JP2018174811A - Analysis method and analysis system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve measuring accuracy.SOLUTION: An analysis method for detecting a biochemical reaction using an analysis device 10 having a base substance part 11 in which a plurality of wells 12 are arranged fills up the well 12 with a sample and reagent to perform the biochemical reaction, photographs only one part of the base substance 11 as a photographing area one time to acquire one image, calculates the ratio of the number of the wells 12, in which signal amplification was performed by biochemical reaction, with respect to the total number of the wells 12 included in the photographing area based on the image, determines the total number of the photograph to photograph the base substance 11 according to the ratio, photographs the base substance 11 until reaching the total number of the photographs when the total number of the photographs is larger than one, and analyzes the sample based on all images acquired by photographing the base substance 11.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an analysis method and an analysis system.

  Conventionally, it is known that diagnosis of a disease or constitution is performed by analyzing a biomolecule. For example, constitution diagnosis by single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism) analysis, administration decision of an anticancer agent by somatic cell mutation analysis, infection disease control by analysis of virus protein or DNA, etc. are known.

  In addition, it is suggested that, in cancer treatment drugs, quantification of copy number of mutant EGFR (epithelial growth factor receptor) before and after administration of EGFR-TKI (tyrosine kinase inhibitor) can be used as an indicator of therapeutic effect. ing. Conventionally, quantification was performed using real-time PCR (Polymerase Chain Reaction), but it has been revealed that the change in the total amount of nucleic acids used in the test affects the quantitativity. For this reason, digital PCR is currently being developed in which the total amount of nucleic acids does not affect the quantitativeness.

  In digital PCR, nucleic acids in a sample are quantified as follows. First, the mixture of PCR reaction reagent and nucleic acid is divided into a large number of microdroplets. These microdroplets are subjected to PCR amplification using a nucleic acid to be detected among the nucleic acids in the mixture as a template, and a signal such as fluorescence by PCR amplification is detected from the microdroplets containing the template nucleic acid. And the nucleic acid in a sample is quantified by calculating | requiring the ratio of the micro droplet in which the signal was detected among the total number of micro droplets. For example, Patent Document 1 and Non-patent Document 1 describe performing a biological substance test using digital PCR by performing an enzyme reaction in a well or a chamber having a very small volume.

  As a method for producing microdroplets used in digital PCR, a method of producing microdroplets by dividing a mixture of a reagent and a nucleic acid with a sealing solution, or a reagent and nucleic acid in which a hole is formed on a substrate The method of producing a microdroplet, etc. is known by sealing a hole with sealing liquid, after putting the mixture of and. In addition, Patent Document 2 describes a method of producing an emulsion (droplet) in a microchamber in order to obtain a large amount of experimental data in a short time with a small amount of reagent.

  In digital PCR, the mixture of PCR reaction reagent and nucleic acid is diluted so that the number of template nucleic acids present in one microdroplet is one or zero. In addition, in the case of digital PCR, the volume of each microdroplet is preferably small in order to increase the sensitivity of nucleic acid amplification and to perform nucleic acid amplification simultaneously on a large number of microdroplets. For example, Non-Patent Document 1 describes a microarray-like reaction container formed so that the volume of each chamber is 6 nl. In addition, in Non-Patent Document 2, a sample is flowed in a flow channel to a microarray in which a large number of microwells having a depth of 3 μm and a diameter of 5 μm are formed in the flow channel, and a sample is introduced into each microwell. Described is a method of introducing a sample into each microwell by extruding the excess reagent therein with a sealing solution.

  Further, for example, Patent Document 3 describes a method of detecting a difference in one base of a gene by performing an invader reaction in a minute space.

Japanese Patent Publication No. 2003-511009 Patent No. 4911592 gazette International Publication No. 2015/115635

"PLOS ONE", 2008, Volume 3, No. 8, e2876 "Lab on a Chip", 2012, No. 12, p. 4986-4991

  Patent Document 1 describes that the concentration of an oligonucleotide to be measured is calculated according to how many of a large number of microspaces are illuminated. However, when the measurement target is low concentration or high concentration, high-precision measurement is difficult.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide an analysis method and an analysis system capable of improving the accuracy of measurement.

