JP2007024742A - Enzyme immunoassay method and enzyme immunosensor for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme immunoassay method capable of speedily, highly sensitively, and easily measuring even a very small amount of molecules present in a living body with a satisfactory S/N ratio and without a part to be detected affected by the effects of nonspecific adsorption or having to use a sandwich method and provide an enzyme immunosensor for the same. <P>SOLUTION: The enzyme immunosensor is provided with both an antibody capture region in which a substance to be tested is immobilized in a channel and an enzyme reaction product measuring region capable of detecting enzyme reaction products of a labeling enzyme. A sample liquid containing the substance to be tested and an anti-substance-to-be-tested antibody labelled with the labelling enzyme is made to flow through the antibody capture region to capture an unreacted antibody in the antibody capture region. A substrate solution containing a substrate of the labelling enzyme is made to flow through the antibody capture region to induce enzyme reaction by the labelling enzyme, and products of the enzyme reaction are brought into contact with the enzyme reaction product measuring region and measured. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an enzyme immunoassay method and an enzyme immunosensor used therefor.

  Conventionally, highly sensitive and selective measurement of peptides in a living body has been actively performed. Until now, immunoassay methods utilizing the molecular recognition ability of antibodies against these peptides have been performed. However, important peptides such as brain natriuretic peptide (BNP, about 10 pg / mL in blood), atrial natriuretic peptide (ANP), insulin, substance P, etc. present in biological samples only at very low concentrations It is difficult to quantitate these extremely low-concentration peptides using enzyme immunoassay methods that have been widely used so far. Among the enzyme immunoassays, the sandwich method is known to be capable of more sensitive measurement, but it is necessary to prepare two monoclonal antibodies that recognize different epitopes on the same antigen. The range was limited, and it was difficult to apply the sandwich method especially when the target molecule was small. On the other hand, although the radioimmunoassay method is capable of extremely high-sensitivity quantification, there is a problem in safety due to the use of radioisotopes, and measurement at the bedside or at home is difficult. For this reason, for example, in a clinical setting where patients with heart disease are competing for a moment, a novel sensor for quickly and easily measuring a marker molecule for heart disease at the bedside or the like is required.

  Currently, immunochromatography is commonly used as a rapid and simple immunoassay and is commercially available from various companies (for example, Patent Document 1). However, generally, the lower limit of detection of the immunochromatography method is about several ng / mL, and the measurement object is limited to molecules present at a relatively high concentration.

  On the other hand, in recent years, attempts to miniaturize and integrate an analysis system on glass, silicon, etc. have been actively carried out, and it is considered effective for simplification of operation, automation, and shortening of analysis time. . Regarding the immunoassay method, it is considered that rapid and simple detection can be performed by performing a technique that has been conventionally performed in bulk using a microchannel, and there are some reports (for example, Patent Document 2 and Patent Document 3).

JP-A-5-133958 JP 2003-114229 A JP 2001-4628 A W.C.Tang, D.P.Bame, T.K.Tang, Sensors and Actuators 83_2000.188193, I. Chakraborty

  At present, although it is possible to perform measurement easily and in a short time with a general immunochromatography method, since the sample feeding is performed using the capillary phenomenon, it is difficult to determine an arbitrary flow rate, There is a problem that the quantitativeness and the detection limit are poor. Even in relatively sensitive fluorescence-labeled antibodies and their fluorescence detection, the porous in which the target sample is developed is generally low in transparency, and the light scattering is severe, and the quantitativeness of the immunochromatography method is poor. . On the other hand, although detection examples of trace biomolecules have been reported by immunoassay methods using microchannels that have been reported so far, sandwich immunoassay is performed in the channel to obtain sufficient sensitivity in very small amounts, Further, a secondary antibody labeled with colloidal gold or the like is used. For this reason, the molecules to be measured are limited to relatively large molecules capable of sandwich immunoassay, and it is necessary to integrate a large number of microchannels on the chip and repeat the reaction and washing multiple times. The problem of requiring a simple liquid feeding procedure remains. In addition, when the sample solution is directly introduced into the detection portion, the influence of non-specific adsorption is very large and the reproducibility is poor, so that the improvement of the S / N ratio is a problem.

  The object of the present invention is in view of the problems of sensitivity, influence of nonspecific adsorption, measurement time, and complexity of measurement operation in the immunochromatography method and immunoassay method having a microchannel that have been reported so far. Enzyme immunoassay method not based on the sandwich method, which can be measured quickly, highly sensitively and easily with a good S / N ratio without affecting the nonspecific adsorption even in molecules present in extremely small amounts, and for the same It is to provide an enzyme immunosensor.

  As a result of earnest research, the inventors of the present application provided an antibody capture region in which the test substance is immobilized in the flow path, and an enzyme reaction product measurement region capable of detecting the enzyme reaction product of the labeled enzyme. And a sample solution containing the enzyme-labeled anti-test substance antibody is circulated in the antibody capture region to capture unreacted antibody in the antibody capture region, and then the substrate solution containing the labeled enzyme substrate is captured in the antibody capture region. The enzyme reaction product is made to contact the enzyme reaction product measurement region and the enzyme reaction product is measured without using the sandwich method. The present invention was completed by finding that even a very small amount of molecules in the body can be measured quickly, highly sensitively and easily with a good S / N ratio without being influenced by non-specific adsorption. did.

  That is, the present invention includes a substrate provided with a flow path, an antibody capture region provided in the flow path and immobilizing an antibody capture substance that undergoes an antigen-antibody reaction with an antibody against a test substance, Using an enzyme immunosensor having an enzyme reaction product measurement region provided in a region other than the antibody capture region, an enzyme-labeled antibody or antigen thereof that reacts with the test substance and the test substance with an antigen antibody A sample solution containing a binding fragment is allowed to flow through the antibody capture region to capture the unreacted antibody or antigen-binding fragment thereof in the antibody capture region, and then a substrate solution containing a substrate for the labeling enzyme is Flowing an antibody capture region and performing an enzyme reaction with the labeled enzyme, and then contacting at least a part of the enzyme reaction product with the enzyme reaction product measurement region to measure the enzyme reaction product. Including, before Providing an enzyme immunoassay of a test substance. The present invention also provides a substrate provided with a flow path, an antibody capture region provided in the flow path and immobilizing an antibody capture substance that undergoes an antigen-antibody reaction with an antibody against a test substance, There is provided an enzyme immunosensor for performing the enzyme immunoassay method of the present invention, comprising an enzyme reaction product measurement region provided in a region other than the antibody capture region.

  According to the enzyme immunoassay method of the present invention, without using the sandwich method, even in a molecule present in a very small amount in a living body, the detection portion is not affected by non-specific adsorption, has a good S / N ratio, is rapid, High sensitivity and simple measurement are possible. In addition, since the immunoassay method of the present invention is an enzyme immunoassay method, it does not use a radiolabel that requires a dangerous and large-scale apparatus and is inconvenient to handle. Therefore, the method of the present invention can be easily performed at the bedside, the patient's home, and the like, and is expected to greatly contribute to the diagnosis and treatment based on various diseases.

  As described above, in the enzyme immunoassay method of the present invention, a substrate provided with a flow path, and an antibody capture region provided in the flow path and immobilizing an antibody capture substance that reacts with an antibody against a test substance with an antigen antibody. And an enzyme immunosensor comprising an enzyme reaction product measurement region provided in a region other than the antibody capture region in the channel. Hereinafter, this enzyme immunosensor will be described first.

