JP2020016460A - Assay program and assay device - Google Patents

Abstract

To perform an assay in consideration of the intensity of a signal due to a certain reaction and its reaction time based on a reaction time of another reference reaction.SOLUTION: An assay program in a microchannel in an assay device configured to be able to flow a sample liquid that may contain a specimen and that contains a known reference substance different from the specimen, makes a computer execute: a step ST4 of taking an image of a first signal produced by a first reaction for specifically detecting the specimen and a second signal produced by a known second reaction for specifically detecting the reference substance; a step ST10 of calculating a reaction time of the second reaction based on pixel data of the second signal in the image; and a step ST11 of calculating the concentration of the specimen based on a regarded reaction time in which the reaction time of the second reaction is regarded as a reaction time of the first reaction and the pixel data of the first signal in the image.SELECTED DRAWING: Figure 4

Description

  The present invention relates to an assay program and an assay device for performing an assay using a liquid.

  In the fields of biology and chemistry, etc., inspections, experiments, assays, etc., using liquids such as reagents and treatment agents on the order of μl (microliter), that is, about 1 μl or more and less than about 1 ml (milliliter). To do so, an assay device including a microchannel is used. As for such an assay device, a lateral flow type assay device, a flow-through type assay device, and the like have been used in recent years in order to improve cost, operability, durability, and liquid control performance.

  In particular, the lateral flow type assay device is configured to perform movement, manipulation, and the like of a liquid using a hydrophilic porous medium such as paper and a capillary phenomenon such as a cellulose membrane, and is simple. Therefore, the lateral flow type assay device can be manufactured at low cost, does not require an external mechanism such as a pump, does not require complicated operations, and can improve durability. The lateral flow-type assay device is particularly used when detecting or quantifying the concentration of an antibody or antigen contained in a sample by an ELISA (Enzyme-Linked Immunosorbent Assay), an immunochromatography method, or the like. Can be

  As an example of such an assay device, a channel and an assay region connected to the channel are provided in a stacked porous medium, and the channel and the assay region are stacked. Assay devices, which are defined by a barrier made of polymerized photoresist absorbed throughout the thickness of the porous medium. (For example, see Patent Document 1)

  Another example of the above-mentioned assay device includes an assay device invented by the present inventor. This assay device has a microchannel, a porous medium disposed at an interval from one end of the microchannel, and a space disposed between one end of the microchannel and the porous medium. Then, after the liquid that has moved in the micro flow channel contacts the porous medium beyond the space portion and is absorbed, the fluid is separated in the space portion so as to be retained in the micro flow channel. It is. (See, for example, Patent Document 2)

JP 2010-515877 A Japanese Patent No. 6037184

  The signal intensity (color development, luminescence, fluorescence amplification, etc.) generated by a reaction such as an enzymatic reaction varies depending on the time of the reaction. Therefore, it is difficult to perform an accurate assay even if only the signal intensity is considered. An object of the present invention is to perform an assay in consideration of the signal intensity due to a certain reaction and the reaction time based on the reaction time of another reference reaction.

  In order to solve the above problem, an assay program according to one embodiment of the present invention is configured to allow a sample liquid containing a known reference substance different from the sample to flow, which may contain a sample. In a microchannel in an assay device, a first signal generated by a first reaction for specifically detecting the analyte and a second signal generated by a known second reaction for specifically detecting the reference substance were copied. An acquisition step of acquiring an image, a reaction time calculation step of calculating a reaction time of the second reaction based on pixel data of the second signal in the image acquired by the acquisition step, and a reaction time calculation step The reaction time of the second reaction calculated by the above is defined as the reaction time of the first reaction only, and the reaction time of the first reaction only, Tsu based on the first signal of pixel data in the image acquired by the flop, to execute the density calculation step of calculating the concentration of the analyte in the computer.

  The assay support device according to one embodiment of the present invention includes a micro flow in an assay device configured to be able to flow a sample liquid that may contain a sample and that includes a known reference substance different from the sample. An acquisition unit for acquiring an image of a first signal generated by a first reaction for specifically detecting the sample and a second signal generated by a known second reaction for specifically detecting the reference substance. A reaction time calculation unit that calculates a reaction time of the second reaction based on pixel data of the second signal in the image acquired by the acquisition unit; and a second response time calculation unit that calculates the second reaction time. The reaction time of the reaction is defined as the reaction time of the first reaction only, and the reaction time of the first reaction only, and the image of the first signal in the image acquired by the acquisition unit. Based on the data, and a concentration calculator that calculates the concentration of the analyte.

  In another aspect, the assay device includes a microchannel configured to allow a sample liquid that may include a sample and that includes a known reference substance different from the sample to flow, and the microchannel includes a microchannel. A first region in which a first reaction for specifically detecting the sample occurs, and a first region in the microchannel which is adjacent to the first region and specifically detects the reference substance. And a second region in which the second reaction occurs.