  According to a first aspect of the present invention, there is provided an analysis method for detecting a biochemical reaction using an analysis device having a base portion in which a plurality of wells are arranged, the method comprising: The sample is filled with the sample and the reagent to perform the biochemical reaction, and one image is taken with only a part of the base portion taken as an imaging range to obtain an image, and the imaging range is included in the imaging range. The ratio of the number of wells in which signal amplification has been performed by the biochemical reaction to the total number of wells is calculated, and according to the ratio, the total number of photographed images for photographing the base portion is determined. If the total number of the images is more than one, the necessary number of images of the base portion up to the total number of photographed images is taken, and the sample is analyzed based on all the images obtained by the imaging of the base portion. Analysis method

  In the above analysis method, when the ratio is 60% or more, the total number of captured images may be at least two.

  In the above analysis method, when the ratio is 70% or more, the total number of photographed images may be at least three.

  In the above analysis method, when the ratio is 80% or more, the total number of captured images may be at least five.

  In the above analysis method, when the ratio is 90% or more, the total number of photographed images may be at least ten.

  In the above analysis method, when the ratio is 1% or less, the total number of captured images may be at least three.

  In the above analysis method, when the ratio is 0.1% or less, the total number of captured images may be at least five.

  In the above analysis method, when the ratio is 0.01% or less, the total number of captured images may be at least ten.

  A second aspect of the present invention is an analysis system for detecting a biochemical reaction, comprising an analysis device having a base portion in which a plurality of wells are arranged, and an imaging portion capable of imaging the base portion. And a controller capable of detecting the biochemical reaction, a calculation unit and a judgment unit, and a control device for controlling the detector. The control device controls the imaging unit to capture an image by capturing only a part of the base unit as an imaging range, and the calculation unit is included in the imaging range of the image. Controlling the ratio of the number of wells in which signal amplification has been performed by the biochemical reaction to the total number of the cells, and determining the number of photographed images of the base according to the ratio. Control to determine the total number.

  According to the above analysis method and analysis system, the measurement accuracy can be improved.

It is a schematic sectional drawing of the analysis device which concerns on one Embodiment of this invention. It is an II-II arrow line view of FIG. It is a figure which shows the filling procedure of the sample in the analysis method which concerns on the said embodiment, and a reagent. It is a figure which shows an example of the image acquired by the said analysis method. It is a flowchart of the imaging procedure in the said analysis method. It is a whole schematic diagram of an analysis system concerning the embodiment.

  Hereinafter, an analysis method and an analysis system according to an embodiment of the present invention will be described with reference to FIGS. 1 to 6.

  The analysis method according to the present embodiment is used for analysis of biomolecules. As an object to be analyzed, cells, exosomes, DNA, RNA, miRNA (microRNA), mRNA (messenger RNA) (hereinafter collectively referred to as RNAs, miRNAs, and mRNAs as appropriate), proteins, etc. It can be mentioned.

  Further, in the analysis method according to the present embodiment, the analysis device 10 is used. Therefore, first, the analysis device 10 according to the present embodiment will be described. Although the analysis device 10 described below is used for nucleic acid quantification, it is not limited to this, and may be used, for example, for analysis of protein.

  FIG. 1 is a schematic cross-sectional view of an analysis device 10. FIG. 2 is a view on arrow II-II in FIG. As shown in FIG. 1 and FIG. 2, the analysis device 10 has a base portion 11 in which a plurality of wells 12 are arranged.

  The base portion 11 is formed in a plate shape. The well 12 is formed on one surface of the base portion 11 and formed in a cylindrical shape having a bottom. In plan view, the wells 12 are arranged on the base 11 in a triangular lattice. The distance between the center lines of the wells 12 is set larger than the diameter of the wells 12. Here, the center line of the well 12 is a straight line that passes through the center of gravity of the opening of the well 12 and is parallel to the depth direction of the well 12. In the present embodiment, the center line of the well 12 is the center axis of the cylinder. The arrangement of the wells 12 is not limited to the triangular lattice, and may be, for example, a square lattice.

  On the side of one surface of the base portion 11, a cover portion 13 disposed so as to cover the base portion 11 is provided. The cover portion 13 is formed in a plate shape. A spacer 14 is provided between the peripheral edge of the base 11 and the peripheral edge of the cover 13. Thus, a space functioning as a flow path 15 through which various liquids flow is formed between the base portion 11 and the cover portion 13. At one end side of the cover 13, an inlet 16 communicating with the flow path 15 is provided. Further, at the other end side of the cover portion 13, a discharge port 17 communicating with the flow path 15 is provided. The inlet 16 is used to introduce liquid into the flow path 15 and the outlet 17 is used to discharge liquid from the flow path 15.

  Subsequently, the configuration of the base portion 11 will be described in more detail. The base 11 includes a substrate 11 a and a wall layer 11 b formed on the substrate 11 a.

