JP7062285B2 - Assay program and assay equipment - Google Patents

Description

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

Mainly in the fields of biology, chemistry, etc., inspections, experiments, assays, etc. are performed on the order of μl (microliter), that is, using a small amount of reagents such as reagents and treatment agents of about 1 μl or more and less than about 1 ml (milliliter). When doing so, an assay device containing a microchannel is utilized. 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 move and operate a liquid by utilizing 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 assay device is particularly used for detecting or quantifying the concentration of an antibody or antigen contained in a sample by an ELISA (Enzyme-Linked Immuno Sorbent Assay) method, an immunochromatography method, or the like. Be done.

As an example of such an assay device, a channel and an assay region connected to the channel are provided in a plurality of laminated porous media, and the channels and the assay region are laminated. An assay device defined by a barrier consisting of a polymerized photoresist absorbed throughout the thickness direction of the porous medium of. (See, for example, Patent Document 1.)

Another example of the assay device is the assay device invented by the inventor of the present application. This assay device has a microchannel, a porous medium arranged at a distance from one end of the microchannel, and a space portion arranged between one end of the microchannel and the porous medium. Then, the liquid that has moved in the microchannel is contacted with the porous medium beyond the space and is absorbed, and then the fluid is separated in the space so as to be indwelled in the microchannel. Is. (See, for example, Patent Document 2.)

Special Table 2010-515877 Japanese Patent No. 6037184

The signal intensity (color development, luminescence, fluorescence amplification, etc.) generated by a reaction such as an enzymatic reaction changes 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 which the intensity of a signal due to one reaction is taken into consideration and the reaction time is taken into consideration based on the reaction time of another reference reaction.

In order to solve the above problems, the assay program according to one aspect of the present invention is configured to be able to flow a sample liquid which may contain a sample and contains a known reference substance different from the sample. In the microchannel in the assay device, the first signal generated by the first reaction that specifically detects the sample and the second signal generated by the known second reaction that specifically detects the reference substance are copied. An acquisition step for acquiring an image, a reaction time calculation step for calculating the reaction time of the second reaction based on the pixel data of the second signal in the image acquired by the acquisition step, and the reaction time calculation step. The reaction time of the second reaction calculated by the above is taken as the deemed reaction time of the first reaction, the deemed reaction time of the first reaction, and the pixel data of the first signal in the image acquired by the acquisition step. Based on the above, a computer is made to perform a concentration calculation step of calculating the concentration of the sample.

The assay support device according to an aspect of the present invention is a micro flow in an assay device configured to contain a sample and to allow a sample liquid containing a known reference substance different from the sample to flow. An acquisition unit that acquires an image of a first signal generated by a first reaction that specifically detects the sample and a second signal generated by a known second reaction that specifically detects the reference substance in the road. The reaction time calculation unit that calculates the reaction time of the second reaction based on the pixel data of the second signal in the image acquired by the acquisition unit, and the second reaction time calculation unit that calculates the reaction time. The reaction time of the reaction is defined as the deemed reaction time of the first reaction, and the sample is based on the deemed reaction time of the first reaction and the pixel data of the first signal in the image acquired by the acquisition unit. It is provided with a concentration calculation unit for calculating the concentration.

In another embodiment, the assay device comprises a microchannel configured to allow the flow of a sample liquid that may contain a sample and that contains a known reference substance that is different from the sample, and said microflow. A first region provided in the pathway where the first reaction for specifically detecting the sample occurs, and a known region provided adjacent to the first region in the microchannel to specifically detect the reference substance. It is provided with a second region in which the second reaction of the above occurs.

According to the present invention, it is possible to perform an assay in consideration of the intensity of a signal due to a reaction such as an enzymatic reaction and the time of the reaction.

