Patent Description:
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 <NUM> billion base pairs of the human genome have been revealed, and it has become evident that the number of human genes is about <NUM>,<NUM> to <NUM>,<NUM>. The base sequence of human beings varies between individuals, and a variation in the base sequence that exists more than <NUM>% 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 NPLs <NUM> and <NUM>). 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 (see NPL <NUM>). 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 <NUM>, 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 <NUM>, 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, PTL <NUM> 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 <NUM> pl.

<CIT> describes a microfluidic device comprising a plurality of reaction chambers in fluid communication with a flow channel formed in an elastomeric substrate, a vapor barrier for preventing evaporation from the plurality of reaction chambers, and a continuous phase fluid for isolation of each of the plurality of reaction chambers.

<CIT> describes a microfluidic device including a plurality of reaction wells; and a plurality of solid supports, and each of the solid supports has a reagent attached thereto. The reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation.

<CIT> describes a technique for detecting target molecules of low concentration with high sensitivity. The method includes (i) a step of introducing a hydrophilic solvent containing beads into a space <NUM>) between (a) a lower layer section including a plurality of receptacles each of which is capable of storing only one of the beads and which are separated from each other by a side wall having a hydrophobic upper surface and (b) an upper layer section facing a surface of the lower layer section on which surface the plurality of receptacles are provided; and (ii) a step of introducing a hydrophobic solvent into the space, the step (ii) being carried out after the step (i).

<CIT> describes an integrated microfluidic device and its usage are provided. The microfluidic device comprises an upper layer and a lower layer, wherein the lower layer is bound to the upper layer. The upper layer comprises a micro-channel and the lower layer comprises a micro-well array. The micro-channel is in fluidic connection with the micro-well array, and the height of the micro-channel is greater than the diameter of the oocyte flowing through the micro-channel.

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.

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 the present invention is defined in claim <NUM>.

The enzymatic reaction is 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 <NUM>.

The nonionic surfactant may be Triton-<NUM>.

The concentration of the surfactant may be equal to or greater than <NUM>% and equal to or less than <NUM>%.

A biomolecule analysis method according to the present invention is defined in claim <NUM>.

The biomolecule analysis method according to 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.

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.

Hereinafter, a biomolecule analysis kit and a biomolecule analysis method according to a first embodiment of the present invention will be described with reference to <FIG>.

<FIG> is a sectional view of the biomolecule analysis kit to which the biomolecule analysis method according to the present embodiment is applicable. In the biomolecule analysis kit according to the reference configuration, as a biomolecule to be analyzed, any of DNA, RNA, miRNA, mRNA (hereinafter, referred to as RNAs in some cases), and a protein is selected.

As shown in <FIG>, a biomolecule analysis kit <NUM> includes a soft flat plate <NUM> and a glass substrate <NUM> that constitute a reaction container <NUM> and a cover glass <NUM> that can seal off the reaction container <NUM>.

The reaction container <NUM> has a base portion <NUM> which is formed to have microspaces <NUM> (container-shaped portions) each having a bottomed cylindrical shape with one open end and low-adsorption structural portions <NUM> which are provided on the surface of the base portion <NUM>.

By preparing the microspaces <NUM> in the soft flat plate <NUM> formed of polydimethylsiloxane (PDMS) by means of imprinting, the reaction container <NUM> is formed.

The microspace <NUM> constituting the reaction container <NUM> is a space having a bottomed cylindrical shape with an opening portion at one end. The microspace <NUM> has a diameter L1 of <NUM> and a depth L2 of <NUM>, for example. For instance, the microspace <NUM> has a volume of about <NUM> femtoliters (fl). In the reaction container <NUM>, an array of a plurality of microspaces <NUM> is formed. That is, the microspaces <NUM> are arrayed in the reaction container <NUM>.

For example, on the <NUM> × <NUM> rectangular surface of the soft flat plate <NUM>, the microspaces <NUM> are arrayed in the form of a lattice along each side of the surface. The size of a gap between the microspaces <NUM> is set according to the resolution by which a signal can be independently detected in each of the microspaces <NUM>.

The volume of each microspace <NUM> may be appropriately set. However, the smaller the volume of the microspace <NUM>, shorter the reaction time until a signal becomes detectable. For example, the volume of the microspace <NUM> is equal to or less than <NUM> picoliters (pl).

Specifically, in a case where the aim is to shorten the time taken for generating a sufficient signal by saturating the signal, the volume of the microspace <NUM> is set based on a liquid amount in which the number of biomolecules as a target of analysis becomes equal to or less than <NUM> per well.

The soft flat plate <NUM> is formed on the glass substrate <NUM>, for example. The thickness of the glass substrate <NUM> is appropriately set in consideration of a point that the glass substrate <NUM> needs to have a sufficient strength in a process of forming the plurality of microspaces <NUM> by means of imprinting by using the soft flat plate <NUM> as a material.

In the present embodiment, the low-adsorption structural portions <NUM> have the following constitution, for example.

In each low-adsorption structural portion <NUM>, a region positioned on the inner surface of the microspace <NUM> of the reaction container <NUM> within the surface of the base portion <NUM> is hydrophobic. For instance, each low-adsorption structural portion <NUM> has a modified portion <NUM> formed by modifying the surface of the base portion <NUM> to be hydrophobic.