  The substrate 11a is a plate-like member made of a substantially transparent material. For example, resin or glass can be used as the material of the substrate 11a. Specifically, the substrate 11a is made of, for example, polystyrene or polypropylene. Further, the substrate 11 a has such rigidity that it does not break when the device for transporting the analysis device 10 is handled by an operator or a worker manually.

  The wall layer 11 b is a layer in which a plurality of through holes are formed. The plurality of through holes are arranged in a triangular lattice. The plurality of through holes formed in the wall layer 11 b and the surface of the substrate 11 a form a well 12 whose bottom is the substrate 11 a. The thickness of the wall layer 11 b is, for example, 3 μm. The distance between the wall layer 11 b and the cover 13 facing the wall layer 11 b is, for example, 100 μm.

  As a material of the wall part layer 11b, resin, glass etc. can be used, for example. The material of the wall layer 11 b may be the same as or different from the material of the substrate 11 a. Examples of the resin constituting the wall layer 11 b include cycloolefin polymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, and amorphous fluorocarbon resin.

  The wall layer 11b may be formed directly on the substrate 11a, or a member on which the wall layer 11b is formed may be fixed to the substrate 11a by adhesion, welding or the like. The wall layer 11b may form a pattern to be a plurality of through holes, for example, by patterning a layer formed on the substrate 11a to form the wall layer 11b until the substrate 11a is exposed. it can. Also, for example, the wall layer 11b forms a hydrophobic film on the entire surface of the substrate 11a on which the wall layer 11b is to be formed, and the hydrophobic film is subjected to processing such as etching, embossing, and cutting. By being formed, a plurality of through holes can be formed.

  The volume of the well 12 is set appropriately. As described later, the biochemical reaction of the object to be analyzed in the well 12 occurs. The smaller the volume of the well 12, the shorter the reaction time until the signal generated by this biochemical reaction becomes detectable. Also, the volume of the well 12 is, for example, 100 pL (picoliter) or less. In the case where biomolecules are identified in the well 12, as the volume of the well 12 increases, the signal is diluted and the detection sensitivity decreases. By setting the volume of the well 12 to 100 pL or less, detection sensitivity can be maintained even when biomolecules are identified in the well 12. Also, more specifically, if the purpose is to reduce the time required to saturate the signal to generate a sufficient signal, the volume of the well 12 is such that the molecule to be analyzed is one in one well 12. It is set on the basis of the amount of liquid that can be entered. One example of a biochemical reaction is a change in fluorescence by real-time PCR using PCR, a change in turbidity using LAMP (trademark registration), a change in turbidity or luminescence intensity using ELISA, etc. .

  The distance between the wells 12 (the shortest distance between the peripheries of the adjacent wells 12) is set in accordance with the resolution at which the signal can be detected in each well 12.

  The wall layer 11b may be integrated with the same material as the substrate 11a. The wall layer 11b may be integrally formed of the same material as the substrate 11a. When the wall layer 11b is integrally formed with the substrate 11a, the substrate 11a is subjected to processing such as etching, embossing, cutting, etc., whereby a portion corresponding to the well 12 is formed in the wall layer 11b. Ru.

  Moreover, the wall part layer 11b may be colored. When the wall layer 11b is colored, the effect of light from another well 12 adjacent to the well 12 to be measured is measured when light such as fluorescence, light emission, or absorption is measured in the well 12. It can be reduced.

  Next, a detection reaction reagent (reagent) used together with the analysis device 10 according to the present embodiment will be described. The detection reaction reagent is a solvent in which particles such as biomolecules and microbeads to be analyzed are dispersed. It is also possible to use cells, bacteria, viruses, gels, emulsions, micelles and the like as particles for capturing an analysis target. The detection reaction reagent is a solution that can be injected from the inlet 16 into the flow path 15 formed between the wall layer 11 b and the cover portion 13. The detection reaction reagent is a reagent for performing a biochemical reaction such as an enzymatic reaction on a template nucleic acid related to a substance to be analyzed. The biochemical reaction to the template nucleic acid is, for example, a reaction in which signal amplification occurs under the conditions in which the template nucleic acid is present. The detection reaction reagent is selected, for example, according to the method capable of detecting the nucleic acid. Specifically, reagents used for the Invader (registered trademark) method, LAMP method (registered trademark), TaqMan (registered trademark) method, fluorescent probe method and the like are included in the detection reaction reagent according to the present embodiment.

  Also, for example, when a specific gene is to be analyzed (detected), the template nucleic acid itself or a part of the template nucleic acid is to be analyzed.

