction site 12 are each bonded to the particle 13a via a sulfur atom.
Examples of such particles 13a include metals such as gold and silver, metal oxides thereof, dyes, pigments, polystyrene, methacrylic acid, and the like. Of these, metal is preferable, and gold is more preferable.
Since gold can be sulfide-bonded to a sulfur atom, the plurality of bonding sites 11 and the first reaction site 12 can be easily bonded via sulfur atoms as shown in FIG. As a result, since many labeled sites 22 are present due to the coupling of the second compound 2, the detection target can be detected with high sensitivity.

When the first connecting portion 13 includes the particle 13a, the abundance ratio of the binding site 11 and the first reaction site 12 binding to the surface of the particle 13a is A for the binding site 11 and B for the first reaction site 12. At this time, A / B is preferably 0.1 to 1.0, and more preferably 0.3 to 0.8.
Thereby, since the presence ratio of the 1st reaction site | part 12 is larger than the binding site | part 11, the 2nd compound 2 which has the label | marker site | part 22 can be connected more. As a result, the presence ratio of the labeled portion 22 increases, and the detection target can be detected with higher sensitivity.

If the abundance ratio of the binding site 11 and the first reaction site 12 is too smaller than the above range, the abundance ratio of the binding site 11 is reduced, and there is a possibility that the detection target is not sufficiently bound.
On the other hand, when the abundance ratio of the binding site 11 and the first reaction site 12 is too larger than the above range, the abundance ratio of the first reaction site 12 becomes too small and the rate of linking the second compound 2 becomes small. There is a fear.

The second compound 2 shown in FIG. 3 includes a second reaction site 21 that reacts with the first reaction site 12, a labeling site 22, and a second reaction site 21 that links the second reaction site 21 and the labeling site 22. And a connecting portion 23.
The second reaction site 21 is a site that reacts with and binds to the first reaction site 12 of the first compound 1. That is, the second reaction site 21 has a function of linking the first compound 1 and the second compound 2.

  Such second reaction site 21 is not particularly limited. For example, polymerizable group such as vinyl group, allyl group, acrylic group, methacryl group, styryl group, N-hydroxysuccinimide group, NHS group, maleimide group And pyridyl disulfide group. Of these, a polymerizable group is preferable, and a vinyl group is more preferable. Since the polymerizable group, particularly the vinyl group, is a highly reactive group, the second reaction site 21 can be efficiently reacted with the first reaction site 12.

The label portion 22 has a function of improving the detection sensitivity of the detection target.
Examples of the label portion 22 include a fluorescent substance, a redox substance, or a dye compound. Two or more of these can be used in combination. By using a plurality of label portions 22, the detection target can be detected with high sensitivity.
When a fluorescent substance is used for the labeling part 22, the fluorescence intensity is obtained by mixing the fluorescence detection reagent 5 in which the first reaction part 12 and the second reaction part 21 are connected in the liquid sample 151 containing the detection target. Since this changes, it is possible to easily detect the detection object.

When a redox substance is used for the labeling site 22, the redox detection reagent 4 in which the first reaction site 12 and the second reaction site 21 are linked is brought into contact with the sensor that captures the detection target, thereby redoxing. Since the reaction occurs, the redox current can be measured.
When a dye compound is used for the labeling portion 22, the presence of the detection target can be confirmed by the change in the color tone of the sample including the detection target, so that the detection target can be detected more easily.
Such a fluorescent substance 52 is not particularly limited, and examples thereof include fluorescein, dansyl chloride, fluorescamine, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-chloride), Aromatic organic compounds such as orthoaminothiophenol and dansyl hydrazine.

  Although it does not specifically limit as the oxidation-reduction substance 42, For example, a ferrocene (chemical formula 3), an osmium-bipyridyl complex (chemical formula 4), a phthalocyanine, a tetrazolium salt, tetramethylbenzidine, diaminobenzidine, a triaminotriphenylmethane, a Trinder reagent, etc. Is mentioned. Of these, ferrocene or osmium-bipyridyl complex is preferable.

Since ferrocene is excellent in redox activity, redox current can be detected more efficiently when a detection target is detected.
Since the osmium-bipyridyl complex is water-soluble, it has a high affinity with the polymer. In addition, the polymer chain also extends in the hydrated structure, and the reaction is likely to proceed. Therefore, when the detection target is detected, the redox current can be detected more efficiently.
The dye compound is not particularly limited, and examples thereof include porphyrin, chlorophyll, neutral red, methylene blue, phenolphthalein, indocyanine green, cyanine dye, azo dye, flavonoid, carotenoid and the like.

The second linking site 23 has a bonding group (functional group) that can link the second reaction site 21 and the labeling site 22. Examples of such a linking group include an amide bond, an ether bond, an ester bond, a sulfide bond, and a carbonyl bond. Two or more of these may be included.
For example, when the labeling site 22 is ferrocene, the linking group preferably contains an ester bond. Thereby, since ferrocene and a bonding group react efficiently, ferrocene and a bonding group can be combined reliably.

Moreover, it is preferable that the 2nd connection site | part 23 contains the polyethyleneglycol (PEG) chain | strand (spacer). Thereby, since the 2nd reaction site | part 21 and the label | marker site | part 22 leave | separate a fixed distance, when detecting a detection target object, the said detection target object can be detected efficiently. Further, as described in connection with the first connecting portion 13, since it becomes easy to take an elongated structure in water, the detection target in the liquid sample 151 can be detected with high sensitivity.
Examples of the second compound 2 having the second reaction site 21, the labeling site 22, and the second linking site 23 as described above include the following compounds (5) to (7).