FIG. 1 is a plan view schematically showing an assay device according to an embodiment. FIG. 2 is an exploded perspective view schematically showing the assay device according to the embodiment. 3 (a) to 3 (d) are cross-sectional views taken along the line AA of FIG. 1 showing a state of a microchannel when a sample liquid is supplied to the assay device according to an embodiment and an assay is performed. be. FIG. 4 is a flowchart showing an assay method according to an embodiment. FIG. 5 is an explanatory diagram showing an example of the functional configuration of the assay support device. FIG. 6 is an explanatory diagram showing an example of 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 product produced by the reaction. FIG. 8 is an explanatory diagram showing an example of a plurality of calibration curves.

An embodiment of the present invention will be described below. However, the present invention is not limited to 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 a sample described below, and may also include a gas, another liquid or solid dissolved, dispersed or suspended in a liquid. can.

For example, the sample liquid may be hydrophilic, and the hydrophilic sample liquid may be, for example, extraction of whole human or animal blood, serum, plasma, blood cells, urine, fecal diluent, saliva, sweat, tears, nails. Examples thereof include liquids, skin extracts, hair extracts, and biological sample liquids such as cerebrospinal fluid. In this case, in the assay device, an in-vitro diagnostic drug for pregnancy test, urine test, stool test, adult disease test, allergy test, infectious disease test, drug test, cancer test, etc., general-purpose test drug, POCT ( It is possible to measure a sample that is effective for clinical examination, diagnosis, or analysis in a sample liquid in applications such as PointOfCareTesting), but the application of the assay device is not particularly limited. Further, the hydrophilic sample liquid is not limited to the sample liquid derived from a living body, and examples thereof include food suspensions, drinking water, river water, and soil suspensions. 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 water-based.

As used herein, "lateral flow" refers to the flow of liquid that moves when gravity settling is the 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 the gravitational sedimentation acts predominantly (dominantly). On the other hand, the movement of the liquid based on the capillary force (capillary phenomenon) refers to the movement of the liquid in which the interfacial tension acts predominantly (predominantly). The movement of liquid based on lateral flow is different from the movement of liquid based on capillary force.

As used herein, "specimen" refers to a compound or composition that is present in the sample liquid and is detected or measured. For example, a "specimen" is a saccharide (eg, glucose), protein or peptide (eg, serum protein, hormone, enzyme, immunomodulator, phosphokine, monokine, cytokine, glycoprotein, vaccine antigen, antibody, growth factor, or proliferation. Factors), fats, amino acids, nucleic acids, cells, steroids, vitamins, pathogens or antigens thereof, natural or synthetic chemicals, contaminants, therapeutic drugs or illegal drugs or poisons, or metabolites or antibodies of these substances. However, it is not limited to a specific sample. The sample liquid may not contain the sample, or may not contain the sample in a detectable amount. A "reference substance" is a known substance different from the sample that is added to the sample liquid in a known amount to detect the sample concentration. The reference substance can be selected from the above options in the same manner as the sample, and can be selected in relation to the sample. In particular, it does not interact with the sample and can be selected from stable substances.

As used herein, a "microchannel" is used to detect or measure a sample on the order of μl (microliter), i.e., with a trace amount of sample liquid greater than or equal to about 0.1 μl and less than about 1 ml (milliliter). Refers to a pathway configured to flow a liquid within an assay device.

In the present specification, "film" refers to a film-like object having a thickness of about 200 μm (micrometer) or less, and “sheet” refers to a film-like object or a plate-like object having a thickness of more than about 200 μm. Point to.

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

In the present specification, the "porous medium" may be a member having a plurality of 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, and may be paper. , A member containing a cellulose film, a non-woven fabric, a glass fiber, a polymer gel, a plastic, or the like. For example, the "porous medium" may be hydrophilic when the sample liquid is hydrophilic, and may be hydrophobic when the sample liquid is hydrophobic. In particular, the "porous medium" is preferably hydrophilic and is preferably paper, cotton wool or the like containing a large number of fibers. Further, the "porous medium" shall be one or more of cellulose, nitrocellulose, cellulose acetate, filter paper, tissue paper, toilet paper, paper towels, fabrics, cotton, or a hydrophilic porous polymer that is permeable to water. Can be done.