  Next, an oil-based sealing solution used together with the analysis device 10 according to the present embodiment will be described. The oily sealing solution is used to seal the detection reaction reagent introduced into the flow path 15 of the analysis device 10 in the well 12. The oily sealing solution is a solution that can be injected from the inlet 16 into the flow path 15 formed between the wall layer 11 b and the cover portion 13. The oil-based sealing liquid can be appropriately selected from materials which are not mixed with the sample containing the substance to be analyzed. As the oily sealing solution, mineral oil, chloroform, squalene, hexadecane, FC-40 as a fluorinated liquid, or the like can be used.

  When the signal to be detected is fluorescence, excitation light for measurement is applied to a fluorescent dye or the like contained in the detection reaction reagent contained in the well 12 to measure the generated fluorescence. At this time, the cover 13 may emit excitation light upon receiving the excitation light. This is called autofluorescence. In this case, due to the autofluorescence of the cover portion 13, the S / N ratio of the fluorescence in the well 12 to be originally measured may be deteriorated, and the measurement accuracy may be lowered. In order to prevent such problems and improve the S / N ratio, a light absorbing substance is used for the oil-based sealing liquid, or a light absorbing substance dissolved in the oil-based sealing liquid is used. You can do it. It is also possible to mix a light absorbing substance with the oily sealing solution.

  The light absorbing substance is a substance having a property of absorbing excitation light or a wavelength to be observed, and can be appropriately selected from materials soluble in the oil-based sealing solution. Examples of the light absorbing substance include pigments, dyes, quenchers (excitation energy absorbers), and the like. The following effects can be obtained by using an oil-based sealing solution containing a light absorbing substance. For example, when irradiating excitation light from the side of the base portion 11 and measuring fluorescence from a detection target present in the well 12, the light is incident on the side of the cover portion 13 by an oil-based sealing solution containing a light absorbing substance. Since the excitation light is absorbed, it is possible to suppress the generation of autofluorescence of the cover portion 13. In addition, since the oily sealing solution containing the light absorbing substance absorbs the light emitted from the cover portion 13 by receiving the excitation light, the autofluorescence of the generated cover portion 13 is measured (the side of the base portion 11) Can be prevented from reaching Therefore, it is possible to prevent the deterioration of the S / N ratio and to increase the measurement accuracy.

  Next, an analysis method according to the present embodiment will be described. First, the well 12 of the analysis device 10 is filled with the sample for the biochemical reaction and the detection reaction reagent to carry out the biochemical reaction. Specifically, a mixed solution 21 is prepared by mixing a sample containing a substance such as a biomolecule such as DNA to be analyzed and a detection reaction reagent according to the substance to be analyzed. The mixed solution 21 is injected from the injection port 16 of the analysis device 10 into the flow path 15, and as shown in FIG. 3A, the well 12 and the flow path 15 are filled with the mixed solution 21. Subsequently, the oil-based sealing liquid 22 is injected from the injection port 16 into the flow path 15, and as shown in FIG. 3B, the flow path 15 is filled with the oil-based sealing liquid 22. At this time, the mixed liquid 21 that has filled the flow path 15 is discharged from the discharge port 17 of the analysis device 10. At the same time, the mixed solution 21 in the well 12 is sealed by the oily sealing solution 22. As a result, as shown in FIG. 3C, the mixed solution 21 is filled in the well 12. In this state, a predetermined biochemical reaction such as an enzyme reaction occurs in each well 12 by heating the analysis device 10, for example. A predetermined signal amplification is performed in the well 12 by this biochemical reaction. Below, the case where the signal to be amplified is fluorescence is described as an example.

  After the biochemical reaction is completed, the base 11 of the analysis device 10 is photographed from the substrate 11a side with a fluorescence microscope (detector), and an image as shown in FIG. 4 is acquired, for example. At this time, the number of wells 12 arranged in the base 11 is usually, for example, one million, and it is huge. Therefore, only a part of the base 11 is photographed as a photographing range. The imaging range is a range including, for example, 10,000 wells 12. Subsequently, the number of light-emitting points (white points in FIG. 4) from the acquired image, that is, the number of wells 12 subjected to signal amplification is measured. The point of light emission is a point having a fluorescence value equal to or more than a predetermined threshold value. Then, the ratio of the number of wells 12 subjected to signal amplification to the total number of wells 12 included in the imaging range is calculated.