  The substrate of the enzyme immunosensor used in the enzyme immunoassay method of the present invention has a flow path through which a liquid flows. The channel is preferably a groove provided on the surface of the substrate, but is not limited to this. For example, a portion other than a position where sample liquid or substrate liquid described later is supplied or discharged is covered. It can also be a tunnel. The substrate may be composed of a single member, but an upper sheet having a through hole as a flow path portion and a simple plate-like lower substrate may be bonded to form a substrate. In this case, the lower substrate constitutes the bottom surface of the flow path. The material constituting the substrate is not limited as long as the material does not non-specifically adsorb the test substance, and various synthetic resins, synthetic rubbers, glass, and the like can be used. In the following example, a silicon rubber sheet (upper sheet) having a flow path as a through hole and a glass plate (lower substrate) are bonded together to form a substrate. However, the present invention is not limited to this. Note that the substrate may be formed of a flexible material.

  The channel preferably has a shape having one branch (for example, a T shape) or a shape having no branch (for example, a straight shape). The shape of the flow path will be described in detail later with reference to the drawings. The size of the flow path is not particularly limited, but if it is too large, a large amount of sample solution or substrate liquid is required, which is disadvantageous, and if it is too small, the liquid is difficult to circulate. About 50 μm to 3 mm, preferably about 500 μm to 2 m, and the depth of the channel is usually about 20 μm to 500 μm, preferably about 100 μm to 200 μm. The length of the flow path is not particularly limited, but in the case of a single flow path without branching, it is usually about 10 mm to 100 mm, preferably about 10 mm to 40 mm.

  An antibody capture region in which an antibody capture substance that undergoes an antigen-antibody reaction with an antibody against a test substance is immobilized in the flow path. Here, the test substance is a substance to be measured by the enzyme immunoassay method of the present invention, and is not limited as long as it can undergo an antigen-antibody reaction as an antigen, and induces an immune response when administered to a living body. It also includes haptens that do not elicit an immune response per se but react with the antibody antigen-antibody. The test substance is a biological substance such as various peptides, proteins, polysaccharides, and polynucleotides contained in a living body, preferably in body fluids such as blood and urine, but is not limited thereto. It may be contained other than living organisms, such as goods and environmental water. Since the method of the present invention has high measurement sensitivity, it is effective when the test substance is a substance contained only in a trace amount in the sample, such as diuretic peptides, particularly brain natriuretic peptide (BNP). . In the antibody capture region, an antibody capture substance that undergoes an antigen-antibody reaction with an antibody against such a test substance is immobilized. Preferably, the test substance itself is immobilized as an antibody capture substance in the antibody capture region, but a derivative of the test substance that cross-reacts with an antibody against the test substance may be immobilized as the antibody capture substance. . For accurate measurement, it is preferable to immobilize an excessive amount of an antibody capture substance capable of capturing the whole amount of an antibody or an antigen-binding fragment thereof contained in a sample solution described later by an antigen-antibody reaction in the antibody capture region. Preferably, about 10 to 100 times the number of moles of the antibody or antigen-binding fragment thereof contained in the sample solution is immobilized.

  The antibody capture region may be formed only on the bottom surface of the flow path, or may be formed on both the bottom surface and the side wall or only on the side wall, but at least the bottom surface in order to ensure sufficient contact with the sample solution and the substrate solution. Preferably, it is formed on the entire surface. When the substrate is formed by bonding the upper sheet and the lower substrate as described above, it is convenient for manufacturing to partially form the surface of the lower substrate. The length of the antibody capture region is not particularly limited as long as such an excessive amount of the antibody capture substance can be immobilized, but is usually about 1 mm to 20 mm, preferably about 5 mm to 10 mm.

  Immobilization of the above-described antibody capture substance to the antibody capture region can be performed by a conventional method. For example, a gold layer is formed in one region in the flow path by sputtering, vapor deposition, etc., and cysteamine is reacted therewith to bond an amino group to the gold layer, and then a peptide using a binding reagent such as carbodiimide. The carboxyl group of the test substance such as can be bound. Alternatively, it is also possible to bind the antibody capture substance in the same manner to a synthetic resin obtained by covalently bonding an amino group or a region coated with lysine. It is also possible to physically adsorb the antibody capture substance.

  In the flow path, an enzyme reaction product measurement region is provided in a region other than the antibody capture region. The enzyme reaction product measurement region is a region for detecting a reaction product of an enzyme reaction with a labeling enzyme used for labeling an antibody or antigen-binding fragment thereof added to a sample solution, which will be described later. For this reason, the structure changes with kinds of labeling enzyme to be used.

  In a preferred embodiment of the enzyme immunoassay method of the present invention, acylthiocholinesterase or the like that generates a thiol compound is used as the labeling enzyme. In this case, a gold thin layer is preferably used as the enzyme reaction product measurement region. Can do. The thiol compound is known to have its SH group bonded to gold, and the binding reaction can be measured in real time by measuring the surface plasmon resonance (SPR) angle. When the binding of the enzyme reaction product to the gold layer is measured by SPR, the enzyme reaction product measurement region may be a simple gold thin layer provided in the flow path. In the case of SPR measurement, since the evanescent wave oozes out from the thin gold layer, the thickness of the gold layer is preferably 200 nm or less. There is no particular lower limit on the thickness of the gold layer, but usually about 50 nm is suitable. It is also possible to measure the enzyme reaction product by configuring the enzyme reaction product measurement region with a thin gold layer and measuring the change in mass of the thin gold layer with a quartz crystal microbalance. In the present specification, “measurement” includes any of quantitative, semi-quantitative and detection. When the enzyme reaction product measurement area is measured by SPR as a thin gold layer, the size of the thin gold layer is not particularly limited, but in order to facilitate measurement, the area should be larger than the width of the channel. For example, it may be a circle having a diameter of about 1 mm to 5 mm. Alternatively, the enzyme reaction product measurement region is composed of a gold layer, and a voltage is applied to the gold layer to reduce and desorb the thiol compound bound to gold, and the thiol compound is also measured by measuring the reduction current at that time. Can be measured. In this case, for example, a gold layer to which a thiol compound is bonded is used as a working electrode, and a counter electrode and preferably further a metal layer serving as a reference electrode is formed in the flow channel in the flow channel to form an electrochemical cell. Then, each electrode can be connected to a potentiostat to measure the reduction current of the working electrode. When measuring the reduction current with an electrochemical cell, it is preferable to stop the liquid feeding. A specific method will be described later. At the time of measurement, it is preferable to measure the reduction current in a state where the flow path is filled with an alkaline solution such as a potassium hydroxide solution and each electrode is immersed in the alkaline solution.

  Alternatively, an enzyme that catalyzes a reaction that generates hydrogen peroxide, such as various oxidases, can be used as the labeling enzyme, and the generated hydrogen peroxide can be measured in the enzyme reaction product measurement region. In this case, a hydrogen peroxide measurement electrode can be preferably employed as the enzyme reaction product measurement region. The hydrogen peroxide measuring electrode itself is well known. In this case as well, a working electrode and a counter electrode, preferably a reference electrode, are formed in the flow channel to form an electrochemical cell, and each electrode is connected to a potentiostat to measure hydrogen peroxide in the solution. it can. Alternatively, the hydrogen peroxide measuring electrode may be composed of a metal to which a substance that is directly or indirectly oxidized by hydrogen peroxide is bound. In this case, a voltage is applied to the electrode to reduce the substance oxidized by hydrogen peroxide, and hydrogen peroxide is measured by measuring a reduction current at that time. Examples of the substance that is directly or indirectly oxidized by hydrogen peroxide include ferrocene and derivatives thereof, polymer compounds containing the same, hydroquinone and derivatives thereof, polymer compounds including the same, osmium bipyridine complexes and derivatives thereof, In addition, at least one redox substance selected from the group consisting of polymer compounds containing the same can be exemplified. Here, the “derivative” preferably includes ferrocene, hydroquinone, and osmium bipyridine complex, and exhibits the same redox properties as these, for example, an alkyl group (preferably having about 1 to 20 carbon atoms). Examples thereof include alkyl derivatives to which is bonded. In order to oxidize these oxidation-reduction substances, the substrate solution is oxidized by hydrogen peroxide such as peroxidase or microperoxidase, or hydrogen peroxide, and then the oxidation-reduction substance is added. Contains hemins that oxidize. In this case, it is preferable that the amount of peroxidase or hemin contained in the substrate solution is an excessive amount sufficient to consume the entire amount of hydrogen peroxide generated by the enzyme reaction with the labeling enzyme.

