JP2017209048A - Method for calculating concentration of target biomolecule, bead for calculating target biomolecule concentration, set of beads, and target biomolecule concentration calculation apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for accurately quantifying the concentration of a target biomolecule in a sample.SOLUTION: According to the present invention, there is provided a method for calculating the concentration of a target biomolecule in a sample, the method comprising the steps of: (a) dividing a sample containing a biomolecule into a plurality of fractions comprising a ligand capable of binding to a target biomolecule; (b) adding to the fractions a reagent necessary for nucleic acid amplification; (c) performing a nucleic acid elongation reaction for each of the fractions; (d) measuring the amount of protons generated in each fraction during the nucleic acid elongation reaction; (e) determining the presence or absence of nucleic acid elongation for each fraction based on the measured proton yield; and (f) calculating the concentration of the target biomolecule in the sample based on the number of fractions judged to have nucleic acid elongation.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a target biomolecule concentration calculation method, a target biomolecule concentration calculation bead, a bead set, and a target biomolecule concentration calculation apparatus.

  For high-throughput nucleic acid analysis, a primer is bound to a bead via a photocleavage site, the primer captures the nucleic acid in the sample, the primer is photoinduced cleaved, PCR is performed, and the target nucleic acid is A technique for amplifying and analyzing an amplification product is known (see Patent Document 1). However, in the technique of Patent Document 1, when analyzing an amplification product, it was necessary to collect a bead emulsion and perform sequence analysis or the like.

US Patent Application Publication No. 2013/0190191

One embodiment of the present invention is a method for calculating the concentration of a target biomolecule,
(A) dividing a sample containing a biomolecule into a plurality of fractions containing a ligand capable of binding to the target biomolecule;
(B) adding a reagent necessary for nucleic acid extension to the fraction;
(C) performing a nucleic acid extension reaction for each of the fractions;
(D) measuring a proton generation amount of each fraction during the nucleic acid extension reaction;
(E) determining the presence or absence of nucleic acid elongation for each fraction based on the measured proton generation amount;
(F) calculating the concentration of the target biomolecule in the sample based on the number of fractions determined to have nucleic acid elongation;
Is a method for calculating the concentration of a target biomolecule including

  One embodiment of the present invention is a target biomolecule concentration calculating bead in which a ligand corresponding to the electrical property is immobilized on a bead body having a predetermined electrical property.

  A set of beads for calculating the concentration of a target biomolecule, including a plurality of beads for calculating the target biomolecule concentration, in which a ligand corresponding to the electric property is immobilized on a bead body having predetermined electric characteristics It is.

  One embodiment of the present invention includes a detection unit that detects protons of each fraction during a nucleic acid extension reaction performed for each fraction of a sample containing a biomolecule divided into a plurality of fractions, and the detection Based on the amount of protons detected by the part, based on the number of fractions determined by the nucleic acid extension determination unit for determining the presence or absence of nucleic acid amplification for each fraction, and the nucleic acid extension determination unit determined to have nucleic acid extension, A target biomolecule concentration calculation apparatus comprising: a target biomolecule concentration calculation unit that calculates a concentration of a target biomolecule in the sample.

It is a schematic diagram of an example of the bead body in 1st Embodiment. It is a schematic diagram of an example of the state which the target nucleic acid sequence annealed to the bead body in 1st Embodiment. It is a schematic diagram of an example of arrangement | positioning of the bead body to the reaction tank in this embodiment. It is a schematic diagram of the modification of arrangement | positioning of the bead body to the reaction tank in this embodiment. It is a schematic diagram of an example of the sensor in this embodiment. It is a figure which shows an example of a structure of the target nucleic acid sequence density | concentration calculation apparatus in this embodiment. It is a schematic diagram which shows an example of the bead body in 2nd Embodiment.

<< First Embodiment >>
As an example, the target biomolecule in the method of the present embodiment is a nucleic acid having a desired target nucleic acid sequence. The ligand capable of binding to the target biomolecule is a primer (hereinafter referred to as “target nucleic acid primer”) that can anneal to the target nucleic acid sequence. In the present embodiment, a case where the target biomolecule is a nucleic acid will be described.

  The method for calculating the concentration of a target nucleic acid sequence according to this embodiment comprises (a1) dividing a sample containing a nucleic acid into a plurality of fractions containing a target nucleic acid primer that can be annealed with the target nucleic acid sequence, and (b1) Adding a reagent necessary for nucleic acid extension to the fraction; (c1) performing a nucleic acid extension reaction of the target nucleic acid sequence for each fraction; and (d1) each fraction in the nucleic acid extension reaction. A step of measuring the amount of proton generation; (e1) a step of determining the presence or absence of nucleic acid extension for each fraction based on the measured amount of proton generation; and (f1) a fraction determined to have nucleic acid extension. And calculating the concentration of the target nucleic acid sequence in the sample based on the number of each. Hereinafter, each step will be described.

<Embodiment 1>
[Split sample into multiple fractions]
Step (a1) in the present embodiment is a step of dividing a sample containing nucleic acid into a plurality of fractions. In this embodiment, a sample containing nucleic acid is brought into contact with a plurality of beads on which a primer capable of annealing to a target nucleic acid sequence is immobilized, and the beads are placed in a reaction vessel one by one, whereby the sample is divided into a plurality of fractions. To divide.
[Annealing with target nucleic acid primer]
In this embodiment, the method includes a step of bringing a sample containing nucleic acid into contact with a plurality of beads on which a target nucleic acid primer is immobilized.

  First, the beads used in this embodiment will be described with reference to FIG. In FIG. 1, 1 is a bead, 2 is a target nucleic acid primer, and 100 is a bead body in which a target nucleic acid primer 2 is immobilized on a bead 1.

  In this embodiment, the beads 1 are used as a carrier for immobilizing the target nucleic acid primer 2. As an example, the shape of the beads 1 is a spherical shape or a shape close thereto. The shape of the beads 1 is not limited to this. As an example, the size of the beads 1 has an average diameter of 1 to 250 μm. Moreover, as an example, the size of the beads 1 has an average diameter of 30 to 80 μm.