  The substance to be analyzed can be quantified by performing calculation taking Poisson distribution into consideration based on the ratio calculated above. For example, even when two or more molecules of the substance to be analyzed are contained in one well, it is counted as one as a point emitting light, but appropriately quantified by performing the statistical calculation described above be able to. However, in the case of a high concentration at which the substance to be analyzed is statistically contained 1.5 molecules or more in one well, the measurement accuracy decreases. This is because the influence of the measurement error increases. For example, in the case where 9850 wells out of 10000 wells emit light, it is calculated as 116 pM (pico molar), and in the case where 9800 wells emit light, it is calculated as 112 pM. Therefore, even with an error of about 0.5% (50 wells to 9850 wells), the concentration changes greatly. In the case of low concentration, the accuracy of measurement also decreases. For example, it is calculated as 0.003 pM when one of the 10000 wells emits light, and is calculated as 0.006 pM when two wells emit light. Therefore, the concentration changes twice with the difference of one well. Thus, the concentration range in which the accuracy can be maintained changes with respect to the measurement error.

  In the analysis method according to the present embodiment, imaging of the base portion 11 of the analysis device 10 is performed according to the flowchart shown in FIG. 5 in order to maintain high measurement accuracy even in the case of the above-described high concentration and low concentration. First, one part of the base 11 is photographed as a photographing range to acquire an image (step S1). The number of light emitting points from the acquired image, that is, the number of wells 12 subjected to signal amplification is counted, and the ratio of the number of wells 12 subjected to signal amplification to the total number of wells 12 included in the imaging range Calculate (step S2).

  Subsequently, based on the calculated ratio, the total number of photographed images for photographing the base portion 11 is determined. In the case where the total number of captured images is more than one, the necessary number of images of the base portion 11 are captured until the total number of captured images is reached. Specifically, first, it is determined whether the calculated ratio is 90% or less (step S3). If the calculated ratio exceeds 90%, the total number of captured images is set to 10 (step S13). If the calculated ratio is 90% or less, it is determined whether the calculated ratio is 80% or less (step S4). If the calculated ratio exceeds 80%, the total number of captured images is set to 5 (step S14). If the calculated ratio is 80% or less, it is determined whether the calculated ratio is 70% or less (step S5). If the calculated ratio exceeds 70%, the total number of captured images is set to 3 (step S15). If the calculated ratio is 70% or less, it is determined whether the calculated ratio is 60% or less (step S6). If the calculated ratio exceeds 60%, the total number of captured images is set to 2 (step S16).

  If the calculated ratio is 60% or less, it is determined whether the calculated ratio is 1% or less (step S7). If the calculated ratio exceeds 1%, the total number of captured images is set to one (step S17). If the calculated ratio is 1% or less, it is determined whether the calculated ratio is 0.1% or less (step S8). If the calculated ratio exceeds 0.1%, the total number of captured images is set to 3 (step S18). If the calculated ratio is 0.1% or less, it is determined whether the calculated ratio is 0.01% or less (step S9). If the calculated ratio exceeds 0.01%, the total number of captured images is set to 5 (step S19). If the calculated ratio is 0.01% or less, the total number of photographed images is set to 10 (step S10). After determining the total number of captured images (steps S10 and S13 to S19), it is determined whether the total number of captured images is greater than one (step S11). If the total number of captured images is more than one, the necessary number of images of the base portion 11 is captured until the total number of captured images is reached (step S12). At this time, imaging is performed in the imaging range different from the imaging range of the first image in the base unit 11 so that the imaging ranges do not overlap each other. After imaging the base portion 11 up to the total number of imagings, imaging of the base portion 11 is ended. When the total number of photographed images is one, the photographing of the base unit 11 is ended as it is.

After the imaging of the base unit 11 is completed, the sample is analyzed based on all the images acquired by the imaging of the base unit 11. Specifically, the following two forms are mentioned as an example.
(1) When Not Using Particles Capturing Analysis Target It is known in advance that a certain number of wells 12 exist in the image capturing target area. The point at which light is emitted, ie, the number of wells 12 subjected to signal amplification, is measured from all the acquired images, and signal amplification is performed on the total number of the wells 12 included in the acquired image (image photographing target area). The ratio of the number of wells 12 is calculated. The substance to be analyzed in the sample is quantified based on the calculated ratio.
(2) When using particles for capturing an analysis target When beads are used as particles for capturing a substance to be analyzed, the number of wells 12 containing beads is counted in an image acquired in a certain image capturing target area ( measure). At this time, it is possible to count the number of beads in the bright field image of the microscope. Next, in the same fluorescent image (dark-field image) of the image capturing target area as the bright-field image, among the wells containing the beads, the light emitting point, that is, the number of wells 12 subjected to signal amplification measure. From the number of wells 12 measured, the substance to be analyzed in the sample is quantified by the following equation.
x = 1000y / (khvNa)
(If the total number of DNA mixed with beads <total number of beads and total number of wells)
Target concentration (MOL / L) = x
Total number of wells emitted = y
Efficiency at which beads capture DNA = k
Entrapment efficiency of beads into wells = h
Target solution volume to be mixed with beads (ml) = v
Avogadro constant = Na