  ADVANTAGE OF THE INVENTION According to this invention, the assay which considered the intensity | strength of the signal by reaction, such as an enzyme reaction, and the time of the reaction can be performed.

FIG. 1 is a plan view schematically showing an assay device according to one embodiment. FIG. 2 is an exploded perspective view schematically showing an assay device according to one embodiment. FIGS. 3A to 3D are cross-sectional views taken along the line AA of FIG. 1 and illustrating a state of a microchannel when an assay is performed by supplying a sample liquid to an assay device according to an embodiment. is there. FIG. 4 is a flowchart illustrating an assay method according to one embodiment. FIG. 5 is an explanatory diagram illustrating an example of a functional configuration of the assay support device. FIG. 6 is an explanatory diagram illustrating an example of a computer hardware configuration of the assay support device. FIG. 7 is a graph showing an example of the relationship between the reaction time and the amount of the product by the reaction. FIG. 8 is an explanatory diagram illustrating an example of a plurality of calibration curves.

  Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited by the following embodiments.

  The sample liquid applicable to the assay device described later is not particularly limited as long as it can flow in the assay device. The sample liquid applicable to such an assay device may typically include the analyte described below, and may also include a gas, another liquid or a solid dissolved, dispersed, or suspended in a liquid. it can.

  For example, the sample liquid may be hydrophilic, such as human or animal whole blood, serum, plasma, blood cells, urine, feces dilution, saliva, sweat, tears, nail extraction. Liquid, a skin extract, a hair extract, or a sample liquid derived from a living body such as cerebrospinal fluid. In this case, in the assay device, a pregnancy test, a urine test, a stool test, an adult disease test, an allergy test, an infectious disease test, a drug test, a cancer test, an in vitro diagnostic drug, a general test drug, a POCT ( In an application such as Point of Care Testing), a sample effective for clinical examination, diagnosis, or analysis in a sample liquid can be measured, but the use of the assay device is not particularly limited. In addition, the hydrophilic sample liquid is not limited to a sample liquid derived from a living body, and includes, for example, food suspension, drinking water, river water, soil suspension, and the like. In this case, the assay device can measure pathogens in food or drinking water, or can measure contaminants in river water or soil. These hydrophilic liquids may typically be those using water as a solvent.

  In the present specification, “lateral flow” refers to a flow of a liquid that moves when gravity settling becomes a driving force. The movement of the liquid based on the lateral flow refers to the movement of the liquid in which the driving force of the liquid due to gravitational settling acts dominantly (dominantly). In contrast, the movement of liquid based on capillary force (capillary action) refers to the movement of liquid in which interfacial tension acts dominantly (dominantly). The movement of liquid based on lateral flow is different from the movement of liquid based on capillary force.

  As used herein, "analyte" refers to a compound or composition that is present in a sample liquid and that is detected or measured. For example, an “analyte” is a saccharide (eg, glucose), protein or peptide (eg, serum protein, hormone, enzyme, immunomodulator, lymphokine, monokine, cytokine, glycoprotein, vaccine antigen, antibody, growth factor, or growth) Factors), fats, amino acids, nucleic acids, cells, steroids, vitamins, pathogens or their antigens, natural or synthetic chemicals, contaminants, therapeutic or illegal drugs or toxins, or metabolites or antibodies of these substances , But is not limited to a specific sample. In some cases, the sample liquid does not contain the sample or does not contain the sample in a detectable amount. A “reference substance” is a known substance different from the analyte, which is added to the sample liquid in a known amount for detecting the analyte concentration. The reference substance can be selected from the above options similarly to the specimen, and can be selected in relation to the specimen. In particular, a stable substance can be selected without interacting with the specimen.

  In the specification of the present application, the “micro flow path” is used to detect or measure a sample using a small amount of a sample liquid on the order of μl (microliter), that is, about 0.1 μl or more and less than about 1 ml (milliliter). Refers to a path configured to flow a liquid in an assay device.

  As used herein, “film” refers to a film-like object having a thickness of about 200 μm (micrometer) or less, and “sheet” refers to a film-like or plate-like object having a thickness of greater than about 200 μm. Point.

  As used herein, "plastic" refers to a polymerizable or polymeric material that has been polymerized or molded to use as an essential component. Plastics also include polymer alloys that combine two or more polymers.

  In the specification of the present application, the “porous medium” may be a member having a plurality and a large number of micropores and capable of sucking and passing a sample liquid, or a member capable of capturing or concentrating a solid substance. , Cellulose membrane, nonwoven fabric, glass fiber, polymer gel, plastic, etc. For example, the “porous medium” may have hydrophilicity when the sample liquid is hydrophilic, and may have hydrophobicity when the sample liquid is hydrophobic. In particular, the “porous medium” is preferably a paper having a hydrophilic property and containing a large number of fibers, absorbent cotton, or the like. Further, the “porous medium” is one or more of cellulose, nitrocellulose, cellulose acetate, filter paper, tissue paper, toilet paper, paper towel, fabric, cotton, or a hydrophilic porous polymer that is permeable to water. Can be.

