JP2022119414A - Detection method, detection substrate, and detection kit - Google Patents

Abstract

To provide a detection method that can easily and efficiently detect a detection object in a sample.SOLUTION: A detection method of detecting a detection object in a sample includes the following steps (A) to (F). (A) is a detection substrate preparation step of preparing a detection substrate including a detection surface, and a first bonding body anchored to the detection surface and specifically bonded to the detection object. (B) is a sample supply step of supplying the sample to the detection surface of the detection substrate. (C) is a second bonding body supply step of supplying a second bonding body specifically boded to the detection object and including a polymerization initiator, to the detection surface of the detection substrate. (D) is a removal step of removing the non-anchored second bonding body from the detection substrate. (E) is a monomer supply step of supplying a monomer to be polymerized by the polymerization initiator to the detection surface of the detection substrate. (F) is a change confirmation step of confirming a change of the detection surface before and after the monomer supply step.SELECTED DRAWING: Figure 1

Description

The present invention relates to detection methods, detection substrates, and detection kits.

In recent years, viral infectious diseases such as human and bird influenza and severe acute respiratory syndrome (SARS) have occurred, and recently, infectious disease (COVID-19) caused by the new coronavirus (SARS-CoV-2) has occurred. It has spread worldwide (pandemic) and has become an extremely serious problem for mankind. For this reason, the importance of virus detection is increasing in order to prevent the spread of viral infections. Patent Document 1 discloses a technique for detecting and quantifying a test substance by oxidation-reduction polymerization.

JP-A-2-259567

Conventional methods for detecting objects to be detected (viruses, etc.) require quantitative manipulation techniques using a pipetman or the like, or use reagents such as enzymes that are difficult to handle. For this reason, a specific technician is required to detect the object to be detected, which poses a problem of poor detection efficiency.

Accordingly, an object of the present invention is to provide a detection method, a detection substrate, and a detection kit that can easily and efficiently detect a detection target in a sample.

In order to achieve the above object, the detection method of the present invention comprises
A detection method for detecting a detection target in a sample,
A method comprising the following steps (A) to (F).
(A) A detection substrate preparation step of preparing a detection substrate including a detection surface and a first binding body immobilized on the detection surface and specifically binding to the detection target.
(B) a sample supply step of supplying the sample to the detection surface of the detection substrate;
(C) a second conjugate supplying step of supplying a second conjugate that specifically binds to the substance to be detected and contains a polymerization initiator to the detection surface of the detection substrate;
(D) a removing step of removing the non-immobilized second binder from the detection substrate;
(E) a monomer supply step of supplying a monomer to be polymerized by the polymerization initiator onto the detection surface of the detection substrate;
(F) a change confirmation step of confirming a change in the detection surface before and after the monomer supply step;

The detection substrate of the present invention is
A detection substrate capable of detecting a detection target in a sample,
including a base layer and a protective film layer;
at least one side of the base layer is a sensing surface;
the detection surface immobilizes a first binding body that specifically binds to the detection target;
The protective film layer is releasably laminated on the sensing surface.

The detection kit of the present invention is
comprising a detection substrate of the present invention, a conjugate-containing solution containing a second conjugate, and a monomer-containing solution containing a monomer;
The second conjugate is a conjugate that specifically binds to the substance to be detected and contains a polymerization initiator,
The monomer is a monomer that is polymerized by the polymerization initiator.

According to the present invention, it is possible to easily and efficiently detect a substance to be detected in a sample because advanced techniques such as quantitative manipulation and reagents such as enzymes are not required. In particular, by not using enzymes, chemical reaction steps can be easily performed. Moreover, according to the present invention, since an expensive device such as a thermal cycler is not required, the detection target can be detected at low cost. Furthermore, according to the detection substrate of the present invention, since it is excellent in portability, the detection target can be detected regardless of the location.

FIG. 1 is a flow chart showing an example of the detection method of the present invention. FIG. 2 is an explanatory diagram showing an example of detection of a detection target using the detection method of the present invention. FIG. 3 is a diagram showing the configuration of an example of the detection substrate of the present invention. FIG. 4 is an explanatory diagram showing an example of how to use the detection kit of the present invention.

In the detection method of the present invention, for example,
The detection surface is a hologram surface that has been finely textured,
In the color tone change confirmation step of the step (F), the color tone change of the detection surface before and after the monomer supply step may be confirmed.

In the detection method of the present invention, for example,
The (E) monomer supply step may further include supplying a polymerization reaction accelerator.

In the detection method of the present invention, for example,
the sensing surface comprises a plurality of compartments;
The plurality of compartments includes a control compartment,
The first conjugate is not immobilized in the control plot,
The first conjugate is immobilized in other sections other than the control section,
In the change confirmation step of the step (F), changes in the control section and the other sections before and after the monomer supply step may be confirmed.