  The analysis method according to the present embodiment is an analysis method for detecting a biochemical reaction using an analysis device 10 having a base portion 11 in which a plurality of wells 12 are arranged, and the biochemical reaction in the wells 12 is performed. Samples and reagents are filled to perform a biochemical reaction, and only one part of the base 11 is photographed as an imaging range to obtain an image, and based on the image, the wells 12 included in the imaging range The ratio of the number of wells 12 in which signal amplification has been performed by the biochemical reaction to the total number of the cells is calculated, and the total number of imaging sheets for imaging the base portion 11 is determined according to the ratio. When the number is larger than the number of sheets, the necessary number of the base body 11 is photographed until the total number of imagings is reached, and the sample is analyzed based on all the images acquired by the photographing of the base body 11.

  According to such an analysis method, the total number of imaging | photography number which image | photographs the base | substrate part 11 according to the ratio calculated based on the 1st image which image | photographed the base | substrate part 11 is determined. For this reason, the measurement accuracy in such a case can be improved by increasing the number of photographed images when the measurement error is considered to be large, for example, in the case of high concentration or low concentration. In addition, when the measurement error is considered to be small, the increase in the imaging time can be suppressed by not increasing the number of imagings.

  In the above-described shooting procedure, when the calculated ratio exceeds 90% in step S13, the total number of shootings is set to 10, but the number is not limited to this and may be more than 10 . In step S14, when the calculated ratio exceeds 80%, the total number of captured images is five, but the number is not limited to this and may be more than five. In step S15, when the calculated ratio exceeds 70%, the total number of captured images is three, but the number is not limited to three, and may be more than three. In step S16, when the calculated ratio exceeds 60%, the total number of captured images is two, but the number is not limited to this and may be more than two. In step S18, when the calculated ratio exceeds 0.1%, the total number of captured images is three, but the number is not limited to three, and may be more than three. In step S19, when the calculated ratio exceeds 0.01%, the total number of captured images is five, but the number is not limited to five, and may be more than five. In step S10, when the calculated ratio is 0.01% or less, the total number of captured images is set to 10, but the number is not limited to this and may be more than 10.

  Next, the analysis system 1 according to the present embodiment will be described with reference to FIG. FIG. 6 is an overall schematic view of the analysis system 1. As shown in FIG. 6, the analysis system 1 includes the analysis device 10 described above, the detector 30 such as the fluorescence microscope described above, and the control device 40.

  The detector 30 is configured to detect the predetermined biochemical reaction described above, such as a fluorescence microscope capable of detecting fluorescence. The detector 30 also has an imaging unit 31 capable of imaging the base unit 11 of the analysis device 10. The photographing unit 31 photographs the base unit 11 to obtain an image. The detector 30 further includes a placement unit 32 on which the analysis device 10 is placed. In the detector 30, in the state where the analysis device 10 is placed on the placement unit 32, the imaging unit 31 can capture the base unit 11 from the substrate 11a side. Further, the placement unit 32 is configured to be able to move the analysis device 10 with the imaging device 31 in focus with the analysis device 10 by a known drive mechanism. Therefore, the imaging | photography range of the base | substrate part 11 by the imaging | photography part 31 can be changed by the mounting part 32 moving the base | substrate part 11 suitably.

  The control device 40 is electrically connected to the detector 30, and can control the detector 30. The control device 40 includes a calculation unit 41 and a determination unit 42. The calculating unit 41 measures the number of wells 12 subjected to signal amplification based on the image acquired by the imaging unit 31 of the detector 30 by imaging, and performs signal amplification with respect to the total number of wells 12 included in the imaging range of the image. Calculate the proportion of the number of wells 12 in which the The determination unit 42 determines the total number of photographed images for photographing the base 11 based on the calculated ratio. The specific determination method of the total number of photographed images is the same as the flowchart shown in FIG. 5 described above.

