FIELD

The present invention relates to a biomolecule analysis kit and a biomolecule analysis method.

BACKGROUND

It is known that a disease or a physical predisposition may be diagnosed through biomolecular analysis. For example, a physical predisposition may be diagnosed through Single Nucleotide Polymorphism (SNP) analysis, whether or not an anticancer drug will be administered may be determined through somatic mutation analysis, and infection control measures may be designed based on the analysis of the protein or DNA of viruses.

In recent years, through human genome analysis performed worldwide, sequences of about 3.1 billion base pairs of the human genome have been revealed, and it has become evident that the number of human genes is about 30,000 to 40,000. The base sequence of human beings varies between individuals, and a variation in the base sequence that exists more than 1% of a specific population is called a genetic polymorphism. Among genetic polymorphisms, SNP is considered to be associated with various diseases.

For example, genetic diseases of human beings are considered to be caused by SNP in a single gene. Furthermore, SNP in a plurality of genes is considered to affect the life style-related diseases, cancer, and the like. Therefore, it is considered that the analysis of SNP is extremely effective in the development of pharmaceutical products such as in the search for potential drug targets or the prediction of side effects. Accordingly, SNP analysis is being pushed forward as a large global project.

As one factor causing a degree of efficacy or a side effect of a drug to vary between individuals, a difference of an enzyme group involved in the drug metabolism of individuals may be exemplified. Recently, it has become evident that such a difference may also result from a slight genetic difference such as an SNP.

In recent years, a method of selecting an optimal drug by prior genetic analysis of a patient and administering it to a patient has been considered. In addition, the significance of genetic diagnosis has rapidly become highly appreciated, not only for single-gene disorders but also for multifactorial disorders. The efficacy of drugs targeting pathogenic bacteria or viruses varies between individuals in some cases even in the same species, and this variation results from minute genetic differences between individuals in many cases. It is expected that the test targets will greatly increase in the future in the genetic diagnosis of pathogenic bacteria or viruses as extrinsic factors described above.

As described above, in medical treatment of the post-genomic era, the ability to analyze for a minute genetic difference between human beings or pathogenic microorganisms, particularly, the ability to analyze for an SNP is important, and it is expected that this importance will increase in the future.

In the related art, various methods for analyzing for a minute difference in a base sequence, particularly, for analyzing for an SNP have been examined (see Landegren, Laboratory protocols for mutation detection, Oxford university press, 1996, and Ahmadian et al., Biotechniques, Vol. 32, pp 1122-1137, 2002). In order to perform analysis at a practical level, such methods are required to be excellent in all of aspects such as low cost, simplicity of the method, a short signal detection time, and accuracy of the detection result. However, a method satisfying all of the above requirements has not become known so far.

In a case where an SNP is analyzed for, generally, the sample contains only a small amount of target gene fragment. In this case, the target gene needs to be amplified in advance by a certain method. As a rapid gene amplification method with high reproducibility, a Polymerase Chain Reaction (PCR) method is well known in the related art.

Generally, in order to detect a difference of a single base of a target gene, it is necessary to perform a two-stage operation consisting of a gene amplification stage using the PCR method or the like and a stage of investigating a difference of a single base of the amplified gene. However, because this method requiring a two-stage operation includes a plurality of operations, the process thereof is complicated. Furthermore, in a PCR method, the temperature needs to be increased or decreased. Therefore, the size of a device is increased, a heat-resistant reaction container is required, and measures for preventing evaporation of the reaction solution need to be provided.

As an SNP detection method not requiring a two-stage reaction, there is an INVADER method. Because an INVADER method does not require PCR amplification and can cause an isothermal reaction, the size of a device can be reduced. However, since the INVADER method does not include a gene amplification step, a signal is slowly amplified, and the reaction needs to be performed for several hours to perform detection and determination. The INVADER method is a detection method using an enzymatic reaction. Particularly, as a method of shortening the time it takes for the signal concentration to become saturated during signal amplification using an enzyme, a method of performing a reaction in a microspace may be considered.

If the INVADER reaction is performed in a microspace, the number of molecules as a target of analysis contained in a single well can become equal to or less than 1, and the molecule as a target of analysis is seemingly in a concentrated state. Accordingly, the time taken for the signal to become saturated can be shortened. In addition, because the number of molecules to be detected that is put into a single well is equal to or less than 1, by counting the number of wells from which signals are obtained, the concentration of the molecules to be detected can be accurately ascertained.

For example, Japanese Unexamined Patent Application, First Publication No. 2004-309405 discloses that a genetic test can be performed by performing an enzymatic reaction in a microspace having a volume of equal to or less than 1 pl.

If PCR is used for SNP analysis, detection can be performed in a short time, but the constitution of the device or the procedure becomes complicated. Furthermore, in the isothermal reaction not using PCR, it takes a long time until the SNP analysis is completed, and the reactivity is low. Therefore, those methods of the related art are impractical.