Each low-adsorption structural portion <NUM> has a low-adsorption substance layer 4A in a region positioned on the inner surface of the reaction container <NUM> within the surface of the base portion <NUM>. The low-adsorption substance layer 4A is formed of a material to which a sample as a target of analysis using the biomolecule analysis kit <NUM> of the present embodiment or a reagent for the analysis exhibits a low adsorption rate. For example, the low-adsorption substance layer 4A is a hydrophobic coat.

Examples of the low-adsorption substance layer 4A also include a polymer coat having a molecular structure that does not allow the permeation of a fluorescent substance. The polymer coat preferably has a molecular structure denser than that of PDMS described above. By inhibiting the permeation of a fluorescent substance, the polymer coat exerts an effect of preventing the decrease of signal intensity. For substances other than PDMS, based on the molecular structure of the substance that becomes the material of the base portion <NUM>, a polymer coat having a molecular structure that can prevent the permeation of a reagent may be selected. Such a polymer coat inhibits a decrease of signal intensity.

The polymer coat in the low-adsorption structural portions <NUM> is not limited to the coat inhibiting the permeation of a fluorescent substance, and a coat that inhibits the permeation of a substance involved in an enzymatic reaction may be appropriately selected according to the reagent to be used.

Next, the composition of the reagent which can be suitably used in the biomolecule analysis kit <NUM> according to the present embodiment will be described.

In the present embodiment, each reagent contains an adsorption inhibitor, and accordingly, the constituents of the reagent can be prevented from being adsorbed onto the inner surface of the reaction container <NUM> of the biomolecule analysis kit <NUM>.

For example, the adsorption inhibitor has a composition containing at least one kind of component among a surfactant, phospholipid, and other polymer compounds, and any materials may be used by being mixed together. Examples of the surfactant include a nonionic surfactant. Examples of the nonionic surfactant include Tween, glycerol, Triton-X100, and the like. Examples of the polymer compounds include polyethylene glycol (PEG), DNA, and a protein.

Examples of the adsorption inhibitor as a mixture of two or more kinds of material include an adsorption inhibitor obtained by mixing phospholipid with PEG.

In a case where a nonionic surfactant is used as the surfactant, the concentration of the nonionic surfactant contained in the reagent is preferably equal to or less than <NUM>%. In a case where Tween <NUM> is used, the concentration of Tween <NUM> contained in the reagent is preferably within a range of equal to or greater than <NUM>% and equal to or less than <NUM>%, and more preferably within a range of equal to or greater than <NUM>% and equal to or less than <NUM>%. If the concentration of Tween <NUM> is equal to or greater than <NUM>%, the reactions caused in the plurality of microspaces <NUM> can be independently detected, and the fluorescence from the microspaces <NUM> can be accurately measured. If the concentration of Tween <NUM> is equal to or less than <NUM>%, a sufficient enzymatic reaction is obtained.

The adsorption inhibitor described above may be a substance adsorbed onto the inner surface of the microspace <NUM> in the reaction container <NUM>. By supplying the adsorption inhibitor-containing reagent into the reaction container <NUM>, the adsorption inhibitor is adsorbed onto the inner surface of the reaction container <NUM>. As a result, compared to a case where the reagent does not contain the adsorption inhibitor, the enzyme used for the enzymatic reaction, the nucleic acid or protein that becomes a target of analysis, the labeling substance used for signal detection, and the like are hardly adsorbed onto the inner surface of the reaction container <NUM>.

In a case where oil is put into the microspaces <NUM>, the adsorption inhibitor may be added to the oil to be used.

It is preferable that the adsorption inhibitor is contained in at least any one of the reagents coming into contact with the inside of the reaction container <NUM> during the period between a time before at least one of the enzyme used for the enzymatic reaction, the nucleic acid or protein that becomes a target of analysis, the labeling substance used for signal detection, and the like is initially supplied into the reaction container <NUM> and a time when the signal detection is ended. For example, the adsorption inhibitor may be mixed with a solvent such as a buffer solution for diluting the reagent at a predetermined concentration.

The adsorption inhibitor may be contained in all of the reagents coming into contact with the inside of the reaction container <NUM> during the period between a time before at least one of the enzyme used for the enzymatic reaction, the nucleic acid or protein that becomes a target of analysis, the labeling substance used for signal detection, and the like is initially supplied into the reaction container <NUM> and a time when the signal detection is ended.

The adsorption inhibitor is preferably a substance that does not hinder an enzymatic reaction or a signal amplification reaction.

Next, a biomolecule analysis method using the biomolecule analysis kit <NUM> according to the present embodiment will be described. <FIG> is a flowchart showing the biomolecule analysis method according to the present embodiment.

First, a reagent containing a substance (DNA in the present embodiment, for example) that becomes a target of analysis is added dropwise to the microspaces <NUM> of the reaction container <NUM> (Step S101 shown in <FIG>). Specifically, the reagent added dropwise in the present embodiment contains an invader reaction reagent (<NUM> allele probe, <NUM> invader oligo, <NUM> FAM-labeled arm, <NUM> MOPS pH <NUM>, <NUM> NaCl, <NUM> MgCl<NUM>, and <NUM> U/µL cleavase) and DNA.

The liquid amount of the reagent added dropwise to the microspaces <NUM> of the reaction container <NUM> may be appropriately set according to the number of microspaces <NUM>. Furthermore, the liquid amount and concentration of the reagent added dropwise to the microspaces <NUM> of the reaction container <NUM> are adjusted such that approximately single DNA molecule is put into each of the microspaces <NUM>. For example, in the present embodiment, the liquid amount of the reagent added dropwise to the microspaces <NUM> of the reaction container <NUM> is <NUM>µL in total, and <NUM>µL of the liquid is distributed into the plurality of microspaces <NUM>.