  The analysis system 1 according to the present embodiment is an analysis system for detecting a biochemical reaction, and is capable of imaging the analysis device 10 having the base portion 11 in which a plurality of wells 12 are arranged, and the base portion 11 The imaging unit 31 is provided, and a detector 30 capable of detecting a biochemical reaction, and the control unit 40 including the calculation unit 41 and the determination unit 42 and controlling the detector 30 are provided. The control device 40 controls the imaging unit 31 to capture an image by capturing only one part of the base 11 as the imaging range. The control device 40 controls the calculation unit 41 to calculate the ratio of the number of wells 12 in which signal amplification is performed by the biochemical reaction to the total number of wells 12 included in the imaging range of the image. The control device 40 controls the determination unit 42 so as to determine the total number of imagings for imaging the base 11 in accordance with the ratio of the number of wells 12 in which signal amplification has been performed by the biochemical reaction.

  According to such a configuration, the analysis method according to the present embodiment described above can be automatically performed by the control of the control device 40. In addition, the same effect as the analysis method according to the present embodiment described above can be obtained.

  Hereinafter, the present invention will be further described by way of examples, but the present invention is not limited thereto.

Example 1
<Preparation of array device for nucleic acid quantification>
CYTOP (registered trademark) (made by Asahi Glass) is spin-coated on a glass substrate having a thickness of 0.5 mm, thermally cured at 180 ° C. for 3 hours, and 1 million wells with a diameter of 5 μm using photolithography technology A base portion having the same was produced. The layer formed on the substrate by spin coating CYTOP (registered trademark) became a wall layer by being formed by photolithography. The thickness of the layer formed on the substrate by spin coating CYTOP® is 3 μm.

Subsequently, a cover glass was installed as a cover portion so that a gap with the base portion was 100 μm. A spacer made of an adhesive tape was disposed between the base portion and the cover portion. Furthermore, an aqueous liquid is injected as a substitution solution into the flow path between the base portion and the cover portion, and the entire 5 μm diameter well and the flow path between the base portion and the cover portion are filled with the aqueous liquid. The In this example, the composition of the aqueous liquid is 20 μM MOPS pH 7.5, 15 mM NaCl, 6.25 mM MgCl ₂ .

<Injection of mixed solution of sample and detection reaction reagent>
Invader reaction reagent (2 μM allele probe, 1 μM invader oligo, 1 μM FAM labeled arm, 20 μM MOPS pH 7.5, 15 mM NaCl, 6.25 mM MgCl ₂ , 50 U / μL CLIBASE) and artificial synthetic DNA for detection are mixed, The mixed solution was injected into the flow path between the base portion and the cover portion. Here, the concentration of the artificial synthetic DNA for detection is 0.003 pM (average of 0.17% of all wells) for detection, which is an average concentration of 1 molecule in 10000 wells with a diameter of 5 μm formed on the substrate portion. Artificially synthesized DNA is added). After injecting the mixed solution of the invader reaction reagent and the artificial synthetic DNA for detection, FC-40 (SIGMA) is injected into the flow path between the base part and the cover part as an oily sealing solution, and the well with a diameter of 5 μm By sealing, to construct an independent nucleic acid detection reaction container.

<Measurement of fluorescence intensity>
Next, the analysis device for nucleic acid quantification of the present example having one million independent nucleic acid detection reaction containers was incubated in an oven at 63 ° C., and removed after 15 minutes. This analysis device was photographed from the bottom of the wells with a fluorescence microscope, and the fluorescence intensity of each well was observed. Here, the reaction container for detecting nucleic acid is a fluorescence microscope (Zeiss AG, AX10), a light source (LEJ, FluoArc001.26A Usable with HBO 10), a sensor (Hamamatsu Photonics, EM-CCD C9100), a filter (Olympus) , U-MNIBA2), analysis software (Hamamatsu Photonics Co., Ltd., AQUACOSMOS 2.6: exposure time 488 ms, EM gain 120, offset 0, binning × 1), and S / N sufficient for autofluorescence The number of nucleic acid detection reaction vessels emitting fluorescence with a ratio was counted. 100 million wells were measured in one shot, and five shots were taken.

Example 2
The concentration of the artificially synthesized DNA for detection is 28 pM which is an average concentration of 10000 molecules in 10000 wells with a diameter of 5 μm formed in the base portion (the artificially synthesized DNA for detection is filled in 64% of all wells. ). The measurement of the wells was performed in the same manner as in Example 1 under the same conditions as in Example 1 except the above.

[Example 3]
The concentration of the artificially synthesized DNA for detection is 56 pM which is an average concentration of 20000 molecules in 10000 wells with a diameter of 5 μm formed in the substrate portion (87% of all wells are filled with artificially synthesized DNA for detection. ). The measurement of the wells was performed in the same manner as in Example 1 under the same conditions as in Example 1 except the above.