  The enzyme immunoassay method of the present invention is performed using the enzyme immunosensor described above. First, a sample solution containing the test substance and an enzyme-labeled antibody or antigen-binding fragment thereof that undergoes an antigen-antibody reaction with the test substance is circulated through the antibody capture region. The test substance is as described above. An enzyme-labeled antibody or antigen-binding fragment thereof that reacts with the test substance for antigen-antibody reaction is added to the sample solution containing the test substance. As the labeling enzyme used for enzyme labeling, as described above, an enzyme that generates a thiol compound or an enzyme that catalyzes a reaction that generates hydrogen peroxide is preferably used. Preferable examples of the enzyme that generates a thiol compound include acylthiocholinesterase, particularly acetylthiocholinesterase. Preferable examples of the enzyme that catalyzes the reaction for generating hydrogen peroxide include various oxidases, particularly glucose oxidase. These enzymes are well known and are also commercially available, so that they can be easily obtained.

The antibody or antigen-binding fragment thereof added to the sample solution may be an antibody or antigen-binding fragment thereof that reacts with the test substance by antigen-antibody reaction, and is not necessarily obtained by immunizing an animal with the test substance as an immunogen. It may not be an antibody or an antigen-binding fragment thereof. The antibody may be an antibody produced by genetic engineering or an antibody that cross-reacts with a test substance. Further, it may be not only a complete antibody but also a fragment capable of undergoing an antigen-antibody reaction with an antigen, such as a Fab fragment or F (ab ′) ₂ fragment (referred to herein as “antigen-binding fragment”). Single-chain antibodies such as ScFv are also included in the antigen-binding fragment for interpretation. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of reproducibility and specificity. The production method of the monoclonal antibody itself is well known. The method of enzyme-labeling an antibody or an antigen-binding fragment thereof is well known in the field of enzyme immunoassay, and one example is specifically described in the following examples.

  An enzyme-labeled antibody or antigen-binding fragment thereof is added to the sample solution. The amount of the enzyme-labeled antibody or antigen-binding fragment thereof to be added is preferably excessive with respect to the test substance contained in the sample. That is, even when the amount of the test substance contained in the sample solution is the upper limit of the expected range, it is possible to add an enzyme-labeled antibody or an antigen-binding fragment thereof in such an amount that the whole amount can undergo an antigen-antibody reaction. preferable. If an excessive amount of enzyme-labeled antibody or antigen-binding fragment thereof is added, the amount of unreacted antibody or antigen-binding fragment after the addition of the antibody or antigen-binding fragment thereof is included in the sample solution. This is because it changes depending on the amount of the test substance.

  When an enzyme-labeled antibody or an antigen-binding fragment thereof is added to a sample solution containing a test substance, the test substance and the enzyme-labeled antibody or an antigen-binding fragment thereof are bound by an antigen-antibody reaction. Although the antigen-antibody reaction occurs rapidly even at room temperature, in order to sufficiently react with the antigen-antibody, after adding the enzyme-labeled antibody or the antigen-binding fragment thereof, it is about 60 to 600 seconds at room temperature before being used for the enzyme immunosensor. It is preferable to leave the time.

  The above sample solution is supplied to the flow path of the enzyme immunosensor and circulated over the antibody capture region. Then, the unreacted enzyme-labeled antibody or the antigen-binding fragment thereof binds to the antibody capture substance on the antibody capture region and is captured on the antibody capture region. The flow rate when circulating on the antibody capture region is a flow rate at which the antigen-antibody reaction between the unreacted enzyme-labeled antibody or its antigen-binding fragment in the sample solution and the antibody capture substance on the antibody capture region is sufficiently caused. It is preferable to set. For example, when the flow path is 200 μm wide and 50 μm deep as in the enzyme immunosensor prepared in the following examples, the flow rate is not particularly limited, but is usually about 0.1 μL / min to 10 μL / min. Preferably, it is about 0.2 μL / min to 0.5 μL / min. Further, the time for passing the liquid is preferably a time for which the reaction occurs sufficiently, and is not particularly limited, but is usually 15 seconds to 5 minutes, preferably about 30 seconds to 2 minutes. The flow rate of the liquid flowing through the flow path can be controlled, for example, by supplying the liquid to the flow path using a micro metering pump such as a syringe pump. The sample liquid after flowing through the antibody capture area is discharged from the flow path or guided to the branch flow path where the enzyme reaction product measurement area is not provided so that it does not mix with the substrate liquid supplied later. It is preferable to make it.

  Next, a substrate solution containing the substrate of the enzyme used for the labeling enzyme is supplied to the flow path of the enzyme immunosensor and circulated over the antibody capture region. The amount of the substrate in the substrate solution is an excess amount, that is, even when an amount of the enzyme-labeled antibody or antigen-binding fragment thereof in the upper limit of the expected amount range to be captured in the antibody capture region is captured. It is preferable that the total amount is a reacting amount. The flow rate when the substrate solution is circulated is preferably a flow rate at which an enzyme reaction by the labeled enzyme of the enzyme-labeled antibody or antigen-binding fragment captured on the antibody-capturing region is sufficiently performed. When the flow path has a width of 200 μm and a depth of 50 μm as in the produced enzyme immunosensor, the flow rate is not particularly limited, but is usually about 0.1 μL / min to 10 μL / min, preferably 0.2 μL. / Min to about 0.5 μL / min. Further, the time for passing the liquid is preferably a time for which the reaction occurs sufficiently, and is not particularly limited, but is usually about 15 seconds to 20 minutes, preferably about 30 seconds to 5 minutes. Since it is necessary that an enzyme reaction occurs when the substrate solution comes into contact with the labeling enzyme, when a plurality of substrates are required for the enzyme reaction, the substrate solution contains the plurality of substrates.

  An enzyme reaction occurs when the substrate solution flows over the antibody capture region, and an enzyme reaction product is generated. This enzyme reaction product flows with the substrate solution in a state of being mixed in the substrate solution. The substrate solution containing the enzyme reaction product is further circulated through the flow path and is measured by contacting with the enzyme reaction product measurement region described above. As described above, the enzyme reaction product is measured by a well-known method such as SPR or electrochemical measurement of hydrogen peroxide.

  Hereinafter, the structure of a preferable enzyme immunosensor used in the enzyme immunoassay method of the present invention will be described with reference to the drawings.

FIG. 1 is a schematic exploded perspective view of a preferred embodiment of the enzyme immunosensor of the present invention. Note that the drawings (the same applies to other drawings described later) are for illustrative purposes, and therefore the ratio of various dimensions may be significantly different from the actual dimension ratio. Enzyme immunoassay sensor shown in FIG. 1 includes a substrate 10, substrate 10 is obtained by bonding the upper sheet 10a and a lower substrate 10b. Note that FIG. 1 shows a state in which the upper sheet 10a and the lower substrate 10b are disassembled for clarity. The upper sheet 10a is made of silicone rubber such as polydimethylsiloxane, and the lower substrate 10b is made of glass, for example. The substrate 10 is provided with a substantially T-shaped flow path 12. The flow path 12 penetrates the upper sheet 10 a in the thickness direction, and therefore the surface of the lower substrate 10 b forms the bottom surface of the flow path 12. Each end of the flow path 12 has a circular shape whose diameter is larger than the width of the flow path in order to facilitate supply and discharge of the liquid, and these form through holes in the upper sheet 10a. And so on. One end of the “T” horizontal bar of the flow path is the liquid supply port 14, the other end of the “T” horizontal bar is the sample solution outlet 16, and the end of the “T” vertical bar is the substrate. A liquid discharge port 18 is provided. Since the sample liquid and the substrate liquid are supplied from the liquid supply port 14 into the flow channel 12, the liquid supply port 14 becomes the uppermost stream in the flow channel 12. An antibody capture region 20 is formed between the liquid supply port 14 and the branch point of the flow path 12 (intersection of “T” -shaped horizontal bar and vertical bar). As described above, the antibody capture region 20 is formed, for example, by forming a gold thin film on the lower substrate 10b by vapor deposition, sputtering, or the like, bonding cysteamine to this to add an amino group, and adding a test substance to this amino group. It can be formed by bonding. In the illustrated example, the antibody capture region 20 is formed only on the bottom surface of the flow path 12. A widened region 22 is provided in the vicinity of the center of the “T” -shaped vertical bar of the flow path 12, and an enzyme reaction product measurement region 24 is provided on the bottom surface of the widened region 22. In the specific example shown in FIG. 1, the enzyme reaction product measurement region 24 is formed from a gold thin film for SPR measurement. This gold thin film can also be formed on the lower substrate 10b by vapor deposition or sputtering.