  The material of the beads 1 is not particularly limited, and examples thereof include silicon, titanium dioxide, aluminum oxide, glass, polystyrene, cellulose, and polyamide. Moreover, you may use a magnetic bead from a viewpoint of arrangement | positioning to a reaction tank. The beads 1 may be composed of not only one kind of substance but also two or more kinds of substances. The beads 1 may be surface-coated. When using dielectrophoresis for arranging the bead body 100 in the reaction vessel, a ferroelectric material having a high dielectric constant is used as the material of the bead 1. Examples of such ferroelectric materials include barium titanate and potassium dihydrogen phosphate.

  As shown in FIG. 1, a target nucleic acid primer 2 is immobilized on a bead 1. Immobilization of these nucleic acids to the beads 1 can be performed using a known method. For example, by immobilizing the target nucleic acid primer 2 with biotin and coating the surface of the bead 1 with avidin, both nucleic acids can be immobilized on the bead 1 using an avidin-biotin bond. Further, the target nucleic acid primer 2 is modified with a functional group such as an amino group, formyl group, or SH group, and the beads 1 are fixed by surface treatment with a silane coupling agent having an amino group, formyl group, epoxy group, or the like. May also be performed.

  The target nucleic acid primer 2 has a sequence that can be annealed to the target nucleic acid sequence. As an example, the target nucleic acid primer 2 is a nucleic acid having a sequence complementary to a part of the target nucleic acid sequence. As an example, the target nucleic acid primer 2 is DNA. The target nucleic acid primer 2 anneals to the target nucleic acid sequence and induces a nucleic acid extension reaction using the target nucleic acid sequence as a template. The sequence of the target nucleic acid primer 2 can be appropriately selected according to the target nucleic acid.

  In FIG. 1, only one molecule of the target nucleic acid primer 2 is immobilized on each bead 1, but the number of molecules of the target nucleic acid primer 2 immobilized on the bead 1 is one. Without limitation, two or more molecules can be immobilized. As an example, in this embodiment, two or more molecules of the target nucleic acid primer 2 are immobilized. The target nucleic acid primer 2 may be immobilized at a density that does not hinder the annealing and extension reaction of the target nucleic acid according to the surface area of the beads 1. When two or more target nucleic acid primers 2 are immobilized on the beads 1, the target nucleic acid primers 2 to be immobilized all target the same target nucleic acid sequence.

  In this step, the bead body 100 in which the target nucleic acid primer 2 is immobilized on the beads 1 is brought into contact with a sample containing nucleic acids. In this embodiment, the sample containing a nucleic acid is not specifically limited, For example, the sample etc. which contain the nucleic acid extract etc. from a biological sample or an environmental sample can be used. As an example, the nucleic acid contained in the sample is DNA. Examples of DNA include cDNA and genomic DNA. The nucleic acid contained in the sample is not limited to DNA, but may be RNA or modified nucleic acid.

  The contact between the bead body 100 and the sample containing the nucleic acid can be performed, for example, by mixing the bead body 100 and the sample containing the nucleic acid. At this time, it is necessary that the number of the bead bodies 100 brought into contact with the sample is sufficiently larger than the number of target nucleic acid sequences predicted to be included in the sample. As an example, the mixture is placed under conditions that allow the target nucleic acid primer 2 to anneal with the target nucleic acid. The annealing conditions can be appropriately set according to the length of the target nucleic acid primer 2 and the like. When the target nucleic acid of the target nucleic acid primer 2 is contained in the sample, the target nucleic acid primer 2 anneals to the target nucleic acid sequence 3 as shown in FIG. Be captured. However, when the number of the bead bodies 100 is sufficiently larger than the number of target nucleic acid sequences, the target nucleic acid primer 2 is mixed with those that anneal to the target nucleic acid sequence 3 and those that do not anneal. When the concentration of the target nucleic acid sequence 3 in the sample is high, the number of target nucleic acid primers 2 that anneal to the target nucleic acid sequence 3 increases. On the other hand, when the concentration of the target nucleic acid sequence 3 in the sample is low, the number of target nucleic acid primers 2 that ligate to the target nucleic acid sequence 3 is reduced. In addition, the number of the bead bodies 100 brought into contact with the sample can be appropriately selected according to the type of the sample. If the target nucleic acid concentration contained in the sample is expected to be high, the sample may be diluted.

[Placement of beads in the reaction vessel]
Next, the bead body 100 brought into contact with the sample containing nucleic acid is placed in a separate reaction tank for each bead. The arrangement of beads in the reaction tank will be described with reference to FIG. In FIG. 3, 20 is a substrate for bead arrangement, and 21 is a reaction vessel. In this example, the bead arrangement substrate 20 includes m × n reaction vessels 21 (m and n are natural numbers). This reaction vessel 21 is also referred to as well W.

  In addition, the reaction vessel 21 includes a sensor 30 for detecting the hydrogen concentration. Of the reaction vessel 21, for example, the well W11 includes a sensor 30-11. The well Wmn includes a sensor 30-mn. As an example, the sensor 30 is disposed on the bottom surface of the reaction vessel 21. As an example, the sensor 30 is an ion sensitive field effect transistor (ISFET).

  In the present embodiment, the material for the bead arrangement substrate 20 is not particularly limited, but may be glass, silicon, polymer, or the like. When an elastomer material is used for the bead arrangement substrate 20, the entire bead arrangement substrate of the reaction tank 21 generated when particles such as fine dust are sandwiched between the reaction tank 21 and the bead arrangement substrate 20. There is an advantage that an adverse effect on the adhesiveness with 20 is avoided by local deformation of the elastomer.

