Biomolecule analysis kit and biomolecule analysis method

Abstract

The biomolecular analysis kit of the present invention is configured as follows: it has a reaction vessel for performing an enzyme reaction, the reaction vessel comprising a base portion having a container-shaped portion and a low-adsorption structure portion disposed on at least the inner surface of the container-shaped portion, wherein the low-adsorption structure portion has a lower adsorption rate with at least one of the sample being analyzed and the reagent used in the enzyme reaction than the adsorption rate of the base portion; and during the enzyme reaction in the reaction vessel, the signal generated by the enzyme reaction is detected.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201580006310.7, filed on February 2, 2015, with a priority date of January 31, 2014, entitled "Biomolecular Analysis Kit and Biomolecular Analysis Method". Technical Field

[0002] This invention relates to biomolecular analysis kits and biomolecular analysis methods.

[0003] This application claims priority based on Japanese Patent Application No. 2014-017942, filed in Japan on January 31, 2014, the contents of which are incorporated herein by reference. Background Technology

[0004] It is known that disease or health status diagnosis (constitutional diagnosis) is performed by analyzing biomolecules. For example, there are health status diagnoses using single nucleotide polymorphism (SNP) analysis, anticancer drug administration decisions using somatic cell mutation analysis, and infectious disease countermeasures using viral protein or DNA analysis.

[0005] Recent global human genome analysis has clarified the sequences of approximately 3.1 billion base pairs, revealing that the human genome contains approximately 30,000 to 40,000 genes. Differences in base sequences exist among individuals; variations in base sequences occurring at a frequency of more than 1% within a specific population group are termed genotypes. This suggests that SNPs are associated with a variety of diseases.

[0006] For example, regarding human genetic diseases, it is believed that a single SNP in a gene is the cause of the disease. Furthermore, it is believed that SNPs in multiple genes also influence lifestyle-related diseases or cancer. Therefore, SNP analysis is considered extremely effective in drug development, such as identifying drug targets and predicting side effects. Consequently, SNP analysis is being advanced as a massive global project.

[0007] One reason for individual differences in drug efficacy or the severity of side effects is the variation in enzyme groups related to drug metabolism in each person. It has recently become clear that this variation is caused by subtle differences in genes such as SNPs.

[0008] In recent years, there has been a growing interest in pre-analyzing patients' genes to select the most suitable medications for treatment. Furthermore, the significance of genetic diagnosis is rapidly increasing, not only for single-gene genetic diseases but also for multifactorial diseases. Additionally, the effectiveness of drugs targeting pathogens or viruses sometimes varies from person to person, largely due to subtle differences in each individual's genes. It is foreseeable that the use of genetic diagnosis for pathogens or viruses as exogenous agents will steadily increase in the future.

[0009] Therefore, in post-genomic medicine, it is important to be able to analyze minute differences in the genes of humans or pathogens, especially SNPs, and their importance is expected to increase in the future.

[0010] Various methods for analyzing minute differences in base sequences, especially SNPs, have been studied (see Non-Patent Literature 1-2). For practical analysis, excellent performance is required in terms of low cost, simplicity, short signal detection time, and accurate results. However, to date, no method has been found that meets all of these requirements.

[0011] When analyzing SNPs, the target gene fragment is typically present in trace amounts in the sample. In this case, it is necessary to amplify the target gene beforehand using any method. PCR (Polymerase Chain Reaction) is widely known as a rapid and highly reproducible gene amplification method.

[0012] Generally, detecting a single base difference in a target gene requires two stages: a gene amplification stage using methods such as PCR; and a stage for detecting the difference in a single base of the amplified gene (see Non-Patent Literature 3). However, methods requiring two stages are complex due to the numerous steps involved. Furthermore, PCR methods require temperature adjustments, necessitating the development of large-scale equipment, heat-resistant reaction vessels, and non-evaporating reaction solutions.

[0013] As a SNP detection method that does not require a two-stage reaction, the intrusion method is an example. The intrusion method does not require PCR amplification, and because the reaction can be promoted isothermally, the device can be miniaturized. However, since the gene amplification step is not included in the intrusion method, signal amplification is slow, requiring several hours of reaction time for detection. The intrusion method is a detection method that uses an enzyme reaction. In the signal amplification using enzymes, methods to shorten the time for the signal concentration to reach saturation are considered, such as reacting within a small space.

[0014] When performing invasive reactions within a small space, the number of analyte molecules contained in a single well can be less than one, resulting in an apparent concentration of the analyte molecules and thus shortening the time it takes for the signal to reach saturation. Furthermore, since the number of detectable molecules entering a single well is less than one, the concentration of the detectable molecules can be accurately studied by counting the wells from which the signal is obtained.

[0015] For example, Patent Document 1 shows that gene detection can be performed by carrying out an enzyme reaction in a tiny space with a volume of less than 1 p1.

[0016] Existing technical documents

[0017] Patent documents

[0018] Patent Document 1: Japanese Patent Application Publication No. 2004-309405

[0019] Non-patent literature 1: Landegren, Laboratory protocols for mutation detection, Oxford University Press, 1996

[0020] Non-patent literature 2: Ahmadian et al., Biotechniques, Vol. 32, pp. 1122-1137, 2002

[0021] Non-patent literature 3: J Biochem Biophys Method, Vol. 70, No. 50, 2007, pp. 789-795 Summary of the Invention

[0022] The problem that the invention aims to solve

[0023] In other words, while PCR can perform SNP analysis quickly, it increases the complexity of the equipment and procedures. Furthermore, in isothermal reactions without PCR, the time required to complete the SNP analysis is long, and the reactivity is low. Therefore, these existing methods are not practical.

[0024] The present invention was made in view of the above-mentioned problems, and its object is to provide a biomolecular analysis kit and a biomolecular analysis method that can perform rapid and quantitative analysis of biomolecules and improve reactivity.

[0025] Methods for solving problems

[0026] The biomolecular analysis kit of the first aspect of the present invention is configured as follows: it has a reaction vessel for performing an enzyme reaction, the reaction vessel comprising a base portion having a container shape portion and a low-adsorption structure portion disposed on at least the inner surface of the container shape portion, wherein the low-adsorption structure portion has a lower adsorption rate with at least one of the sample as the analyte and the reagent used in the enzyme reaction than the adsorption rate of the base portion; and during the enzyme reaction in the reaction vessel, the signal generated by the enzyme reaction is detected.

[0027] The low-adsorption structure has a lower adsorption rate with the sample than the matrix, and the background during signal detection can be lower compared to the case where the matrix is ​​exposed inside the reaction vessel.

[0028] The low-adsorption structure has a lower adsorption rate with the sample than the matrix, and the signal intensity during signal detection can be higher compared to the case where the matrix is ​​exposed inside the reaction vessel.

[0029] In order to make the adsorption rate lower than that of the substrate, the reaction vessel may further have a modified portion on the inner surface of the vessel shape portion that is a surface modified by the substrate portion. The vessel shape portion may be a bottomed cylindrical shape with a generally circular opening having a diameter of less than 5 micrometers.