In the detection method of the present invention, for example,
the first conjugate is an ACE2 (Angiotensin-converting enzyme 2) receptor or a part thereof;
In another aspect, the second conjugate is an ACE2 receptor or a portion thereof that includes a polymerization initiator.
In the above aspect, for example, the detection target may be SARS-CoV-2.

In the detection substrate of the present invention, for example,
The detection surface may be a hologram surface on which fine concavo-convex processing is applied.

In the detection substrate of the present invention, for example,
the sensing surface comprises a plurality of compartments;
A different first conjugate is fixed for each of the plurality of compartments,
The first conjugates may be conjugates that specifically bind to different detection targets.

In the detection substrate of the present invention, for example,
the sensing surface comprises a plurality of compartments;
The plurality of compartments includes a control compartment,
The first binding body may not be immobilized on the control section.

In the detection substrate of the present invention, for example,
The detection substrate from which the protective film layer has been peeled off may be the detection substrate used in the detection method of the present invention.

In the detection substrate of the present invention, for example,
The base layer may be either a film or a sheet.

In the detection kit of the present invention, for example,
The monomer-containing solution may further include a polymerization reaction accelerator.

In the present invention, the "sample" is not particularly limited, and may be in the form of liquid, for example. The samples are obtained from, for example, living organisms such as animals including humans and plants, environments such as air, seawater, and soil, wastewater after cleaning, sewage, treated sewage, and the like. Specifically, the sample includes, for example, saliva, nasal discharge, blood, fecal suspension, sewage, treated sewage water, and diluted solutions thereof.

In the present invention, the "object to be detected" is not particularly limited, and includes, for example, viruses, proteins, bacteria, fungi, microorganisms, products produced from these, and the like. Specifically, the detection target includes, for example: coronaviruses (for example, SARS-CoV which is the causative virus of SARS, SARS-CoV-2 which is the causative virus of COVID-19, MERS which is the causative virus of MERS -CoV), influenza virus, norovirus, hepatitis B virus, hepatitis C virus, mycoplasma, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, ESBL-producing bacteria, Neisseria gonorrhoeae, Mycobacterium tuberculosis, Streptococcus hemolyticus, Clostridium difficile , enterobacteria, enterococci, acinetobacter, pseudomonas aeruginosa, staphylococcus aureus, campylobacter, candida, non-typhoid salmonella, typhi, shigella, group A streptococci, group B streptococci, nontuberculous mycobacteria, Spirochetes, fungi, and their products, and the like; proteins such as B cells, T cells, and antibodies in a sample;

Hereinafter, the present invention will be specifically described using examples.

(Detection method)
An example of the detection method of the present invention will be described based on the flowchart of FIG. The detection method of the present invention ends (END) after performing the following (A) to (F).

(A) Detection Substrate Preparing Step First, a detection substrate including a detection surface and a first binder that is immobilized on the detection surface and specifically binds to the detection target is prepared (S11). The detection substrate is not particularly limited as long as it includes the detection surface and the first binding body. The detection substrate is, for example, the detection substrate of the present invention, which will be described later, and the description of the detection substrate of the present invention can be used. The first conjugate is not particularly limited as long as it specifically binds to the detection target, and is, for example, an antibody. Specifically, when the object to be detected is SARS-CoV-2, the first binding body includes ACE2 (Angiotensin-converting enzyme 2) receptor or a portion thereof. Further, when the detection target is Sendai virus (SeV), the first conjugate includes an anti-SeV antibody, and the detection target is Norovirus (NoV). when the first conjugate includes an anti-NoV antibody, and when the detection target is an influenza virus (Influenza virus: IFV), the first conjugate includes an anti-IFV antibody. be done.

(B) Sample supply step Next, the sample is supplied to the detection surface of the detection substrate (S12). Specifically, the sample supply step is, for example, a step of bringing the sample into direct contact with the detection surface. The amount of the sample to be supplied is not particularly limited, and is, for example, 0.5 ml, 1 ml, 1.5 ml, 3 ml, and the like. In addition, the time for which the sample is continuously supplied to the detection surface (contact time) can be appropriately adjusted depending on the reagent and the first binding body. For example, 5 minutes. In the detection method of the present invention, for example, the detection surface of the detection substrate is washed with a washing liquid (eg, water, etc.) after the sample supply step and before the start of the second conjugate supply step, which will be described later. May be washed.