  As shown in FIGS. 1 to 3, the assay device AS has three microchannel assemblies MC. The micro flow channel assembly MC has a micro flow channel 1 configured to allow a sample liquid to flow. The microchannel 1 has a distal end 1a located downstream and a proximal end 1b located upstream. Hereinafter, a direction (indicated by an arrow C) along the flow of the sample liquid in the micro flow channel 1 is referred to as a “flow direction”.

  The micro flow channel assembly MC has a porous medium for absorption 2 arranged at the tip 1 a of the micro flow channel 1. The absorbing porous medium 2 is configured to be able to absorb the sample liquid flowing from the base end 1b of the micro flow channel 1 to the front end 1a.

  The microchannel assembly MC has a top surface and a bottom surface facing each other in a height direction (indicated by an arrow H) orthogonal to the flow direction C and the width direction W of the microchannel. In the use state, the microchannel assembly MC may be arranged so that the height direction is directed vertically, and in this case, the top surface and the bottom surface of the microchannel assembly MC face upward and downward, respectively. . The micro channel assembly MC has a first layer member S1, a second layer member S2, and a third layer member S3 arranged in order from the top surface to the bottom surface. The first layer member S1, the second layer member S2, and the third layer member S3 are preferably formed in a substantially layered shape.

  The first layer member S1 and the third layer member S3 are made of a material that does not transmit a sample liquid and does not absorb signals from an assay region and a confirmation region described later. The contact angle between the first layer member S1 and the third layer member S3 may be smaller than 90 degrees. The first layer member S1 is preferably transparent or translucent. At least one of the first layer member S1 and the third layer member S3 is desirably elastically deformable by the pressure of the sample liquid when the sample liquid is passed through the microchannel assembly MC. Not limited.

  Each of the first layer member S1 and the third layer member S3 may be manufactured using plastic. Further, the material of each of the first layer member S1 and the third layer member S3 is preferably a plastic sheet or film. For example, such plastics include polyethylene (PE), high-density polyethylene (HDPE), ABS resin (ABS), AS resin (SAN), polypropylene (PP), polyvinylidene chloride (PVDC), polystyrene (PS), Polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyolefin (PO), nylon, polymethyl methacrylate (PMMA), cycloolefin copolymer (COC), cycloolefin polymer (COP), polycarbonate (PC), polydimethylsiloxane (PDMS), polyacrylonitrile (PAN), polylactic acid (PLA) and other biodegradable plastics or other polymers or combinations thereof. However, as long as at least one of the first layer member S1 and the third layer member S3 is a material through which a fluid does not penetrate, it can be made of a material other than plastic. , Glass, metal and the like. Materials or materials used for the first layer member S1 and the third layer member S3 may be the same or different.

  The second layer member S2 is preferably a double-sided adhesive tape. The top surface and the bottom surface of the second layer member S2 have adhesiveness. The bottom surface of the first layer member S1 is joined to the top surface of the second layer member S2, and the bottom surface of the second layer member S2 is joined to the top surface of the third layer member S3. However, the second layer member can also be manufactured using materials or materials shown as materials that can be used for the first and third layer members as described above. In this case, the second layer member may be joined to the first and third layer members by using a joining means such as an adhesive or welding, and the material or the material used for the second layer member may be the first or third material. It may be the same as or different from that used for the three-layer member.

  As shown in FIG. 2, the micro flow channel 1 is formed so as to penetrate the second layer member S2. That is, the two side surfaces of the microchannel 1 facing each other in the width direction are defined by the second layer member S2. The top surface and the bottom surface of the microchannel 1 are defined by the first layer member S1 and the third layer member S3, respectively. As shown in FIG. 2, the microchannel 1 is preferably formed in a substantially straight line. However, in the present invention, the microchannel can be formed to be curved or bent.

  The surface defining the microchannel 1 may be subjected to a hydrophilic treatment. Such a hydrophilic treatment includes a treatment using a blocking agent that makes it possible to prevent the specific conjugate from being adsorbed on these surfaces when the specific liquid is contained in the sample liquid. Examples of the blocking agent include commercially available blocking agents such as Block Ace, bovine serum albumin, casein, skim milk, gelatin, surfactant, polyvinyl alcohol, globulin, serum (for example, fetal calf serum or normal rabbit serum), ethanol, and MPC polymer. And the like. Such blocking agents can be used alone or in combination of two or more.