Example 4
The concentration of artificially synthesized DNA for detection is 112 pM, which is an average concentration of 40000 molecules in 10000 wells with a diameter of 5 μm formed in the base portion (the artificially synthesized DNA is filled in 98.5% of all wells. ). The measurement of the wells was performed in the same manner as in Example 1 under the same conditions as in Example 1 except the above.

  Tables 1 and 2 show the results of Examples 1 to 4. In Tables 1 and 2, the sample concentration is the concentration of artificially synthesized DNA for detection. Further, in Tables 1 and 2, the number of molecules per 10000 wells (average number of molecules) corresponding to the sample concentration and the ratio of filling in the wells are displayed. In Table 1, the number of wells measured indicates the result of counting the number of wells emitting fluorescence for each of the five images acquired in five times of imaging. In Table 2, the calculated concentration is the concentration of the artificial synthetic DNA for detection calculated by statistical calculation taking Poisson distribution into consideration based on the number of wells shown in Table 1, and is displayed for each number of images used doing. For example, in the first to third sheets of Table 2, based on the first to third images, that is, the number of the first measured wells in Table 1 and the number of the second measured wells, 3 It shows the concentration of artificially synthesized DNA for detection calculated by statistical calculation based on all of the number of wells measured.

  As shown in Tables 1 and 2, it was found that in the case of an average concentration of 10000 molecules in 10000 wells, it is better to perform multiple imaging. In addition, it was found that it is better to carry out imaging several times also in the case where the concentration is such that the average 1 molecule is contained in 10000 wells and the concentration is such that the average is 20000 molecules or more. From this result, it was found that when the concentration of the measurement object is extremely high or low, quantitative analysis with high accuracy can be performed in a short time by determining and calculating the number of photographed images from the first photographed result.

  As mentioned above, although the preferable embodiment of this invention was described, this invention is not limited to the said embodiment. Additions, omissions, substitutions, and other modifications of the configuration are possible without departing from the spirit of the present invention.

DESCRIPTION OF SYMBOLS 1 analysis system 10 analysis device 11 base 11a substrate 11b wall layer 12 well 13 cover 14 spacer 15 flow path 16 inlet 17 discharge port 21 mixed liquid 22 oil-based sealing liquid 30 detector 31 imaging unit 32 placement unit 40 Control device 41 Calculation unit 42 Determination unit

Claims (9)

An analysis method for detecting a biochemical reaction using an analysis device having a base portion in which a plurality of wells are arranged,
Filling the wells with samples and reagents for the biochemical reaction to perform the biochemical reaction;
Taking an image by capturing only one part of the base portion as a shooting range;
Based on the image, a ratio of the number of the wells in which signal amplification is performed by the biochemical reaction to the total number of the wells included in the imaging range is calculated;
According to the above ratio, the total number of photographed images for photographing the base portion is determined;
In the case where the total number of the photographed number is more than one, the necessary number of the base portion is photographed until the total number of the photographed number is reached,
The analysis method which analyzes the said sample based on all the images acquired by imaging | photography of the said base | substrate part. The analysis method according to claim 1, wherein
When the ratio is 60% or more, the total number of photographed images is at least two. The analysis method according to claim 2, wherein
When the ratio is 70% or more, the total number of photographed images is at least three. The analysis method according to claim 3, wherein
When the ratio is 80% or more, the total number of photographed images is at least five. Analysis method. The analysis method according to claim 4, wherein
When the ratio is 90% or more, the total number of photographed images is at least ten. The analysis method according to claim 1, wherein
When the ratio is 1% or less, the total number of captured images is at least three. The analysis method according to claim 6, wherein
When the ratio is 0.1% or less, the total number of photographed images is at least five. The analysis method according to claim 7, wherein
When the ratio is 0.01% or less, the total number of photographed images is at least 10. Analysis method. An analysis system for detecting a biochemical reaction, comprising
An analysis device having a base portion in which a plurality of wells are arranged;
A detector having an imaging unit capable of imaging the base unit and capable of detecting the biochemical reaction;
A control device that has a calculation unit and a determination unit and controls the detector;
Equipped with
The controller is
The imaging unit is controlled so as to capture an image by capturing one image taking only a part of the base unit as an imaging range;
Controlling the calculation unit to calculate a ratio of the number of wells in which signal amplification has been performed by the biochemical reaction to the total number of the wells included in the imaging range of the image;
An analysis system that controls the determination unit to determine the total number of captured images for capturing the base according to the ratio.

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