[0030] In order to make the adsorption rate lower than that of the substrate, the reaction vessel may further have a low-adsorption material layer stacked on the substrate on the inner surface of the container shape portion. The container shape portion may be a bottomed cylindrical shape with a generally circular opening with a diameter of less than 5 micrometers.

[0031] The biomolecular analysis kit of the second aspect of the present invention is configured as follows: it has a reaction vessel for performing an enzyme reaction and a reagent that can be supplied to the reaction vessel and used in the enzyme reaction; the reaction vessel has a container-shaped portion that can supply a sample as the analyte and a base portion on which the container-shaped portion is formed; the reagent contains an anti-adsorption agent for reducing the adsorption rate of at least one of the sample and the reagent on the base portion; and when the enzyme reaction is performed in the reaction vessel, the signal generated by the enzyme reaction is detected.

[0032] The enzyme reaction can be an isothermal reaction.

[0033] The sample used as the analyte may contain any one of DNA, RNA, miRNA, mRNA, or protein. The analyte may be any one of DNA, RNA, miRNA, mRNA, or protein.

[0034] The analyte can be nucleic acid, and the enzyme reaction can be an invasive reaction.

[0035] The reagent can generate a signal through any one of fluorescence, luminescence, pH, absorption, or potential.

[0036] The anti-adsorption agent can be a surfactant.

[0037] The surfactant may be a nonionic surfactant.

[0038] The nonionic surfactant may be Tween 20.

[0039] The nonionic surfactant may be Triton-100.

[0040] The concentration of the surfactant can be greater than 0.0005% and less than 5%.

[0041] The third-party biomolecular analysis method of the present invention uses the biomolecular analysis kit of the first or second method described above.

[0042] The biomolecular analysis kit of the fourth aspect of the present invention is configured as follows: it has a reaction vessel for performing an enzyme reaction and a reagent that can be supplied to the reaction vessel and used in the enzyme reaction; the reaction vessel has a container-shaped portion that can supply a sample through a flow path and a base portion on which the container-shaped portion is formed; the reagent contains a surfactant for reducing the surface tension of the reagent; and during the enzyme reaction in the reaction vessel, the fluorescence or color signal generated by the enzyme reaction is detected.

[0043] The fifth aspect of the biomolecular analysis method of the present invention comprises the following steps: in a reaction vessel having a flow path and multiple container-shaped portions, a reagent is fed into the flow path, the reagent is filled into the multiple pores, an oily sealing liquid is fed into the flow path, and the reagent in the multiple pores is sealed by the oily sealing liquid, thereby forming multiple independent nucleic acid detection reaction vessels; wherein, either the reagent or the oily sealing liquid contains a surfactant.

[0044] The biomolecular analysis method of the fifth aspect of the present invention may further include a step of filling the plurality of container-shaped portions with a washing buffer through the flow path before filling the reagents into the plurality of wells.

[0045] The sixth method of the present invention is a biomolecular analysis method using the biomolecular analysis kit of the first, second or fourth method described above, wherein the reagent is supplied to the container shape portion after the washing buffer is supplied to the container shape portion.

[0046] Invention Effects

[0047] According to the above-described manner of the present invention, a biomolecular analysis method and a biomolecular analysis kit that can perform rapid and quantitative analysis of biomolecules and improve reactivity can be provided. Attached Figure Description

[0048] Figure 1 A cross-sectional view of a biomolecular analysis kit for applying the biomolecular analysis method of the first embodiment of the present invention.

[0049] Figure 2 This is a flowchart of the biomolecular analysis method according to the first embodiment of the present invention.

[0050] Figure 3 A fluorescence image illustrating the results of a fluorescence measurement experiment according to the first embodiment of the present invention.

[0051] Figure 4 A graph showing the results of a fluorescence intensity measurement experiment according to the first embodiment of the present invention.

[0052] Figure 5 This table shows the results of the reaction time measurement experiment according to the first embodiment of the present invention.

[0053] Figure 6 A cross-sectional view of a biomolecular analysis kit for applying the biomolecular analysis method of the second embodiment of the present invention.

[0054] Figure 7 A cross-sectional view of a biomolecular analysis kit for applying the biomolecular analysis method of the second embodiment of the present invention.

[0055] Figure 8 A cross-sectional view of a biomolecular analysis kit for applying the biomolecular analysis method of the second embodiment of the present invention.

[0056] Figure 9 This is a flowchart illustrating the biomolecular analysis method according to the second embodiment of the present invention.

[0057] Figure 10 Microscopic photograph showing the hole in the second embodiment of the present invention.

[0058] Figure 11A This is a fluorescence image illustrating the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention, under conditions where the concentration of Tween 20 was 0% and the heating time was 10 minutes.

[0059] Figure 11B The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.0005% and a heating time of 10 minutes.

[0060] Figure 11C The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.001% and a heating time of 10 minutes.

[0061] Figure 11D The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.005% and a heating time of 10 minutes.

[0062] Figure 11E This is a fluorescence image illustrating the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention, under the conditions of a Tween 20 concentration of 0.05% and a heating time of 10 minutes.

[0063] Figure 11F This is a fluorescence image illustrating the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention, under the conditions of a Tween 20 concentration of 0.5% and a heating time of 10 minutes.

[0064] Figure 11G This is a fluorescence image illustrating the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention, under the conditions of a Tween 20 concentration of 5% and a heating time of 10 minutes.

[0065] Figure 11H The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.0005% and a heating time of 20 minutes.

[0066] Figure 11I The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.001% and a heating time of 20 minutes.

[0067] Figure 11J The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.005% and a heating time of 20 minutes.

[0068] Figure 11K This is a fluorescence image illustrating the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention, under the conditions of a Tween 20 concentration of 0.05% and a heating time of 20 minutes.

[0069] Figure 11LThe fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 0.5% and a heating time of 20 minutes.

[0070] Figure 11M The fluorescence image is shown to represent the results of a fluorescence measurement experiment conducted in the second embodiment of the present invention under the conditions of a Tween 20 concentration of 5% and a heating time of 20 minutes.

[0071] Figure 12 A graph showing the results of a fluorescence intensity measurement experiment according to the second embodiment of the present invention.

[0072] Figure 13 This is a diagram illustrating the effect of the biomolecular analysis method according to the second embodiment of the present invention.

[0073] Figure 14A A fluorescence image is shown to illustrate the results of the fluorescence measurement test of sample 1 according to the third embodiment of the present invention.

[0074] Figure 14B A fluorescence image is shown to illustrate the results of the fluorescence measurement test of sample 2 in the third embodiment of the present invention.

[0075] Figure 14C A fluorescence image is shown to illustrate the results of the fluorescence measurement test of sample 3 in the third embodiment of the present invention.