Then, the microspaces <NUM> of the reaction container <NUM> are covered with the cover glass <NUM> (Step S102 shown in <FIG>). As a result, each of the microspaces <NUM> becomes an independent reaction chamber filled with the invader reaction reagent and DNA.

Thereafter, the reaction container <NUM> in which the microspaces <NUM> are filled with the invader reaction reagent and DNA is incubated in an oven at <NUM>, for example (Step S103 shown in <FIG>). Through the incubation, signal amplification performed in an isothermal invader reaction suitably proceeds.

Subsequently, the reaction container <NUM> in which each of the microspaces <NUM> is filled with the invader reaction reagent and DNA is taken out after a preset time, and the number of wells emitting fluorescence and the fluorescence amount thereof are measured (Step S104 shown in <FIG>).

In the present embodiment, a detection system may also be adopted which detects, in addition to fluorescence, emission of visible light, color development, a change in pH, a change in electric potential, or the like as a signal. In addition, the constitution of the present embodiment can be adopted for analyzing proteins.

Hereinafter, a biomolecule analysis kit and a biomolecule analysis method according to an embodiment of the present invention will be described with reference to <FIG>. A biomolecule analysis kit 100A according to the present embodiment includes an array device <NUM> for nucleic acid quantification, reagent, and an oil sealing solution.

<FIG> is a sectional view of the array device <NUM> for nucleic acid quantification according to the present embodiment. In the biomolecule analysis kit according to the present embodiment, as a biomolecule to be analyzed, any of DNA, RNA, miRNA, mRNA (hereinafter, referred to as RNAs in some cases), and a protein is selected.

As shown in <FIG>, the array device <NUM> for nucleic acid quantification includes a reaction container <NUM>, a cover portion <NUM>, an inlet portion (not shown in the drawing), and an outlet portion (not shown in the drawing). The reaction container <NUM> includes a base portion <NUM> and a flow channel <NUM>. In the base portion <NUM>, a plurality of wells (container-shaped portions) <NUM>, a substrate <NUM>, and a micropore array layer <NUM> are formed.

The micropore array may be formed directly in the substrate <NUM>. Alternatively, a member in which the micropore array is formed may be fixed to the substrate <NUM> by means of adhesion, welding, or the like.

The substrate <NUM> is a plate-like member formed of a material that is substantially transparent. The material of the substrate <NUM> is a resin or glass, for example. Specifically, the substrate <NUM> may be formed of polystyrene or polypropylene. The substrate <NUM> should have such a stiffness that the substrate is not broken at the time of handling due to a device for transporting the array device <NUM> for nucleic acid quantification or a manual operation of an operator.

The micropore array layer <NUM> is a layer in which a plurality of through holes 25a arranged in a line. The thickness of the micropore array layer <NUM> is <NUM>, and there is a clearance of <NUM> between the micropore array layer <NUM> and the cover portion <NUM>. Each of the through holes 25a is a bottomed cylindrical space having an opening portion at one end, and has a diameter of <NUM> (the through hole 25a has a cylindrical shape <NUM> long in the centerline direction). For example, the volume of the through hole 25a is about <NUM> femtoliters (fl).

The volume of each through hole 25a may be appropriately set. However, the smaller the volume of the through hole 25a, the further the reaction time taken until a signal becomes detectable can be shortened.

For example, the volume of each through hole 25a is equal to or less than <NUM> picoliters.

The distance (pitch) between the center lines of the through holes 25a should be longer than the diameter of each of the through holes 25a.

The size of the interval (gap) between the through holes 25a is set according to the resolution by which a signal can be independently detected in each of the through holes 25a.

The through holes 25a are arranged to form a triangular lattice shape with respect to the micropore array layer <NUM>.

The way the through holes 25a are arranged is not particularly limited. In the base portion <NUM>, bottomed cylindrical micro-wells <NUM> (container-shaped portions) in which the substrate <NUM> becomes a bottom surface portion 26a are formed by the through holes 25a formed in the micropore array layer <NUM> and a surface 24a of the substrate <NUM>.

Specifically, in a case where the aim is to shorten the time taken for generating a sufficient signal by saturating the signal, the volume of each well <NUM> is set based on a liquid amount in which the number of biomolecules as a target of analysis becomes equal to or less than <NUM> per well.

The material of the micropore array layer <NUM> may be a resin, glass, and the like, and may be the same as or different from the material of the substrate <NUM>. Furthermore, the micropore array layer <NUM> and the substrate <NUM> may be integrated by the same material. In addition, the micropore array layer <NUM> and the substrate <NUM> may be integrally molded with the same material. Examples of the material of the micropore array layer <NUM> formed of a resin include a cycloolefin polymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, a fluorine resin, an amorphous fluorine resin, and the like. These are merely examples of the material of the micropore array layer <NUM>, and the material of the micropore array layer <NUM> is not limited thereto.

The micropore array layer <NUM> may be colored. If the micropore array layer <NUM> is colored, in a case where optical measurement such as the measurement of fluorescence, light emission, absorbance, and the like is performed in the wells <NUM>, the influence of light from other wells <NUM> adjacent to a well <NUM> that becomes a measurement target can be reduced.