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

By including the first compound 1 and the second compound 2 described above, a detection kit is obtained.
This detection kit may include a substrate 190 on which a capture object 8 that can selectively capture the detection target is supported, for example, what is necessary for detection of the detection target.
Examples of such a substrate 190 include a working electrode 121 used for a biosensor, a substrate 120, a carrier 124, and the like. Thus, the detection target can be detected with high sensitivity using the first compound 1 and the second compound 2 after the detection target is once captured by the capture target 8 of the substrate 190.

The shape of the substrate 190 is not particularly limited, but when the carrier 124 is used, it is preferably spherical (granular). Thereby, since the surface becomes smooth, the liquid sample 151 can settle and float with a small resistance.
In addition, the detection kit of the present invention may include a spatula, a reaction container, a dropper, and the like. Thereby, a detection target can be detected more quickly regardless of the measurement location.
In addition, the detection kit of the present invention can be provided by containing the first compound 1, the second compound 2, and the like in a container in order to facilitate carrying.
Each of the first compound 1 and the second compound 2 contained in such a detection kit can be produced as follows.

  Hereinafter, the production method of the first compound 1 and the production method of the second compound 2 will be described in detail by taking two methods as examples. In the following description, the case where an antibody is used for the binding site 11 will be described as a representative.

<Method for producing first compound>
[1] First Production Method First, the following compound having an antibody as the binding site 11 and a succinimide at one end of the PEG via an ester group and the first reaction site 12 at the other end via an ester group Prepare (8).

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

  Next, as shown in the following reaction formula, the antibody and compound (8) are reacted.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of the compound (8) used in the reaction is preferably 2 to 100 times, more preferably 5 to 50 times the amount of 1 μg of antibody.
On the surface of the antibody, there is a free amino group derived from lysine, which is one of amino acids. Therefore, the reaction proceeds between the amino group and compound (8). Therefore, when the amount of the compound (8) is as described above, the compound (8) is present in excess, so that it can react with the amino group of the antibody without excess or deficiency.

The reaction time is preferably 0.5 to 10 hours, and more preferably 1 to 5 hours.
The reaction temperature is preferably 0 to 60 ° C, and more preferably 20 to 40 ° C.
The pH of the reaction is preferably 6-8, and more preferably 6.5-7.5.
By setting the reaction conditions as described above as preferable conditions, the reaction between the antibody and the compound (8) proceeds efficiently, so that the first compound 1 (compound (9)) can be obtained in a high yield. .

[2] Second Manufacturing Method Next, a second manufacturing method of the first compound 1 when the first connecting portion 13 contains gold fine particles will be described.
[A1] First, gold fine particles and aminoethanethiol are prepared.
The outer diameter of the gold fine particles is preferably 0.01 to 10 μm, and more preferably 0.05 to 1 μm.
The surface area of the gold fine particles is preferably 1 × 10 ⁻¹⁵ to 1 × 10 ⁻¹¹ m ² , and more preferably 5 × 10 ⁻¹⁵ to 8 × 10 ⁻¹³ m ² .

When the outer diameter and surface area of the gold fine particles are within such ranges, a plurality of compounds can be bonded to the surface of the gold fine particles.
The amount of aminoethanethiol used is preferably 0.1 to 1 mmol / L, and more preferably 0.5 to 0.8 mmol / L. By using the amount of aminoethanethiol in such a range, the amount of aminoethanethiol is larger than the amount of gold fine particles, so that a plurality of aminoethanethiols can be bonded to the surface of the gold fine particles.

[A2] The prepared gold fine particles and aminoethanethiol are mixed and reacted as shown in the following reaction formula.

As reaction conditions at this time, it is preferable to carry out the reaction in a reaction solvent while stirring at 25 ° C. for 1 hour.
The reaction solvent is not particularly limited, and examples thereof include various solvents such as dichloroethane, acetonitrile, methylene chloride, and methanol.
As a result, a compound (10) in which aminoethane was bonded to gold via a sulfur atom was obtained.

[A3] The compound (10) obtained is mixed with the compound (11) and reacted as shown in the following reaction formula.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of compound (11) to be used is preferably 1 to 100 equivalents, more preferably 5 to 50 equivalents, relative to compound (10). Since the amount of the compound (11) used is within such a range, the amount of the compound (11) is large relative to the gold fine particles, so that a plurality of compounds (11) are bonded to the surface of the gold fine particles via sulfur atoms. can do.
As reaction conditions at this time, it is preferable to carry out the reaction in a reaction solvent while stirring at 25 ° C. for 1 hour.
The reaction solvent is the same as that mentioned in the above [A2].
As a result, a compound (12) in which an amino group and the first reaction site 12 were bonded to gold fine particles via a sulfur atom was obtained.

[A4] Next, as shown in the following reaction formula, the compound (12) obtained in [A3] and the compound (13) having succinimide at both ends are mixed and reacted.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of compound (13) to be used is preferably 1 to 10 equivalents, more preferably 1.5 to 5 equivalents, relative to compound (12).
As reaction conditions at this time, it is preferable to carry out the reaction in a reaction solvent while stirring at 25 ° C. for 1 hour.
The reaction solvent is the same as that mentioned in the above [A2].
This obtained the compound (14) with which the amino group and the succinimide group reacted.

[A5] Finally, as shown in the following reaction formula, the compound (14) obtained in [A4] and an antibody are mixed and reacted.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of compound (14) to be used is preferably 2 to 100 times, more preferably 5 to 50 times the amount of 1 μg of antibody. As described above, the amino group on the antibody surface reacts with the succinimide group of the compound (14). Therefore, since the compound (14) is in an amount as described above, the compound (14) is excessively present, so that it can react with the amino group of the antibody without excess or deficiency.
As reaction conditions at this time, it is preferable to carry out the reaction in a reaction solvent while stirring at 25 ° C. for 1 hour.
The reaction solvent is the same as that mentioned in the above [A2].
Thereby, the 1st compound 1 (compound (15)) which has a granular gold fine particle in the 1st connection part 13 was obtained.