[0076] Figure 14D A fluorescence image illustrating the results of a fluorescence measurement experiment of sample 4 according to the third embodiment of the present invention.

[0077] Figure 14E A fluorescence image is shown to illustrate the results of the fluorescence measurement test of sample 5 according to the third embodiment of the present invention.

[0078] Figure 14F A fluorescence image illustrating the results of a fluorescence measurement experiment of sample 6 according to the third embodiment of the present invention. Detailed Implementation

[0079] (First Implementation)

[0080] The following is a reference Figure 1 and Figure 2 The first embodiment of the present invention describes the biomolecular analysis kit and biomolecular analysis method.

[0081] Figure 1This is a cross-sectional view of a biomolecular analysis kit that enables the application of the biomolecular analysis method of this embodiment. In the biomolecular analysis kit of this embodiment, the biomolecule to be analyzed is selected from DNA, RNA, miRNA, mRNA (hereinafter sometimes referred to as RNA-like molecules), and protein.

[0082] like Figure 1 As shown, the biomolecular analysis kit 100 includes a flexible plate 12 and a glass substrate 14 constituting a reaction vessel 10, and a coverslip 13 capable of sealing the reaction vessel 10.

[0083] The reaction vessel 10 has a base portion 2 formed into a small space 11 (vessel shape portion) with a bottomed cylindrical shape and an opening at one end, and a low adsorption structure portion 3 disposed on the surface of the base portion 2.

[0084] Microspaces 11 are created by imprinting a flexible plate 12 made of polydimethylsiloxane (PDMS), thereby forming a reaction vessel 10.

[0085] The micro-spaces 11 constituting the reaction vessel 10 are cylindrical spaces with an opening at one end. For example, the micro-space 11 has a diameter L1 of 5 μm and a depth L2 of 5 μm. For example, the capacity of the micro-space 11 is approximately 100 feli (fl). An array of multiple micro-spaces 11 is formed in the reaction vessel 10. That is, the micro-spaces 11 are arranged in a row within the reaction vessel 10.

[0086] For example, in the flexible plate 12, the micro-spaces 11 are arranged in a grid pattern along each side of the surface having a rectangle with dimensions of 5 mm in both directions. The size of the gap between each micro-space 11 is set in each micro-space 11 according to the decomposition capability that enables independent signal detection.

[0087] The volume of the microspace 11 can be appropriately set, but a smaller volume of the microspace 11 can shorten the response time to a level where the signal can be detected. For example, the volume of the microspace 11 is 100 picoliters (pl) or less.

[0088] Specifically, in order to shorten the time required to saturate the signal and generate a sufficient signal, the volume of the microspace 11 is set according to the amount of liquid in one well for the analyte molecules.

[0089] The flexible flat plate 12 is formed, for example, on a glass substrate 14. The thickness of the glass substrate 14 is appropriately set with consideration of ensuring sufficient strength when the flexible flat plate 12 is used as the material to form multiple micro-spaces 11 by imprinting.

[0090] In this embodiment, the low-adsorption structure 3 has, for example, the following configuration.

[0091] (Example 1)

[0092] The low-adsorption structure 3 has a hydrophobic region on the surface of the substrate 2 located within the microspace 11 of the reaction vessel 10. For example, the low-adsorption structure 3 has a modified section 4 formed by modifying the surface of the substrate 2 to be hydrophobic.

[0093] (Example 2)

[0094] The low-adsorption structure 3 has a low-adsorption material layer 4A in the region located on the inner surface of the reaction vessel 10 within the surface of the substrate 2. The low-adsorption material layer 4A is formed of a material with low adsorption rate for the sample or analytical reagent used as the analyte of the biomolecular analysis kit 100 of this embodiment. For example, the low-adsorption material layer 4A is a hydrophobic coating.

[0095] Another example of a low-adsorption layer 4A is a polymer coating with a molecular structure that prevents fluorescent substances from passing through. This polymer coating preferably has a denser molecular structure than PDMS, thus preventing signal intensity reduction by inhibiting the permeation of fluorescent substances. Furthermore, for materials other than PDMS, polymer coatings with molecular structures that prevent reagent permeation can be selected based on the molecular structure of the material forming the substrate 2. These polymer coatings can suppress signal intensity reduction.

[0096] In addition, the polymer coating of the low adsorption structure 3 is not limited to the coating that inhibits the transmission of fluorescent substances, but can also be selected to inhibit the transmission of substances related to enzyme reactions, depending on the reagents used.

[0097] Next, the composition of the reagents that can be preferably used in the biomolecular analysis kit 100 of this embodiment will be described.

[0098] In this embodiment, by including an anti-adsorption agent in each reagent, the constituent components of the reagent can be prevented from adsorbing onto the inner surface of the reaction vessel 10 of the biomolecular analysis kit 100.

[0099] The composition of an anti-adsorption agent may include at least one of the following: a surfactant, a phosphate lipid, or another polymeric compound; any materials may also be mixed. For example, nonionic surfactants may be cited as surfactants. Examples of nonionic surfactants include Tween or glycerol, Triton-X100, etc. Additionally, examples of polymeric compounds include polyethylene glycol (PEG), DNA, and proteins.

[0100] In addition, as an anti-adsorption agent that combines two or more materials, an example of an anti-adsorption agent that combines phosphate lipids and PEG can be cited.

[0101] When using a nonionic surfactant as a surfactant, the concentration of the nonionic surfactant contained in the reagent is preferably 5% or less. When using Tween 20, the concentration of Tween 20 contained in the reagent is preferably in the range of 0.0005% or more and 5% or less, particularly preferably in the range of 0.001% or more and 0.5% or less. When the concentration of Tween 20 is 0.0005% or more, the reaction in multiple microspaces 11 can be detected independently, and the fluorescence of the microspaces 11 can be accurately measured. When the concentration of Tween 20 is 5% or less, sufficient enzyme reaction can be obtained.

[0102] These anti-adsorption agents can even adsorb substances onto the inner surface of the tiny spaces 11 within the reaction vessel 10. By supplying a reagent containing the anti-adsorption agent into the reaction vessel 10, the anti-adsorption agent adsorbs onto the inner surface of the reaction vessel 10. As a result, compared to the case without the anti-adsorption agent, the inner surface of the reaction vessel 10 becomes a state where enzymes used in enzyme reactions, nucleic acids or proteins used as analytes, and labeling substances used in signal detection are less likely to be adsorbed.

[0103] In addition, when oil is added to the microspace 11, the aforementioned anti-adsorption agent can also be added to the oil.

[0104] During the period from the initial supply of at least one of the enzyme used in the enzyme reaction, the nucleic acid or protein being analyzed, and the labeling substance used in signal detection to the end of signal detection, preferably at least one of the reagents in contact with the interior of the reaction vessel 10 contains an anti-adsorption agent. For example, the anti-adsorption agent may be mixed in a solvent such as a buffer solution used to dilute the reagents to a specified concentration.