  A plurality of reaction vessels 21 are arranged on the bead arrangement substrate 20. The number of reaction vessels 21 to be arranged needs to be equal to or more than the number of bead bodies 100 used in the annealing step with the target nucleic acid primer. As an example, the size of the reaction vessel 21 is slightly larger than the bead 1. As an example, the size of the reaction tank 21 is about 1 to 2 times the size of the beads 1 and is large enough to accommodate one bead 1. Note that the surface of the bead-arranging substrate 20 and the inner wall of the reaction vessel 21 are blocking agents that prevent non-specific adsorption of nucleic acids, such as polyethylene glycol (PEG), 2-metheoyloxyoxyphosphorylcholine (MPC), etc. It may be coated with. By providing such a coating, nonspecific adsorption of nucleic acids to the surface of the bead arrangement substrate 20 and the inner wall of the reaction vessel 21 can be suppressed. Moreover, as an example, the inner wall of the reaction vessel 21 is hydrophilized. Since the inner wall is hydrophilized, the efficiency of filling the bead body 100 into the reaction vessel 21 is improved when the dispersion liquid of the bead body 100 is dropped onto the bead placement substrate 20.

In the step (a1) of the present embodiment, as shown in FIG. 3, a plurality of bead bodies 100 brought into contact with a sample containing nucleic acid are arranged one by one in the reaction vessel 21. The method for disposing the bead body 100 in the reaction vessel 21 is not particularly limited. For example, a liquid in which the bead body 100 is dispersed is dropped onto the bead disposition substrate 20 and stirred, shaken, centrifuged, or the like. Thus, the bead body 100 may be guided to the reaction vessel 21. Alternatively, the bead arrangement substrate 20 may be immersed in the dispersion of the bead body 100, and means such as stirring, shaking, and centrifugation may be used.
In the reaction vessel 21, for example, a bead body 100-11 is disposed in the well W11. A bead body 100-mn is disposed in the well Wmn.

  When magnetic beads are used for the beads 1, a magnetic plate and a magnet can be arranged under the substrate material of the bead arrangement substrate 20, and the bead body 100 can be guided to the reaction vessel by the magnetic force of the magnet. . In this case, by moving the magnet under the substrate material in the direction parallel to the bead placement substrate 20, the bead bodies are dispersed, and the efficiency of filling the bead bodies into the reaction vessel 21 is improved. The strength of the magnetic field applied to the bead arranging substrate 20 by the magnet is not particularly limited, but is 100 to 10,000 gauss. In addition, since the magnetization of the magnetic plate remains after the magnet is removed, the bead body 100 can continue to maintain a stable arrangement. As a material of such a magnetic body, metals such as nickel, nickel alloy, iron and iron alloy can be used.

[Placement of beads in reaction vessel: modified example]
In this modification, the bead body 100 has different electrical characteristics for each type of sequence of the target nucleic acid primer 2. The electrical characteristics include impedance, dielectric constant, conductivity, and the like. In this modification, “dielectric constant” will be described as an example of electrical characteristics. In this example, in the step of placing the bead body 100 in the reaction vessel 21, sorting by dielectrophoresis may be performed as shown in FIG. For example, the dielectric constant of the bead body 100A is the dielectric constant εA, the dielectric constant of the bead body 100B is the dielectric constant εB, the dielectric constant of the bead body 100C is the dielectric constant εC, and the dielectric constant of the bead body 100D is the dielectric constant εD. is there.
A method for producing beads having different dielectric constants can be realized, for example, by producing beads having different ferroelectric content in the bead material.

Here, a method of performing sorting according to the dielectric constant using dielectrophoresis will be described. The dielectrophoretic force acting on the dielectric particles depends on the strength of the external electric field applied, the frequency, and the dielectric constant of the particles. Considering the case where a constant voltage and frequency are applied between the electrodes, dielectrophoretic forces of different magnitudes act on beads having different dielectric constants. If an appropriate frequency is set, the dielectrophoretic force acts in the direction of exclusion from the strong electric field portion (negative dielectrophoresis). For details of the dielectrophoresis theory, refer to “AC Electrokinetic: Colloids and Nanoparticles, Research Studies Pr” and the like. Therefore, as shown in FIG. 4, when a pair of electrodes are arranged in the flow path and a frequency at which a negative dielectrophoretic force acts is applied, a force acts on the beads in an oblique direction with respect to the flow. As a result, the trajectory of the beads in the flow path changes. Furthermore, since the magnitude of the dielectrophoretic force that works depending on the dielectric constant of the beads changes, the beads can be moved along different trajectories for each type of beads, and as a result, sorting is possible for each type of bead. At this time, by providing a plurality of electrode pairs to which different voltages and frequencies are applied (for example, by providing the electrode pair EPA and the electrode pair EPB), the types of beads that can be sorted can be increased.
That is, according to this modification, the type of sequence of the target nucleic acid primer 2 fixed to the bead body 100 can be selected based on the dielectric constant.

  By using the dielectrophoresis and arranging the bead body 100 in the reaction vessel 21 for each type of the target nucleic acid primer 2, a plurality of target nucleic acid sequences can be simultaneously quantified.

[Addition of reagents necessary for nucleic acid extension reaction]
Step (b1) is a step of adding a reagent necessary for the nucleic acid extension reaction to the fraction.
In the present embodiment, examples of reagents necessary for the nucleic acid extension reaction include DNA polymerase, deoxynucleoside triphosphate, an appropriate buffer, and the like. As an example, the DNA polymerase is a heat-resistant DNA polymerase such as Taq polymerase or a strand displacement DNA polymerase. The reagent necessary for the nucleic acid extension reaction can be appropriately selected according to the method of the nucleic acid extension reaction performed in the step (d1) described later. As an example, some reagents such as DNA polymerase may not be added in this step but may be bonded to the inner wall of the reaction vessel 21 in advance.

  As an example, when a nucleic acid amplification reaction such as a PCR method or an isothermal amplification method is performed as a nucleic acid extension reaction, a reverse primer can be added in this step. In addition, when the bead body 100 is arranged in the reaction vessel 21 using dielectrophoresis as in [Placement of beads in the reaction vessel: Modification], a plurality of types corresponding to the dielectric constant of the beads 1 are used. A target nucleic acid primer is immobilized on the beads 1. That is, in this case, there are a plurality of target nucleic acid sequences corresponding to the dielectric constant of the beads 1. Thus, when there are a plurality of target nucleic acid sequences, the reverse primer may be a universal primer common to all target nucleic acid sequences, or may be a primer specific to each target nucleic acid sequence.