In addition, by bonding a plurality of aminoethanethiol and / or compound (11) to the surface of the gold fine particle, as shown in FIG. 2, the bonding site 11 and / or the first 1 A plurality of reaction sites 12 can be bonded. In other words, a plurality of binding sites 11 and / or first reaction sites 12 can be formed radially from the surface of the gold fine particles.
As a result, the plurality of detection objects and the plurality of binding sites 11 react with the plurality of second reaction sites 21 and the plurality of first reaction sites, thereby further improving the detection sensitivity of the detection objects. be able to.

<Method for producing second compound>
[1] First production method First, a labeled compound (16) shown below, and a compound (17) having a second reaction site 21 at one end of PEG and a hydroxyl group at the other end shown below are prepared. .

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

  Next, as shown in the following reaction formula, the compound (16) and the compound (17) are reacted in the presence of a catalyst.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of compound (16) used in the reaction is preferably 1 to 10 equivalents, more preferably 1.5 to 5 equivalents, relative to compound (17).
The reaction time is preferably 0.5 to 10 hours, and more preferably 1 to 5 hours.
The reaction temperature is preferably 0 to 60 ° C, and more preferably 20 to 40 ° C.
By setting the reaction conditions as described above as preferable conditions, the reaction between the compound (16) and the compound (17) proceeds without excess or deficiency, so that the second compound 2 (compound (18)) can be obtained in good yield. Obtainable.

[2] Second Production Method Next, a second production method of the second compound 2 will be described.
First, a compound (19) having a second reaction site 21 at one end and a compound (20) having a labeling site 22 at one end are prepared.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

  Next, as shown in the following reaction formula, the compound (19) and the compound (20) are radically polymerized in the presence of a catalyst.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

The amount of the compound (19) used for the reaction is preferably 1 to 10 equivalents, more preferably 1.5 to 5 equivalents, relative to the compound (20).
The radical polymerization reaction time is preferably 0.5 to 10 hours, and more preferably 1 to 5 hours.
The radical polymerization reaction temperature is preferably 50 to 300 ° C, and more preferably 100 to 250 ° C.
The reaction pressure is preferably about normal pressure.
Examples of the catalyst include radical catalysts such as benzoyl peroxide. The amount of the catalyst is preferably 0.1 to 5 mmol, and more preferably 0.5 to 1 mmol.

By setting the reaction conditions as described above as preferable conditions, the reaction between the compound (19) and the compound (20) proceeds without excess or deficiency, so that the second compound 2 (compound (21)) is produced in good yield. Obtainable.
Since this compound (21) has a plurality of labeling sites 22, the detection target can be detected with higher sensitivity by binding to the first compound 1.
By the manufacturing method as described above, the first compound 1 and the second compound 2 are obtained, and a detection kit containing them is obtained.

<How to use the detection kit>
The detection kit containing the first compound 1 and the second compound 2 thus obtained can be used, for example, as follows.
Hereinafter, a method for using the detection kit (a method for detecting a detection target) will be described in detail with reference to the drawings. In the following description, the case where the antigen 3 is used as the detection target substance and the antibody is used as the binding site 11 will be described as a representative.

[1] First Detection Method First, a first detection method for a detection target using the detection kit of the present invention will be described.
FIG. 4 is a longitudinal sectional view schematically illustrating the first detection method.
In the following description, the upper side in FIG. 4 is referred to as “upper” and the lower side is referred to as “lower”.
In the first detection method, the step [A1] of preparing the fluorescence detection reagent 5 by reacting the first compound 1 and the second compound 2 and the step of supplying the fluorescence detection reagent 5 into the liquid sample 151 [ A2] and a step [A3] for detecting a change in fluorescence intensity caused by the binding between the fluorescence detection reagent 5 and the antigen 3.

[A1] Detection reagent preparation step First, the fluorescence detection reagent 5 is prepared by reacting the first compound 1 and the second compound 2. In the following description, the binding site 11 of the fluorescence detection reagent 5 will be described as the antibody 51, and the labeling site 22 as the fluorescent material 52.
For example, as shown in the following reaction formula, the fluorescence detection reagent 5 has the first compound 1 (compound (22)) having a vinyl group at the first reaction site 12 and the fluorescent substance 52 at the labeling site 22. The second compound 2 (compound (23)) is subjected to a photopolymerization reaction by ultraviolet irradiation with a high-pressure mercury lamp.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

In addition, 1 mol of compound (22) and 5 mol of compound (23) were used. The ultraviolet rays were irradiated with light having an intensity of 1600 mJ / cm ² and a wavelength of 365 nm at 25 ° C. for 15 minutes. This prepared the fluorescence detection reagent 5 (compound (24)).
In addition, by controlling the degree of polymerization (ultraviolet irradiation time) of the fluorescence detection reagent 5, a large number of fluorescent substances 52 can be introduced to the surface of the antibody 51 molecule via the first connecting portion 12.
For example, 100 fluorescent substances 52 can be introduced per chain length of the first connecting portion 12. Assuming that 10 polymer chains cause a growth reaction from one antibody, 1000 fluorescent substances 52 can be introduced per 51 molecules of one antibody. Thereby, since the fluorescent substance 52 can be introduced in a large amount, the detection sensitivity of the antigen can be greatly amplified.