  Two double-sided adhesive tapes T are arranged between the absorbing porous medium 2 and the first layer member S1. The two double-sided adhesive tapes T are spaced from each other in the width direction. The absorbent porous medium 2 is joined to the first layer member S1 by these double-sided adhesive tapes T. However, the absorbing porous medium may be joined by joining means other than the double-sided adhesive tape. For example, the joining means may be an adhesive, welding, or the like. Further, the absorbent porous medium may be joined to the third layer member by a double-sided adhesive tape or a joining means other than the double-sided adhesive tape.

  At the tip of the first layer member S1 that defines the top surface of the tip 1a of the micro flow channel 1, a vent 6 configured to allow air to flow is provided. The vent 6 is preferably configured to allow communication between the outside of the micro flow channel assembly MC and the tip 1a of the micro flow channel 1. When the liquid flows through the micro flow path 1 from the base end 1b to the tip end 1a, the air in the micro flow path 1 goes out of the micro flow path assembly MC through the ventilation port 6.

  The microchannel assembly MC has an inlet 8 configured to allow a liquid to flow into the microchannel 1. The inlet 8 is formed so as to penetrate the base end of the first layer member S1 that defines the top surface of the base end 1b of the micro flow channel 1.

  The microchannel assembly MC further has a developing porous medium 9 disposed in the base end 1 b of the microchannel 1 so as to correspond to the inlet 8. The material of the developing porous medium 9 and the material of the absorbing porous medium 2 may be the same or different. However, if the lateral flow of the liquid can be generated in the micro flow channel as described later, the micro flow channel assembly MC can be configured so as not to have the developing porous medium 9. In FIG. 3, the illustration of the developing porous medium 9 is omitted.

  As shown in FIGS. 1 and 3, an assay region 10 is provided at an intermediate portion of the microchannel 1 in the flow direction C. In the assay region 10, a reagent that specifically binds to a specimen in an assay is fixed. Hereinafter, in the present specification, a reagent involved in signal generation derived from a specimen and a reference substance is referred to as an assay reagent. Assay reagents include an immobilized reagent that is used by being previously fixed to the microchannel, and an additional reagent that is used by being added to the microchannel in the assay step. The immobilized reagent provided in the assay region 10 specifically reacts with the sample in the sample liquid, and produces a result capable of detecting the sample together with the added reagent. The result that can detect the analyte may be visible to the naked eye, for example, based on a change in color, or the result that can detect the analyte may be detectable only by a spectrometer or other measurement means. You may. This assay area is also called a first area.

  The immobilized reagent provided in the assay region 10 includes enzymes, antibodies, epitopes, nucleic acids, cells, aptamers, peptides, molecular imprinted polymers, adsorbed polymers, adsorbed gels, and iron (III) ions that are colored by reaction with a sample. Of the present invention, or any other substance that produces a detectable result by reacting with an analyte, typically an immobilized reagent is an antibody. The immobilization reagent may be immobilized on the assay region 10 by a well-known immobilization technique such as a physical adsorption method or a chemical adsorption method. Immobilized reagents include colored molecules such as radioisotopes, enzymes, gold colloids, color reagents, quantum dots, latex, dyes, electrochemical reactants, fluorescent substances, or luminescent substances in order to analyze or amplify the detection signal. Any labeling substance such as a substance may be bound. Alternatively, these labeling substances may be bound to additional reagents to be used by being added to the microchannel in the assay step. Specifically, the immobilization reagent can be fixed to one or both of the first layer member S1 and the third layer member S3.

  In the assay of such an assay device, the assay is performed in a state where the sample liquid flows in the micro flow channel 1 or in a state where the sample liquid is left standing or temporarily stopped in the micro flow channel 1. Typically, the analyte concentration in the sample liquid can be detected.

  As shown in FIG. 1, a color sample CS can be arranged on the top surface of the assay device AS and near the assay region 10. By comparing the color sample with the signal generated in the assay region 10, the intensity of the signal can be confirmed.

  Further, in the microchannel assembly according to the present embodiment, as shown in FIGS. 1 and 3, a confirmation region 12 is provided adjacent to the assay region 10 and downstream of the assay region 10. It is sufficient that the assay region 10 and the confirmation region 12 are separated from each other to such an extent that a signal generated from each region can be distinguishably detected. The confirmation area 12 is configured to generate a known second reaction (described later) that can be considered to have the same reaction time as a first reaction (described below) occurring in the assay area 10. This confirmation area 12 is also called a second area. The confirmation region 12 is provided with an immobilization reagent that specifically binds to the reference substance. The immobilization reagent in the confirmation area 12 may also be an antibody or the like, like the immobilization reagent in the assay area 10, and the immobilization reagent may have any labeling substance bound thereto. It can be fixed to one or both of the first layer member S1 and the third layer member S3.