  As an example, the reverse primer can be immobilized on the beads 1. Further, when the target nucleic acid sequence is one kind, the reverse primer can be immobilized on the inner wall of the reaction tank 21. Even when there are a plurality of target nucleic acid sequences, when the reverse primer is a universal primer, for example, the universal reverse primer can be immobilized on the inner wall of the reaction vessel 21. When the reverse primer is immobilized on the bead 1 or the inner wall of the reaction vessel 21, the reverse primer has, for example, a light cutting site on the bead 1 side or the inner wall side of the reaction vessel 21. The photocleavable site refers to a group having a property of being cleaved when irradiated with light such as ultraviolet rays. Examples of the group using such a group include PC Linker Phosphoramidite (Glen research) and a nucleic acid containing fullerene. The composition for photocleavage of (Nucleic acid photocleavage composition: Japanese Patent Application Laid-Open No. 2005-245223), Patent Document 1, and the like can be mentioned. Alternatively, the reverse primer may have a single-stranded nucleic acid cleaving enzyme cleavage site. The single-stranded nucleic acid cleaving enzyme cleavage site refers to a nucleic acid group that can be cleaved by a single-stranded nucleic acid cleaving enzyme such as deoxyribonuclease and ribonuclease, and includes nucleotides and derivatives thereof. Examples of such a nucleic acid group include deoxyiocin recognized by endonuclease V. Prior to the start of the nucleic acid amplification reaction, it is possible to efficiently carry out the nucleic acid amplification reaction by carrying out light irradiation or single-stranded nucleic acid cleaving enzyme treatment to separate the reverse primer from the bead 1.

[Nucleic acid elongation reaction]
Step (c1) is a step of performing a nucleic acid extension reaction for each fraction.
The nucleic acid extension reaction can be performed by a known method using DNA polymerase or the like. The conditions for the nucleic acid extension reaction can be appropriately set according to the method used. For example, the nucleic acid extension reaction may be performed while maintaining the temperature in the reaction vessel 21 at about 55 to 70 ° C.

  One bead body 100 is arranged in the reaction vessel 21. In the bead body 100 in which the target nucleic acid primer 2 is annealed to the target nucleic acid sequence 3, a nucleic acid extension reaction occurs using the target nucleic acid sequence 3 as a template. On the other hand, in the bead body 100 in which the target nucleic acid primer 2 is not annealed to the target nucleic acid sequence 3, no nucleic acid extension reaction occurs.

  In the present embodiment, the nucleic acid extension reaction is not necessarily a nucleic acid amplification reaction. When the density of the target nucleic acid primer 2 immobilized on the beads 1 is high, protons sufficient for detection are generated in one extension reaction. When the nucleic acid extension reaction is performed only once, it is not necessary to add a reverse primer to the reaction tank 21.

  On the other hand, in this embodiment, a nucleic acid amplification reaction can also be performed. A publicly known method can be used for the method of nucleic acid amplification reaction. As an example, a PCR method is used as a method for nucleic acid amplification reaction. As another example, an isothermal amplification method such as the LAMP method is used. As an example, digital PCR or digital isothermal amplification is used as a method for nucleic acid amplification reaction. In addition, when performing nucleic acid amplification reaction, it is necessary to make a reverse primer exist in the reaction tank 21 as mentioned above.

[Measurement of proton generation amount]
Step (d1) is a step of measuring the proton generation amount of each fraction during the nucleic acid extension reaction. When the nucleic acid is extended by one base by the nucleic acid extension reaction, one molecule of proton is generated. The amount of proton generation in the reaction vessel 21 is detected by the sensor 30. As an example, the sensor 30 is an ISFET. Examples of the ISFET that can be used in the present embodiment include those described in International Publication No. WO2008 / 107014.

  A measurement example of the proton generation amount when the sensor 30 is an ISFET will be described. In FIG. 5, the structure of the sensor 30 with which each reaction tank 21 is equipped is shown. The sensor 30 includes a gate insulating film GIF, an N-type semiconductor SN (source src), a source terminal TS, an N-type semiconductor SN (drain drn), a drain terminal TD, a P-type semiconductor SP, and a reference electrode ER. , A gate power supply VG, a bias power supply VB, and a current detector CD. Since the configuration of each part is the same as that of a known ISFET, description thereof is omitted. The sensor 30 measures the amount of proton generation by detecting a drain current having a current value corresponding to the interface potential generated between the solution and the gate insulating film GIF by the current detector CD.

[Determination of nucleic acid elongation]
Step (e1) is a step of determining the presence or absence of nucleic acid elongation for each fraction based on the measured proton generation amount. The presence or absence of nucleic acid elongation in the reaction vessel 21 is determined based on whether or not the measured value of the proton generation amount in the reaction vessel 21 by the sensor 30 has increased. When the amount of proton generation in the reaction tank 21 increases, it is determined that nucleic acid elongation has occurred. When no increase in the amount of proton generation in the reaction tank 21 is detected, it is determined that no nucleic acid extension has occurred. .

In the present embodiment, a sensor 30 arranged for each reaction tank 21 detects a change in the amount of protons generated in each reaction tank 21, and determines whether or not there is nucleic acid extension for each reaction tank 21.
The sensor 30 may be configured to detect the number of the bead bodies 100 in which the nucleic acid elongation has occurred in the reaction vessel 21 according to the magnitude of the drain current detected by the current detector CD.