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

The microchannel assembly MC has an absorbing porous medium 2 arranged at the tip portion 1a of the microchannel 1. The absorbing porous medium 2 is configured to be able to absorb the sample liquid that has flowed from the base end portion 1b of the microchannel 1 to the tip end portion 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 its use state, the microchannel assembly MC is preferably arranged so that the height direction faces vertically, and in this case, the top surface and the bottom surface of the microchannel assembly MC face upward and downward, respectively. .. The microchannel 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. It is preferable that the first layer member S1, the second layer member S2, and the third layer member S3 are formed substantially in layers.

The first layer member S1 and the third layer member S3 are configured by using a material that does not allow the sample liquid to permeate and does not absorb signals from the assay region and the confirmation region described later. The contact angles of the first layer member S1 and the third layer member S3 are preferably smaller than 90 degrees. The first layer member S1 may be transparent or translucent. It is desirable that at least one of the first layer member S1 and the third layer member S3 be 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 made of plastic. Further, each material of the first layer member S1 and the third layer member S3 may be 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), and the like. Polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyolefin (PO), nylon, polymethylmethacrylate (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, if at least one of the first layer member S1 and the third layer member S3 is a material that does not allow fluid to permeate, it can be manufactured using a material other than plastic, and such a material other than plastic is a resin. , Glass, metal, etc. The materials or materials used for such a first layer member S1 and a third layer member S3 may be the same or different.

The second layer member S2 may be 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 by using the material or the material shown as the material 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 material used for the second layer member is the first or first layer member. It may be the same as or different from that used for the three-layer member.

As shown in FIG. 2, the microchannel 1 is formed so as to penetrate the second layer member S2. That is, the two side surfaces of the micro flow path 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 micro flow path 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 linear shape. However, in the present invention, the microchannel can also be formed to be curved or bent.

The surface defining the microchannel 1 may be treated with hydrophilicity. Such hydrophilic treatments include treatments with blocking agents that allow the specific conjugates to be prevented from adsorbing on their surfaces when the sample liquid contains specific conjugates. Examples of the blocking agent include commercially available blocking agents such as Block Ace, bovine serum albumin, casein, skim milk, gelatin, surfactants, polyvinyl alcohol, globulin, serum (for example, fetal bovine serum or normal rabbit serum), ethanol, and MPC polymer. And so on. Such blocking agents can be used alone or in admixture of two or more.

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

A vent 6 configured to allow air to flow is provided at the tip of the first layer member S1 that defines the top surface of the tip 1a of the microchannel 1. The vent 6 may be configured to communicate the outside of the microchannel assembly MC with the tip portion 1a of the microchannel 1. When the liquid flows through the microchannel 1 from the proximal end 1b to the distal end 1a, the air in the microchannel 1 exits the microchannel assembly MC through the vent 6.

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

The microchannel assembly MC further has a deployable porous medium 9 disposed within the proximal end portion 1b 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 microchannel as described later, the microchannel assembly MC can be configured so as not to have the developing porous medium 9. In FIG. 3, the development porous medium 9 is not shown.

As shown in FIGS. 1 and 3, an assay region 10 is provided in the middle portion of the microchannel 1 in the flow direction C. In the assay region 10, a reagent that specifically binds to the sample in the assay is immobilized. Hereinafter, in the present specification, reagents involved in signal generation derived from a sample and a reference substance are referred to as assay reagents. Assay reagents include immobilization reagents that are previously immobilized in the microchannel and used, and additive reagents that are added and used in the microchannel in the assay step. The immobilization reagent provided in the assay region 10 specifically reacts with the sample in the sample liquid and, together with the additive reagent, produces a detectable result of the sample. Specimen detectable results may be visible to the naked eye, for example based on color changes, etc., or specimen detectable results may be detectable only by a spectroscope or other measuring means. You may. This assay region is also referred to as the first region.