[0105] From the moment at least one of the enzyme used in the enzyme reaction, the nucleic acid or protein being analyzed, and the labeling substance used in signal detection is supplied to the interior of the reaction vessel 10 until the end of signal detection, all reagents in contact with the interior of the reaction vessel 10 may contain an adsorbent.

[0106] In addition, the anti-adsorption agent is preferably a substance that does not hinder enzyme reactions or signal amplification reactions.

[0107] Next, the biomolecular analysis method using the biomolecular analysis kit 100 of this embodiment will be described. Figure 2 This is a flowchart illustrating the biomolecular analysis method of this embodiment.

[0108] First, a reagent containing the substance to be analyzed (e.g., DNA in this embodiment) is dropped into the small space 11 of the reaction vessel 10. Figure 2 (See step S101 shown). Specifically, in this embodiment, the reagents added contain an intrusion reaction reagent (1 μM Allele Probe, 0.4 μM Invader Oligo, 1 μM FAM Labelling Arm, 20 mM MOPS pH 7.5, 15 mM NaCl, 6.25 mM MgCl2, 50 U / μL Cleacase) and DNA.

[0109] The volume of reagent added to the micro-spaces 11 of the reaction vessel 10 can be appropriately set according to the number of micro-spaces 11. Furthermore, the volume and concentration of the reagent added to the micro-spaces 11 of the reaction vessel 10 are adjusted such that one DNA molecule is added to each micro-space 11. For example, in this embodiment, the total volume of reagent added to the micro-spaces 11 of the reaction vessel 10 is 0.5 μL, and this 0.5 μL of liquid is distributed into multiple micro-spaces 11.

[0110] Next, cover the small space 11 of the reaction vessel 10 with a coverslip 13. Figure 2 (See step S102). Thus, each microspace 11 becomes an independent reaction chamber sealed with intrusive reaction reagents and DNA.

[0111] Next, the reaction vessel 10, which contains the intruding reaction reagents and DNA, is sealed within the micro-space 11 and incubated, for example, in an oven at 62°C. Figure 2 (See step S103). Through this incubation, the signal amplification, which is isothermally induced during the invasion reaction, is appropriately carried out.

[0112] Next, the reaction containers 10, each sealed within a micro-space 11 containing the intruding reaction reagents and DNA, are removed after a pre-set time, and the number of fluorescent wells and their fluorescence intensity are measured. Figure 2 Step S104 is shown.

[0113] It should be noted that, in this embodiment, in addition to fluorescence detection, detection systems that use visible light emission, color development, pH changes, and potential changes as signals can also be applied. Furthermore, the configuration of this embodiment can also be used for protein analysis.

[0114] (Second Implementation)

[0115] The following is a reference Figure 6The biomolecular analysis kit and biomolecular analysis method of the second embodiment of the present invention will be described below. The biomolecular analysis kit 100A of this embodiment contains a nucleic acid quantification array device 20, reagents, and an oily sealing solution.

[0116] Figure 6 This is a cross-sectional view of the nucleic acid quantification array device 20 of this embodiment. In the biomolecular analysis kit of this embodiment, the biomolecule to be analyzed is selected from DNA, RNA, miRNA, mRNA (hereinafter sometimes referred to as RNA-like molecules) and protein.

[0117] like Figure 6 As shown, the nucleic acid quantification array device 20 includes a reaction container 30, a cap 27, an injection port (not shown), and an outlet port (not shown). The reaction container 30 has a substrate 23 and a flow path 31. The substrate 23 has a plurality of holes (container-shaped portions) 26, a substrate 24, and a micropore array layer 25 formed thereon.

[0118] The micro-hole array can be formed directly on the substrate 24, or the component with the micro-hole array can be fixed on the substrate 24 by means of bonding, fusion bonding or other methods.

[0119] The substrate 24 is a plate-shaped component made of a substantially transparent material. The material of the substrate 24 may be, for example, resin or glass. Specifically, the substrate 24 may be formed of polystyrene or polypropylene. The substrate 24 only needs to be a device for transporting the nucleic acid quantification array device 20 or have sufficient rigidity to prevent damage during manual handling by an operator.

[0120] The micro-pore array layer 25 is a layer formed by arranging multiple through-holes 25a. The micro-pore array layer 25 has a thickness of 3 μm, and there is a 100 μm gap between the micro-pore array layer 25 and the cap portion 27. The through-hole 25a is a bottomed cylindrical space with an opening at one end. The through-hole 25a is a cylindrical shape with a diameter of 5 μm and a length of 3 μm in the centerline direction. For example, the volume of the through-hole 25a is approximately 60 fl.

[0121] The volume of each through hole 25a can be set appropriately, but when the volume of the through hole 25a is small, the response time can be shortened to the point where the signal can be detected.

[0122] As an example, the volume of each through hole 25a is 100 picoliters or less.

[0123] In addition to detecting fluorescence, this embodiment can also utilize detection systems that use visible light emission, color development, pH changes, and potential changes as signals for detection. Furthermore, the configuration of this embodiment can be applied to protein analysis.

[0124] The distance (spacing) between the center lines of each through hole 25a only needs to be larger than the diameter of each through hole 25a.

[0125] The size of the interval (gap) between each through hole 25a is set independently in each through hole 25a according to the decomposition capability that enables signal detection.

[0126] Each through hole 25a is arranged in a triangular lattice pattern relative to the micro-hole array layer 25.

[0127] There is no particular limitation on the arrangement of the through holes 25a. Through the through holes 25a formed in the micro-hole array layer 25 and the surface 24a of the substrate 24, a bottomed cylindrical micro-hole 26 (container-shaped part) with the substrate 24 as the bottom part 26a is formed on the substrate part 23.

[0128] Specifically, in order to shorten the time required to generate a sufficient signal to saturate the signal, the volume of the well 26 is set according to the liquid volume to achieve a minimum of one molecule of the analyte in one well.

[0129] The micro-via array layer 25 can be made of resin or glass, etc. The material of the micro-via array layer 25 can be the same as or different from the material of the substrate 24. Alternatively, the micro-via array layer 25 can be integrally formed using the same material as the substrate 24. Examples of materials for the resin-based micro-via array layer 25 include cyclic olefin polymers or silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluoropolymers, and amorphous fluoropolymers. However, these are merely examples, and the material of the micro-via array layer 25 is not limited to these.

[0130] In addition, the micro-pore array layer 25 can also be colored. If the micro-pore array layer 25 is colored, the influence of light from other pores 26 adjacent to the pore 26 being measured is reduced when measuring light such as fluorescence, emission, and absorbance in the pores 26.

[0131] The micro-hole array layer 25 forms through-holes 25a by etching, embossing, or cutting the entire pattern of the hydrophobic film stacked on the substrate 24. Alternatively, when the micro-hole array layer 25 is integrally formed with the substrate 24, a portion corresponding to the through-holes 25a of the micro-hole array layer 25 is formed by etching, embossing, or cutting the substrate 24. Thus, a pattern with both hydrophobic and hydrophilic portions can be formed on the substrate.