[Calculation of concentration of target nucleic acid sequence]
Step (f1) is a step of calculating the concentration of the target nucleic acid sequence in the sample based on the number of fractions determined to have nucleic acid amplification. In the step (a1), one bead body 100 is disposed in each reaction vessel 21. That is, the number of reaction vessels 21 corresponds to the number of bead bodies 100. By determining the number of reaction tanks 21 determined to have nucleic acid amplification with respect to the number of reaction tanks 21 in which the bead bodies 100 are arranged, the presence of nucleic acid amplification with respect to the number of bead bodies 100 brought into contact with a sample containing nucleic acids. The ratio of the determined bead body 100 can be obtained. The ratio of the bead body 100 determined to have nucleic acid amplification to the number of bead bodies 100 brought into contact with the sample containing nucleic acid indicates the concentration of the target nucleic acid sequence in the sample. That is, the concentration of the target nucleic acid sequence in the sample can be obtained by counting the number of reaction vessels 21 determined to have nucleic acid amplification in step (e1).

  In addition, when arranging the bead body 100 in the reaction vessel 21 using dielectrophoresis as in [Placement of beads in the reaction vessel: modification], a plurality of types of targets corresponding to the dielectric constant of the beads 1 are used. Nucleic acid primers are immobilized on the beads 1. That is, in this case, there are a plurality of target nucleic acid sequences corresponding to the dielectric constant of the beads 1. Thus, when there are a plurality of target nucleic acid sequences, the concentration of the target nucleic acid sequence in the sample is calculated for each type of target nucleic acid sequence. For example, in the example of FIG. 4, target nucleic acid primers 2 that can anneal to different target nucleic acid sequences 3 are immobilized on the bead bodies 100A, 100B, 100C, and 100D. Here, the target nucleic acid sequences 3 that can be annealed by the target nucleic acid primer 2 immobilized on the bead bodies 100A, 100B, 100C, and 100D are referred to as target nucleic acid sequences 3A, 3B, 3C, and 3D, respectively. In this case, the concentration of the target nucleic acid sequence 3A in the sample is calculated by obtaining the ratio of the number of reaction tanks 21 determined to have nucleic acid amplification in all the reaction tanks 21 in which the bead body 100A is arranged. be able to. Similarly, the concentration of the target nucleic acid sequence 3B in the sample is calculated by obtaining the ratio of the number of reaction tanks 21 determined to have nucleic acid amplification in all the reaction tanks 21 in which the bead body 100B is arranged. be able to. Similarly, the concentration of the target nucleic acid sequences 3C and D in the sample is the same as the number of reaction vessels 21 determined to have nucleic acid amplification in each of the reaction vessels 21 in which the bead bodies 100C and 100D are arranged. It can be calculated by obtaining the ratio.

<Embodiment 2>
In the first embodiment, the beads 1 are used when the sample is divided. However, the sample may be divided without using the beads 1. For example, the sample can be divided by adding the target nucleic acid primer 2 to a sample containing nucleic acid, performing an annealing reaction, and then distributing the sample to each reaction tank 21. Alternatively, the target nucleic acid primer 2 may be immobilized in each reaction tank 21 in advance, and the sample may be distributed to each reaction tank 21. In this case, the capacity of the reaction vessel 21 can be 1 fL to 1 mL, and can be 1 pL to 1 μL.

  Nucleic acid elongation reaction, measurement of proton generation amount, determination of presence / absence of nucleic acid amplification, and calculation of target nucleic acid sequence concentration can be performed in the same manner as in the first embodiment.

  In the method of this embodiment, sample division, nucleic acid extension reaction, and target nucleic acid sequence concentration calculation may be performed using standard digital PCR or digital isothermal amplification techniques.

  According to the method of this embodiment, the concentration of the target nucleic acid sequence of a sample containing nucleic acid can be accurately measured without using a technique such as fluorescent staining.

  The method of this embodiment can be used for detection / identification of pathogen genes causing infectious diseases, detection / identification of disease-related genes such as cancer, expression analysis of disease-related genes, etc. It is useful for diagnosis of diseases and monitoring of therapeutic effects.

<< Second Embodiment >>
As an example, the target biomolecule in the method of the present embodiment is a desired protein. The ligand capable of binding to the target biomolecule is an antibody capable of binding to the target protein (hereinafter referred to as “target antigen detection antibody”). In this embodiment, a case where the target biomolecule is a protein will be described.

The target protein concentration calculation method of the present embodiment includes (a2) a step of dividing a sample containing a protein into a plurality of fractions containing an antibody capable of binding to the target protein, and (b2) a nucleic acid in the fraction A step of adding a reagent necessary for extension; (c2) a step of performing a nucleic acid extension reaction of a target nucleic acid sequence for each fraction; and (d2) measuring a proton generation amount of each fraction during the nucleic acid extension reaction. And (e2) determining the presence or absence of nucleic acid extension for each fraction based on the measured amount of proton generation; and (f2) based on the number of fractions determined to have nucleic acid extension. And calculating the concentration of the target protein in the sample. Hereinafter, each step will be described.
<Embodiment 1>
[Split sample into multiple fractions]
Step (a2) in the present embodiment is a step of dividing a sample containing protein into a plurality of fractions. In this embodiment, a sample containing a protein is brought into contact with a plurality of beads on which an antibody capable of binding to a target protein is immobilized, and the beads are placed in a reaction tank one by one, thereby dividing the sample into a plurality of fractions. To do.
[Antigen-antibody reaction with target protein]
In this embodiment, the method includes a step of bringing a sample containing a protein into contact with a plurality of beads on which a target antigen detection antibody is immobilized.

  First, the beads used in this embodiment will be described with reference to FIG. In FIG. 7, 1 is a bead, 5 is an antibody for target antigen detection, 100a is a bead body in which the antibody 5 for target antigen detection is immobilized on the bead 1, and 400 is a target protein.

In this embodiment, instead of the target nucleic acid primer 2 of the first embodiment, a target antigen detection antibody 5 is immobilized on the beads 1. The target antigen detection antibody 5 is an antibody that can specifically bind to the target protein of interest. The target antigen detection antibody 5 can be obtained by a known antibody acquisition method. The target antigen detection antibody 5 may be derived from any animal. Antibodies generally used in the art such as mouse antibody, chicken antibody, rabbit antibody and the like can be used. The target antigen detection antibody 5 may be a recombinant antibody such as a chimeric antibody or a humanized antibody. Further, the target antigen detection antibody 5 may be an antibody fragment such as a Fab fragment, an F (ab ′) ₂ fragment, an Fv fragment, or a single chain antibody. As used herein, the term “antibody” encompasses those antibody fragments in which specificity and binding to the antigen are maintained.