(C) Second conjugate supply step Next, a second conjugate that specifically binds to the detection target and contains a polymerization initiator is supplied to the detection surface of the detection substrate (S13 ). Specifically, the step of providing a second conjugate is, for example, a step of contacting the detection surface with the second conjugate. The second conjugate may be of one type or two or more types. The amount of the second conjugate to be supplied is not particularly limited, and is, for example, 0.5 ml, 1 ml, 1.5 ml, 3 ml, and the like. In addition, the time (contact time) during which the second conjugate is continuously supplied to the detection surface can be appropriately adjusted depending on the substance to be detected and the second conjugate. 5 minutes, 3 minutes, 5 minutes, and so on. The second conjugate supply step (C) may be performed, for example, after the sample supply step (B), or may be performed in parallel with the sample supply step (B). The second conjugate is not particularly limited as long as it specifically binds to the substance to be detected and contains a polymerization initiator, and is, for example, an antibody containing a polymerization initiator. The second conjugate containing the polymerization initiator may be present, for example, in a solvent. Specifically, when the substance to be detected is SARS-CoV-2, the second binding body includes an ACE2 (Angiotensin-converting enzyme 2) receptor containing a polymerization initiator or a portion thereof. Further, when the detection target is Sendai virus (SeV), the second conjugate includes an anti-SeV antibody containing a polymerization initiator, and the detection target is Norovirus (Norovirus : NoV), the second conjugate includes an anti-NoV antibody containing a polymerization initiator, and the detection target is an influenza virus (Influenza virus: IFV), the second Conjugates of include anti-IFV antibodies containing polymerization initiators. The polymerization initiator is not particularly limited, and examples thereof include initiators (radical polymerization initiators) that generate radicals by photopolymerization, heat, oxidation-reduction (redox), and the like. Types of radical polymerization initiators include, for example, azo initiators, peroxide initiators, alkylphenone radical polymerization initiators, acylphosphine oxide radical polymerization initiators, oxime ester radical polymerization initiators, persulfate initiators, and the like. There is

Specifically, the polymerization initiator includes, for example, Azobis(isobutyronitrile) (AIBN), ammonium persulfate (APS), benzoyl peroxide, benzenesulfonate, alkylsulfonium salts, dicyandiamide, 2,2 '-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(isobutyronitrile), 2,2' -azobis(2-methylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, 2,2'-azobis[2-(-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2, 2′-azobis(2-methylpropionamidine dihydrochloride, 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, 2,2′-azobis[2- methyl-N-(2-hydroxyethyl)propionamide], 2,2′-azobis[N-(2-propenyl)-2-methylpropionamide], 2,2′-azobis(N-butyl-2-methyl propionamide), 2,2′-azobis(isobutyrate)dimethyl, 4,4′-azobis(4-cyanovaleric acid), 2,2′-dimethoxy-1,2-diphenylethan-1-one, 2- Hydroxy-2-methyl-1-phenylpropan-1-one, 1-hydroxycyclohexylphenyl ketone, 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propane-1- one, 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methylpropan-1-one, 2-methyl-1-(4-methylthiophenyl) -2-morpholinopropan-1-one, 2-benzylmethyl 2-dimethylamino-1-(4-morpholinophenyl)-1-butanone, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, bis(2 ,4,6-trimethylbenzoyl)-phenylphosphine oxide, (2E)-2-(benzoyloxyimino)-1-[4-(phenylthio)phenyl]octan-1-one, derivatives thereof, etc. is mentioned. However, the polymerization initiator is not limited to these. The polymerization initiator may have, for example, a functional group capable of reacting with protein (eg, 4,4'-Azobis-4-cyanovaleric acid, etc.).

In the present invention, oxidation-reduction (redox) is not particularly limited, and may be aqueous redox, non-aqueous redox, or the like. Aqueous redoxes include, for example, combinations of peroxides, such as persulfates, hydrogen peroxide, and hydroperoxides, and reducing agents, such as divalent iron salts, monovalent copper salts, and amines. Examples of non-aqueous redox include combinations of oxidizing agents such as hydroperoxides, dialkyl peroxides, and diacyl peroxides, and reducing agents such as tertiary amines, naphthenates, thiols, and organometallic compounds. be.

(D) Removal Step Next, the non-immobilized second conjugate is removed from the detection substrate (S14). Specifically, for example, the non-immobilized second binding body is removed by washing with a washing solution (eg, water, etc.). Here, in the present invention, the “non-immobilized second conjugate” means, for example, the second conjugate that is not immobilized on the detection surface via the detection target and the first conjugate. means a combination of On the other hand, in the present invention, for example, by hydrogen bonding or the like, the second conjugate in a state of being immobilized on the detection surface is referred to as an "immobilized second conjugate" via the detection target and the first conjugate. It is also called "combination of 2".