  FIG. 3 is a diagram schematically showing an assay region 10 and a confirmation region 12 when an antibody is used as an immobilizing reagent. With reference to FIG. 3, the reaction between the assay reagent and the sample, the reaction between the reference substance, and the mechanism for detecting the concentration of the sample using the reaction will be described. As shown in FIG. 3A, a first antibody Y11 as an immobilization reagent is immobilized on the assay area 10, and an immobilization reagent different from the first antibody Y11 is immobilized on the confirmation area 12. Certain second antibody Y12 is immobilized. Then, a sample liquid that may contain the first antigen X11 as a specimen and contains the second antigen X12 in a known amount is injected from the injection port 8. The second antigen X12 is a known reference substance different from the first antigen X11 that is the specimen, and is intentionally included in the sample liquid by the assay performer in a predetermined known amount. The first antigen X11 is specifically recognized by the first antibody Y11, and the second antigen X12 is specifically recognized by the second antibody Y12.

  Next, as shown in FIG. 3B, a first reagent, which is an additional reagent including the first detection antibody Y21 and the second detection antibody Y22, is injected from the injection port 8. The first detection antibody Y21 specifically recognizes the first antigen X11, and the second detection antibody Y22 specifically recognizes the second antigen X12. The components contained in the first reagent, the first detection antibody Y21 and the second detection antibody Y22, can be determined based on the relationship between the sample and the reference substance.

  Subsequently, as shown in FIG. 3 (c), an additional reagent containing a first labeled antibody Y31 labeled with a first labeling enzyme and a second labeled antibody Y32 labeled with a second labeling enzyme Is injected from the inlet 8. The first labeled antibody Y31 specifically recognizes the first detection antibody Y21, and the second labeled antibody Y32 specifically recognizes the second detection antibody Y22.

  The first labeling enzyme and the second labeling enzyme may be the same or different. When the two labeling enzymes are different, it is preferable that the reaction conditions with the substrate, particularly the pH (hydrogen ion index), be the same. Preferably, the first labeling enzyme and the second labeling enzyme are the same. As the labeling enzyme, horseradish peroxidase, alkaline phosphatase, and the like, which are generally used in an ELISA reaction, can be used, but are not limited thereto.

  As shown in FIG. 3D, a substrate solution, which is an additive reagent containing the first substrate Z11 and the second substrate Z12, is injected from the injection port 8. Thereafter, the first enzyme reaction involving the first substrate Z11 and catalyzed by the first labeling enzyme, and the second enzyme reaction involving the second substrate Z12 and catalyzing by the second labeling enzyme are performed. proceed. The first enzyme reaction yields a first signal SG1 having a certain signal intensity (coloring, emission, fluorescence amplification, etc.), and the second enzyme reaction yields a second signal SG2 having a different signal intensity. The substrate can be determined in relation to the labeling enzyme. For example, as a substrate of horseradish peroxidase, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) which develops blue, O-phenylenediamine dihydrochloride (OPD) which develops orange yellow, and green which develops green 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt (ABTS). In addition, examples of the substrate for alkaline phosphatase include p-nitrophenyl phosphate and disodium salt (PNPP).

  As described above, the first enzymatic reaction is a reaction in which a substance that specifically recognizes the first antigen X11, which is a sample, is involved, and the sample is quantitatively detected. The second enzyme reaction is a known reaction in which a substance that specifically recognizes the second antigen X12 as a reference substance is involved and quantitatively detects the reference substance. These reactions are based on the principle of the enzyme-linked immunosorbent assay, and the components of the specific immobilization reagent and additive reagent are appropriately selected by those skilled in the art so as to be compatible with a desired specimen and reference substance. be able to. The first and second enzyme reactions are also referred to as a first reaction and a second reaction, respectively.

In the confirmation region 12, the amounts of antigen, antibody, and enzyme involved in the second reaction are constant and known. Then, the reaction time of the second reaction can be considered to be the same as the reaction time of the first reaction.
In addition, the reaction mode of the assay reagent, the specimen, and the reference substance in the present invention is not limited to the embodiment described with reference to FIG. In each of the assay region and the confirmation region, the reaction mode is such that each component of the assay reagent reacts quantitatively and specifically with the sample and the reference substance, and can generate a signal of an amount corresponding to the sample and the reference substance. Any mode may be used. Therefore, the present invention does not require an assay using all of the first reagent, the second reagent, and the substrate solution as described above.

  FIG. 4 shows the flow of the assay in one embodiment. As described later, some steps in this flow are performed by the assay support device 100 shown in FIG. The assay support device 100 includes an acquisition unit 110, a reaction time calculation unit 120, and a concentration calculation unit 130. The assay support device 100 can be, for example, a smartphone.

  FIG. 6 shows a computer hardware configuration example of the assay support apparatus 100. The assay support device 100 includes a CPU 181, an interface device 182, a display device 183, an input device 184, a drive device 185, an auxiliary storage device 186, a memory device 187, and a camera 190. They are interconnected by a bus 188.