  The specific example of the enzyme immunosensor shown in FIG. 1 can be used as follows. First, the above-described sample solution (as described above, to which an enzyme-labeled antibody or antigen-binding fragment thereof is added to the test substance) is supplied from the solution supply port 14. The sample liquid can be supplied by inserting the tip of a capillary connected to a micro metering pump such as a syringe pump into the liquid supply port 14. On the other hand, suction is performed from the sample solution outlet 16. Similarly, suction can be performed by inserting the tip of the capillary connected to the pump into the sample solution discharge port 16. Then, the supplied sample solution passes through the antibody capture region 20 and flows toward the sample solution discharge port 16, and is discharged from the sample solution discharge port 16. At this time, the unreacted antibody or the antigen-binding fragment thereof contained in the sample solution reacts with the test substance immobilized in the antibody capture region 20 and is captured in the antibody capture region 20. Next, the enzyme substrate solution is supplied from the solution supply port 14 and sucked from the substrate solution discharge port 18. Then, the substrate solution passes over the antibody capture region 20, changes its direction at the branch point of the flow path 12, and moves toward the substrate solution discharge port 18. At this time, it passes over the enzyme reaction product measurement region 24. When passing over the antibody capture region 20, the substrate undergoes an enzyme reaction with the enzyme labeled antibody or the antigen-binding fragment thereof captured in the antibody capture region 20, and an enzyme reaction product is generated. When the enzyme reaction product is bonded to gold and measured by SPR, the enzyme reaction product is a thiol compound as described above. When the enzyme reaction product, which is a thiol compound, passes over the enzyme reaction product measurement region 24 composed of a gold thin film, it reacts with and binds to gold. The enzyme reaction product can be measured by measuring the refractive index change at this time by the SPR method. In FIG. 1, an arrow 26 indicates incident light for SPR measurement, and an arrow 28 indicates reflected light for SPR measurement.

  When a certain amount of enzyme-labeled antibody is added to the sample solution, the amount of unreacted labeled antibody decreases as the amount of the test substance contained in the sample solution increases. As a result, in the enzyme reaction product measurement region 20. The amount of the enzyme-labeled antibody to be captured decreases, the amount of the substrate that undergoes the enzyme reaction decreases, and the amount of the enzyme reaction product decreases accordingly. Therefore, the test substance contained in the sample can be measured by measuring the enzyme reaction product by the above method.

  In the above example, the flow direction of the liquid from the branch point is defined by suction from the sample liquid discharge port 16 or the substrate liquid discharge port 18, but for example, by providing a micro valve at the branch point, It is also possible to define the distribution direction from the branch point. Various kinds of microvalves themselves provided in the narrow channel are well known, and are described in Non-Patent Document 1, for example. In addition, when using a micro valve, since suction from each discharge port becomes unnecessary, a porous water-absorbing material such as a sponge may be disposed at each discharge port to absorb the liquid.

  In the above example, the enzyme reaction product bound to the enzyme reaction product measurement region 24 was measured by the SPR method. As described above, the mass change in the enzyme reaction product measurement region 24 is measured using the quartz crystal microbalance ( It is also possible to measure the enzyme reaction product by measuring by (not shown).

  A preferred second specific example of the enzyme immunosensor of the present invention is shown in FIG. The configuration of the enzyme immunosensor shown in FIG. 2 is similar to that of the enzyme immunosensor shown in FIG. 1, and members corresponding to those of the enzyme immunosensor shown in FIG. Is attached. The specific example shown in FIG. 2 is different from the specific example shown in FIG. 1 in that the shape of the enzyme reaction product measurement region 24 made of a gold thin film has a strip shape extending to one end of the substrate. The counter electrode 32 and the reference electrode 30 made of a metal thin film are arranged in parallel with the enzyme reaction product measurement region 24 at an interval. In the specific example shown in FIG. 2, the widened region 22 is not provided in the T-shaped vertical bar portion of the flow path 12 (however, the widened region may be provided).

  The method for using the enzyme immunosensor shown in FIG. 2 is the same as the method for using the enzyme immunosensor shown in FIG. 1 except for the method for measuring the enzyme reaction product bound to the enzyme reaction product measurement region 24. In the enzyme immunosensor shown in FIG. 2, an enzyme reaction product measurement region 24 is used as a working electrode, an electrochemical cell is formed together with the counter electrode 32 and the reference electrode 30, and a voltage is applied to the enzyme reaction product measurement region 24 (working electrode). Then, the enzyme reaction product bonded to the enzyme reaction product measurement region 24 is reduced and desorbed. By measuring the reduction current at this time, the amount of the enzyme reaction product bound to the enzyme reaction product measurement region 24, and hence the amount of the test substance in the sample solution can be measured. The reduction current is measured when the enzyme reaction product measurement region 24 (working electrode), the counter electrode 32 and the reference electrode 30 are connected to a potentiostat, and the working electrode potential is swept with respect to the reference electrode potential. This can be done by measuring the current with an ammeter (see Examples below).

  Alternatively, in the specific example shown in FIG. 2, the enzyme reaction product measurement region 24 may be the hydrogen peroxide measurement electrode described above (third specific example). In this case, as described above, an enzyme that catalyzes a reaction that generates hydrogen peroxide, such as various oxidases, is used as the labeling enzyme. In the case where the hydrogen peroxide measuring electrode is one in which a redox substance such as a ferrocene derivative is bound, as described above, hemin and peroxidases are added to the substrate solution. Even in the case of the third specific example, the hydrogen peroxide generated by the enzyme reaction is the potentiostat in the enzyme reaction product measurement region 24 (working electrode), the counter electrode 32 and the reference electrode 30 as in the second specific example. It can be performed by connecting, sweeping the working electrode potential with respect to the reference electrode potential, and measuring the current generated at that time with an ammeter (see Examples below).

  A preferred fourth specific example of the enzyme immunosensor of the present invention is shown in FIG. In the specific example shown in FIG. 3 as well, members corresponding to those of the enzyme immunosensor shown in FIG. In the specific example shown in FIG. 3, the flow path 12 is not branched, and one linear flow path 12 is formed. A sample solution outlet 16 is formed near the center of the flow path 12. The antibody capture region 20 is formed between the liquid supply port 14 and the sample solution discharge port 16, and the enzyme reaction product measurement region 24 is formed between the sample solution discharge port 16 and the substrate solution discharge port 18. Other configurations are the same as the specific example shown in FIG.