(E) Monomer Supply Step Next, a monomer that is polymerized by the polymerization initiator is supplied to the detection surface of the detection substrate (S15). The monomer is not particularly limited as long as it is polymerized by the polymerization initiator, and is, for example, a water-soluble compound. Specific examples include, but are not limited to, acrylamide, dimethylacrylamide, 2-hydroxyethyl methacrylate (HEMA), and the like. One type of the monomer may be used, or two or more types may be used. Specifically, the monomer supply step is, for example, a step of contacting the detection surface with the monomer. The amount of the monomer to be supplied is not particularly limited, and is, for example, a concentration generally used in polymer polymerization (eg, 0.5 mol/L, etc.). In addition, the time during which the monomer is continuously supplied to the detection surface (contact time) can be appropriately adjusted depending on the second conjugate and the monomer. The monomer supply step may, for example, further supply a polymerization reaction accelerator. The polymerization reaction accelerator is not particularly limited, and examples thereof include amines, sulfinic acid and its salts, borate compounds, barbituric acid derivatives, triazine compounds, copper compounds, tin compounds, vanadium compounds, halogen compounds, aldehydes, thiols. compounds, hydroperoxides, metal salts, and the like. However, it is not limited to these. The monomer and the polymerization accelerator may be present, for example, in a solvent.

Specifically, examples of the amines include aliphatic amines and aromatic amines. Examples of the aliphatic amine include primary aliphatic amines such as n-butylamine, n-hexylamine and n-octylamine; secondary aliphatic amines such as diisopropylamine, dibutylamine and N-methyldiethanolamine; N-methylethanolamine, N-ethyldiethanolamine, Nn-butyldiethanolamine, N-lauryldiethanolamine, 2-(dimethylamino)ethyl methacrylate, N-methyldiethanolamine dimethacrylate, N-ethyldiethanolamine dimethacrylate, triethanolamine mono tertiary aliphatic amines such as methacrylate, triethanolamine dimethacrylate, triethanolamine trimethacrylate, triethanolamine, trimethylamine, triethylamine and tributylamine;

Examples of the aromatic amine include N,N-bis(2-hydroxyethyl)-3,5-dimethylaniline, N,N-di(2-hydroxyethyl)-p-toluidine, N,N-bis( 2-hydroxyethyl)-3,4-dimethylaniline, N,N-bis(2-hydroxyethyl)-4-ethylaniline, N,N-bis(2-hydroxyethyl)-4-isopropylaniline, N,N -bis(2-hydroxyethyl)-4-t-butylaniline, N,N-bis(2-hydroxyethyl)-3,5-di-isopropylaniline, N,N-bis(2-hydroxyethyl)-3 ,5-di-t-butylaniline, N,N-dimethylaniline, N,N-dimethyl-p-toluidine, N,N-dimethyl-m-toluidine, N,N-diethyl-p-toluidine, N,N -dimethyl-3,5-dimethylaniline, N,N-dimethyl-3,4-dimethylaniline, N,N-dimethyl-4-ethylaniline, N,N-dimethyl-4-isopropylaniline, N,N-dimethyl -4-t-butylaniline, N,N-dimethyl-3,5-di-t-butylaniline, 4-N,N-dimethylaminobenzoic acid ethyl ester, 4-N,N-dimethylaminobenzoic acid methyl ester , N,N-dimethylaminobenzoic acid-n-butoxyethyl ester, 4-N,N-dimethylaminobenzoic acid-2-(methacryloyloxy)ethyl ester, 4-N,N-dimethylaminobenzophenone, 4-dimethylamino butyl benzoate, N,N,N',N'-tetra(TEMED)methylethyldiamine and the like.

Examples of the sulfinic acid and salts thereof include p-toluenesulfinic acid, sodium p-toluenesulfinate, potassium p-toluenesulfinate, lithium p-toluenesulfinate, calcium p-toluenesulfinate, benzenesulfinic acid, and benzene. sodium sulfinate, potassium benzenesulfinate, lithium benzenesulfinate, calcium benzenesulfinate, 2,4,6-trimethylbenzenesulfinic acid, sodium 2,4,6-trimethylbenzenesulfinate, 2,4,6-trimethylbenzene potassium sulfinate, lithium 2,4,6-trimethylbenzenesulfinate, calcium 2,4,6-trimethylbenzenesulfinate, 2,4,6-triethylbenzenesulfinic acid, sodium 2,4,6-triethylbenzenesulfinate , 2,4,6-triethylbenzenesulfinate potassium, 2,4,6-triethylbenzenesulfinate lithium, 2,4,6-triethylbenzenesulfinate calcium, 2,4,6-triisopropylbenzenesulfinate, 2 , Sodium 4,6-triisopropylbenzenesulfinate, Potassium 2,4,6-triisopropylbenzenesulfinate, Lithium 2,4,6-triisopropylbenzenesulfinate, Calcium 2,4,6-triisopropylbenzenesulfinate etc.