Immobilization reagents provided in the assay region 10 include enzymes, antibodies, epitopes, nucleic acids, cells, aptamers, peptides, molecular imprint polymers, adsorption polymers, adsorption gels, iron (III) ions that develop color by reaction with a sample, and the like. It may be a chemical, color reagent, or any other substance that produces detectable results by reacting with a sample, typically the immobilization reagent is an antibody. The immobilization reagent may be immobilized in the assay region 10 by a well-known immobilization technique such as a physical adsorption method or a chemisorption method. Immobilization reagents include radioactive isotopes, enzymes, gold colloids, color-developing reagents, quantum dots, colored molecules such as 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 attached to an additive reagent used in addition to the microchannel in the step of the assay. Specifically, the immobilization reagent can be immobilized on either 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 with the sample liquid flowing in the microchannel 1 or the sample liquid being allowed to stand in the microchannel 1 or temporarily stopped. Typically, the sample concentration in the sample liquid can be detected.

As shown in FIG. 1, the color swatch CS can be placed on the top surface of the assay device AS and in the vicinity of the assay region 10. The intensity of the signal can be confirmed by comparing this color swatch with the signal generated in the assay region 10.

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. The assay region 10 and the confirmation region 12 may be separated from each other to such an extent that the signals generated from the respective regions can be distinguished and detected. The confirmation region 12 is configured to generate a known second reaction (described below) that can be considered to have the same reaction time as the first reaction (described below) that occurs in the assay region 10. This confirmation area 12 is also referred to as 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 region 12 may also be an antibody or the like, like the immobilization reagent in the assay region 10, and any labeling substance may be bound to the immobilization reagent. It can be fixed to either 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 immobilization reagent. With reference to FIG. 3, the reaction between the assay reagent and the sample, and the reference substance and the mechanism of detecting the sample concentration by the reaction will be described. As shown in FIG. 3A, a first antibody Y11, which is an immobilization reagent, is immobilized in the assay region 10, and an immobilization reagent different from that of the first antibody Y11 is used in the confirmation region 12. A second antibody, Y12, is immobilized. Then, a sample liquid which may contain the first antigen X11 which is a sample and contains a known amount of the second antigen X12 is injected from the injection port 8. The second antigen X12 is a known reference substance different from the first antigen X11 which is a sample, and is intentionally included in the sample liquid in a predetermined known amount by the assayer. 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, the first reagent, which is an additive reagent containing 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 first detection antibody Y21 and the second detection antibody Y22, which are components contained in the first reagent, can be determined in relation to the sample and the reference substance.

Subsequently, as shown in FIG. 3 (c), an additive reagent containing the first labeled antibody Y31 labeled with the first labeling enzyme and the second labeled antibody Y32 labeled with the second labeling enzyme. The second reagent is injected from the injection port 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 both labeling enzymes are different, it is preferable that the reaction conditions with the substrate, particularly pH (hydrogen ion index), are about 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 the ELISA reaction, can be used, but the labeling enzyme is 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. Then, a first enzymatic reaction involving the first substrate Z11 and being catalyzed by the first labeling enzyme and a second enzymatic reaction involving the second substrate Z12 and being catalyzed by the second labeling enzyme are performed. proceed. The first enzymatic reaction gives the first signal SG1 having a certain signal intensity (color development, light emission, fluorescence amplification, etc.), and the second enzymatic reaction gives the second signal SG2 having another signal intensity. The substrate can be determined in relation to the labeling enzyme. For example, horseradish peroxidase substrates include 3,3', 5,5'-tetramethylbenzidine (TMB), which develops a blue color, O-phenylenediamine dihydrochloride (OPD), which develops an orange-yellow color, and green color. 2,2'-Azinobis [3-ethylbenzothiazolin-6-sulfonic acid] -diammonium salt (ABTS) and the like. 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 to quantitatively detect the sample. The second enzymatic reaction is a known reaction in which a substance that specifically recognizes the second antigen X12, which is a reference substance, is involved to quantitatively detect the reference substance. These reactions are based on the principle of the enzyme-bound immunoadsorption method, and each component of the specific immobilization reagent and additive reagent is appropriately selected by those skilled in the art so as to be compatible with the desired sample and reference substance. be able to. The first enzymatic reaction and the second enzymatic reaction are also referred to as a first reaction and a second reaction, respectively.