  Immobilization of the target antigen detection antibody 5 to the beads 1 can be performed using a known method. For example, as with the target nucleic acid primer 2 of the first embodiment, immobilization using an avidin-biotin bond can be performed. Further, by modifying the target antigen detection antibody 5 with a functional group such as an amino group, formyl group, or SH group, and surface-treating the beads 1 with a silane coupling agent having an amino group, formyl group, epoxy group, or the like, Immobilization may be performed.

  The target antigen detection antibody 5 can fix a plurality of molecules to the beads 1. As an example, two or more molecules of the target antigen detection antibody 5 are immobilized on the beads 1. The target antigen detection antibody 5 only needs to be immobilized at a density that does not hinder the binding with the target protein 400 according to the surface area of the beads 1. When two or more molecules of target antigen detection antibody 5 are immobilized on beads 1, target antigen detection antibodies 5 immobilized on one bead 1 all target the same protein.

  The beads 1 are the same as in the first embodiment. When the bead body 100a is arranged in the reaction tank using dielectrophoresis, the beads 1 may have different dielectric constants for each type of target antigen detection antibody 5 to be immobilized.

  In the step (a2) of this embodiment, the bead body 100a in which the target antigen detection antibody 5 is immobilized on the beads 1 is brought into contact with a sample containing a protein. In this embodiment, the sample containing protein is not specifically limited, For example, the sample containing the protein extract from a biological sample, an environmental sample, etc. can be used. The protein contained in the sample is not limited to a natural protein, and may be a modified protein or a recombinant protein.

  The contact between the bead body 100a and the sample containing the protein can be performed, for example, by mixing the bead body 100a and the sample containing the protein. As an example, the mixture of the bead body 100 and the sample containing the protein is placed under conditions that allow the target antigen detection antibody 5 to bind to the target protein. Binding conditions can be appropriately set according to the type of target antigen detection antibody 5 and the like.

  In FIG. 7, when the bead body 100 a is brought into contact with a sample containing a protein, the target antigen detection antibody 5 binds to the target protein 400 when the target protein 400 is contained in the sample. That is, the target protein 400 binds to the target antigen detection antibody 5 and is captured by the bead body 100a. On the other hand, when the target protein 400 is not contained in the sample, nothing binds to the target antigen detection antibody 5. Therefore, when a plurality of bead bodies 100a are brought into contact with the sample, the bead body 100a in which the target antigen detection antibody 5 is bound to the target protein 400 and the target antigen detection antigen 5 are not bound to the target protein 400. A mixture with the bead body 100a is formed. Note that the number of bead bodies 100a to be brought into contact with the sample can be appropriately selected according to the number of target proteins to be detected.

[Placement of beads in the reaction vessel]
Next, the bead body 100a brought into contact with the sample containing the protein is placed in a separate reaction tank for each bead. The arrangement of the bead body 100a in the reaction vessel can be performed in the same manner as in the step (a1) of the first embodiment. It can carry out similarly about the modification described by the process (a1) of 1st Embodiment.

[Addition of reagents necessary for nucleic acid extension reaction]
Step (b2) is a step of adding a reagent necessary for the nucleic acid extension reaction to the fraction.

  In step (b2) of the present embodiment, in addition to the reagent added in step (b1) of the first embodiment, secondary antibody 301 to which signal generating nucleic acid 310 is bound is added. The secondary antibody 301 is an antibody that can specifically bind to the target protein 400. The secondary antibody 301 may be the same antibody as the target antigen detection antibody 5 or a different antibody. As an example, the secondary antibody 301 is an antibody that binds to the same protein as the target antigen detection antibody 5 and has a different epitope.

  For example, as shown in FIG. 7, a signal generating nucleic acid 310 is bound to the secondary antibody 301. As an example, the binding between the secondary antibody 301 and the signal generating nucleic acid 310 can be performed using an avidin-biotin bond.

  As an example, the signal generating nucleic acid 310 includes a primer annealing region 311 and a signal generating sequence 312 as shown in FIG. The primer annealing region 311 is located on the 3 ′ side of the signal generating nucleic acid 310. For example, the signal generating nucleic acid 310 can have the same sequence for all types of secondary antibodies. In addition, in the signal generating nucleic acid 310, for example, the primer annealing region 311 has the same sequence for all types of secondary antibodies. The sequence of the signal generating nucleic acid 310 is not particularly limited and can be appropriately selected. In FIG. 7, reference numeral 300 denotes a complex probe in which the signal generating nucleic acid 310 is bound to the secondary antibody 301.

  In this step, as an example, the complex probe 300 is added to the reaction vessel 21 before adding other reagents necessary for the nucleic acid extension reaction. In this case, as an example, if the bead 1 is a magnetic bead, the complex probe 300 that is not bound to the target protein 400 is removed from the reaction vessel 21 by fixing the bead 1 to the reaction vessel 21 and washing the reaction vessel 21. 21 can be removed. The complex probe 300 may be added to the sample or the reaction vessel 21 at any timing as long as it is before the nucleic acid extension reaction in the step (d2) described later.

  In this step, in addition to the complex probe 300, a signal generating primer 320 is further added. The signal generating primer 320 is added to the reaction vessel 21 after the complex probe 300 is added. As an example, the complex probe 300 not bound to the target protein 400 is removed from the reaction vessel 21 by washing before the addition of the signal generating primer 320. As an example, the signal generating primer 320 can be added to the reaction vessel 21 together with other reagents necessary for nucleic acid extension.

  The reagents necessary for the nucleic acid extension reaction other than the above are the same as those in the step (b1) of the first embodiment.