  The enzyme immunosensor shown in FIG. 3 is different from the specific example shown in FIG. 1 only in the shape of the flow path 12 and the position of the sample liquid discharge port 16, and the usage method is the same as the specific example shown in FIG. is there. That is, the sample liquid is supplied from the liquid supply port 14, sucked from the sample liquid discharge port 16, and discharged from the sample liquid discharge port 16. At this time, an unreacted enzyme-labeled antibody or an antigen-binding fragment thereof binds to the antibody capture region 20. Next, the substrate liquid is supplied from the liquid supply port 14 and sucked and discharged from the substrate liquid discharge port 18. Then, the enzyme reaction product generated by the enzyme reaction with the labeling enzyme bonded to the antibody capture region 20 is bonded to the enzyme reaction product measurement region 24. This is measured by the SPR method or the like.

  A preferred fifth specific example of the enzyme immunosensor of the present invention is shown in FIGS. FIG. 5 is an end view of the schematic cut portion of FIG. 4 (cut along the longitudinal direction of the flow path of FIG. 4). Also in the specific examples shown in FIGS. 4 and 5, members corresponding to the members of the enzyme immunosensor shown in FIG. 1 are denoted by the same reference numerals as in FIG.

  In the enzyme immunosensor shown in FIGS. 4 and 5 as well, as in the specific example shown in FIG. 3, the flow path 12 is not branched and has a single linear shape. In the specific examples shown in FIGS. 4 and 5, the liquid supply port is separated into the sample liquid supply port 14a and the substrate liquid supply port 14b, and the antibody capture region 20 is disposed between them. The enzyme reaction product measurement region 24 is disposed between the sample solution supply port 14 a and the substrate solution discharge port 18. Further, a first flow direction regulating region 34 is formed between the sample solution supply port 14a and the enzyme reaction product measurement region 24, while a second flow direction defining region 34 is formed between the substrate solution supply port 14b and the sample solution discharge port 16. A flow direction defining region 36 is formed, and the first and second flow direction defining regions 34 and 36 are formed by bonding a substance whose hydrophilicity changes depending on an electric potential on an electrode made of a metal thin film. The substance whose hydrophilicity changes depending on the electric potential does not need to be bonded to the entire surface of the electrode, but may be bonded to the bottom surface of the channel 12. Examples of the substance whose hydrophilicity changes with potential include ferrocene and derivatives thereof. Here, the “derivative” preferably includes a ferrocene portion, and similarly to ferrocene, the one whose hydrophilicity changes depending on the electric potential is preferable. For example, an alkyl thiol group (preferably having about 1 to 20 carbon atoms) bonded thereto. Examples thereof include thiol derivatives. An electrode to which a substance whose hydrophilicity varies depending on the potential is bonded is obtained by, for example, immersing the electrode in a solution of ferrocene alkyl thiol, and self-assembling the ferrocene alkyl thiol on the electrode surface, as specifically described in the following examples. It can be formed by coating with a molecular film.

  In use, the first flow direction defining area 34 is grounded (potential 0 V), and a positive potential (for example, +0.4 V) is applied to the second flow direction defining area 36. Then, the ferrocene derivative on the second flow direction defining region 36 becomes hydrophilic, and the ferrocene derivative on the first flow direction defining region 34 remains hydrophobic. In this state, the sample solution is supplied from the sample solution supply port 14a. Then, since the first flow direction defining region 34 is hydrophobic, the sample liquid does not travel over the first flow direction defining region 34. On the other hand, since the second flow direction defining region 36 is hydrophilic, the sample liquid passes over the second flow direction defining region 36 and reaches the sample liquid discharge port 16. In this process, since the sample solution passes over the antibody capture region 20, unreacted enzyme-labeled antibody or antigen-binding fragment thereof is bound onto the antibody capture region 20. Next, the second flow direction defining region 36 is grounded (potential 0 V), and a positive potential (for example, +0.4 V) is applied to the first flow direction defining region 34. In this state, the substrate solution is supplied from the substrate solution supply port 14b. Then, since the second flow direction defining region 36 is hydrophobic due to the potential change, the substrate liquid does not travel over the second flow direction defining region 36. On the other hand, since the first flow direction defining region 34 becomes hydrophilic due to a change in potential, the substrate liquid proceeds over the first flow direction defining region 34 and reaches the substrate liquid discharge port 18. In this process, since the substrate solution passes over the antibody capture region 20 and the enzyme reaction product measurement region 24, the enzyme reaction product is transferred over the enzyme reaction product measurement region 24 as in the other specific examples described above. To join. This is measured by the SPR method or the like as in the case of other specific examples. In this specific example, as described above, since the flow direction of the liquid is defined by the potential applied to the first and second flow direction defining regions 34 and 36, suction from each discharge port is unnecessary. Therefore, a porous water-absorbing material such as a sponge may be disposed at each outlet to absorb the liquid.

  A preferred sixth specific example of the enzyme immunosensor of the present invention is shown in FIG. Also in the specific example shown in FIG. 6, members corresponding to the members of the enzyme immunosensor shown in FIG.

  In the enzyme immunosensor shown in FIG. 6 as well, as in the specific example shown in FIG. In the specific example shown in FIG. 6, the upper sheet is separated into two upper and lower stages by the semipermeable membrane 38. Therefore, the flow path is also separated into two upper and lower stages by the semipermeable membrane 38. The upper sheet is denoted by 10a1, the lower sheet is denoted by 10a2, the upper flow path is denoted by 12a, and the lower flow path is denoted by 12b. The semipermeable membrane 38 is permeable to enzyme reaction products such as acetylthiocholine but not the enzyme-labeled antibody or antigen-binding fragment thereof. The antibody capture region 20 is formed only in the upper stage. This can be achieved by providing the antibody capture region 20 on the semipermeable membrane 38, or the antibody capture region 20 may be provided on the side wall of the upper flow path 12a. In the specific example shown in FIG. 6, the sample liquid outlet and the substrate liquid outlet are the same outlet 18a. One end of the lower flow path 12b is a dialysate supply port 40, and the other end is a dialysate discharge port. The upper sheet as described above can be easily manufactured by bonding two sheets with the semipermeable membrane 38 interposed therebetween. Further, similarly to the other specific examples, a lower substrate (not shown) is attached to the lower side of the lower sheet 10a2, and a gold film is formed on the surface of the lower substrate to form the enzyme reaction product measurement region 24. The enzyme reaction product measurement region 24 is formed downstream of the antibody capture region 20 as in the specific example of FIG. 3 (however, the enzyme reaction product measurement region 24 is in the lower flow path 12b, and the antibody capture region 20 is In the upper flow path 12a).

  A method of using the specific example shown in FIG. 6 will be described. First, the dialysate is supplied from the dialysate supply port 40 and sucked from the dialysate discharge port 42 to fill the lower flow path 12b with the dialysate. In this state, the suction is stopped, and the lower flow path 12b is maintained in a state where the dialysate is filled. As the dialysate, for example, 0.1M phosphate buffer can be used. Next, the sample liquid is supplied from the liquid supply port 14 and sucked from the liquid discharge port 18a. Then, the sample liquid flows toward the liquid discharge port 18a, and the unreacted enzyme-labeled antibody or the antigen-binding fragment thereof in the sample liquid is bound to the antibody capturing region 20. At this time, since the enzyme-labeled antibody or the antigen-binding fragment thereof does not permeate the semipermeable membrane 38, it does not migrate to the lower flow path 12b. When the sample solution is a body fluid such as blood, various proteins and the like are included, but these also do not pass through the semipermeable membrane 38 and therefore do not migrate to the lower flow path 12b. Next, the substrate liquid is supplied from the liquid supply port 14 and sucked from the liquid discharge port 18a. Then, the substrate liquid flows toward the liquid discharge port 18a and passes over the antibody capture region 20 during that time, and an enzyme reaction occurs by the labeling enzyme bound to the antibody capture region 20 at that time, and the enzyme reaction product Produces. Since the generated enzyme reaction product permeates the semipermeable membrane 38, at least a part of the enzyme reaction product moves to the lower flow path 12 b through the semipermeable membrane 38 and is bonded to the enzyme reaction product measurement region 24. At this time, suction may be performed from the dialysate discharge port 42. The enzyme reaction product bound to the enzyme reaction product measurement region 24 can be measured by the SPR method or the like, as in other specific examples.