By performing processing such as etching, embossing, or cutting on the solid pattern of a hydrophobic coat laminated on the substrate <NUM>, the through holes 25a are formed on the micropore array layer <NUM>. In a case where the micropore array layer <NUM> and the substrate <NUM> are integrally molded, by performing processing such as etching, embossing, or cutting on the substrate <NUM>, the portions corresponding to the through holes 25a of the micropore array layer <NUM> are formed. In this way, a pattern having a hydrophobic portion and a hydrophilic portion can be formed on the substrate.

The cover portion <NUM> is superposed on the base portion <NUM> such that the cover portion <NUM> covers opening portions of the plurality of wells <NUM> in a state where a gap is formed between the base portion <NUM> and the cover portion <NUM>. The space between the base portion <NUM> and the cover portion <NUM> becomes a flow channel <NUM> through which various liquids flow. In the present embodiment, through the space between the base portion <NUM> and the cover portion <NUM>, various liquids flow from the inlet portion toward the outlet portion.

Next, the composition of the reagent which can be suitably used in the biomolecule analysis kit 100A according to the present embodiment will be described.

As shown in <FIG> and <FIG>, a detection reaction reagent <NUM> is a solution which can be fed into the space between the base portion <NUM> and the cover portion <NUM> from the inlet portion. The detection reaction reagent <NUM> is a reagent for causing a biochemical reaction such as an enzymatic reaction with regard to a target substance of analysis.

The biochemical reaction to a target substance of analysis is a reaction by which signal amplification may occur in the presence of a nucleic acid in a case where the target substance of analysis is DNA (nucleic acid), for example. The detection reaction reagent <NUM> is selected according to the method which can detect a nucleic acid, for example. For instance, the reagents used in an invader (registered trademark) method, a LAMP method (registered trademark), a TaqMan (registered trademark) method, a fluorescent probe method, or other methods are included in the detection reaction reagent <NUM> of the present embodiment.

In the present embodiment, when the target substance of analysis is a nucleic acid, the nucleic acid can be detected without performing a nucleic acid amplification step as in the PCR method as in the related art. However, if necessary, a product obtained by amplifying the nucleic acid as a target of analysis by the PCR method or the like may be used as a sample.

Furthermore, even when the target substance of analysis is other than a nucleic acid, the present embodiment can be applied after the substance is appropriately pre-treated as necessary such that it becomes applicable to the present embodiment.

In the present embodiment, at least one of the reagents contains the adsorption inhibitor, and as a result, the constituents of the reagent can be prevented from being adsorbed onto the inner surface of the wells <NUM> of the biomolecule analysis kit 100A. All of the reagents may contain the adsorption inhibitor.

Examples of the reagents include a buffer, a detection reaction reagent, a sample (a substance as a target of analysis: DNA, RNAs, a protein, or the like) solution, a sealing solution, and a solvent for diluting a reagent or a sample.

Examples of the adsorption inhibitor as a mixture of two or more kinds of materials include an adsorption inhibitor obtained by mixing phospholipid with PEG.

In a case where a nonionic surfactant is used as the surfactant, the concentration of the nonionic surfactant contained in a reagent is preferably equal to or less than <NUM>%. In a case where Tween <NUM> is used, the concentration of Tween <NUM> contained in a reagent is preferably within a range of equal to or greater than <NUM>% and equal to or less than <NUM>%, and more preferably within a range of equal to or greater than <NUM>% and equal to or less than <NUM>%. If the concentration of Tween <NUM> is equal to or greater than <NUM>%, the reactions caused in the plurality of wells <NUM> can be independently detected, and the fluorescence from the wells <NUM> can be accurately measured. If the concentration of Tween <NUM> is equal to or less than <NUM>%, a sufficient enzymatic reaction is obtained.

The surfactant is not limited to the nonionic surfactant, and an ionic surfactant (an anionic, cationic, or amphoteric surfactant) may be used as the surfactant. A mixture of ionic surfactants or a mixture of an ionic surfactant and a nonionic surfactant may also be used.

Furthermore, a mixture of a surfactant and a polymer compound can also be used as the adsorption inhibitor.

Next, the composition of the oil sealing solution <NUM> which can be suitably applied to the biomolecule analysis kit 100A according to the present embodiment will be described.

In the present embodiment, for the purpose of preventing the constituents of the reagent from being adsorbed onto the inner surface of the wells <NUM> of the biomolecule analysis kit 100A, the adsorption inhibitor may be contained in the oil sealing solution <NUM>.

The oil sealing solution <NUM> (see <FIG>) is a solution which can be fed into the space between the base portion <NUM> and the cover portion <NUM> from the inlet portion. The oil sealing solution <NUM> can be selected from the materials which are immiscible with the sample containing the target substance of analysis. As the oil sealing solution <NUM>, mineral oil, FC40 as a fluorine-based liquid, or the like can be used.

Furthermore, in the present embodiment, for the purpose of preventing the constituents of the reagent from being adsorbed onto the inner surface of the wells <NUM> of the biomolecule analysis kit 100A, a wash buffer for wells may be fed into the kit before the reagent is fed into the kit. The buffer may contain the adsorption inhibitor.