When reacting the compound (2) and a compound obtained by substituting the ferrocene of the compound (7) with the fluorescent substance 52, first, a compound in which the compound is dispersed in a buffer solution is prepared. Next, 0.1 mol / L of 2-mercaptoethanol is put into the buffer solution to cleave the disulfide. When this solution and compound (2) are mixed, the thiol at the polymer end reacts with the maleimide molecule on the surface of the antibody 51 molecule. Thereby, a covalent bond is formed, and a large number of fluorescent substances 52 can be immobilized on the surface of the antibody 51 molecule.
Similarly to the second detection method described later, the fluorescence detection reagent 5 containing gold fine particles can also be prepared by a reaction between the compound (28) and the compound (23).

[A2] Detection Reagent Supply Step As shown in FIG. 4A, a liquid sample 151 containing the antigen 3 that is the detection target is placed in the reaction container 152.
Next, as shown in FIG. 4A, the fluorescence detection reagent 5 is supplied into the liquid sample 151 (sample supply space 150). Thereby, the antigen 3 and the fluorescence detection reagent 5 contact.

When the antigen 3 and the fluorescence detection reagent 5 come into contact with each other, as shown in FIG. 4B, the antibody 51 of the fluorescence detection reagent 5 reacts with the antigen 3, and the antigen 3 and the antibody 51 are combined.
Here, examples of the liquid sample (subject) 151 include body fluids such as blood, urine, sweat, lymph, cerebrospinal fluid, bile, saliva, processed fluids obtained by performing various treatments on these body fluids, alcohol, and the like. Foods and beverages, pharmaceuticals, cosmetics and the like.
In addition, the supply concentration of the fluorescence detection reagent 5 is not particularly limited, but is preferably 0.01 to 1 mmol / L, and more preferably 0.05 to 0.5 mmol / L.

[A3] Antigen detection step When the antibody 51 is bound to the antigen 3, the fluorescence intensity of the antigen 3 is increased by the fluorescent substance 52 of the fluorescence detection reagent 5. That is, in a state where the antibody 51 is not bound to the antigen 3, the fluorescence intensity of the antigen 3 is 0 or an extremely low value. However, when the antibody 51 of the fluorescence detection reagent 5 binds to the antigen 3, the fluorescence intensity of the antigen 3 increases. The antigen 3 can be detected by measuring the fluorescence intensity with HPLC using a fluorometer or a fluorescence detector.
When measuring the concentration of the antigen 3 in the liquid sample 151, a plurality of antigen 3-containing liquid samples 151 with known concentrations are prepared in stages. And the antigen 3 in the liquid sample 151 of each density | concentration is measured with the said detection method. A calibration curve is prepared with the fluorescence intensity of antigen 3 in each liquid sample 151 obtained by the measurement as the vertical axis and the concentration of antigen 3 as the horizontal axis.

Next, the antigen 3 in the liquid sample 151 is measured by the method described above. Then, the concentration of the antigen 3 present in the liquid sample 151 can be quantified from the fluorescence intensity obtained by the measurement and the calibration curve.
By the method as described above, the presence of the antigen 3 can be detected, and the concentration of the antigen 3 can also be quantified.

[2] Second Detection Method Next, a second detection method of the detection object using the detection kit of the present invention will be described.
FIG. 5 is a schematic diagram (perspective view) showing a state in which the biosensor is mounted on the measuring device, FIG. 6 is a plan view schematically showing the biosensor shown in FIG. 5, and FIG. 7 is a biosensor shown in FIG. FIG. 8 is a partially enlarged view of the AA line sectional view shown in FIG.
In the following description, the front side of the paper in FIG. 6 is referred to as “up” and the back side of the paper is referred to as “down”. Further, the upper side in FIGS. 7 and 8 is referred to as “upper” and the lower side is referred to as “lower”.

First, the biosensor of FIG. 5 will be described.
A measurement apparatus (electronic device) 101 illustrated in FIG. 5 includes a biosensor 100, an arithmetic device 102 including a processing circuit 200 that analyzes a current value (oxidation reduction current) obtained by the biosensor 100, and a biosensor 100. A connector 131 to be mounted and a wiring 132 for connecting the processing circuit 200 and the connector 131 are provided.
As illustrated in FIG. 6, the biosensor 100 includes a detection unit 110 including a working electrode 121, a counter electrode 122, and a reference electrode 123 on a substrate 120.

Each of these electrodes 121, 122, and 123 is independently electrically connected to the processing circuit 200 via the wiring 130, the connector 131, and the wiring 132. The biosensor 100 is detachable at the connector 131.
Further, the range on the substrate 120 other than the detection unit 110 is covered with an insulating film 160 as shown in FIG. That is, the insulating film 160 is provided on the substrate 120, and the detection unit 110 is exposed from the opening 165 provided in a part of the insulating film 160.

  Such a biosensor 100 is provided on the working electrode 121 by supplying a liquid sample 151 to a sample supply space 150 defined by a substrate 120 and an insulating film 160, as shown in FIG. A reaction layer 140 described later and the liquid sample 151 come into contact with each other. Then, when the antigen 3 in the liquid sample 151 reacts with the reaction layer 140, the current value changes. Thereby, the amount of the antigen 3 in the liquid sample 151 can be measured based on the change in the oxidation-reduction current from the current value and the voltage.

The substrate 120 supports each part constituting the biosensor 100, and insulates the electrodes 121, 122, 123 and the wiring 130 from each other.
As a constituent material of the substrate 120, for example, various resin materials such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate (PET), polyethylene naphthalate (PES), polyimide (PI), various glass materials such as quartz glass, alumina, Various ceramic materials such as zirconia can be mentioned, and one or more of these can be used in combination.