  According to this specific example, the enzyme reaction product can be measured in the absence of a high-molecular substance such as various proteins in the sample solution, and more accurate measurement is possible.

  In the fourth to sixth specific examples described above, the enzyme reaction product measurement region 24 is a gold thin film for the SPR method, but instead of this, as in the second or third specific example, A chemical cell can also be employed.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Preparation of acetylthiocholinesterase-labeled BNP
(1) A brain natriuretic peptide (BNP) antibody (Anti-BNP-AchE) using acetylthiocholinesterase as a labeling enzyme was prepared as follows. A 10 mg / mL S-acetylcaptosuccinyl hydride solution was reacted with 1 mg / mL acetylthiocholinesterase to introduce a thiol group into acetylthiocholinesterase. On the other hand, it was reacted with 0.1 mg / mL sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate and 400 μg / mL anti-human BNP antibody (source: Phoenix Pharmaceuticals) to give anti-anti Maleimide groups were introduced into human BNP antibody. Thereafter, anti-BNP-AchE was prepared by reacting thiol-modified acetylthiocholinesterase with an anti-human BNP antibody into which a maleimide group was introduced.

2. Production of enzyme immunosensor An enzyme immunosensor as a first specific example shown in FIG. 1 was produced. The upper sheet 10a was made of a polydimethylsiloxane (PDMS) sheet (16 mm × 16 mm, thickness 50 μm), and the lower substrate 10b was a glass plate. The upper sheet 10a was produced by pouring PDMS and a curing agent using a negative pattern having a convex structure formed on a stainless steel substrate by a sandblasting method as a mold, and peeling off after curing. The flow path had a width of 200 μm and a depth of 50 μm. The length of the T-shaped horizontal bar was 12 mm, and the length of the vertical bar was 12 mm. Further, the diameter of the widened region 22 in the flow channel 12 was 3 mm. Thereafter, the liquid supply port 14, the sample liquid discharge port 16 and the substrate liquid discharge port 18 were opened by a drill, and capillaries for introducing and discharging the liquid were connected. The diameter of each hole drilled was 0.6 mm.

  On the other hand, the lower substrate 10b was made of a glass plate (16 mm × 16 mm, thickness 0.15 mm, manufactured by Matsunami Glass Co., Ltd.), and a gold thin film was formed as the antibody capture region 20 and the enzyme reaction product measurement region 24, respectively. The gold thin film was formed by depositing 5 nm of titanium on a glass plate using a magnetron sputtering apparatus (manufactured by Nippon Seed Co., Ltd.) and then depositing a 50 nm thick gold thin film. The length of the antibody capture region 20 was 5 mm, and the diameter of the enzyme reaction product measurement region 24 was 3 mm. Thereafter, a 0.1 mM cystamine solution was dropped onto the gold thin film in the antibody capture region 20 and allowed to stand for 2 hours. After introducing an amino group on the gold surface, it was washed with pure water, and further 2.5 mg / mL BNP and 0. By reacting in a solution containing 1 g / L of water-soluble carbodiimide for 1 hour, BNP was immobilized on the gold thin film surface. The obtained lower substrate 10b was washed and then bonded to the upper sheet 10a to produce an enzyme immunosensor as a first specific example of the present invention.

3. Quantification method BNP was quantified by the following procedure. Anti-BNP-AchE was added to an aqueous solution containing BNP at each concentration so that the final concentration was 10 ng / mL. The sample liquid is supplied from the liquid supply port 14 using a syringe pump, and is sucked from the sample liquid discharge port 16 and flows in the direction from the liquid supply port 14 to the sample liquid discharge port 16 at a flow rate of 1 μL / min for 1 minute. I let you. Thereby, the unreacted antibody was immobilized on the antibody capture region 20 on which BNP was immobilized. Thereafter, a substrate solution containing 1 mM acetylthiocholine is supplied from the solution supply port 14, while being sucked from the substrate solution discharge port 18, in the direction from the solution supply port 14 to the substrate solution discharge port 18 at a flow rate of 1 μL / min. While continuously circulating, the refractive index change on the surface of the enzyme reaction product measurement region 24 made of a gold thin film and disposed in the widening region 22 was read by the SPR method. The SPR angle was measured using a commercially available Handy-SPR (trade name, manufactured by NTT-AT).

4). Results The results are shown in FIGS. FIG. 7 shows how the SPR angle increases for each BNP concentration. When a large amount of BNP is contained in the sample solution, the amount of unreacted antibody immobilized on the antibody capture region 20 decreases, so that the acetylcholinesterase activity is low and the amount of thiocholine that is an enzyme reaction product is generated. Less is. For this reason, the SPR angle change became small with the increase in BNP in the sample solution. On the other hand, when the amount of BNP in the sample solution is small, the amount of unreacted antibody increases, so the amount of unreacted antibody immobilized on the antibody capture region 20 increases, and the immobilized acetylcholinesterase activity is high. As the BNP decreases, the SPR angle change increases. FIG. 8 shows the amount of change in the SPR angle after 5 minutes in FIG. 7 for each BNP concentration. As the BNP concentration in the sample solution increases, the rate of change of the SPR angle decreases. That is, it is shown that BNP can be quantified by the method of the present invention.

1. Production of enzyme immunosensor An enzyme immunosensor as a second specific example shown in FIG. 2 was produced. The upper sheet 10a was produced in the same manner as in Example 1 (however, the widened region 22 of Example 1 was not formed). The lower substrate 10b is also a glass plate similar to that in Example 1, and the enzyme reaction product measurement region 24 (working electrode) and the reference electrode 30 are gold thin films produced in the same manner as in Example 1. The counter electrode 32 was a silver thin film. The width of each electrode was 1 mm.

2. Quantification After producing Anti-BNP-AchE in the same manner as in Example 1, Anti-BNP-AchE was added to an aqueous solution containing each concentration of BNP in the same manner as in Example 1 so that the final concentration was 10 ng / mL. This sample liquid is supplied into the flow path 12 at a flow rate of 1 μL / min in the direction from the liquid supply port 14 to the sample liquid discharge port 16 using a syringe pump, and unreacted antibody is immobilized with gold immobilized on BNP. Immobilized on a thin film (antibody capture region) 20. Thereafter, a substrate solution containing 1 mM acetylthiocholine is supplied from the solution supply port 14 toward the substrate solution discharge port 18 at a flow rate of 1 μL / min, and an enzyme reaction product is generated on the surface of the enzyme reaction product measurement region (working electrode) 24. The product thiocholine was bound. Thereafter, a 0.1 M potassium hydroxide solution was fed from the liquid supply port 14 toward the substrate liquid discharge port 18, the inside of the detection channel 19 was filled with a 0.1 M potassium hydroxide solution, and the liquid feeding was stopped.

  Thereafter, when the working electrode 24, the reference electrode 30 and the counter electrode 32 of the sensor are connected to a potentiostat (manufactured by ALS), the working electrode potential is swept from -0.4V to -1.4V with respect to the reference electrode potential. , A reduction current peak having a peak in the vicinity of −1.1 V was confirmed. This reduction current is a current when the thiocholine adsorbed on the working electrode 24 is reduced and desorbed. This reduction current value decreased as the BNP concentration increased. This is because when there is a large amount of BNP in the sample, the amount of unreacted antibody decreases, so the amount of unreacted antibody immobilized on the gold thin film 20 decreases, and the immobilized acetylcholinesterase activity is low. As the BNP increases, the reduction current value decreases. Thus, the present invention shows that BNP can be quantified even by an electrochemical method.

1. Preparation of glucose oxidase-labeled anti-BNP antibody A glucose oxidase-labeled anti-human BNP antibody was prepared in the same manner as in Example 1 except that glucose oxidase was used instead of acetylthiocholinesterase as the labeling enzyme.