The adsorption inhibitor may be a substance adsorbed onto the inner surface of the wells <NUM> in the reaction container <NUM>. By supplying the adsorption inhibitor-containing reagent into the reaction container <NUM>, the adsorption inhibitor is adsorbed onto the inner surface of the reaction container <NUM>. As a result, compared to a case where the reagent does not contain the adsorption inhibitor, the enzyme used for the enzymatic reaction, the nucleic acid or protein that becomes a target of analysis, the labeling substance used for signal detection, and the like are hardly adsorbed onto the inner surface of the reaction container <NUM>.

The adsorption inhibitor contained in the wash buffer may be a nonionic surfactant. Examples of the nonionic surfactant include Tween, glycerol, Triton-X100, and the like. Furthermore, the wash buffer may constitute a portion of the reagent.

It is preferable that the adsorption inhibitor is contained in at least one of the reagents coming into contact with the inside of the reaction container <NUM> during the period between a time before at least one of the enzyme used for the enzymatic reaction, the nucleic acid or protein that becomes a target of analysis, the labeling substance used for signal detection, and the like is initially supplied into the reaction container <NUM> and a time when the signal detection is ended. For example, the adsorption inhibitor may be mixed with a solvent such as a buffer solution for diluting the reagent at a predetermined concentration.

Next, a biomolecule analysis method using the biomolecule analysis kit 100A according to the present embodiment will be described. <FIG> is a flowchart showing the biomolecule analysis method according to the present embodiment.

First, the inlet portion and the outlet portion not shown in the drawing are opened, and a wash buffer <NUM> containing the adsorption inhibitor is fed into the gap between the base portion <NUM> and the cover portion <NUM> through the inlet portion by a dispensing pipette, for example (Step S201 shown in <FIG>). The buffer <NUM> spreads across the gap between the base portion <NUM> and the cover portion <NUM> so as to cover all of the plurality of wells <NUM> (see <FIG>). As a result, within the surface of the base portion <NUM>, low-adsorption structural portions <NUM> having a low-adsorption substance layer <NUM> is formed in a region positioned on the inner surface of the through hole 25a and a region <NUM> positioned between wells adjacent to each other.

The reaction container <NUM> may be filled in advance with the buffer <NUM> instead of being fed with the buffer <NUM>. In this case, the inlet portion and the outlet portion may be sealed with a film or the like such that the buffer <NUM> is sealed in the reaction container <NUM>.

Then, a reagent containing a substance (in the present embodiment, DNA for example) that becomes a target of analysis is fed into the gap between the base portion <NUM> and the cover portion <NUM> through the inlet portion by a dispensing pipette, for example (Step S202 shown in <FIG>). Specifically, the reagent that fills the kit in the present embodiment contains an invader reaction reagent (the detection reaction reagent <NUM>) (<NUM> allele probe, <NUM> of invader oligo, <NUM> FAM-labeled arm, <NUM> MOPS pH <NUM>, <NUM> MgCl<NUM>, and <NUM> U/µL cleavase, and Tween <NUM>) and DNA which is a target substance of analysis. The reagent spreads across the gap between the base portion <NUM> and the cover portion <NUM> so as to cover all of the plurality of wells <NUM> (see <FIG>). The reagent is fed into the gap between the base portion <NUM> and the cover portion <NUM>, and as a result, the buffer <NUM> is discharged from the outlet portion. At this time, if the color of the reagent is different from that of the buffer <NUM>, it is easy to ascertain into which portion the reagent has been fed within the space between the base portion <NUM> and the cover portion <NUM>.

As shown in <FIG>, in the flow channel <NUM> formed by the base portion <NUM> and the cover portion <NUM>, the plurality of wells <NUM> formed by the substrate <NUM> and the micropore array layer <NUM> is arranged. As the reagent is flowed into the wells <NUM>, the buffer <NUM> that fills the plurality of wells <NUM> is sequentially replaced with the reagent.

However, some of the wells <NUM> retain the buffer <NUM> on the inner surface thereof. In this case, the buffer <NUM> that fills the plurality of wells <NUM> is not replaced with the reagent, and the reagent is superposed on the buffer <NUM>. However, because the buffer <NUM> and the reagent are easily intermixed, after the reagent is superposed on the buffer <NUM>, the solute in the reagent is diffused into the buffer <NUM>. Therefore, in both of the well in which the buffer is replaced with the reagent and the well in which the reagent is superposed on the buffer <NUM>, substantially the same reaction occurs.

The amount of liquid that fills the wells <NUM> may be appropriately set according to the number of through holes 25a. Furthermore, the amount and concentration of the liquid added dropwise to the wells <NUM> are adjusted such that approximately single DNA molecule is put into each well <NUM>. For example, in the present embodiment, the amount of liquid that fills the wells <NUM> is <NUM>µL for the entirety of the reaction container, and <NUM>µL of the liquid is distributed into the plurality of wells <NUM>.

Thereafter, as shown in <FIG>, from the inlet portion, the oil sealing solution <NUM> is fed into the flow channel <NUM> formed by the base portion <NUM> and the cover portion <NUM>. In a state where the reagent has been distributed into the buffer, the oil sealing solution <NUM> seals the liquid in the plurality of wells <NUM>, and as a result, the plurality of well <NUM> becomes a plurality of independent reaction chambers <NUM> (reaction containers for nucleic acid detection). That is, in the present embodiment, because the oil sealing solution <NUM> covers each of the wells <NUM>, each of the wells <NUM> becomes independent from each other similarly to the microspaces disclosed in the reference configuration. Furthermore, in the gap between the base portion <NUM> and the cover portion <NUM>, the oil sealing solution <NUM> pushes the liquid on the outside of the plurality of wells <NUM> out of the outlet portion (Step S203 shown in <FIG>).