  An assay support program for realizing the function of the assay support apparatus 100 (hereinafter, also simply referred to as “assay program” or “program”) is provided by a recording medium 189 such as a memory card. When the recording medium 189 on which the program is recorded is set in the drive device 185, the program is installed from the recording medium 189 to the auxiliary storage device 186 via the drive device 185. Alternatively, the program need not always be installed on the recording medium 189, and can be downloaded from another computer via a network. The auxiliary storage device 186 stores installed programs and also stores necessary files and data.

  The memory device 187 reads the program from the auxiliary storage device 186 and stores it when there is an instruction to start the program. The CPU 181 implements the functions of the assay support device 100 according to a program stored in the memory device 187. The interface device 182 is used as an interface for connecting to another computer through a network. The display device 183 displays a GUI (Graphical User Interface) by a program. The input device 184 is a keyboard, a mouse, and the like. Camera 190 can be, for example, a digital camera.

  FIG. 4 is referred to again. In step ST <b> 1, the assay practitioner drops a sample liquid that may contain the first antigen X <b> 11 as a specimen and contains the second antigen X <b> 12 as a reference substance in a known amount into the inlet 8. The state inside the microchannel in this step is as described above with reference to FIG. This step can also be performed in each of all the microchannel assemblies. The sample liquid dropped into the inlet of each microchannel assembly may be the same or different from each other.

  In step ST2, the assay practitioner drops the first reagent, which is a liquid containing the first detection antibody Y21 and the second detection antibody Y22, into the inlet 8. In this step, further, the assay practitioner determines that the second labeled antibody Y31 labeled with the first labeling enzyme and the second labeled antibody Y32 labeled with the second labeling enzyme are second liquids. Is dropped into the inlet 8. The mode of the reaction in the microchannel in this step is as described above with reference to FIGS. 3B and 3C. This step can also be performed in each of all the microchannel assemblies. The first reagent dropped at the inlet of each microchannel assembly may be the same or different from each other. The same applies to the second reagent.

  In step ST3, the assay operator drops a substrate solution, which is a liquid containing the first substrate Z11 and the second substrate Z12, into the inlet 8. The inside of the microchannel in this step is as described above with reference to FIG. In this step ST3, if the specimen is contained in the sample liquid dropped in step ST1, a first enzymatic reaction occurs and a first signal SG1 is obtained. Further, in this step ST3, a second enzyme reaction occurs and a second signal SG2 is obtained irrespective of the presence or absence of the sample. Both signals are visible through the transparent or translucent first layer member S1. This step can also be performed in each of all the microchannel assemblies. The substrate solution dropped into the inlet of each microchannel assembly may be the same or different from each other.

  In step ST4, the assay practitioner uses the assay support device 100 provided with the camera 190 to align the assay device AS with the assay support device 100 and then take a picture. This alignment is performed such that a rectangular guide region (not shown) displayed on the display device 183 of the assay support device 100 overlaps the outer shape of the top surface of the assay device AS in FIG. The image of the photograph taken in this step is acquired by the acquisition unit 110 of the assay support device 100. That is, in this image, the first signal SG1 generated in the assay area 10 in step ST3 and the second signal SG2 generated in the confirmation area 12 in the same step are shown.

  In step ST5, the assayer confirms the imaging environment (external light, brightness). For example, the assay practitioner may determine whether the color swatch CS is located near the assay area 10, the difference in reflected light between each color swatch (white) is within 5%, or the brightness of the color swatch (white). Is checked to see if the gradient is within a predetermined allowable range.

  In step ST6, the assay performer determines whether or not the image acquired in step ST4 needs to be corrected based on the result of checking the imaging environment in step ST5. If it is determined that correction is necessary, step ST8 is performed after step ST7 is performed, otherwise step ST8 is performed without performing step ST7.

  In step ST7, the obtaining unit 110 corrects the image obtained in step ST4. This correction is performed, for example, so that the difference in reflected light between the color samples (white) is within 5% and the brightness gradient of the color sample (white) is within a predetermined allowable range.

  Steps ST5 to ST7 can be omitted.

  In step ST8, the assay operator visually checks the second signal SG2 in the image (the image after correction in the case where the correction in step ST7 is performed). Hereinafter, the corrected image when the correction in step ST7 is performed is also simply referred to as an “image”.

  In step ST9, the assay executor determines whether or not the above-described second enzyme reaction has been performed based on the result of the visual observation in step ST8. When it is determined that the second enzyme reaction has been performed, step ST10 is performed. If it is not determined that the second enzyme reaction has been performed, the subsequent steps are not performed, and the flow ends. Thereafter, this flow can be performed again from step ST1.

  In step ST10, the reaction time calculation unit 120 of the assay support device 100 calculates the intensity of the second signal SG2 based on the pixel data (color information) of the pixel representing the second signal SG2 in the image. Specifically, a difference obtained by subtracting pixel data of a color sample (white) from pixel data (color information) of a pixel representing the second signal SG2 can be used as an intensity of the second signal SG2.