[Nucleic acid elongation reaction]
Step (c2) is a step of performing a nucleic acid extension reaction for each fraction.
This process can be performed similarly to the process (c1) of 1st Embodiment. In this step, as shown in FIG. 7, a nucleic acid extension reaction using the signal generating sequence 312 as a template occurs. Similar to the first embodiment, when the target protein 400 is not bound to the target antigen detection antibody 5, the nucleic acid elongation reaction does not occur.

[Detection of proton concentration]
Step (d2) is a step of measuring the amount of proton generation in each reaction tank of the nucleic acid extension reaction. This process can be performed similarly to the process (e1) of 1st Embodiment.

[Calculation of concentration of target nucleic acid sequence]
Step (f2) is a step of calculating the concentration of the target protein in the sample based on the number of fractions determined to have nucleic acid elongation. This process can be performed similarly to the process (f1) of 1st Embodiment. That is, by obtaining the number of reaction tanks 21 determined to have nucleic acid amplification with respect to the number of reaction tanks 21 in which the bead bodies 100a are arranged, nucleic acid amplification with respect to the number of bead bodies 100 brought into contact with the sample containing protein. The ratio of the bead body 100 determined to be present can be obtained. The ratio of the bead body 100 determined to have nucleic acid amplification to the number of the bead bodies 100a brought into contact with the sample containing the protein indicates the concentration of the target protein in the sample. That is, the concentration of the target protein in the sample can be determined by counting the number of reaction vessels 21 determined to have nucleic acid amplification in step (e2).

  In addition, when the bead body 100 is arranged in the reaction vessel 21 by using dielectrophoresis as in [Placement of beads in the reaction vessel: modification] described in the first embodiment, the dielectric constant of the beads 1 is used. A plurality of target antigen detection antibodies 5 corresponding to the above are immobilized on the beads 1. That is, in this case, there are a plurality of target proteins corresponding to the dielectric constant of the beads 1. Thus, when there are a plurality of target nucleic acid sequences, the concentration of the target protein in the sample is calculated for each type of target antigen detection antibody 5 as in the target nucleic acid sequence of the first example.

<Embodiment 2>
Similar to the first embodiment, the sample may be divided without using the beads 1. For example, the sample can be divided by adding the target antigen detection antibody 5 to the sample containing the protein, performing the antigen-antibody reaction, and then distributing the sample to each reaction tank 21. Alternatively, the target antigen detection antibody 5 may be immobilized in each reaction tank 21 in advance, and the sample may be distributed to each reaction tank 21. In this case, the capacity of the reaction vessel 21 can be 1 fL to 1 mL, and can be 1 pL to 1 μL as an example.

  Addition of reagents necessary for nucleic acid extension reaction, nucleic acid extension reaction, measurement of proton generation amount, determination of presence / absence of nucleic acid amplification, and calculation of target protein concentration can be performed in the same manner as in the first embodiment.

  In the method of the present embodiment, sample division, nucleic acid extension reaction, and target protein concentration calculation may be performed by a method according to a standard digital ELISA technique.

  According to the method of the present embodiment, the concentration of a target protein in a sample containing a protein can be accurately measured without using a technique such as fluorescent staining.

  The method of this embodiment can be used for detection / identification of pathogen genes causing infectious diseases, detection / identification of disease-related genes such as cancer, expression analysis of disease-related genes, etc. It is useful for diagnosis of diseases and monitoring of therapeutic effects.

<Target biomolecule concentration calculation device>
[Configuration example of target nucleic acid sequence concentration calculation apparatus]
An example of the configuration of the target nucleic acid sequence concentration calculation apparatus 200 that realizes the method of the first embodiment described above will be described with reference to FIG.
FIG. 6 is a diagram showing an example of the configuration of the target nucleic acid sequence concentration calculation apparatus 200 in the present embodiment. The target nucleic acid sequence concentration calculation device 200 includes a bead arrangement substrate 20 and an arithmetic device 210.
The bead arrangement substrate 20 includes a plurality of reaction vessels 21 and sensors 30. The reaction vessel 21 is divided into a plurality of fractions, and a sample containing biomolecules is arranged. The sensor 30 is an example of a detection unit that detects each fraction during a nucleic acid amplification reaction, that is, a proton in the reaction tank 21.
One bead 1 is disposed in one reaction vessel 21. The target nucleic acid primer 2 described above is immobilized on the bead 1.

The arithmetic device 210 includes, for example, a CPU (Central Processing Unit), and includes a nucleic acid amplification determination unit 211 and a target nucleic acid sequence concentration calculation unit 212 as functional units thereof.
The nucleic acid amplification determination unit 211 determines the presence or absence of nucleic acid amplification in the reaction tank 21 for each reaction tank 21 based on the amount of protons generated for each reaction tank 21 detected by the sensor 30. That is, the nucleic acid amplification determination unit 211 is an example of a nucleic acid amplification determination unit that determines the presence or absence of nucleic acid amplification for each fraction based on the amount of protons detected by the detection unit. Specifically, the nucleic acid amplification determination unit 211 has information in advance indicating correspondence between the output current value of the sensor 30 and the amount of proton generation. The nucleic acid amplification determination unit 211 acquires the output current value of the sensor 30, and estimates the generation amount of protons based on the acquired output current value. The nucleic acid amplification determination unit 211 determines the presence or absence of nucleic acid amplification based on the estimated generation amount of protons. Here, the sensor 30 is provided for each reaction vessel 21. The nucleic acid amplification determination unit 211 determines the presence or absence of nucleic acid amplification for each reaction tank 21 by acquiring the output current value of the sensor 30 for each reaction tank 21.

  The target nucleic acid sequence concentration calculation unit 212 calculates the concentration of the target nucleic acid sequence in the sample based on the number of reaction vessels 21 that the nucleic acid amplification determination unit 211 determines to have nucleic acid amplification. That is, the target nucleic acid sequence concentration calculation unit 212 is a target biomolecule concentration calculation unit that calculates the concentration of the target biomolecule in the sample based on the number of fractions that the nucleic acid amplification determination unit 211 determines to have nucleic acid amplification. It is an example. The specific procedure for specifying the sequence of the bead specifying nucleic acid 10 has been described in detail in the above-mentioned [Calculation of the concentration of the target nucleic acid sequence], and will not be described here.