Then, the array device <NUM> in which each of the wells <NUM> is filled with the invader reaction reagent and DNA is incubated in an oven at <NUM>, for example (Step S204 shown in <FIG>). By the incubation, signal amplification performed in an isothermal invader reaction suitably proceeds.

Subsequently, the array device <NUM> in which each of the wells <NUM> is filled with the invader reaction reagent and DNA is taken out after a preset time, and the number of wells giving off fluorescence and the amount of fluorescence are measured (Step S205 shown in <FIG>).

That is, the biomolecule analysis method using the biomolecule analysis kit 100A according to the present embodiment includes a step (reagent feeding step) of feeding a reagent into a flow channel in a reaction container having the flow channel and a plurality of container-shaped portions such that the plurality of wells is filled with the reagent, and a step (sealing step) of sealing the reagent in the plurality of wells with an oil sealing solution by feeding the oil sealing solution into the flow channel after the reagent feeding step such that the plurality of wells becomes a plurality of independent reaction containers for nucleic acid detection.

In the present embodiment, each reagent contains the adsorption inhibitor, and accordingly, the constituents of the reagent can be prevented from being adsorbed onto the inner surface of the reaction container <NUM> of the biomolecule analysis kit 100A. The adsorption inhibitor may be contained in all of the reagents or some of the reagents.

The reagents may contain a substance reducing the surface tension of the constituents of the reagents instead of the adsorption inhibitor. For example, a surfactant reduces the surface tension of the reagents. Accordingly, in order to fill each of the wells with the reagents, it is effective for the reagents to contain a surfactant.

Next, an example demonstrated for checking the operation and effect of the biomolecule analysis method according to the reference configuration of the present invention will be described. <FIG> shows fluorescence images showing the results of a fluorescence amount measurement test in the present example. <FIG> is a graph showing the results of a fluorescence intensity measurement test in the present example. In <FIG>, the abscissa shows the reaction time, and the ordinate shows the fluorescence intensity. <FIG> is a table showing the results of a reaction time measurement test in the present example. In <FIG>, "Excellent" means that the quantification properties are excellent, and "Poor" means that the quantification properties are poor in the present example.

First, the microspaces <NUM> of the reaction container <NUM> were filled with the invader reaction reagent and <NUM> kinds of artificial synthetic DNA. At this time, the concentration of the artificial synthetic DNA was set to be <NUM> pM at which a single molecule was put into a single well, <NUM> at which <NUM>,<NUM> molecules were put into a single well, and <NUM> at which no molecule was put into a single well.

Then, the reaction container <NUM> was incubated in an oven at <NUM>, and after <NUM> minute (<NUM>), <NUM> minutes (<NUM>), and <NUM> minutes (<NUM>), the condition of the reaction container <NUM> was checked.

As shown in <FIG>, if the DNA concentration is equal to or greater than <NUM> pM, the fluorescence amount varies in almost all of the microspaces <NUM> with respect to the background observed in a case where the DNA concentration is <NUM>, and it is understood that DNA is present.

Next, the reaction container <NUM> filled with the artificial synthetic DNA set as above was incubated in an oven at <NUM>. Furthermore, in order to check the condition of the reaction container after <NUM> minutes, <NUM> minutes, and <NUM> minutes, images of <NUM> wells were selected at each DNA concentration, and the average of the fluorescence amount of <NUM> pixels in each image was determined. Herein, the wells after the reaction were measured using a fluorescence microscope (ZEISS, AX10), an object lens (EC Plan-Neofluar 40x oil NA <NUM>), a light source (LEJ, FluoArc00 <NUM>. 26A Usable with HBO <NUM>), a sensor (Hamamatsu Photonics K. , EM-CCD C9100), a filter (OLYMPUS CORPORATION, U-MNIBA2), and analysis software (Hamamatsu Photonics K. , AQUACOSMOS <NUM>: exposure time <NUM>, EM gain <NUM>, offset <NUM>, binning X1).

As shown in <FIG>, when the DNA concentration was <NUM> pM at which a single molecule was accommodated in a single microspace <NUM>, the fluorescence with intensity distinguishable from the case where the DNA concentration was <NUM> was detected.

Thereafter, the reaction time in the analysis method of the related art was compared with the reaction time in the analysis method of the present invention. For the reaction time measurement test, as methods compared with the present invention, a method of performing a digital PCR reaction using a reagent in an amount of <NUM> nanoliter (nl) × multiple wells (Comparative example <NUM>), a method of performing a PCR reaction using a reagent in an amount of <NUM> microliters (µl) (Comparative example <NUM>), a method of performing PCR + invader reaction using a reagent in an amount of <NUM>µl (Comparative example <NUM>), a method of performing an invader reaction using a reagent in an amount of <NUM>µl (Comparative example <NUM>), and a method of performing a digital ELISA reaction using a reagent in an amount of <NUM> femtoliters (fl) × multiple wells (Comparative example <NUM>) were adopted.