2. Preparation of enzyme immunosensor Except that the enzyme reaction product measurement region 24 (working electrode) was immersed in an ethanol solution of 1 mM ferroceneundecanethiol for 2 hours and the electrode surface was modified with a self-assembled monolayer of ferroceneundecanethiol. An enzyme immunosensor similar to that of Example 2 was produced.

3. Quantification As in Example 1, glucose oxidase-labeled anti-human BNP antibody was added to an aqueous solution containing BNP at each concentration so that the final concentration was 10 ng / mL, and this sample solution was then added to the liquid supply port 14 using a syringe pump. To the sample solution outlet 16 at a flow rate of 1 μL / min for 1 minute into the flow path 12 to immobilize the unreacted antibody on the gold thin film (antibody capture region) 20 on which BNP is immobilized. Thereafter, a solution containing 10 mM glucose and 1 μM hemin as a substrate solution was fed in the same manner as in Example 2. As a result, hydrogen peroxide was generated in the flow path 12 and a part of the ferroceneundecanethiol modified on the working electrode 24 was oxidized via hemin. Thereafter, a reduction current peak could be observed by sweeping the potential of the working electrode modified with ferroceneundecanethiol with respect to the reference electrode 30 in the same manner as in Example 2. This is because a molecule in which the ferroceneundecanethiol modified on the working electrode was partially oxidized via hemin was reduced again. The hydrogen peroxide concentration can be determined from the reduction current peak, and the BNP concentration that is the target antigen can be determined.

1. Production of enzyme immunosensor An enzyme immunosensor of the fourth specific example shown in FIG. 3 was produced. It was produced in the same manner as in Example 1 except that the shape of the flow path of the upper sheet 10a was changed as shown in FIG. However, the diameter of the gold thin film constituting the enzyme reaction product measurement region 24 was 10 mm.

2. Determination The sample solution and the substrate solution were prepared in the same manner as in Example 1. The sample liquid outlet 16 and the substrate liquid outlet 18 were connected to a syringe, and the syringe was further attached to a syringe pump. Thereafter, by sucking only the syringe pump connected to the sample liquid discharge port 16, the sample liquid containing BNP and Anti-BNP-AchE was sucked and fed from the liquid supply port 14 toward the sample liquid discharge port 16. During this time, the unreacted antibody is immobilized on the gold thin film 20 on which BNP is immobilized. Thereafter, by sucking a syringe pump connected to the substrate liquid discharge port 18, a 1 mM acetylthiocholine solution was sucked and fed from the liquid supply port 14 toward the substrate liquid discharge port 18. The conditions for feeding the sample solution and the substrate solution were the same as in Example 1. At this time, a part of acetylthiocholine is decomposed into thiocholine by Anti-BNP-AchE immobilized on the gold thin film 20 and concentrated on the gold thin film 24. This thiocholine concentration was detected by the SPR method in the same manner as in Example 1.

1. Production of enzyme immunosensor An enzyme immunosensor of the fifth specific example shown in FIGS. 4 and 5 was produced. The upper sheet 10a was produced in the same manner as in Example 1 except that the shape of the flow path was changed as shown in FIGS. In the lower substrate 10b, the antibody capture region 20 and the enzyme reaction product measurement region 24 were formed in the same manner as in Example 4. Furthermore, the base layer of the 1st distribution direction regulation area | region 34 and the 2nd distribution direction regulation area | region 36 was each formed with the gold thin film. These gold thin films were immersed in an ethanol solution of 1 mM ferrocene undecanethiol for 2 hours, and the electrode surface was modified with a self-assembled monolayer of ferrocene undecanethiol to provide first and second flow direction defining regions 34 and 36. Formed.

2. Determination The sample solution and the substrate solution were prepared in the same manner as in Example 1. First, the electrode potentials of the second flow direction defining region 36 and the first flow direction defining region 34 are respectively applied to 0.4 V and 0 V, and the sample liquid containing BNP and Anti-BNP-AchE is supplied to the sample liquid supply port 14a. The liquid was sent from. The introduced sample liquid is fed in the direction of the second flow direction defining region 36 having high hydrophilicity, and unreacted antibodies are immobilized on the antibody capturing region 20 on which BNP is immobilized. Next, the electrode potentials of the second flow direction defining region 36 and the first flow direction defining region 34 were applied to 0V and 0.4V, respectively, and a 1 mM acetylthiocholine solution was introduced from the substrate solution supply port 14b. At this time, the acetylthiocholine solution is fed in the direction of the first flow direction defining region 34 having high hydrophilicity, and further contacts the enzyme reaction product measurement region (gold thin film) 24. The conditions for feeding the sample solution and the substrate solution were the same as in Example 1. At this time, a part of acetylthiocholine is decomposed into thiocholine by Anti-BNP-AchE immobilized on the gold thin film 24 and concentrated on the gold thin film 24. By detecting the concentration of thiocholine by the SPR method as in Example 1 or by detecting it electrochemically as in Example 2, BNP in the sample could be quantified. Thus, by arranging the gold thin film on which the redox species are immobilized at the boundary of each flow path, the liquid feeding direction is controlled, and the detection can be easily performed with a high S / N ratio.

1. Production of enzyme immunosensor An enzyme immunosensor of the sixth specific example shown in FIG. 6 was produced. The upper sheet was bonded by sandwiching a semipermeable membrane 38 between two PDMS sheets prepared in the same manner as in Example 1 except that the shape of the flow path was as shown in FIG. The semipermeable membrane 38 was a cellulose membrane having an average pore diameter of 4 nm and a thickness of 20 μm. A gold thin film was formed on the side wall of the upper stage 10a1 of the upper sheet by sputtering in the same manner as in Example 1, and BNP was bonded to this to form the antibody capture region 20 in the same manner as in Example 1. The lower substrate was the same as in Example 1. Only the gold thin film that becomes the enzyme reaction product measurement region 24 was formed on the lower substrate.

2. Determination The sample solution and the substrate solution were prepared in the same manner as in Example 1. First, 0.1 M phosphate buffer as a dialysate was sent from the dialysate supply port 40 toward the dialysate discharge port 42, and the lower flow path 12b was filled with the dialysate. Thereafter, the sample liquid containing BNP and Anti-BNP-AchE was sucked and fed from the liquid supply port 14 toward the discharge port 18a using a syringe pump. Thereafter, similarly, 1 mM acetylthiocholine, which is a substrate solution, was fed. The conditions for feeding the sample solution and the substrate solution were the same as in Example 1. A portion of the thiocholine thus produced passes through the semipermeable membrane 38 and is adsorbed and concentrated on the enzyme reaction product measurement region (gold thin film) 24. Concentrated thiocholine can be confirmed by the SPR method as in Example 1. Thus, by selectively feeding molecules having a small molecular weight such as thiocholine, which is an enzyme product, to the detection channel, measurement can be performed without the influence of adsorption of macromolecules such as proteins in the sample.

It is a disassembled perspective view which shows typically one preferable specific example of the enzyme immunosensor of this invention. It is a disassembled perspective view which shows typically another one specific example of the enzyme immunosensor of this invention typically. It is a disassembled perspective view which shows typically another preferable one specific example of the enzyme immunosensor of this invention. It is a disassembled perspective view which shows typically another preferable one specific example of the enzyme immunosensor of this invention. FIG. 5 is an end view of the cut portion in FIG. 4. It is a disassembled perspective view which shows typically another preferable one specific example of the enzyme immunosensor of this invention. It is a figure which shows the time change of the SPR angle about each standard sample liquid containing the test substance of various density | concentration measured in Example 1 of this invention. FIG. 8 is a calibration curve showing the relationship between the BNP concentration in the sample solution and the rate of change of the SPR angle, derived from the results shown in FIG.