In the confirmation region 12, the amount of antigen, the amount of antibody, and the amount of enzyme involved in the second reaction are constant and known. Then, the reaction time of the second reaction can be regarded as the same as the reaction time of the first reaction.
The reaction mode between the assay reagent in the present invention and the sample and the reference substance is not limited to the mode described with reference to FIG. A reaction mode capable of quantitatively and specifically reacting each component of the assay reagent with the sample and the reference substance in each of the assay region and the confirmation region to generate a signal in an amount corresponding to the sample and the reference substance. For example, any aspect 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 will be 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 an example of the computer hardware configuration of the assay support device 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 connected to each other by bus 188.

The assay support program (simply also referred to as "assay program" or "program") that realizes the function of the assay support device 100 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 in the auxiliary storage device 186 from the recording medium 189 via the drive device 185. Alternatively, the installation of the program does not necessarily have to be performed by the recording medium 189, and can be downloaded from another computer via the network. The auxiliary storage device 186 stores the installed program and also stores necessary files, data, and the like.

The memory device 187 reads and stores the program from the auxiliary storage device 186 when the program is instructed to start. The CPU 181 realizes the function of the assay support device 100 according to the 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) or the like by a program. The input device 184 is a keyboard, a mouse, or the like. The camera 190 can be, for example, a digital camera.

See again FIG. In step ST1, the assayer drops a sample liquid into the inlet 8 that may contain the first antigen X11 which is the sample and which contains the second antigen X12 which is the reference substance in a known amount. The state of the inside of the microchannel in this step is as described above with reference to FIG. 3A. It should be noted that this step can also be performed in each of all the microchannel assemblies. The sample liquids dropped onto the inlet of each microchannel assembly may be the same or different from each other.

In step ST2, the assayer drops a 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, the assayer further comprises a second labeled antibody Y31 labeled with the first labeling enzyme and a second labeled antibody Y32 labeled with the second labeling enzyme. The reagent of is dropped into the injection port 8. The mode of the reaction in the microchannel in this step is as described above with reference to FIGS. 3 (b) and 3 (c). It should be noted that this step can also be performed in each of all the microchannel assemblies. The first reagents dropped into 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 assayer drops a substrate solution, which is a liquid containing the first substrate Z11 and the second substrate Z12, into the inlet 8. The state of the inside of the microchannel in this step is as described above with reference to FIG. 3 (d). By this step ST3, if the sample liquid dropped in step ST1 contains a sample, the first enzymatic reaction occurs and the first signal SG1 is obtained. Further, in this step ST3, a second enzymatic reaction occurs regardless of the presence or absence of the sample, and the second signal SG2 is obtained. Both signals are visible through the transparent or translucent first layer member S1. It should be noted that this step can also be performed in each of all the microchannel assemblies. The substrate solutions dropped onto the inlet of each microchannel assembly may be the same or different from each other.

In step ST4, the assay performer uses the assay support device 100 provided with the camera 190 to align the assay device AS and the assay support device 100, and then take a picture. This alignment is performed so that the 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 of 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, this image shows the first signal SG1 generated in the assay region 10 in step ST3 and the second signal SG2 generated in the confirmation region 12 in the same step.

In step ST5, the assayer confirms the imaging environment (external light, brightness). For example, the assayer may determine whether the color swatch CS is located near assay region 10, the difference in reflected light between each color swatch (white) is within 5%, or the brightness of the color swatch (white). Check if the gradient of is within the specified allowable range.