As shown in <FIG>, the reaction time measurement test revealed that in Comparative example <NUM> in which a digital PCR reaction was performed using a reagent in an amount of <NUM> nl × multiple wells, <NUM> minutes were consumed as the reaction time, the temperature condition was nonisothermal, and the quantification properties were excellent. In Comparative example <NUM> in which a PCR reaction was performed using a reagent in an amount of <NUM>µl, <NUM> minutes were consumed as the reaction time, the temperature condition was nonisothermal, and the quantification properties were not excellent. In Comparative example <NUM> in which PCR + invader reaction were performed using a reagent in an amount of <NUM>µl, <NUM> minutes were consumed as the reaction time, the temperature condition was nonisothermal, and the quantification properties were not excellent.

In Comparative example <NUM> in which an invader reaction was performed using a reagent in an amount of <NUM>µl, <NUM> minutes were consumed as the reaction time, the temperature condition was isothermal, and the qualification properties were not excellent. In Comparative example <NUM> in which a digital ELISA reaction was performed using a reagent in an amount of <NUM> fl × multiple wells, <NUM> minutes were consumed as the reaction time, the temperature condition was isothermal, and the quantification properties were excellent.

In contrast, in the present example in which a digital invader reaction was performed using a reagent in an amount of <NUM> fl × multiple wells, only <NUM> minutes were consumed as the reaction time, the temperature condition was isothermal, and the quantification properties were excellent. Therefore, it has become evident that because a digital invader reaction was performed using a reagent in an amount of <NUM> f1 × multiple wells in the present example, the above excellent results were obtained.

Next, an example demonstrated for checking the operation and effect of the biomolecule analysis method according to the embodiment of the present invention will be described. <FIG> is a fluorescence image showing wells in the present example. <FIG> are fluorescence images showing the results of a fluorescence amount measurement test in the present example. <FIG> is a graph showing the results of a fluorescence intensity measurement test in the present example. In <FIG>, the abscissa shows the concentration of Tween <NUM>, and the ordinate shows the fluorescence intensity.

A glass substrate having a thickness of <NUM> was spin-coated with CYTOP (registered trademark) (manufactured by ASAHI GLASS CO. ) and then baked for <NUM> hour at <NUM>. The thickness of the formed CYTOP was <NUM>. After being spin-coated with CYTOP, the substrate was coated with a positive photoresist, and a pattern was formed thereon by using a photomask. Then, by using O<NUM> plasma, CYTOP was dry-etched. In order to remove the residual photoresist on the surface, the surface was washed and rinsed with acetone and ethanol.

As shown in <FIG>, each of the wells (microspaces) formed of CYTOP had a diameter of <NUM> and had a volume that makes it possible to detect a signal within several minutes by the invader reaction. In a single base portion, a well array consisting of <NUM> blocks was provided, and each of the blocks had <NUM>,<NUM> wells. Therefore, a total of <NUM>,<NUM>,<NUM> wells were formed. As shown in <FIG>, a glass plate having a feeding port (inlet portion: not shown in the drawing) was bonded to the base portion by using a double-sided tape which had a thickness of <NUM> and was processed to have a flow channel shape.

The ease of forming liquid droplets by the concentration of Tween <NUM> as a surfactant was checked.

First, through the feeding port, a wash buffer containing the surfactant was fed into the array device for nucleic acid quantification. Then, <NUM>µl of an invader reaction reagent (detection reaction reagent <NUM>: <NUM> allele probe, <NUM> invader oligo, <NUM> FAM-labeled arm, <NUM> MOPS pH <NUM>, <NUM> MgCl<NUM>, <NUM> U/µL cleavase, Tween <NUM>) and DNA which was a substance as a target of analysis were fed into the array device for nucleic acid quantification.

Thereafter, through the feeding port, <NUM>µl of FC40 (oleaginous sealing solution <NUM>) as a fluorine-based liquid was fed into the array device such that the reagent was distributed into and filled the respective wells. By heating the array device on a hot plate at <NUM>, the invader reaction was performed.

Subsequently, by using a fluorescence microscope (manufactured by OLYMPUS CORPORATION), at points in time when <NUM> minutes and <NUM> minutes elapsed at <NUM>, the fluorescence from each well was detected. The exposure time was set to be <NUM> msec for a bright field, <NUM>,<NUM> msec for NIBA, and <NUM>,<NUM> msec for mCherry.

<FIG> show the results obtained by observing each well with a microscope after <NUM> minutes of heating. <FIG> shows the results obtained by observing each well with a microscope after <NUM> minutes of heating.

When the concentration of Tween <NUM> was <NUM>%, fluorescence was also detected from the region positioned between the adjacent wells, and accordingly, digital measurement could not be accurately performed. Presumably, this is because the droplets of the reaction solution in the adjacent wells were bonded to each other, and thus the sample reacted in the region positioned between the adjacent wells. As another reason, it is considered that although the droplets of the reaction solution in the adjacent wells were not bonded to each other, the residual sample on the surface of the base portion positioned between the adjacent wells reacted. In contrast, it was confirmed that if the concentration of Tween <NUM> contained is equal to or greater than <NUM>%, the droplets of the reaction solution are separated from each other.

Furthermore, it was confirmed that if the concentration of Tween <NUM> contained is equal to or greater than <NUM>% in the wells having undergone <NUM> minutes of heating, the droplets of the reaction solution are separated from each other. That is, it is considered that in a case where heating is performed for a long period of time, if the concentration of Tween <NUM> contained is equal to or greater than <NUM>%, the reproducibility is further improved compared to a case where heating is performed for a short period of time.