Examples of the constituent material of the working electrode 121 include a metal material such as gold, silver, copper, platinum or an alloy containing these, a metal oxide material such as ITO, and a carbon material such as graphite. . Of these, a metal material is preferable, and gold or silver is more preferable. Since the constituent material of the working electrode 121 is gold or silver, the capture object 8 to be described later can be easily bonded via a sulfur atom, so that the biosensor 100 can be manufactured more easily.
A reaction layer 140 is provided on the upper side of the working electrode 121.

As shown in FIG. 7, a part of the reaction layer 140 is exposed to the sample supply space 150. The liquid sample 151 can be brought into contact with the reaction layer 140 by supplying the liquid sample 151 to the sample supply space 150.
The counter electrode 122 is an electrode that applies a voltage to the working electrode 121. When a voltage is applied between the working electrode 121 and the counter electrode 122 in a state where the liquid sample 151 is supplied to the sample supply space 150 so that the working electrode 121 side has a high potential, the binding between the antigen 3 and the captured substance 8 is performed. Thus, the change in the current value can be reliably captured.

Examples of the constituent material of the counter electrode 122 include the same materials as those of the working electrode 121 described above.
Further, the area of the counter electrode 122 is preferably at least twice that of the working electrode 121, and more preferably at least 10 times. Thereby, the current value can be measured with higher accuracy.

The reference electrode 123 is an electrode that applies a voltage to the counter electrode 122. A voltage is applied between the reference electrode 123 and the counter electrode 122 while the liquid sample 151 is supplied to the sample supply space 150. Then, by comparing the current value flowing between these electrodes with the current value flowing between the working electrode 121 and the counter electrode 122 described above, the current value generated by the reaction between the antigen 3 and the trap 8 is obtained. Can be measured with higher accuracy.
Examples of the constituent material of the reference electrode 123 include silver-silver chloride, mercury-mercury sulfate, and the like.

  Further, the working electrode 121, the counter electrode 122, the reference electrode 123, and the wiring 130 described above may be formed of an aggregate of conductive material powder. Thereby, these electrodes 121, 122, 123 and the wiring 130 can be easily formed by using various printing methods. As a result, the manufacturing process of the biosensor 100 can be greatly simplified, and the cost of the biosensor 100 can be reduced.

As described above, the insulating film 160 has the opening 165 opening near the detection unit 110, and the sample supply space 150 is formed by the opening 165.
Such an insulating film 160 is made of an insulating material. There is no particular limitation on the type of the insulating film 160, and either an organic material or an inorganic material can be used.
Examples of the organic material include polymer compounds such as polymethyl methacrylate, polyvinyl phenol, polyimide, polystyrene, polyvinyl alcohol, and polyvinyl acetate. These can be used alone or in combination of two or more.

Inorganic materials include metal oxides such as silicon oxide, aluminum oxide, tantalum oxide, zirconium oxide, cerium oxide, zinc oxide, and cobalt oxide, silicon nitride, aluminum nitride, zirconium nitride, cerium nitride, zinc nitride, cobalt nitride, and nitride. Examples thereof include metal nitrides such as titanium and tantalum nitride, and metal composite oxides such as barium strontium titanate and lead zirconium titanate. These can be used alone or in combination of two or more.
The average thickness of the insulating film 160 is not particularly limited, but is preferably about 10 to 5000 nm, and more preferably about 50 to 1000 nm. By setting the thickness of the insulating film 160 within the above range, the electrodes 121, 122, 123 and the wirings 130 can be reliably insulated.

In the reaction layer 140, a captured object 8 that specifically captures a detection target is supported on the surface side of the working electrode 121. The reaction layer 140 has a function of binding the captured substance 8 and the antigen 3 as a detection target.
The capture 8 is not particularly limited as long as it specifically reacts with the antigen 3, and examples thereof include an antibody. This antibody can be the same antibody as the antibody of the first compound 1.

In order to support (immobilize) the captured substance 8 on the working electrode 121, for example, an ELISA method can be used. At that time, in order to directly bind the trapped substance 8 to the surface of the working electrode 121, it is preferable to use a cross-linking agent.
Such a cross-linking agent may be any one having a functional group capable of binding to the metal surface of the working electrode 121 and a functional group capable of binding to the captured product 8.

Examples of the functional group capable of binding to the metal surface include sulfur (S), mercapto group (—SH), disulfide group (—S—S—) and the like.
Examples of groups capable of binding to the captured product 8 include amide bond forming groups such as carboxyl groups, acid halides, carboxyl group reactive derivatives such as active esters (phthalic acid imide ester, succinimide ester, etc.), ester bond forming groups, Examples include a thioester bond-forming group.

Next, a second detection method of the present invention using such a biosensor 100 will be described.
The second detection method includes a step [B1] of preparing the oxidation-reduction detection reagent 4 by reacting the first compound 1 and the second compound 2, and a step of supplying the liquid sample 151 to the sample supply space 150 [ B2], a step [B3] of supplying the redox detection reagent 4 into the liquid sample 151, and a step [B4] of detecting a change in the redox current caused by the binding between the redox detection reagent 4 and the antigen 3. Have.

[B1] Redox Detection Reagent Preparation Step First, the first compound 1 and the second compound 2 are reacted to prepare the redox detection reagent 4.
In the following description, the binding site of the redox detection reagent 4 is described as the antibody 41 and the labeling site 22 is described as ferrocene (redox substance) 42.
The redox detection reagent 4 is prepared by, for example, using the first compound 1 (compound (22)) and the second compound 2 (compound (25)) having a ferrocene 42 at the labeling site 22 in the first detection method. The reaction is carried out under the same conditions as in.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

Thereby, redox detection reagent 4 (compound (26)) was prepared.
Similar to the first detection method, a large number of ferrocenes 42 are introduced to the surface of the antibody 41 molecule via the first linking part 12 by controlling the degree of polymerization (ultraviolet irradiation time) of the redox detection reagent 4. can do.
Moreover, the oxidation-reduction detection reagent 4 (compound (27)) can also be obtained by reacting the compound (2) and the compound (7) by the same method as in the first detection method.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

  For example, as shown in the following reaction formula, the compound (28) and the compound (5) are subjected to a photopolymerization reaction in the same manner as in the first detection method, whereby the redox detection reagent 4 (compound (29)). Can also be prepared.