Examples of the borate compounds include trialkylphenylboron, trialkyl(p-chlorophenyl)boron, trialkyl(p-fluorophenyl)boron, trialkyl(3,5-bistrifluoromethyl)phenylboron, dialkyldiphenylboron, Dialkyldi(p-chlorophenyl)boron, dialkyldi(p-fluorophenyl)boron, dialkyldi(3,5-bistrifluoromethyl)phenylboron, monoalkyltriphenylboron, monoalkyltri(p-chlorophenyl)boron, monoalkyltri( p-fluorophenyl)boron, monoalkyltri(3,5-bistrifluoromethyl)phenylboron, tetraphenylboron, tetrakis(p-chlorophenyl)boron, tetrakis(p-fluorophenyl)boron, tetrakis(3,5-bistri fluoromethyl)phenyl boron, and the like.

Examples of the barbituric acid derivatives include barbituric acid, 1,3-dimethylbarbituric acid, 1,3-diphenylbarbituric acid, 1,5-dimethylbarbituric acid, 5-butylbarbituric acid, 5-ethyl Barbituric acid, 5-isopropyl barbituric acid, 5-cyclohexyl barbituric acid, 1,3,5-trimethylbarbituric acid, 1,3-dimethyl-5-ethylbarbituric acid, 1,3-dimethyl-n- Butyl barbituric acid, 1,3-dimethyl-5-isobutylbarbituric acid, 1,3-dimethylbarbituric acid, 1,3-dimethyl-5-cyclopentylbarbituric acid, 1,3-dimethyl-5-cyclohexylbarbituric acid turic acid, 1,3-dimethyl-5-phenylbarbituric acid, 1-cyclohexyl-1-ethylbarbituric acid, 1-benzyl-5-phenylbarbituric acid, 5-methylbarbituric acid, 5-propylbarbituric acid acid, 1,5-diethylbarbituric acid, 1-ethyl-5-methylbarbituric acid, 1-ethyl-5-isobutylbarbituric acid, 1,3-diethyl-5-butylbarbituric acid, 1-cyclohexyl- 5-methylbarbituric acid, 1-cyclohexyl-5-ethylbarbituric acid, 1-cyclohexyl-5-octylbarbituric acid, 1-cyclohexyl-5-hexylbarbituric acid, 5-butyl-1-cyclohexylbarbituric acid , 1-benzyl-5-phenylbarbituric acid and thiobarbituric acid, and salts thereof (especially alkali metal or alkaline earth metals are preferred).

Examples of the triazine compound include 2,4,6-tris(trichloromethyl)-s-triazine, 2,4,6-tris(tribromomethyl)-s-triazine, 2-methyl-4,6-bis (trichloromethyl)-s-triazine, 2-methyl-4,6-bis(tribromomethyl)-s-triazine, 2-phenyl-4,6-bis(trichloromethyl)-s-triazine, 2-(p -methoxyphenyl)-4,6-bis(trichloromethyl)-s-triazine, 2-(p-methylthiophenyl)-4,6-bis(trichloromethyl)-s-triazine, 2-(p-chlorophenyl)- 4,6-bis(trichloromethyl)-s-triazine, 2-(2,4-dichlorophenyl)-4,6-bis(trichloromethyl)-s-triazine, 2-(p-bromophenyl)-4,6 -bis(trichloromethyl)-s-triazine, 2-(p-tolyl)-4,6-bis(trichloromethyl)-s-triazine, 2-n-propyl-4,6-bis(trichloromethyl)-s -triazine, 2-(α,α,β-trichloroethyl)-4,6-bis(trichloromethyl)-s-triazine, 2-styryl-4,6-bis(trichloromethyl)-s-triazine, 2- [2-(p-methoxyphenyl)ethenyl]-4,6-bis(trichloromethyl)-s-triazine, 2-[2-(o-methoxyphenyl)ethenyl]-4,6-bis(trichloromethyl)- s-triazine, 2-[2-(p-butoxyphenyl)ethenyl]-4,6-bis(trichloromethyl)-s-triazine, 2-[2-(3,4-dimethoxyphenyl)ethenyl]-4, 6-bis(trichloromethyl)-s-triazine, 2-[2-(3,4,5-trimethoxyphenyl)ethenyl]-4,6-bis(trichloromethyl)-s-triazine, 2-(1- naphthyl)-4,6-bis(trichloromethyl)-s-triazine, 2-(4-biphenylyl)-4,6-bis(trichloromethyl)-s-triazine, 2-[2-{N,N-bis (2-hydroxyethyl)amino}ethoxy]-4,6-bis(trichloromethyl)-s-triazine, 2-[2-{N-hydroxyethyl-N-ethylamino}ethoxy]-4,6-bis( trichloromethyl)-s-triazine, 2-[2-{N-hydroxyethyl-N-methylamino}ethoxy]-4,6-bis(trichloromethyl n)-s-triazine, 2-[2-{N,N-diallylamino}ethoxy]-4,6-bis(trichloromethyl)-s-triazine and the like.