  For example, when the second substrate Z12 is TMB, the second enzyme reaction is a TMB reaction. The amount of the product of this TMB reaction is reflected in the value of R-R0, where R is the intensity of the second signal SG2 (red reflected light) and R0 is the red reflected intensity of the color sample (white). The purpose of subtracting the red reflection intensity R0 of the color sample (white) from the intensity R of the second signal SG2 is to remove the influence of the imaging conditions.

  In step ST10, the reaction time calculation unit 120 further calculates the amount of the product of the second reaction based on the calculated intensity of the second signal SG2. In this calculation, the reaction time calculation unit 120 refers to a known relationship between the intensity of the second signal SG2 and the amount of the product of the second reaction. This relationship is stored in the auxiliary storage device 186 of the assay support device 100 in advance.

  Similarly, in step ST10, the reaction time calculation unit 120 calculates the reaction time of the second reaction (the time spent in the second reaction) based on the calculated amount of the product of the second reaction. In this calculation, the reaction time calculation unit 120 refers to a known relationship between the reaction time of the second reaction and the amount of the product of the second reaction. An example of this relationship is shown in FIG. This known relationship is stored in the auxiliary storage device 186 of the assay support device 100 in advance.

  In step ST11, the concentration calculation unit 130 sets the reaction time of the second reaction obtained in step ST10 as the reaction time for only the first reaction, and then, based on the deemed reaction time, from among a plurality of calibration curves. Select a single calibration curve. The plurality of calibration curves represent the relationship between the intensity of the first signal SG1 and the concentration of the sample according to the reaction time of the first reaction. FIG. 8 shows examples of a plurality of calibration curves. The reaction times corresponding to the plurality of calibration curves WC1 to WC6 are 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, and 7 minutes, respectively.

  In step ST11, the density calculator 130 further calculates the intensity of the first signal SG1 based on the pixel data (color information) of the pixel representing the first signal SG1 in the image. In this step, the concentration calculator 130 further calculates the concentration of the first antigen X11 as a specimen based on the calculated intensity of the first signal SG1 and the single calibration curve previously selected. .

  For example, assume that the reaction time of the second reaction obtained in step ST10 is “5 minutes” (FIG. 7). In step ST11, this “5 minutes” is determined as the reaction time for only the first reaction. In the same step, a calibration curve WC4 corresponding to “5 minutes”, which is the reaction time for only the first reaction, is selected from the plurality of calibration curves WC1 to WC6 shown in FIG. Then, the intensity of the first signal SG1 is calculated, and the concentration of the sample is calculated based on the calculated intensity of the first signal SG1 and the calibration curve WC4.

  Further, as another example, when the reaction time for only the first reaction is “7.2 minutes”, the reaction time is “7 minutes” from the plurality of calibration curves WC1 to WC6 shown in FIG. The calibration curve WC6 is selected. In the case where only the first reaction is performed and the reaction time is “6.8 minutes”, the calibration curve WC6 having the reaction time of “7 minutes” is selected. When only the first reaction is completed and the reaction time is "6.4 minutes", the calibration curve WC5 having the reaction time of "6 minutes" is selected.

  As described above, the calibration curve corresponding to the reaction time having the minimum difference from the reaction time for the first reaction alone is selected. When the reaction time for the first reaction alone is "6.5 minutes", either the calibration curve WC5 having a reaction time of "6 minutes" or the calibration curve WC6 having a reaction time of "7 minutes" is used. One can be appropriately selected.

  Alternatively, in step ST11, a plurality of calibration curves can be selected. For example, when the reaction time for the first reaction alone is "7.2 minutes", a calibration curve WC6 having a reaction time of "7 minutes" and a calibration curve (not shown) having a reaction time of "8 minutes" And you can choose. Then, the concentration A obtained from the calibration curve WC6 and the concentration B obtained from the calibration curve having a reaction time of “8 minutes” are prorated, and the value of (A × 0.8 + B × 0.2) is determined for the sample. It can also be obtained as a concentration.

  As described above, in step ST11, the concentration of the sample can be obtained after selecting at least one calibration curve.

  The assay device according to the present embodiment includes a micro flow channel 1 configured to allow a sample liquid containing a known reference substance different from the sample, which may contain a sample, to flow therethrough. A first region 10 in which a first reaction for specifically detecting a sample is generated, and a known region which is provided adjacent to the first region 10 in the microchannel 1 and specifically detects a reference substance. And a second region 12 where two reactions occur. According to this device, since the second reaction is a known reaction that specifically detects the reference substance, the reaction time of the second reaction can be calculated based on the second signal from the second reaction. The reaction time of the second reaction can be regarded as the reaction time of the first reaction. A more accurate assay can be performed in consideration of the reaction time of the first reaction alone and the intensity of the first signal due to the first reaction.