[0132] A gap exists between the cover portion 27 and the base portion 23, and the cover portion 27 overlaps the base portion 23 in such a way that it covers the opening portions of the plurality of holes 26. The base portion 23 and the cover portion 27 form a flow path 31 through which various liquids flow. In this embodiment, various liquids flow from the injection port to the discharge port through the space between the base portion 23 and the cover portion 27.

[0133] Next, the composition of the reagents suitable for use in the biomolecular analysis kit 100A of this embodiment will be described.

[0134] like Figure 7 and Figure 8 As shown, the detection reaction reagent 21 is a solution that can be delivered from the injection port to the space between the base portion 23 and the cap portion 27. The detection reaction reagent 21 is a reagent used to perform biochemical reactions such as enzyme reactions on the analyte.

[0135] The biochemical reaction targeting the analyte, for example, when DNA (nucleic acid) is the analyte, occurs in the presence of nucleic acid, resulting in signal amplification. The detection reaction reagent 21 is selected, for example, according to a method capable of detecting nucleic acids. For example, reagents used in the Invader method, LAMP method, TaqMan method, fluorescent probe method, or other methods are included in the detection reaction reagent 21 of this embodiment.

[0136] In this embodiment, when the analyte is nucleic acid, the detection can be performed without the nucleic acid amplification step in the conventional PCR method, or the product obtained by amplifying the nucleic acid of the analyte using PCR or other methods can be used as a sample as needed.

[0137] In addition, when the analyte is not nucleic acid, this embodiment can be applied after necessary pretreatment in order to be applicable to this embodiment.

[0138] In this embodiment, by including an anti-adsorption agent in at least one of the reagents, the constituent components of the reagents can be prevented from adsorbing onto the inner surface of the pores 26 of the biomolecular analysis kit 100A. Alternatively, all reagents may contain an anti-adsorption agent.

[0139] Examples of reagents include buffer solutions, detection reaction reagents, sample (analyte: DNA, RNA, protein, etc.) solutions, sealing solutions, and solvents for diluting reagents or samples.

[0140] The composition of the anti-adsorption agent may include at least one of the following: a surfactant, a phosphate lipid, or another polymeric compound, and it can be mixed with any material. For example, nonionic surfactants can be cited as surfactants. Examples of nonionic surfactants include Tween or glycerol, Triton-X100, etc. Additionally, examples of polymeric compounds include polyethylene glycol (PEG), DNA, or proteins.

[0141] In addition, as an anti-adsorption agent that is a mixture of two or more materials, an anti-adsorption agent that is a mixture of phosphate lipids and PEG can be cited as an example.

[0142] When using a nonionic surfactant as a surfactant, the concentration of the nonionic surfactant contained in the reagent is preferably 5% or less. When using Tween 20, the concentration of Tween 20 contained in the reagent is preferably in the range of 0.0005% or more and 5% or less, particularly preferably in the range of 0.001% or more and 0.5% or less. When the concentration of Tween 20 is 0.0005% or more, the reaction in multiple wells 26 can be detected independently, and the fluorescence of well 26 can be accurately measured. When the concentration of Tween 20 is 5% or less, sufficient enzyme reaction can be obtained.

[0143] Surfactants are not limited to nonionic surfactants. Ionic surfactants (anionic, cationic, and amphoteric) can also be used. Mixtures of ionic surfactants or mixtures of ionic and nonionic surfactants can be used.

[0144] Alternatively, a mixture of surfactants and polymers can be used as an anti-adsorption agent.

[0145] Next, the composition of the oily sealing liquid 22 suitable for use in the biomolecular analysis kit 100A of this embodiment will be described.

[0146] In this embodiment, in order to prevent the components of the reagent from adsorbing onto the inner surface of the pores 26 of the biomolecular analysis kit 100A, the oily sealing liquid 22 may also contain an anti-adsorption agent.

[0147] Oily sealant 22 (reference) Figure 8 The solution 22 is capable of being delivered from the injection port to the space between the base portion 23 and the cap portion 27. The oily sealing fluid 22 can be selected from materials that do not mix with the sample containing the analyte. Mineral oil or fluorinated liquids such as FC40 can be used as the oily sealing fluid 22.

[0148] In addition, in this embodiment, to prevent reagent components from adsorbing onto the inner surface of the wells 26 of the biomolecular analysis kit 100A, a buffer solution for washing the wells can be dispensed before dispensing the reagents. The buffer solution may contain an anti-adsorption agent.

[0149] These anti-adsorption agents are effective even for substances adsorbed on the inner surface of the pores 26 of the reaction vessel 30. By supplying a reagent containing the anti-adsorption agent into the reaction vessel 30, the anti-adsorption agent is adsorbed onto the inner surface of the reaction vessel 30. As a result, compared to the case without the anti-adsorption agent, the inner surface of the reaction vessel 30 becomes less prone to adsorbing enzymes used in enzymatic reactions, nucleic acids or proteins used as analytes, and labeling substances used in signal detection.

[0150] The anti-adsorption agent contained in the washing buffer can be a nonionic surfactant. Examples of nonionic surfactants include Tween or glycerol, Triton-X100, etc. Additionally, the washing buffer can also be part of the reagent.

[0151] During the period from the initial supply of at least one of the enzyme used in the enzyme reaction, the nucleic acid or protein being analyzed, and the labeling substance used in signal detection to the interior of the reaction vessel 30 until the end of signal detection, preferably at least one of the reagents in contact with the interior of the reaction vessel 30 contains an anti-adsorption agent. For example, the anti-adsorption agent may be mixed in a solvent such as a buffer solution used to dilute the reagents to a specified concentration.

[0152] In addition, during the period from the initial supply of at least one of the enzyme used in the enzyme reaction, the nucleic acid or protein being analyzed, and the labeling substance used in signal detection to the interior of the reaction vessel 10 until the end of signal detection, all reagents in contact with the interior of the reaction vessel 10 may also contain an adsorbent.

[0153] In addition, the anti-adsorption agent is preferably a substance that does not hinder enzyme reactions or signal amplification reactions.

[0154] Next, the biomolecular analysis method using the biomolecular analysis kit 100A of this embodiment will be described. Figure 9 This is a flowchart illustrating the biomolecular analysis method of this embodiment.

[0155] First, open the injection port and discharge port (not shown), and use a dispensing pipette to deliver a washing buffer 33 containing an anti-adsorption agent through the injection port into the gap between the base 23 and the cap 27. Figure 9 (Step S201 shown). Buffer 33 diffuses within the gap between the base portion 23 and the cap portion 27 in a manner that completely covers the plurality of wells 26 (see reference). Figure 6Thus, a low-adsorption structure 32 having a low-adsorption material layer 35 is formed in the region on the inner surface of the through hole 25a and in the region 34 between adjacent holes in the surface of the substrate 23.