<FIG> is a graph showing the values of fluorescence intensity associated with the concentration of Tween <NUM>. As the concentration of Tween <NUM> increased, the fluorescence intensity weakened. That is, a behavior in which the increase in the concentration of Tween <NUM> hindered the reaction was confirmed. Therefore, an optimal concentration of Tween <NUM> is assumed to be less than about <NUM>%. In addition, it is considered that, from the viewpoint of costs, the concentration of Tween <NUM> is more preferably equal to or less than <NUM>%.

<NUM>µl of the same reagent was dispensed into a <NUM>-well plate, and the reactivity at a volume of <NUM>µl was detected using LightCycler LC480 (manufactured by Roche Life Science). The temperature condition of the LightCycler was kept constant at <NUM>. With the LightCycler, a reaction was performed in the same composition as described above. As a result, it was confirmed that the increase of the fluorescence signal of the invader reaction was constant regardless of the concentration of Tween <NUM>. Therefore, it was understood that the surfactant contributes not to the improvement of the reactivity of the enzyme but to the stability of the liquid droplets.

The surfactant should be added at such a concentration that can prevent the substance as a target of detection contained in the reagent from being adsorbed onto CYTOP, glass, or the like. In a case where other surfactants such as Triton-X100 are used, the optimal concentration may be changed, but the case of Tween <NUM> can be taken into consideration.

Next, an example demonstrated for checking the operation and effect of the biomolecule analysis method according to the embodiment of the present invention will be described. <FIG> is a view showing the operation and effect of the biomolecule analysis method according to the second embodiment of the present invention. <FIG> are fluorescence images showing the results of a fluorescence amount measurement test in the present example. In <FIG>, "Excellent" in the column of reactivity means that the reactivity is excellent, and "O" in the column of liquid droplet means that fluorescence was not observed in the region between two adjacent wells. Furthermore, in <FIG>, "Δ" in the column of liquid droplet means that, although fluorescence was observed in the region between two adjacent wells, the measurement of concentration was not affected by the fluorescence. In addition, in <FIG>, "X" in the column of liquid droplet means that fluorescence was observed in the region between two adjacent wells, and thus the digital measurement could not be accurately performed in some cases when the region between the two adjacent wells is used.

In the present example, by using the invader reaction reagent and DNA used in the first example, as shown in <FIG>, a reaction was performed by changing the conditions such as whether or not the well will be washed, whether or not a surfactant will be added to the wash buffer, and whether or not a surfactant will be added to the reaction reagent, and from the obtained fluorescence image, the state and reactivity of the liquid droplets were checked. As a surfactant, <NUM>% Tween <NUM> was added to the wash buffer or the reaction reagent. Other conditions were the same as in the first example.

In samples <NUM> and <NUM>, the wells were washed by adding the surfactant to the wash buffer. In samples <NUM> and <NUM>, the wells were washed without adding the surfactant to the wash buffer. In samples <NUM> and <NUM>, the wells were not washed. Furthermore, the surfactant was added to the reaction reagent for the samples <NUM>, <NUM>, and <NUM>. In contrast, the surfactant was not added to the reaction reagent for the samples <NUM>, <NUM>, and <NUM>. The reactivity was excellent in all of the samples. In addition, as is evident from the sample <NUM>, it was understood that, even in a case where the surfactant is not added to the reaction reagent, as long as the wash buffer contains the surfactant, liquid droplets are excellently formed, and the reactivity becomes excellent.

As described above, with the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and with the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, it is possible to rapidly and quantitatively analyze biomolecules by performing an enzymatic reaction in the microspaces <NUM> or the wells <NUM>.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and with the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, an invader method is used as an enzymatic reaction. As a result, PCR amplification is not required, and an isothermal reaction can be performed. Therefore, the device constitution and the analysis procedure can be simplified.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and in the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, a reaction is performed in the microspaces <NUM> and the wells <NUM> each of which has a volume to accommodate a single molecule as a target of analysis. Therefore, the time taken until the signal is saturated can be shortened.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and in the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, the reaction time is shorter and the SN ratio is higher, compared to the related art.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and in the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, the enzymatic reaction is an isothermal reaction. Therefore, the enzymatic reaction that is more stable compared to a nonisothermal reaction can be carried out, and the reproducibility is high.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and in the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, the enzymatic reaction is an invader reaction. Therefore, the time taken for signal detection/determination can be further shortened compare to the process requiring PCR.

In the biomolecule analysis method and the biomolecule analysis kit <NUM> according to the reference configuration and in the biomolecule analysis method and the biomolecule analysis kit 100A according to the embodiment of the present invention, each of the microspaces <NUM> or the wells <NUM> has a volume of equal to or less than <NUM> picoliters. Therefore, the amount of the reagent consumed for analysis can be reduced.

Claim 1:
A biomolecule analysis method comprising:
feeding a reagent (<NUM>) into a flow channel (<NUM>) in a reaction container (<NUM>) which includes a plurality of wells (<NUM>) such that a wash buffer (<NUM>) filled in the plurality of wells (<NUM>) is replaced with the reagent (<NUM>), the reagent (<NUM>) causing an enzymatic reaction which is an isothermal reaction with regard to a target substance of analysis;
sealing the reagent (<NUM>) into the plurality of wells (<NUM>) with an oil sealing solution (<NUM>), thereby forming the plurality of wells (<NUM>) into a plurality of independent reaction containers; and
detecting a signal amplified by the enzymatic reaction.