  According to the assay program in the present embodiment, the reaction time of the second reaction for specifically detecting the reference substance intentionally included in the sample liquid by the assay operator is calculated. The reaction time of the second reaction is regarded as the reaction time of the first reaction for specifically detecting the sample. Then, the concentration of the sample is calculated based on the first signal due to the first reaction and the reaction time when only the first reaction is present. By considering not only the intensity of the first signal but also the reaction time of the first reaction alone, a more accurate assay can be performed as compared to the case where only the signal intensity is considered.

[Others]
The assay device AS may include one or more microchannel assemblies.

  Steps ST5 to ST9 in FIG. 4 can be omitted.

  Regarding the calculation of the reaction time of the second reaction in step ST10, the relationship between the intensity of the second signal and the reaction time of the second reaction can be determined in advance and stored in the auxiliary storage device 186. Then, the reaction time calculation unit 120 can refer to this relationship when calculating the reaction time of the second reaction. In this case, the reaction time calculator 120 does not need to calculate the amount of the product of the second reaction. Therefore, the amount of calculation can be reduced.

  FIG. 3 shows an example in which one assay region (first region) 10 is provided. However, the present invention is not limited to this, and a plurality of assay regions 10 may be provided.

AS: assay device, MC: microchannel assembly, 1: microchannel, 1a: distal end, 1b: proximal end, 2: porous medium for absorption, 6: vent, C: flow direction, W: Width direction, H: Height direction, S1: First layer member, S2: Second layer member, S3: Third layer member, 8: Injection port, 9: Porous medium for development, 10: Assay area, 12 ... Confirmation area 100: assay support device, 110: acquisition unit, 120: reaction time calculation unit, 130: concentration calculation unit

Claims (7)

A microchannel in an assay device configured to be able to flow a sample liquid containing a known reference substance different from the sample, which may include the sample, and specifically detecting the sample. An obtaining step of obtaining an image in which a first signal generated by one reaction and a second signal generated by a known second reaction for specifically detecting the reference substance are obtained;
A reaction time calculating step of calculating a reaction time of the second reaction based on pixel data of the second signal in the image acquired by the acquiring step;
The reaction time of the second reaction calculated in the reaction time calculation step is defined as the reaction time of the first reaction only, and the reaction time of the first reaction only, and the first reaction in the image acquired in the acquisition step. A concentration calculating step of calculating the concentration of the analyte based on the pixel data of the signal. The reaction time calculation step,
Based on the pixel data of the second signal in the image obtained in the obtaining step, calculate the intensity of the second signal, and based on the intensity of the second signal, determine the product of the second reaction. A first sub-step of calculating an amount;
The second sub-step of calculating a reaction time of the second reaction based on the amount of the product calculated in the first sub-step. The concentration calculating step includes:
From a plurality of calibration curves representing the relationship between the intensity of the first signal and the concentration of the sample in accordance with the reaction time of the first reaction, at least one calibration curve based on the reaction time without the first reaction. A third sub-step of selecting;
Calculating the intensity of the first signal based on the pixel data of the first signal in the image obtained in the obtaining step, and selecting the calculated intensity of the first signal by the third sub-step; A fourth sub-step of calculating the concentration of the sample based on the at least one calibration curve thus obtained. A microchannel in an assay device configured to be able to flow a sample liquid containing a known reference substance different from the sample, which may include the sample, and specifically detecting the sample. An acquisition unit that acquires an image in which a first signal generated by one reaction and a second signal generated by a known second reaction that specifically detects the reference substance are captured;
A reaction time calculation unit that calculates a reaction time of the second reaction based on pixel data of the second signal in the image acquired by the acquisition unit;
The reaction time of the second reaction calculated by the reaction time calculation unit is the reaction time of the first reaction only, and the reaction time of the first reaction only, and the first reaction in the image acquired by the acquisition unit. A concentration calculator for calculating the concentration of the sample based on the pixel data of the signal. The reaction time calculation unit,
Based on the pixel data of the second signal in the image acquired by the acquisition unit, calculate the intensity of the second signal, based on the intensity of the second signal, the product of the second reaction Calculate the amount,
The assay support device according to claim 4, wherein a reaction time of the second reaction is calculated based on the calculated amount of the product. The concentration calculator,
From a plurality of calibration curves representing the relationship between the intensity of the first signal and the concentration of the sample in accordance with the reaction time of the first reaction, at least one calibration curve based on the reaction time without the first reaction. Selected,
Based on the pixel data of the first signal in the image acquired by the acquisition unit, calculate the intensity of the first signal, and the calculated intensity of the first signal, the selected at least one of the The assay support device according to claim 4, wherein the concentration of the sample is calculated based on a calibration curve. A microchannel configured to allow a sample liquid containing a known reference substance different from the sample, which may contain the sample, and
A first region provided in the microchannel, where a first reaction for specifically detecting the sample occurs,
A second region, which is provided adjacent to the first region in the microchannel, and in which a known second reaction for specifically detecting the reference substance occurs.