[0156] Alternatively, instead of delivering the buffer solution 33, the reaction vessel 30 can be pre-filled with the buffer solution 33. In this case, the inlet and outlet can be sealed using a membrane or similar device to pre-seal the buffer solution 33 within the reaction vessel 30.

[0157] Next, using a dispensing pipette or similar device, a reagent containing the substance to be analyzed (e.g., DNA in this embodiment) is dispensed through the injection port into the gap between the base portion 23 and the cap portion 27. Figure 9 (See step S202 shown). Specifically, the reagents filled in this embodiment contain an intrusion reaction reagent (detection reaction reagent 21) (1 μM AlleleProbe, 1 μM Invader Oligo, 1 μM FAM Labelling Arm, 10 mM MOPS at pH 7.5, 6.25 mM MgCl2, 50 U / μL Cleacase, Tween 20) and DNA as the analyte. The reagents diffuse in the gap between the base portion 23 and the cap portion 27 in a manner that completely covers the multiple wells 26 (see step S202). Figure 7 Additionally, by delivering the reagent into the gap between the base portion 23 and the cap portion 27, the buffer solution 33 is discharged from the outlet. Furthermore, when the reagent and the buffer solution 33 are different colors, it is easy to determine whether the reagent has been delivered to the portion between the base portion 23 and the cap portion 27.

[0158] like Figure 6 As shown, a plurality of holes 26 formed by a substrate 24 and a micropore array layer 25 are disposed in a flow path 31 formed by a substrate portion 23 and a cap portion 27. Buffer solution 33 filled in the plurality of holes 26 is replaced with reagent sequentially within the holes by allowing reagent to flow in.

[0159] However, wells 26 are also maintained with buffer solution 33 on the inner surface of well 26. In this case, the reagent does not replace the buffer solution 33 filling the multiple wells 26, but rather becomes a layer of reagent on top of buffer solution 33. However, since buffer solution 33 and reagent are easily miscible, after becoming a layer of reagent on top of buffer solution 33, the solute in the reagent diffuses into buffer solution 33. Therefore, the reaction in the wells where buffer solution and reagent are replaced is substantially the same as the reaction in the wells where buffer solution 33 and reagent are layered.

[0160] The volume of liquid filling the well 26 can be appropriately set according to the number of through-holes 25a. Furthermore, the volume and concentration of liquid added to the well 26 are adjusted such that one DNA molecule is contained in each well 26. For example, in this embodiment, the volume of liquid filling the well 26 is 0.5 μL in the entire reaction vessel, and 0.5 μL of liquid is dispensed into multiple wells 26.

[0161] Next, as Figure 8 As shown, the oily sealing liquid 22 is delivered from the injection port into the flow path 31 formed by the base portion 23 and the cap portion 27. The oily sealing liquid 22 seals the liquid in the multiple holes 26 in a state where the reagent diffuses into the buffer solution, making the multiple holes 26 multiple independent reaction chambers (nucleic acid detection reaction containers) 36. That is, in this embodiment, each hole 26 is covered by the oily sealant 22, and like the small space disclosed in the first embodiment, each hole 26 becomes independent. In addition, the oily sealing liquid 22 squeezes out the liquid outside the multiple holes 26 from the discharge port within the gap between the base portion 23 and the cap portion 27. Figure 9 Step S203 (as shown).

[0162] Then, the array device 20, with each well 26 filled with the penetration reaction reagent and DNA, is incubated, for example, in an oven at 62°C. Figure 9 (See step S204). Through this incubation, the signal amplification that occurs isothermally during the invasion reaction is properly carried out.

[0163] Next, the array device 20, with each well filled with the intrusion reaction reagent and DNA, was removed after a predetermined time, and the number of fluorescent wells and their fluorescence intensity were measured. Figure 9 (Step S205 shown).

[0164] That is, the biomolecular analysis method using the biomolecular analysis kit 100A of this embodiment has the following steps: a step of delivering reagents into the flow path and filling the multiple wells with reagents in a reaction vessel having a flow path and multiple container-shaped parts (reagent delivery step); after the reagent delivery step, delivering an oily sealing liquid into the flow path and sealing the reagents in the multiple wells with the oily sealing liquid, thereby making the multiple wells into multiple independent nucleic acid detection reaction vessels (sealing step).

[0165] In addition to detecting fluorescence, this embodiment can also utilize a detection system that uses visible light emission, color development, pH changes, and potential changes as signals for detection. Furthermore, the configuration of this embodiment can be applied to protein analysis.

[0166] Furthermore, in this embodiment, by including an anti-adsorption agent in each reagent, it is possible to prevent the components of the reagent from adsorbing onto the inner surface of the reaction vessel 30 of the biomolecular analysis kit 100A. The anti-adsorption agent may be contained in all the reagents or in a portion of the reagents.

[0167] Alternatively, instead of such anti-adsorption agents, the reagent can contain substances that reduce the surface tension of its components. For example, surfactants reduce the surface tension of the reagent. Therefore, including surfactants in the reagent is also effective in ensuring that the reagent fills the pores.

[0168] Example

[0169] (First Embodiment)

[0170] Next, examples for confirming the effectiveness of the biomolecular analysis method according to the first embodiment of the present invention will be described. Figure 3 A fluorescence image is shown to illustrate the results of the fluorescence measurement experiment in this embodiment. Figure 4 This is a graph showing the results of the fluorescence intensity measurement experiment in this embodiment. Wherein, Figure 4 The horizontal axis represents reaction time, Figure 4 The vertical axis represents fluorescence intensity. Figure 5 This is a table showing the results of the reaction time measurement experiment in this embodiment. Wherein, Figure 5 In this context, "good" indicates good quantitative performance, and "poor" indicates that the quantitative performance is worse than that of this embodiment.

[0171] <Experiment for measuring the number of fluorescent holes>

[0172] First, reaction reagents are sealed into the microspace 11 of the reaction vessel 10, along with three types of artificially synthesized DNA. Here, the concentration of each artificially synthesized DNA is set as follows: 30 pM for one molecule in one well, 50 nM for 1666 molecules in one well, and 0 M for no molecules in one well.

[0173] Then, the reaction vessel 10 was incubated in an oven at 62°C to confirm its state after 0 minutes, 10 minutes, and 15 minutes.

[0174] like Figure 3 As shown, if the DNA concentration is above 30 pM, the fluorescence intensity differs from the background at 0 pM in almost all the tiny spaces 11, indicating the presence of DNA.

[0175] <Measurement Experiment of Fluorescence Intensity>

[0176] Next, the reaction vessel 10, sealed with the artificially synthesized DNA as described above, was incubated in an oven at 62°C. To confirm the state after 0, 10, and 15 minutes, images of 5 wells were selected for each DNA concentration, and the average fluorescence intensity of 21 pixels in each image was calculated. Here, the wells after the reaction were measured using a fluorescence microscope (Zeiss, AX10), objective lens (EC Plan-Neofluar 40×oil NA1.3), light source (LEJ, FluoArc00 1.26A Usable with HBO 10), sensor (Hamamatsu Photonics, EM-CCD C9100), filter (Olympus, U-MNIBA2), and analysis software (Hamamatsu Photonics, AQUACOSMOS 2.6: exposure time 64.3ms, EM gain 180, offset 0, pixel combination ×1).