Examples of the copper compound include copper acetylacetone, cupric acetate, copper oleate, cupric chloride, and cupric bromide.

Examples of the tin compound include di-n-butyltin dimaleate, di-n-octyltin dimalate, di-n-octyltin dilaurate, and di-n-butyltin dilaurate.

Examples of the vanadium compounds include divanadium tetroxide (IV), vanadium oxide acetylacetonate (IV), vanadyl oxalate (IV), vanadyl sulfate (IV), oxobis(1-phenyl-1,3-butanedio phosphate) vanadium (IV), bis(maltrate) oxovanadium (IV), vanadium pentoxide (V), sodium metavanadate (V), ammonium metavanadate (V), and the like.

Examples of the halogen compound include dilauryldimethylammonium chloride, lauryldimethylbenzylammonium chloride, benzyltrimethylammonium chloride, tetramethylammonium chloride, benzyldimethylcetylammonium chloride, and dilauryldimethylammonium bromide.

Examples of the aldehydes include terephthalaldehyde and benzaldehyde derivatives. Benzaldehyde derivatives include dimethylaminobenzaldehyde, p-methyloxybenzaldehyde, p-ethyloxybenzaldehyde, pn-octyloxybenzaldehyde and the like.

Examples of the thiol compound include 3-mercaptopropyltrimethoxysilane, 2-mercaptobenzoxazole, decanethiol, thiobenzoic acid and the like.

Examples of the hydroperoxide include t-butyl hydroperoxide (TBH), cumyl hydroperoxide, p-isopropyl cumyl hydroperoxide, methyl ethyl ketone hydroperoxide, 1,1,3,3-tetramethylbutyl Hydroperoxide, cyclohexane hydroperoxide, mixtures thereof, and the like.

Examples of the metal salt include cobalt naphthenate, cobalt octoate (2-ethyl caproate), cobalt acetoacetonate, copper naphthenate, iron naphthenate, iron octoate (2-ethyl caproate), and mixtures thereof. mentioned.

(F) Change Confirmation Step A change in the detection surface before and after the monomer supply step is confirmed (S16). When the detection surface is the hologram surface, the change confirmation step confirms a change in color tone of the detection surface before and after the monomer supply step, for example. Moreover, when the detection surface includes a plurality of sections, the change confirmation step confirms changes in the control section and the other sections before and after the monomer supply step, for example. In the change confirmation step, for example, the change is confirmed by directly viewing the detection surface with the naked eye. By confirming the change in the detection surface, the detection object in the sample can be detected.

In the detection method of the present invention, for example, after the (E) monomer supply step described later and before the (F) change confirmation step, a film (for example, a protective film in the detection substrate of the present invention) is pressed onto the detection surface by pressure bonding. By laminating the film layer 12), even if the amount of the sample, etc. supplied onto the detection surface is small (1 ml, etc.), the sample, etc. can be supplied over a wide area of the detection surface (or a section described later). can do.

A case where the detection target is SARS-CoV-2 will be described below as an example with reference to FIG. FIG. 2 shows an example of detection of a detection target. In FIG. 2 the detection surface is shown as a holographic surface. First, in the detection substrate preparation step (A), a detection substrate in which the ACE2 receptor or a part thereof (first binding body) is immobilized on the surface of the hologram is prepared (FIG. 2(A)). The detection substrate may be blocked with, for example, BSA (Bovine serum albumin) or the like, except for the portion where the ACE2 receptor or a portion thereof is immobilized. Next, when SARS-CoV-2 is present in the sample, the ACE2 receptor immobilized on the hologram surface or a portion thereof (first binding body ) and the SARS-CoV-2 spike protein (Fig. 2(B)). Next, in the second conjugate supply step (C), the spike protein of SARS-CoV-2 and the polymerization initiator-bound ACE2 receptor or a portion thereof (second conjugate) bound to the polymerization initiator A hydrogen bond or the like occurs between (Fig. 2(C)). Thereafter, in the monomer supply step (E), a polymerization reaction between the immobilized polymerization initiator-bound ACE2 receptor or a portion thereof (second conjugate) and the monomer occurs, as shown in FIG. 2(D). Then, a polymer is formed on the hologram surface. Then, in the change confirmation step (F), a visual change of the hologram surface caused by the polymer is confirmed. Specifically, since the color emitted from the hologram surface changes only in the portion where the polymer is formed, the change in color is confirmed in the change confirmation step (F). SARS-CoV-2 has a different spike protein than other coronaviruses (such as SARS-CoV). Therefore, in the present invention, by using a conjugate that specifically binds to the spike protein as the first conjugate and the second conjugate, the accuracy of detection of SARS-CoV-2 is improved. Note that this is an example, and the detection method of the present invention is not limited to the detection of SARS-CoV-2.