Explanation of symbols

10 substrate 10a upper sheet 10a1 upper sheet upper stage 10a2 upper sheet lower sheet 12 channel 12a upper channel 12b lower channel 14 liquid supply port 14a sample solution supply port 14b substrate solution supply port 16 sample solution discharge port 18 substrate solution discharge port 18a discharge port 20 antibody capture region 22 widening region 24 enzyme reaction product measurement region 26 incident light for SPR measurement 28 reflected light for SPR measurement 30 reference electrode 32 counter electrode 34 first flow direction defining region 36 second flow direction defining region 36 Area 38 Semipermeable membrane 40 Dialysate supply port 42 Dialysate discharge port

Claims (24)

  A substrate provided with a channel, an antibody capture region provided in the channel and immobilizing an antibody capture material that reacts with an antibody against a test substance with an antigen antibody, and other than the antibody capture region in the channel Using an enzyme immunosensor having an enzyme reaction product measurement region provided in the region, the test substance, and an enzyme-labeled antibody or antigen-binding fragment thereof that reacts with the test substance with an antigen-antibody A sample solution is circulated in the antibody capture region to capture the unreacted antibody or antigen-binding fragment thereof in the antibody capture region, and then a substrate solution containing the labeling enzyme substrate is circulated in the antibody capture region. And subjecting the enzyme reaction product to measurement by measuring at least a part of the enzyme reaction product in contact with the enzyme reaction product measurement region. Enzyme immunity Constant method.   The enzyme reaction product measurement region comprises a gold thin film, and the enzyme reaction generates a thiol compound, and the binding of the thiol compound generated by the enzyme reaction to the gold thin film is measured by a surface plasmon resonance method. The method according to claim 1.   The enzyme reaction product measurement region is composed of a gold thin film, and the enzyme reaction generates a thiol compound. After the thiol compound generated by the enzyme reaction is bonded to the gold thin film, a voltage is applied to the metal thin film. The method according to claim 1, wherein the thiol compound is reductively desorbed from the gold thin film by applying and the reduction current at that time is measured.   The method according to claim 2 or 3, wherein the labeling enzyme is acylthiocholinesterase and the substrate is acylthiocholine.   The method according to claim 4, wherein the labeling enzyme is acetylthiocholinesterase and the substrate is acetylthiocholine.   The method according to claim 1, wherein the enzyme reaction generates hydrogen peroxide, and the enzyme reaction product measurement region is a hydrogen peroxide measurement electrode.   The method according to claim 6, wherein the labeling enzyme is an oxidase, and the substrate is a compound that generates hydrogen peroxide by the action of the oxidase.   The method according to claim 7, wherein the labeling enzyme is glucose oxidase and the substrate is glucose.   The hydrogen peroxide measuring electrode is made of a metal bonded with a substance that is directly or indirectly oxidized by hydrogen peroxide, and a voltage is applied to the electrode to reduce the substance oxidized by hydrogen peroxide. The method according to any one of claims 6 to 8, wherein hydrogen peroxide is measured by measuring a reduction current at that time.   The hydrogen peroxide measuring electrode includes the group consisting of ferrocene and derivatives thereof, polymer compounds including the same, hydroquinone and derivatives thereof, polymer compounds including the same, osmium bipyridine complexes and derivatives thereof, and polymer compounds including the same. The method according to claim 9, wherein at least one selected redox substance is bound, and the substrate solution contains a substance or an enzyme that oxidizes the redox substance in the presence of hydrogen peroxide.   The method according to claim 10, wherein the substance or enzyme that oxidizes the redox substance in the presence of hydrogen peroxide is at least one selected from the group consisting of hemin, iron porphyrin complex, microperoxidase, and peroxidase.   12. The method according to claim 11, wherein an alkylated derivative of ferrocene is bound to the hydrogen peroxide measuring electrode, and the substrate solution contains hemin.   The flow path is branched, the antibody capture region is located upstream from the branch point, and the enzyme reaction product measurement region is located in one flow path downstream from the branch point, After passing through the branch point, the sample solution flows through the flow path where the reaction product detection region is not formed, and after passing through the branch point, the substrate solution is formed with the enzyme reaction product measurement region. The method according to any one of claims 1 to 12, wherein the method flows through one of the flow paths.   The flow path is composed of a single flow path without branching, and the sample solution is supplied to the flow path from a position upstream of the antibody capture region and passes through the antibody capture region, and then the enzyme reaction product is generated. The substrate solution is removed from the flow path before reaching the product measurement region, and the substrate solution is supplied to the flow channel from a position upstream of the antibody capture region, and after passing through the antibody capture region, the enzyme reaction product measurement 13. A method according to any one of claims 1 to 12 in contact with a region.   The flow path is composed of a single flow path without branching, and the sample solution is supplied to the flow path from a position between the antibody capture region and the enzyme reaction product measurement region and flows to the antibody capture region side. Pass through the antibody capture region, the substrate solution is supplied to the flow channel from a position opposite to the enzyme reaction product measurement region across the antibody capture region, and flows to the antibody capture region side. The method according to any one of claims 1 to 12, wherein the method passes through the antibody capture region and contacts the enzyme reaction product measurement region.   The immunosensor includes a first flow direction regulating region formed between a conductor that is formed between the antibody capture region and the enzyme reaction product measurement region and is bonded with a substance whose hydrophilicity changes depending on a potential, and the antibody capture A second flow direction regulating region formed of a conductor formed by bonding a substance whose hydrophilicity changes depending on an electric potential, formed at a position opposite to the first flow direction defining region across the region; Is supplied to the flow path from a position between the antibody capture region and the first flow direction defining region, and the substrate liquid is flowed from the position between the second flow direction defining region and the antibody capture region. The flow direction when the sample solution is supplied and the flow direction when the substrate solution is supplied are switched by changing the potential applied to the first and second flow direction defining regions. Claim 15 Law.   The method according to claim 16, wherein the substance that changes the hydrophilicity according to the electric potential and is bonded to the first and second flow direction defining regions is ferrocene or a derivative thereof.   The flow path is separated into two upper and lower stages by a semipermeable membrane that allows the enzyme reaction product to permeate but does not permeate the enzyme-labeled antibody or antigen-binding fragment thereof, and the antibody capture region is formed in the upper stage of the flow path. The method according to any one of claims 1 to 17, wherein the enzyme reaction product measurement region is formed in a lower stage.   A substrate provided with a channel, an antibody capture region provided in the channel and immobilizing an antibody capture material that reacts with an antibody against a test substance with an antigen antibody, and other than the antibody capture region in the channel An enzyme immunosensor for performing the method according to claim 1, further comprising an enzyme reaction product measurement region provided in the region.   The said flow path is branched, The said antibody capture area | region is located upstream from a branch point, and the said enzyme reaction product measurement area | region is located in one flow path downstream from a branch point. Enzyme immunosensor.   The enzyme immunosensor according to claim 19, wherein the flow path comprises a single flow path without branching.   The immunosensor includes a first flow direction regulating region formed between a conductor that is formed between the antibody capture region and the enzyme reaction product measurement region and is bonded with a substance whose hydrophilicity changes depending on a potential, and the antibody capture 23. A second flow direction defining region, which is formed at a position opposite to the first flow direction defining region across the region and is made of a conductor to which a substance whose hydrophilicity is changed by an electric potential is combined. The enzyme immunosensor described.   23. The enzyme immunosensor according to claim 22, wherein the substance that is coupled to the first and second flow direction defining regions and whose hydrophilicity is changed by an electric potential is ferrocene or a derivative thereof. The flow path is separated into two upper and lower stages by a semipermeable membrane that allows the enzyme reaction product to permeate but does not permeate the enzyme-labeled antibody or antigen-binding fragment thereof, and the antibody capture region is formed in the upper stage of the flow path. The enzyme immunosensor according to any one of claims 19 to 23, wherein the enzyme reaction product measurement region is formed in a lower stage.

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