  According to the target nucleic acid sequence concentration calculation apparatus 200 of the present embodiment, the concentration of the target nucleic acid sequence of a sample containing nucleic acid can be accurately measured without using a technique such as fluorescent staining.

[Configuration example of target protein concentration calculation apparatus]
The configuration of the target protein concentration calculation device 200-2 that realizes the method of the second embodiment described above includes a target protein concentration calculation unit in place of the target nucleic acid sequence concentration calculation unit 212 in the target nucleic acid sequence concentration calculation device 200 described above. It can be provided.

  In addition, the program for performing each process of the target nucleic acid sequence concentration calculation apparatus 200 and the target protein concentration calculation apparatus 200-2 in the embodiment of the present invention is recorded on a computer-readable recording medium, and is recorded on the recording medium. The above-described various processes may be performed by causing the computer system to read and execute the program.

  Here, the “computer system” may include an OS and hardware such as peripheral devices. Further, the “computer system” includes a homepage providing environment (or display environment) if a WWW system is used. The “computer-readable recording medium” means a flexible disk, a magneto-optical disk, a ROM, a writable nonvolatile memory such as a flash memory, a portable medium such as a CD-ROM, a hard disk built in a computer system, etc. This is a storage device.

  Further, the “computer-readable recording medium” means a volatile memory (for example, DRAM (Dynamic DRAM) in a computer system that becomes a server or a client when a program is transmitted through a network such as the Internet or a communication line such as a telephone line. Random Access Memory)), etc., which hold programs for a certain period of time. The program may be transmitted from a computer system storing the program in a storage device or the like to another computer system via a transmission medium or by a transmission wave in the transmission medium. Here, the “transmission medium” for transmitting the program refers to a medium having a function of transmitting information, such as a network (communication network) such as the Internet or a communication line (communication line) such as a telephone line. The program may be for realizing a part of the functions described above. Furthermore, what can implement | achieve the function mentioned above in combination with the program already recorded on the computer system, what is called a difference file (difference program) may be sufficient.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design etc. of the range which does not deviate from the summary of this invention are included.

  DESCRIPTION OF SYMBOLS 1 ... Bead, 2 ... Primer for target nucleic acid, 3 ... Target nucleic acid sequence, 5 ... Antibody for target antigen detection, 20 ... Substrate for bead arrangement, 21 ... Reaction tank, 30 ... Sensor, 100 ... Bead body, 200 ... Target nucleic acid Sequence concentration calculator, 211 ... Nucleic acid amplification determination unit, 212 ... Target nucleic acid sequence concentration calculation unit, 300 ... Complex probe, 301 ... Secondary antibody, 310 ... Signal generation nucleic acid, 311 ... Primer annealing region, 312 ... Signal generation Sequence, 320 ... primer for signal generation, 400 ... target protein

Claims (11)

A method for calculating the concentration of a target biomolecule,
(A) dividing a sample containing a biomolecule into a plurality of fractions containing a ligand capable of binding to the target biomolecule;
(B) adding a reagent necessary for nucleic acid amplification to the fraction;
(C) performing a nucleic acid extension reaction for each of the fractions;
(D) measuring a proton generation amount of each fraction during the nucleic acid extension reaction;
(E) determining the presence or absence of nucleic acid elongation for each fraction based on the measured proton generation amount;
(F) calculating the concentration of the target biomolecule in the sample based on the number of fractions determined to have nucleic acid elongation;
A method for calculating the concentration of a target biomolecule comprising   The target biomolecule concentration calculation method according to claim 1, wherein the target biomolecule is a nucleic acid, and the ligand is a primer capable of annealing with a nucleic acid that is the target biomolecule.   The target biomolecule concentration calculation method according to claim 1, wherein the target biomolecule is a protein, and the ligand is an antibody that can bind to a protein that is the target biomolecule. Step (a) is
(A-1) contacting the sample with a target nucleic acid sequence of a primer immobilized on the beads and a plurality of groups of beads having different dielectric constants, and the sample;
(A-2) placing the plurality of groups of beads in a predetermined reaction tank for each bead by dielectrophoresis;
The concentration calculation method of the target biomolecule as described in any one of Claims 1-3 containing these. The method for calculating the concentration of a target biomolecule according to any one of claims 1 to 4, wherein a primer having the same sequence is immobilized on beads having the same electrical characteristics.   The method for calculating the concentration of a target biomolecule according to any one of claims 1 to 5, wherein the nucleic acid amplification reaction in the step (c) is performed by a PCR method or an isothermal amplification method.   The method for calculating the concentration of a target biomolecule according to claim 6, wherein the nucleic acid amplification reaction in the step (c) is performed by digital PCR or digital isothermal amplification.   The method for calculating a concentration of a target biomolecule according to any one of claims 1 to 6, wherein the measurement of the amount of proton generation in the step (d) is performed by an ISFET.   A target biomolecule concentration calculation bead in which a ligand corresponding to the electrical property is immobilized on a bead body having a predetermined electrical property.   A set of beads for calculating the concentration of a target biomolecule, including a plurality of beads for calculating the concentration of a target biomolecule, in which a ligand corresponding to the electric property is immobilized on a bead body having predetermined electric characteristics. A detection unit for detecting protons of each fraction during a nucleic acid amplification reaction performed for each fraction of the sample containing the biomolecule divided into a plurality of fractions;
A nucleic acid amplification determination unit that determines the presence or absence of nucleic acid amplification for each fraction based on the amount of protons detected by the detection unit;
Based on the number of fractions that the nucleic acid amplification determination unit determines to have nucleic acid amplification, a target biomolecule concentration calculation unit that calculates the concentration of the target biomolecule in the sample;
A target biomolecule concentration calculation apparatus comprising:

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