(Detection board)
FIG. 3A shows an example of a cross-sectional view of the detection substrate of the present invention. The sensing substrate 10 of the present invention includes a base layer 11 and a protective film layer 12, as shown in FIG.

The size of the detection substrate 10 is not particularly limited, and is, for example, a portable size.

The detection substrate 10 may be, for example, a flat type such as a film, a sheet, or a card as shown in FIG. 3, or may be, for example, a stick type. In the case of the stick type, for example, the second conjugate containing the polymerization initiator, the monomer to be polymerized by the polymerization initiator, the cleaning liquid, etc. are placed in a syringe, and the syringe can be incorporated in the detection substrate 10 together. . The tip of the syringe is arranged, for example, toward a detection surface of a base layer 11, which will be described later. Since the detection substrate 10 is of a stick type in this way, for example, the reaction can proceed on the base layer 11 without taking time and effort.

At least one surface of the base layer 11 is a detection surface, on which a first binder that specifically binds to the detection target is immobilized. The first conjugate is the same as described above. A technique for fixing the first conjugate is not particularly limited, and for example, it is fixed by hydrogen bonding or the like. The detection surface may be, for example, colored and changeable in color. Specifically, as shown in the figure, it may be a holographic surface that is finely textured. . Base layer 11 may be in the form of, for example, a film, sheet, card, or the like. The detection substrate may be blocked with, for example, BSA (Bovine serum albumin) or the like, except for the portion where the first conjugate is immobilized.

FIG. 3B shows an example of the detection substrate of the present invention viewed from the detection surface side. Said detection surface may, for example, comprise a plurality of compartments. In the figure, the divisions are indicated by "+", "-", "A" to "D". When the plurality of compartments are included, for example, a different first binding body may be fixed to each of the plurality of compartments. The different first conjugates referred to here are the same as those described above, except that they are conjugates that specifically bind to different detection targets. In addition, when the plurality of compartments are included, a control area may be included in which the first conjugate is not immobilized. A first conjugate is immobilized in sections other than the control section. Examples of the control section include a positive control section (“+” in the figure) that serves as a positive control for the reaction (polymerization reaction described later) in the other section, and a negative control section that serves as a negative control (“ -"), etc. In the other compartments, for example, different first binders are immobilized on the detection surface, and the first binders may be binders that specifically bind to different detection targets. good. Furthermore, as shown in FIG. 3B, the detection surface may have a different pattern for each section. Thus, when the detection surface includes a plurality of sections, one detection substrate 10 can be used to detect multiple types of detection targets at once.

A protective film layer 12 is releasably laminated on the sensing surface. A method for laminating the protective film layer 12 on the base layer 11 is not particularly limited, and a known method can be used. The protective film layer 12 may be transparent, for example, so that the detection surface can be visually recognized when the detection substrate 10 is viewed from the laminated protective film layer 12 side. The detection substrate 10 from which the protective film layer 12 has been removed is, for example, the detection substrate used in the detection method of the present invention described above.

(detection kit)
The detection kit of the present invention includes the aforementioned detection substrate 10 of the present invention, a conjugate-containing solution containing a second conjugate, and a monomer-containing solution containing a monomer. The kit of the present invention may be packaged together, for example. The second conjugate and the monomer are the same as described above. The monomer-containing solution may, for example, further contain a polymerization reaction accelerator. The polymerization reaction accelerator is the same as described above.

(How to use the detection kit)
A method for using the detection kit of the present invention will be described with reference to FIG. The detection surface of the detection substrate 10 (base layer 11) shown in FIG. It is Hereinafter, the division is also referred to as a character surface. Then, in FIG. 4, it is assumed that, for example, the first binding body that specifically binds to the detection target is immobilized on the letter face as shown below.
A: Influenza virus type A B: Influenza virus type B C: Rhinovirus D: Human cold coronavirus E: SARS-CoV-2 type S type F: SARS-CoV-2 type K G: SARS-CoV-2 type G H : SARS-CoV-2 Spain variant In addition, the character face "+" is a positive control plot, and instead of the first conjugate, the second conjugate containing the polymerization initiator bond is used on the detection surface Suppose that it is a ward that is fixed above. Furthermore, the character face "-" is a negative control plot, in which neither the first binder nor the second binder is immobilized on the detection surface, and the non-specific one and the second conjugate are masked so as not to bind together.