In step ST6, the assayer determines whether correction of the image acquired in step ST4 is necessary based on the result of confirmation of 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 step ST7.

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

It is also possible to omit steps ST5 to ST7.

In step ST8, the assayer visually confirms the second signal SG2 shown in the image (or the corrected image if 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 assayer determines whether or not the above-mentioned second enzymatic reaction has taken place based on the visual results of step ST8. If it is determined that the second enzymatic reaction has occurred, step ST10 is performed. If it is not determined that the second enzymatic reaction has taken place, no subsequent steps are taken and the flow ends. After that, 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, the difference obtained by subtracting the pixel data of the color sample (white) from the pixel data (color information) of the pixel representing the second signal SG2 can be used as the intensity of the second signal SG2.

For example, when the second substrate Z12 is TMB, the second enzymatic reaction is a TMB reaction. The amount of the product produced by 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 reflection 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 photographing conditions.

In step ST10, the reaction time calculation unit 120 further calculates the amount of the product due to 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 produced by the second reaction. This relationship is pre-stored in the auxiliary storage device 186 of the assay support device 100.

Similarly, in step ST10, the reaction time calculation unit 120 calculates the reaction time of the second reaction (time spent in the second reaction) based on the calculated amount of the product produced by 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 produced by the second reaction. An example of this relationship is shown in FIG. This known relationship is pre-stored in the auxiliary storage device 186 of the assay support device 100.

In step ST11, the concentration calculation unit 130 sets the reaction time of the second reaction obtained in step ST10 as the deemed reaction time of the first reaction, and then selects from a plurality of calibration curves based on this deemed reaction time. 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. An example of a plurality of calibration curves is shown in FIG. 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 calculation unit 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 calculation unit 130 further calculates the concentration of the first antigen X11 as a sample based on the calculated intensity of the first signal SG1 and the previously selected single calibration curve. ..

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 defined as the deemed reaction time of the first reaction. In the same step, the calibration curve WC4 corresponding to the deemed reaction time of the first reaction, "5 minutes", is further 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.

As another example, when the deemed reaction time of 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. Even when the deemed reaction time of the first reaction is "6.8 minutes", the calibration curve WC6 having a reaction time of "7 minutes" is selected. When the deemed reaction time of the first reaction is "6.4 minutes", the calibration curve WC5 having a reaction time of "6 minutes" is selected.

In this way, the calibration curve corresponding to the reaction time having the minimum difference from the deemed reaction time of the first reaction is selected. When the deemed reaction time of the first reaction 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". One can be selected as appropriate.

Alternatively, a plurality of calibration curves can be selected in step ST11. For example, when the deemed reaction time of the first reaction is "7.2 minutes", the calibration curve WC6 having a reaction time of "7 minutes" and the calibration curve having a reaction time of "8 minutes" (not shown). And can be selected. 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 divided into two, and the value of (A × 0.8 + B × 0.2) is used as 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 in this embodiment has a microchannel 1 and a microchannel 1 configured to allow a sample liquid containing a sample and containing a known reference substance different from the sample to flow. A first region 10 in which a first reaction for specifically detecting a sample occurs, and a known first region 10 provided adjacent to the first region 10 in the microchannel 1 to specifically detect a reference substance. It includes a second region 12 where two reactions occur. According to this apparatus, since the second reaction is a known reaction that specifically detects a reference substance, the reaction time of the second reaction can be calculated based on the second signal of 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 deemed reaction time of the first reaction and the intensity of the first signal due to the first reaction.

According to the assay program in this embodiment, the reaction time of the second reaction for specifically detecting the reference substance intentionally contained in the sample liquid by the assayer is calculated. The reaction time of this second reaction is regarded as the reaction time of the first reaction that specifically detects the sample. Then, the concentration of the sample is calculated based on the first signal by the first reaction and the deemed reaction time of the first reaction. By considering not only the intensity of the first signal but also the deemed reaction time of the first reaction, an accurate assay can be performed as compared with the case where only the signal intensity is considered.