（式中、ｎは３〜１５を示す。）

(In the formula, n represents 3 to 15.)

For example, when gold fine particles having a diameter of about 1.5 μm are used, the surface area is about 7 × 10 ⁻¹² m ² . Therefore, assuming that surface binding molecules (PV) are immobilized at about 0.5 nm square, about 3 × 10 ⁷ oxidation-reduction substances 42 are immobilized on the surface as a monomolecular film. And in order to further enhance the amplification effect, the block is composed of one in which PV is immobilized on the monomer terminal (ester part) of the acrylate ester and one in which the redox substance 42 (ferrocene or osmium-bipyridyl complex, etc.) is immobilized. Create a polymer. Furthermore, in order to improve water solubility, a polymer site as a carboxylate is also introduced. Thereby, the oxidation-reduction detection reagent 4 can be obtained.

[B2] Liquid Sample Supply Step As shown in FIG. 8A, the liquid sample 151 is supplied to the sample supply space 150.
When the liquid sample 151 is supplied to the sample supply space 150, the reaction layer 140 and the liquid sample 151 come into contact with each other.
When the reaction layer 140 and the liquid sample 151 come into contact with each other, as shown in FIG. 8B, the trapped material 8 present on the surface of the reaction layer 140 reacts with and binds to the antigen 3 contained in the liquid sample 151.

At this time, if a voltage is applied between the working electrode 121 and the counter electrode 122 so that the working electrode 121 side is at a high potential, the current value changes due to the binding between the trap 8 and the antigen 3, The change is detected by the processing circuit 200.
However, the sensitivity may be low even if the captured substance 8 and the antigen 3 are combined, and the processing circuit 200 may not be able to sufficiently detect the change in the current value. In such a case, the redox detection reagent 4 (detection kit) of the present invention is used to increase the detection sensitivity of the antigen 3.

[B3] Redox Detection Reagent Supply Step As shown in FIG. 8C, the redox detection reagent 4 is supplied into the liquid sample 151 in which the captured product 8 and the antigen 3 are bound.
When the redox detection reagent 4 is supplied into the liquid sample 151, the antibody 41 of the redox detection reagent 4 comes into contact with the antigen 3 bound to the captured product 8.
When the antibody 41 comes into contact with the antigen 3, the antigen 3 and the antibody 41 react and bind as shown in FIG. 8 (d).
The supply amount of the redox detection reagent 4 is the same as that of the fluorescence detection reagent 5.

[B4] Antigen Detection Step When a voltage is applied between the working electrode 121 and the counter electrode 122 so that the working electrode 121 side is at a high potential, the redox substance 42 due to the binding of the antigen 3 and the antibody 41 is obtained. The oxidation-reduction current value changes due to the oxidation-reduction reaction. The antigen 3 can be detected by the processing circuit 200 detecting this change in the redox current.
Moreover, when measuring the density | concentration of the antigen 3 in the liquid sample 151, it can carry out by the method similar to a 1st detection method.

By the operation as described above, since the redox current value varies greatly depending on whether the redox detection reagent 4 is bound to the antigen 3 bound to the captured substance 8 or not, the antigen 3 in the liquid sample 151 is changed. Can be detected with high sensitivity.
When about 1000 redox substances 42 (ferrocene molecules) are present in the polymer chain, there are about 3 × 10 ¹⁰ ferrocene molecules. Therefore, the molar concentration of ferrocene molecules per molecule is 0.5 × 10 ⁻¹³ mol / L, and high sensitivity electrochemical measurement (CV measurement) enables detection per antibody molecule at the nano level. It becomes.

Further, when a small ferrocene-modified gold fine particle (10 nmφ) is separately introduced into the ferrocene (redox) polymer-modified surface by, for example, electrostatic interaction or chemical bonding, the measurement current can be further amplified.
For example, if there are about 1600 ferrocenes in a state in which the same ferrocene polymer is introduced per gold fine particle, and this can be finely immobilized on the antigen 3, for example, 5 × 10 ⁴ gold fine particles are contained in the antigen. 3 is present. The number of total ferrocene molecules immobilized is about 8 × 10 ¹¹ and about 1.2 × 10 ⁻¹² mol / L, and the single-digit detection sensitivity is further improved.
As described above, according to the antibody reaction amplification method using this functional polymer, the result of the single molecule antibody reaction can be monitored in principle, and a great effect can be expected in improving the measurement speed and sensitivity.

[3] Third Detection Method Next, a third detection method of the detection target using the detection kit of the present invention will be described.
Hereinafter, the third detection method will be described, but the description will focus on the differences from the second detection method, and the description of the same matters will be omitted.
FIG. 9 is a plan view schematically showing the biosensor, and FIG. 10 is a cross-sectional view of the biosensor shown in FIG.
In the following description, the upper side in FIG. 10 is referred to as “upper” and the lower side is referred to as “lower”.

The biosensor 100 used in the third detection method is based on the mass change of the reaction layer 140 instead of detecting the difference between the non-capture state and the capture state based on the change in the current value of the reaction layer 140. The second detection method is the same as that of the second detection method except that the second detection method is configured.
That is, in the biosensor 100 shown in FIGS. 9 and 10, each detection unit 110 is provided with a piezoelectric element 180.