First, the film layer 12 is peeled off from the base layer 11 in the detection substrate 10 . Next, the sample is brought into contact with the detection surface of the detection substrate 10 (base layer 11), particularly the character surface. Next, the detection substrate 10 (base layer 11) is washed with tap water or the like and drained. Next, the detection surface of the detection substrate 10 (base layer 11), particularly the character surface, is brought into contact with the conjugate-containing solution. Next, the detection substrate 10 (base layer 11) is washed with tap water or the like, drained, and the detection surface of the detection substrate 10 (base layer 11), particularly the character surface, is brought into contact with the monomer-containing solution. Then, the detection target present in the sample is detected and determined from the change in the color of the character face. Suppose that the colors of "+" and "D" are changed in the character face shown in FIG. In this case, it can be determined to be positive for human cold coronavirus and negative for other viruses.

Although the present invention has been described with reference to the embodiments, the present invention is not limited to the above embodiments. Various changes can be made to the configuration and details of the present invention within the scope of the present invention that can be understood by those skilled in the art.

INDUSTRIAL APPLICABILITY According to the present invention, an object to be detected in a sample can be detected easily and efficiently, and it is useful, for example, for prevention and countermeasures against infectious diseases.

10 detection substrate 11 base layer 12 film layer

Claims (14)

A detection method for detecting a detection target in a sample,
A detection method comprising the following steps (A) to (F).
(A) A detection substrate preparation step of preparing a detection substrate including a detection surface and a first binding body immobilized on the detection surface and specifically binding to the detection target.
(B) a sample supply step of supplying the sample to the detection surface of the detection substrate;
(C) a second conjugate supplying step of supplying a second conjugate that specifically binds to the substance to be detected and contains a polymerization initiator to the detection surface of the detection substrate;
(D) a removing step of removing the non-immobilized second binder from the detection substrate;
(E) a monomer supply step of supplying a monomer to be polymerized by the polymerization initiator onto the detection surface of the detection substrate;
(F) a change confirmation step of confirming a change in the detection surface before and after the monomer supply step; The detection surface is a hologram surface that has been finely textured,
2. The detection method according to claim 1, wherein in the step (F) of confirming a color tone change, a color tone change of the detection surface before and after the monomer supply step is confirmed. 3. The detection method according to claim 1, wherein said (E) monomer supply step further supplies a polymerization reaction accelerator. the sensing surface comprises a plurality of compartments;
The plurality of compartments includes a control compartment,
The first conjugate is not immobilized in the control plot,
The first conjugate is immobilized in other sections other than the control section,
4. The detection method according to any one of claims 1 to 3, wherein in the change confirmation step of step (F), changes in the control section and the other sections before and after the monomer supply step are confirmed. the first conjugate is an ACE2 (Angiotensin-converting enzyme2) receptor or a part thereof;
5. The detection method according to any one of claims 1 to 4, wherein said second conjugate is an ACE2 receptor or part thereof comprising a polymerization initiator. 6. The detection method according to claim 5, wherein the detection target is SARS-CoV-2. A detection substrate capable of detecting a detection target in a sample,
including a base layer and a protective film layer;
at least one side of the base layer is a sensing surface;
the detection surface immobilizes a first binding body that specifically binds to the detection target;
The detection substrate, wherein the protective film layer is detachably laminated on the detection surface. 8. The detection substrate according to claim 7, wherein said detection surface is a hologram surface on which fine irregularities are processed. the sensing surface comprises a plurality of compartments;
A different first conjugate is fixed for each of the plurality of compartments,
9. The detection substrate according to claim 7, wherein said first binders are binders that specifically bind to different detection targets. the sensing surface comprises a plurality of compartments;
The plurality of compartments includes a control compartment,
10. The detection substrate according to any one of claims 7 to 9, wherein the first binding body is not immobilized on the control plot. The detection substrate according to any one of claims 7 to 10, wherein the detection substrate from which the protective film layer has been peeled off is a detection substrate used in the detection method according to any one of claims 1 to 6. . 12. A sensing substrate according to any one of claims 7 to 11, wherein the base layer is one of a film, sheet or card. comprising a detection substrate according to any one of claims 7 to 12, a conjugate-containing solution containing a second conjugate, and a monomer-containing solution containing a monomer;
The second conjugate is a conjugate that specifically binds to the substance to be detected and contains a polymerization initiator,
A detection kit for an object to be detected, wherein the monomer is a monomer that is polymerized by the polymerization initiator. 14. The detection kit according to claim 13, wherein the monomer-containing solution further contains a polymerization reaction accelerator.

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