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

It is also possible to omit steps ST5 to ST9 in FIG.

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 calculation unit 120 does not need to calculate the amount of the product produced by 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, but 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 ... Tip, 1b ... Base end, 2 ... Absorption porous medium, 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 expansion, 10 ... assay region, 12 ... Confirmation area 100 ... Assay support device, 110 ... Acquisition unit, 120 ... Reaction time calculation unit, 130 ... Concentration calculation unit

Claims (7)

A first that specifically detects the sample in a microchannel in an assay device configured to allow the flow of a sample liquid that may contain the sample and contains a known reference substance that is different from the sample. An acquisition step of acquiring an image of a first signal generated by one reaction and a second signal generated by a known second reaction that specifically detects the reference substance, and an acquisition step.
A reaction time calculation step for calculating the reaction time of the second reaction based on the 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 reaction time calculation step is defined as the deemed reaction time of the first reaction, and the deemed reaction time of the first reaction and the first reaction in the image acquired by the acquisition step. An assay program that causes a computer to perform a concentration calculation step of calculating the concentration of the sample based on the pixel data of the signal. The reaction time calculation step
The intensity of the second signal is calculated based on the pixel data of the second signal in the image acquired by the acquisition step, and the product of the second reaction is calculated based on the intensity of the second signal. The first substep to calculate the amount and
The assay program of claim 1, comprising a second substep of calculating the reaction time of the second reaction based on the amount of the product calculated by the first substep. The concentration calculation step
From a plurality of calibration curves representing the relationship between the intensity of the first signal and the concentration of the sample according to the reaction time of the first reaction, at least one calibration curve is obtained based on the deemed reaction time of the first reaction. The third substep to select and
The intensity of the first signal is calculated based on the pixel data of the first signal in the image acquired in the acquisition step, and the calculated intensity of the first signal is selected by the third sub-step. The assay program of claim 1 or 2, comprising a fourth substep of calculating the concentration of the sample based on the at least one calibration curve. A first that specifically detects the sample in a microchannel in an assay device configured to allow the flow of a sample liquid that may contain the sample and contains a known reference substance that is different from the sample. An acquisition unit that acquires an image of a first signal generated by one reaction and a second signal generated by a known second reaction that specifically detects the reference substance.
A reaction time calculation unit that calculates the reaction time of the second reaction based on the 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 defined as the deemed reaction time of the first reaction, and the deemed reaction time of the first reaction and the first reaction in the image acquired by the acquisition unit are used. An assay support device including a concentration calculation unit that calculates the concentration of the sample based on the pixel data of the signal. The reaction time calculation unit
The intensity of the second signal is calculated based on the pixel data of the second signal in the image acquired by the acquisition unit, and the product of the second reaction is calculated based on the intensity of the second signal. Calculate the amount,
The assay support device according to claim 4, wherein the reaction time of the second reaction is calculated based on the calculated amount of the product. The concentration calculation unit
From a plurality of calibration curves representing the relationship between the intensity of the first signal and the concentration of the sample according to the reaction time of the first reaction, at least one calibration curve is obtained based on the deemed reaction time of the first reaction. selection,
The intensity of the first signal is calculated based on the pixel data of the first signal in the image acquired by the acquisition unit, the calculated intensity of the first signal, and at least one selected. The assay support device according to claim 4 or 5, which calculates the concentration of the sample based on the calibration curve. A microchannel configured to allow the flow of a sample liquid that may contain a sample and that contains a known reference substance that is different from the sample.
A first region provided in the microchannel where the first reaction for specifically detecting the sample occurs, and
An assay device provided adjacent to the first region in the microchannel and comprising a second region in which a known second reaction that specifically detects the reference substance occurs.

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