The piezoelectric element 180 includes a plate-like piezoelectric body 181 and an upper electrode 182 and a lower electrode 183 provided on both sides of the piezoelectric body 181. Each of the electrodes 182 and 183 is independently electrically connected to the processing circuit 200 via the wiring 130 and the wiring 130.
Further, the substrate 120 is provided with a recess 126. The edge of the piezoelectric body 181 is fixed (adhered) to the substrate 120 so that the lower electrode 183 (and the upper electrode 182) correspond to the recess 126.

As the constituent material of each of the electrodes 182 and 183, the same material as that of the working electrode 121 can be used.
As a material of the piezoelectric body 181, for example, a piezoelectric material such as crystal, lithium niobate, lithium tantalate, and lithium borate can be used. In addition, these materials may be used independently and can be used combining 2 or more types (for example, as a laminated body).

The insulating film 160 has an opening 165, and the upper electrode 182 is exposed from the opening 165.
A reaction layer 140 is provided on the upper surface of the upper electrode 182.
The insulating film 160 and the reaction layer 140 can have the same configuration as the insulating film 160 and the reaction layer 140 described in the second detection method, respectively, and are formed in the same manner as the second detection method. Can do.

Next, a third detection method of the present invention using such a biosensor 100 will be described.
[C1] Detection reagent preparation step: The same as step B1.
[C2] Liquid sample supply step Performed in the same manner as in step B2.

[C3] Redox Detection Reagent Supply Step Performed in the same manner as step B3.
[C4] Antigen Detection Step When a voltage is applied between the working electrode 121 and the counter electrode 122 so that the working electrode 121 side is at a high potential, the antigen 3 and the antibody 41 bind to each other to cause the reaction layer 140 to The mass changes, and the frequency detected from the piezoelectric element 180 also changes. The antigen 3 can be detected by the processing circuit 200 detecting this change in frequency.
By the operation as described above, the vibration frequency changes greatly depending on whether the redox detection reagent 4 is bound to the antigen 3 bound to the captured substance 8 or not. Therefore, the amount of the antigen 3 in the liquid sample 151 Can be easily detected with high sensitivity.

[4] Fourth Detection Method Next, a fourth detection method of the detection object using the detection kit of the present invention will be described.
FIG. 11 is a longitudinal sectional view for schematically explaining the fourth detection method.
In the following description, the upper side in FIG. 11 is referred to as “upper” and the lower side is referred to as “lower”.
In this detection method, instead of supporting the captured substance 8 on the working electrode 121, the captured substance carrier 7 supporting the captured object 8 on a granular carrier 124 is used.

First, prior to the description of the detection method, the trapped substance carrier 7 will be described.
The captured substance carrier 7 has a function of capturing the antigen 3 in the liquid sample 151.
The carrier 124 of the trapped carrier 7 is formed in a spherical shape (granular shape) in the present embodiment. Thereby, since 151 in sample solution moves with little resistance, detection of antigen 3 can be performed rapidly. Further, since the surface area is increased due to the spherical shape, more trapped matter 8 can be carried on the surface of the carrier 124.
The shape of the carrier 124 is not limited to a spherical shape, and may be a prismatic shape, a cylindrical shape, or the like.
The material constituting the carrier 124 is not particularly limited, and examples thereof include the same materials as those constituting the substrate 120 and the working electrode 121 described above.

In this detection method, the antigen 3 is captured using such a captured substance carrier 7, and detection is performed. However, the trapping substance carrier 7 does not use an electrode and does not have the fluorescent material 42. For this reason, it is difficult to detect the antigen 3 even if the captured substance carrier 7 captures the antigen 3.
Therefore, the presence of the antigen 3 is detected using the color tone detection reagent 6 obtained by the detection kit of the present invention.

Next, a fourth detection method of the present invention using such a trapped substance carrier 7 will be described.
The fourth detection method includes a step [D1] of preparing the color tone detection reagent 6 by reacting the first compound 1 and the second compound 2, and a step of supplying the liquid sample 151 to the sample supply space 150 [D2]. And a step [D3] of supplying the color tone detection reagent 6 into the liquid sample 151, and a step [D4] of detecting a change in color tone caused by the binding between the color tone detection reagent 6 and the antigen 3.

[D1] Color Tone Detection Reagent Preparation Step First, the color tone detection reagent 6 is prepared by reacting the first compound 1 and the second compound 2.
In the following description, the binding site 11 of the color tone detection reagent 6 is described as the antibody 61 and the labeling site 22 is described as the dye compound 62.
The color tone detection reagent 6 is prepared, for example, by using the first compound 1 (compound (22)) and the second compound 2 (compound (30)) having the dye compound 62 at the labeling site 22 in the first detection method. The reaction is carried out under the same conditions as in.

（式中、ｎは３〜１５を示す。）
これにより、色調検出試薬６（化合物（３１））を調製した。

(In the formula, n represents 3 to 15.)
This prepared the color tone detection reagent 6 (compound (31)).

[D2] Liquid Sample Supply Step First, as shown in FIG. 11A, the liquid sample 151 is supplied to the reaction vessel 152.
Next, as shown in FIG. 11A, the trapped substance carrier 7 is supplied into the liquid sample 151 (sample supply space 150).
When the capture object carrier 7 is supplied into the liquid sample 151, the antigen 3 and the capture object 8 of the capture object carrier 7 come into contact with each other.
When the antigen 3 and the captured product 8 come into contact with each other, the captured product 8 of the captured product carrier 7 captures and binds the antigen 3 as shown in FIG.