[0177] like Figure 4 As shown, at a concentration of 30 pM, which contains one molecule within a tiny space 11, fluorescence of distinguishable intensity was detected compared to that at 0 M.

[0178] <Reaction Time Measurement Experiment>

[0179] Next, the reaction times in existing analytical methods and the analytical method of the present invention were compared. In the reaction time determination experiment, as a comparison object with the present invention, the following methods were used: a method for digital PCR reaction with a reagent volume of 1 nanoliter (nl) × multiple wells (Comparative Example 1), a method for PCR reaction with a reagent volume of 20 microliters (μl) (Comparative Example 2), a method for PCR + invasion reaction with a reagent volume of 20 μl (Comparative Example 3), a method for invasion reaction with a reagent volume of 20 μl (Comparative Example 4), and a method for digital ELISA reaction with a reagent volume of 100 fl × multiple wells (Comparative Example 5).

[0180] like Figure 5 As shown in the reaction time measurement experiment, in Comparative Example 1, where a digital PCR reaction was performed with a reagent volume of 1 nmol × multiple wells, a reaction time of 60 minutes was required, the temperature control was variable, and the quantification was good. In Comparative Example 2, where a PCR reaction was performed with a reagent volume of 20 μl, a reaction time of 60 minutes was required, the temperature control was variable, and the quantification was poor. In Comparative Example 3, where a PCR + invasion reaction was performed with a reagent volume of 20 μl, a reaction time of 60 minutes was required, the temperature control was variable, and the quantification was poor.

[0181] In Comparative Example 4, where the invasive reaction was performed with a reagent volume of 20 μl, a reaction time of 120 minutes was required. Although the temperature control was isothermal, the quantification was poor. In Comparative Example 5, where the digital ELISA reaction was performed with a reagent volume of 100 fl × multiple wells, a reaction time of 15 minutes was required. The temperature control was isothermal, and the quantification was good.

[0182] In contrast, in this embodiment, where a digital intrusion reaction was performed with a reagent volume of 100 fl × porous, the reaction time was only 10 minutes, with isothermal temperature control and good quantification. This is because the digital intrusion reaction in this embodiment was performed with a reagent volume of 100 fl × porous.

[0183] (Second Embodiment)

[0184] Next, examples for confirming the effectiveness of the biomolecular analysis method according to the second embodiment of the present invention will be described. Figure 10 This is a fluorescence image of the pores in this embodiment. Figures 11A to 11M A fluorescence image is shown to illustrate the results of the fluorescence intensity measurement experiment in this embodiment. Figure 12 This table shows the results of the fluorescence intensity measurement experiment in this embodiment. (The table includes information about the results of the fluorescence intensity measurement experiment.) Figure 12 The horizontal axis in the figure represents the concentration of Tween 20. Figure 12 The vertical axis represents fluorescence intensity.

[0185] <Fabrication of Array Devices for Nucleic Acid Quantification>

[0186] After spin-coating CYTOP (registered trademark) (manufactured by Asahi Glass) onto a 0.5 mm thick glass substrate, it was baked at 180°C for 1 hour. The resulting CYTOP thickness was 3 μm. Following spin-coating the CYTOP onto the substrate, positive photoresist was applied, and a pattern was formed using a photomask. The CYTOP was then dry-etched using O2 plasma. To remove any remaining photoresist, the surface was washed and rinsed with acetone and ethanol.

[0187] like Figure 10 As shown, the pores (micro-spaces) formed by CYTOP have a diameter of 5 μm and a volume capable of detecting signals generated by the invasive reaction within minutes. An array of 100 pore blocks is provided on a single substrate. Each block has 10,000 pores. Therefore, a total of 1 million pores are formed. Figure 6 As shown, a glass plate with a liquid inlet (injection port: not shown) is bonded to the substrate using double-sided adhesive tape with a thickness of 50 μm and processed into a flow path shape.

[0188] <Dispensing of the mixture of sample and detection reagent>

[0189] Confirm the ease of droplet formation resulting from the concentration of the surfactant, Tween 20.

[0190] First, a washing buffer containing surfactant is fed into the nucleic acid quantification array device through the inlet. Then, 22 μl of the infiltration reaction reagent (detection reaction reagent 21: 1 μM Allele Probe, 1 μM Invader Oligo, 1 μM FAM Labelling Arm, 10 mM MOPS pH 7.5, 6.25 mM MgCl2, 50 U / μL Cleacase, Tween 20) and the DNA to be analyzed are fed into the nucleic acid quantification array device through the inlet.

[0191] Next, 80 μl of fluorine-based liquid FC40 (oil-based sealing fluid 22) was injected through the injection port to seal the reagent into each well. The solution was then heated on a heating plate at 63°C to carry out the intrusion reaction.

[0192] Next, fluorescence was detected in each well at 63°C for 10 and 20 minutes using a fluorescence microscope (Olympus). Exposure times were: bright field: 100 ms, NIBA: 2000 ms, mCherry: 2000 ms.

[0193] The results of microscopic observation of each well after heating for 10 minutes are shown below. Figures 11A to 11G The results of microscopic observation of each well after heating for 20 minutes are shown in the figure. Figures 11H to 11M .

[0194] When the concentration of Tween 20 is 0%, accurate digital measurements cannot be performed because fluorescence is detected even in the region between adjacent wells. This is because the reaction solution droplets in adjacent wells are believed to have combined, thus causing the sample to react in the region between them. Additionally, even if the reaction solution droplets in adjacent wells are not combined, the sample remaining on the substrate surface between adjacent wells is considered to have reacted. On the other hand, if Tween 20 contains 0.0005% or more, it is confirmed that the reaction solution droplets are individually separated.

[0195] Furthermore, if the Tween 20 content in the well after heating for 20 minutes is 0.001% or more, the separation of droplets in the reaction solution is confirmed. That is, if the Tween 20 content is 0.001% or more during prolonged heating, the reproducibility is considered to be further improved compared to short-time heating.

[0196] Figure 12This graph shows the fluorescence intensity values ​​corresponding to different concentrations of Tween 20. The fluorescence intensity decreases as the concentration of Tween 20 increases. That is, as the concentration of Tween 20 increases, behavior that inhibits the reaction is confirmed. Therefore, it is speculated that the optimal concentration of Tween 20 is around 5%. Furthermore, from a cost perspective, a concentration of Tween 20 of 0.5% or less is more preferable.