SUMMARY

The present invention has been made to solve the above problems, and an object thereof is to provide a biomolecule analysis kit and a biomolecule analysis method which make it possible to rapidly and quantitatively analyze biomolecules and to improve reactivity.

A biomolecule analysis kit according to a first aspect of the present invention includes a reaction container configured to perform an enzymatic reaction, the reaction container including a base portion which has a container-shaped portion and a low-adsorption structural portion which is provided on at least the inner surface of the container-shaped portion, the low-adsorption structural portion having an adsorption rate lower than the base portion at which at least one of a sample which becomes a target of analysis in the enzymatic reaction and a reagent for the enzymatic reaction is adsorbed thereonto, wherein a signal resulting from the enzymatic reaction is configured to be detected when the enzymatic reaction is performed in the reaction container.

The low-adsorption structural portion may have a lower adsorption rate than that of the base portion at which the sample is absorbed thereonto, and a background in the signal detection may be lower than a case in which the base portion is exposed in the reaction container.

The low-adsorption structural portion may have a lower adsorption rate than that of the base portion at which the sample is absorbed thereonto, and a signal intensity in the signal detection may be higher than a case in which the base portion is exposed in the reaction container.

The reaction container may further have a modified portion formed by modifying the surface of the base portion such that the adsorption rate in the base portion within the inner surface of the container-shaped portions becomes lower than the adsorption rate in the base portion, and the container-shaped portions may have a bottomed cylindrical shape having an approximately circular opening portion having a diameter of equal to or less than 5 μm.

The reaction container may further have a low-adsorption substance layer laminated on the base portion such that the adsorption rate within the inner surface of the container-shaped portions becomes lower than the adsorption rate in the base portion, and the container-shaped portions may have a bottomed cylindrical shape having an approximately circular opening portion having a diameter of equal to or less than 5 μm.

A biomolecule analysis kit according to a second aspect of the present invention includes a reaction container configured to perform an enzymatic reaction, the reaction container including a container-shaped portion configured so that a sample that becomes a target of analysis is supplied to the container-shaped portion and a base portion in which the container-shaped portion is formed, and a reagent configured to be supplied to the reaction container and is used for the enzymatic reaction, wherein the reagent contains an adsorption inhibitor for reducing an adsorption rate of at least one of the sample and the reagent with respect to the base portion, and a signal resulting from the enzymatic reaction is configured to be detected when the enzymatic reaction is performed in the reaction container.

The enzymatic reaction may be an isothermal reaction.

The sample which becomes a target of analysis may include any one of DNA, RNA, miRNA, mRNA, and a protein, and a target substance of analysis may be any one of DNA, RNA, miRNA, mRNA, and a protein.

The target substance of analysis may be a nucleic acid, and the enzymatic reaction may be an INVADER reaction.

The reagent may generate a signal by any one of fluorescence, light emission, pH, light absorption, and electric potential.

The adsorption inhibitor may be a surfactant.

The surfactant may be a nonionic surfactant.

The nonionic surfactant may be TWEEN 20.

The nonionic surfactant may be TRITON-100.

The concentration of the surfactant may be equal to or greater than 0.0005% and equal to or less than 5%.

A biomolecule analysis method according to a third aspect of the present invention uses the biomolecule analysis kit according to the first or second aspect.

A biomolecule analysis kit according to a fourth aspect of the present invention includes a reaction container configured to perform an enzymatic reaction, the reaction container including a container-shaped portion configured so that a sample is supplied through a flow channel to the container-shaped portion and a base portion in which the container-shaped portion is formed, and a reagent configured to be supplied to the reaction container and used for the enzymatic reaction, wherein the reagent contains a surfactant for reducing the surface tension of the reagent, and fluorescence or a colorimetric signal resulting from the enzymatic reaction is configured to be detected when the enzymatic reaction is performed in the reaction container.

A biomolecule analysis method according to a fifth aspect of the present invention includes feeding a reagent into a flow channel in a reaction container which has the flow channel and a plurality of wells such that the plurality of wells is filled with the reagent; and feeding the oil sealing solution into the flow channel and sealing the reagent into the plurality of wells with an oil sealing solution, thereby forming the plurality of wells into a plurality of independent reaction containers for nucleic acid detection, wherein any one of the reagent and the oil sealing solution contains a surfactant.

The biomolecule analysis method according to the fifth aspect of the present invention may further include a step of filling the plurality of container-shaped portions with a wash buffer through the flow channel before filling the plurality of wells with the reagent.

A biomolecule analysis method according to a sixth aspect of the present invention is a biomolecule analysis method using the biomolecule analysis kit according to the first, second, or fourth aspect, in which after the wash buffer is supplied to the container-shaped portion, the reagent is supplied to the container-shaped portion.

According to the above aspects of the present invention, a biomolecule analysis kit and a biomolecule analysis method which makes it possible to rapidly and quantitatively analyze biomolecules and to improve reactivity can be provided.