[D3] Color Tone Detection Reagent Supply Step Next, as shown in FIG. 11C, the color tone detection reagent 6 is supplied into the liquid sample 151.
When the color tone detection reagent 6 is supplied into the liquid sample 151, the antibody 61 of the color tone detection reagent 6 comes into contact with the antigen 3 bound to the captured product 8 of the captured product carrier 7.
When the antibody 61 comes into contact with the antigen 3, the antigen 3 and the antibody 61 react and bind as shown in FIG. 11 (d).

At this time, since the color tone detection reagent 6 has the dye compound 62 at the labeling portion 22, the color tone of the antigen capture product 9 in which the color tone detection reagent 6 and the captured product carrier 7 are bound to the antigen 3 is the color tone of the dye compound 62. Discolor.
The specific gravity of the color tone detection reagent 6 with respect to the liquid sample 151 is preferably 1 g / cm ³ or less.

[D4] Antigen detection step Antigen 3 contained in the liquid sample 151 is insolubilized by binding to the captured substance carrier 7, and becomes an antigen captured substance 9 by binding to the color tone detection reagent 6, and the color tone changes. To do.
Since the antigen-captured substance 9 has a higher specific gravity with respect to the liquid sample 151, it gradually settles and agglomerates to the lower part of the liquid sample space 150 over time. Therefore, when the reaction container 152 is visually recognized from the outside, the color tone gradually moves to the lower part of the reaction container 152.

Then, as shown in FIG. 11 (e), the antigen capture product 9 reaches the bottom of the reaction container 152, and all of the antigen capture product 9 is aggregated and precipitated.
At this time, as for the color tone in the reaction container 125, the color tone of the dye compound 62 of the antigen capture product 9 is recognized at the lower end portion in the reaction container 152, and the color tone of the liquid sample 151, for example, colorless in the upper side in the reaction container 152. Transparency is recognized.

By the method as described above, since the color tone is changed by binding the antigen 3 bound to the captured substance carrier 7 to the color tone detection reagent 6, the presence of the antigen 3 in the liquid sample 151 can be detected with high sensitivity. it can.
In addition, when measuring the density | concentration of the antigen 3 in the liquid sample 151, it can carry out by the method similar to a 1st detection method.

[5] Fifth Detection Method Next, a fifth detection method for a detection target using the detection kit of the present invention will be described.
FIG. 12 is a longitudinal sectional perspective view schematically illustrating the fifth detection method.
In the following description, the upper side in FIG. 12 is referred to as “upper” and the lower side is referred to as “lower”.
Hereinafter, the fifth detection method will be described, but the description will focus on differences from the first detection method, and the description of the same matters will be omitted.
This detection method is performed using the substrate 120 having the well 125.
The substrate 120 has a plurality of wells 125. Then, the trap 8 is carried on the bottom surface of the well 125.
The antigen 3 is detected by the same method as the second detection method. However, when the well 125 does not have an electrode, it is detected by fluorescence, for example, as in the first detection method.
The detection kit of the present invention has been described above, but the present invention is not limited to this.
For example, in the detection kit of the present invention, the configuration of each part can be replaced with any configuration that exhibits the same function, and any configuration can be added.

  DESCRIPTION OF SYMBOLS 1 ... 1st compound 11 ... Binding site 12 ... 1st reaction site 13 ... 1st connection site 13a ... Particle 2 ... 2nd compound 21 ... 2nd reaction site 22 ... Labeling part 23 …… Second linking part 3 …… Antigen 4 …… Redox detection reagent 41 …… Antibody 42 …… Redox substance 5 …… Fluorescence detection reagent 51 …… Antibody 52 …… Fluorescent substance 6 …… Color tone Detection reagent 61 …… Antibody 62 …… Dye compound 7 …… Captured carrier 8 …… Captured product 9 …… Antigen captured product 100 …… Biosensor 101 …… Measuring device 102 …… Arithmetic device 110 …… Detection unit 120 …… ... Substrate 121 ... Working electrode 122 ... Counter electrode 123 ... Reference electrode 124 ... Carrier 125 ... Well 126 ... Recess 130 ... Wiring 131 ... Connector 132 ... Wiring 140 ... Layer 150 ... Sample supply space 151 ... Liquid sample 152 ... Reaction vessel 160 ... Insulating film 165 ... Opening 180 ... Piezoelectric element 181 ... Piezoelectric element 182 ... Upper electrode 183 ... Lower electrode 190 ... ... Base 200 ... Processing circuit

Claims (8)

A detection kit capable of selectively detecting a detection object,
Between the binding site that specifically binds to the detection target, a reactive first reaction site, and the binding site and the first reaction site, the binding site and the first reaction site. A first compound comprising a first linking site linking
A second reaction site that reacts with the first reaction site and a second compound comprising a labeling site;
The first linking site includes a linear polyethylene glycol chain,
The first reaction site and the second reaction site are each composed of a vinyl group,
A plurality of second reaction sites are connected to one first reaction site by reacting one first compound with a plurality of second compounds. Detection kit.   The detection kit according to claim 1, wherein the binding site is an antigen or an antibody.   The detection kit according to claim 1 or 2, wherein the labeling site comprises at least one of a redox substance, a fluorescent substance, and a dye compound.   The detection kit according to any one of claims 1 to 3, comprising a plurality of the labeling sites.   5. The method according to claim 1, wherein the second compound has a second linking site for linking the second reaction site and the labeling site between the second reaction site and the labeling site. A detection kit according to claim 1.   The detection kit according to any one of claims 1 to 5, further comprising a substrate on which a captured substance capable of selectively capturing an object to be detected is supported. The substrate comprises a well;
The detection kit according to claim 6, wherein the captured substance is carried on the inner surface of the well.   The detection kit according to claim 6, wherein the substrate is formed in a granular shape.

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