[0197] In addition, 10 μl of the same reagent was dispensed into 96-well plates, and the reactivity of the 10 μl volume was detected using a LightCycler LC480 (Roche). The LightCycler was kept at a constant temperature of 63 °C. The reaction was confirmed using the LightCycler with the same composition, and the results were independent of the concentration of Tween 20; the increase in fluorescence signal of the intrusion reaction was consistent. This indicates that the surfactant is not used to enhance enzyme reactivity, but rather to contribute to droplet stability.

[0198] Surfactants only need to be added at a concentration sufficient to prevent the analyte contained in the reagent from adsorbing onto CYTOP or glass. The optimal concentration may vary for other surfactants such as Triron-X100, but Tween 20 can be considered as an example.

[0199] (Third Embodiment)

[0200] Next, other embodiments for confirming the effectiveness of the biomolecular analysis method of the second embodiment of the present invention will be described. Figure 13 This is a diagram illustrating the effect of the biomolecular analysis method according to the second embodiment of the present invention. Figures 14A to 14F A fluorescence image is shown to illustrate the results of the fluorescence intensity measurement experiment in this embodiment. Figure 13 In the reactivity column, "Good" indicates good reactivity. Figure 13 In the droplet column, “○” indicates that no fluorescence was observed between two adjacent wells. Figure 13 In the droplet section, the "△" indicates the degree to which fluorescence was observed between two adjacent wells, but had no effect on the concentration measurement. Figure 13 In the droplet column, the "×" indicates that due to the observation of fluorescence between two adjacent wells, there is a situation where digital measurements cannot be accurately performed when using the area between two adjacent wells.

[0201] This embodiment uses the invasive reaction reagent and DNA used in the second embodiment, such as Figure 13As shown, the reaction conditions were varied by altering whether washing was performed, whether surfactant was added to the washing buffer, and whether surfactant was added to the reaction reagent. The state and reactivity of the droplets were confirmed by the resulting fluorescence images. 0.05% Tween 20 was added to the washing buffer or reaction reagent as a surfactant. Other conditions were the same as in the second embodiment.

[0202] For samples 1 and 2, a surfactant was added to the washing buffer for washing. For samples 3 and 4, no surfactant was added to the washing buffer for washing. For samples 5 and 6, no washing was performed. Furthermore, a surfactant was added to the reaction reagents for samples 1, 3, and 5. On the other hand, no surfactant was added to the reaction reagents for samples 2, 4, and 6. Reactivity was good in all samples. Moreover, as shown in sample 2, even when the reaction reagent did not contain a surfactant, droplet formation was good and reactivity was good when the washing buffer contained a surfactant.

[0203] As explained above, the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention can perform rapid and quantitative analysis of biomolecules by performing an enzyme reaction in the microspace 11 or well 26.

[0204] Furthermore, the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention, by using an intrusion method as an enzyme reaction, eliminates the need for PCR amplification and makes isothermal reactions possible, thus simplifying the machine configuration and analysis steps.

[0205] Furthermore, according to the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention, since the reaction is carried out in a tiny space 11 or well 26 containing a molecule of analyte, the time required for the signal to reach saturation can be shortened.

[0206] Furthermore, compared with the conventional method and reagent kit 100 of the first embodiment and the method and reagent kit 100A of the second embodiment of the present invention, the reaction time is shorter and the SN ratio is higher.

[0207] Furthermore, according to the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention, since the enzyme reaction is an isothermal reaction, a stable enzyme reaction can be obtained and the reproducibility is higher compared with the temperature-variable reaction.

[0208] Furthermore, according to the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention, since the enzyme reaction is an invasive reaction, the time for signal detection and determination can be shortened compared with the step that requires PCR.

[0209] Furthermore, in the biomolecular analysis method and biomolecular analysis kit 100 of the first embodiment and the biomolecular analysis method and biomolecular analysis kit 100A of the second embodiment of the present invention, since the microspace 11 or well 26 is less than 100 picoliters, the amount of reagents consumed can be reduced for analysis.

[0210] Furthermore, the above embodiments disclose examples of using a low-adsorption structure and an anti-adsorption agent in combination. However, by employing at least one of the low-adsorption structure and the anti-adsorption agent, rapid and quantitative analysis can be performed compared to the case where neither of the low-adsorption structure nor the anti-adsorption agent is employed.

[0211] Symbol Explanation

[0212] 100, 100A Biomolecular Analysis Kit

[0213] 2.23 Base part

[0214] 3.32 Low Adsorption Structure

[0215] 4 Modification Department

[0216] 4A, 35 Low-adsorption material layer

[0217] 10, 30 reaction vessels

[0218] 11. Tiny Space (Container Shape)

[0219] 12 Soft flat plate

[0220] 13 Coverslip

[0221] 14 Glass substrate

[0222] 22 Oily sealant

[0223] 24 substrate

[0224] 26 holes (container-shaped part)

[0225] 31 flow path

[0226] 33 Buffer

Claims

1. A biomolecular analysis method for non-diagnostic purposes, characterized in that, It has the following processes: The process of feeding a washing buffer containing an anti-adsorption agent into a reaction vessel having multiple pores. The reagent delivery process involves feeding the reagent into the reaction vessel, replacing the washing buffer with the reagent in the plurality of wells, and filling the wells with the reagent. A sealing liquid is supplied to the reaction vessel, and the reagents in the plurality of holes are sealed by the sealing liquid, thereby forming a sealing process for multiple independent reaction vessels. The reagent mentioned above is used to perform an enzymatic reaction on the analyte. The enzyme reaction is an isothermal reaction. The signal is amplified through the enzyme reaction, and then detected.

2. A biomolecular analysis method for non-diagnostic purposes, characterized in that, It has the following processes: In a reaction vessel having multiple wells and filled with a washing buffer containing an anti-adsorption agent, a reagent delivery process is performed: reagent is fed into the reaction vessel; the washing buffer is replaced with the reagent in the multiple wells; and the reagent is filled into the multiple wells. A sealing liquid is supplied to the reaction vessel, and the reagents in the plurality of holes are sealed by the sealing liquid, thereby forming a sealing process for multiple independent reaction vessels. The reagent mentioned above is used to perform an enzymatic reaction on the analyte. The enzyme reaction is an isothermal reaction. The signal is amplified through the enzyme reaction, and then detected.

3. The biomolecular analysis method according to claim 1 or 2, wherein, The reagent is added dropwise into a tiny space of less than 100 picoliters.

4. The biomolecular analysis method according to claim 1 or 2, wherein, The reaction vessel has a flow path, and the reagent and the sealing liquid are delivered to the reaction vessel by being fed into the flow path.

5. The biomolecular analysis method according to claim 1 or 2, wherein, The biomolecule in question is a nucleic acid.

6. The biomolecular analysis method according to claim 5, wherein, The signal is measured without performing the nucleic acid amplification process.

7. The biomolecular analysis method according to claim 1 or 2, wherein, The enzyme reaction is an invasive reaction.

8. The biomolecular analysis method according to claim 1 or 2, wherein, The number of holes that detected the signal is counted.

Images