JP2010236945A - Method for immobilization of specimen, and micro analysis device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immobilization method of a specimen and a micro analysis device, capable of performing quick and highly-sensitive measurement plurally and simultaneously with a trace amount of sample. <P>SOLUTION: This method includes: a nonspecific adsorption prevention step of performing nonspecific adsorption prevention treatment to the inside of a microchannel and to an immobilization electrode 13 by bringing a nonspecific adsorption prevention treatment agent 12 into contact with the microchannel; a mixed liquid preparation step of adjusting pH of a solution containing the specimen material 10, electrifying the specimen material 10, and mixing a crosslinking agent, to thereby prepare mixed liquid; and an immobilization step of bringing the mixed liquid into contact with the microchannel, sending a current between the immobilization electrode 13 and a counter electrode provided in the microchannel, applying a variable voltage for supplying a potential opposite to an electrified potential of the specimen material 10 to the immobilization electrode 13 to impart activation energy to the immobilization electrode 13, and immobilizing the specimen material 10 to the immobilization electrode 13 by being crosslinked with the nonspecific adsorption prevention treatment agent 12. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for immobilizing an analyte and a microanalyzer. In particular, a method for immobilizing an analyte substance that immobilizes an analyte substance that can bind to the analyte in order to detect an analyte such as an allergen, adiponectin, cytokine, or immune antibody that is a specific substance contained in the analyte , And a microanalyzer using the immobilization method.

  An immunoassay method using an antigen-antibody reaction is useful as an analysis / measurement method in the fields of medicine, biochemistry, and measurement fields such as allergens. However, the conventional immunoassay has problems such as a long time required for analysis and complicated operation.

  Under such circumstances, in recent years, a micro technology (Micro Electro Mechanical System, MEMS) using semiconductor microfabrication technology has been developed, and in the analysis in the biochemistry field of proteins, genes, etc., antigen-antibody reaction has been performed. The micro technology to be used (Micro Total Analytical System, μ-TAS) is rapidly developing.

  For example, Patent Document 1 discloses an analysis time for analyzing a specimen such as an antigen by forming a micro-order channel (hereinafter also referred to as “microchannel”) on a substrate and immobilizing an antibody or the like on the microchannel. Microchannel device technology has been proposed to shorten and simplify analysis operations.

  Specifically, Patent Document 1 discloses a technique related to a microchannel device including a ligand fixing part and a ligand such as an antibody or an artificial antibody fixed to the ligand fixing part in the microchannel.

  FIG. 13 shows the structure of the microchannel device disclosed in Patent Document 1. As shown in FIG. 13, in this microchannel device, a microchannel 101, an injection hole 102, a liquid reservoir 103, and a discharge hole 104 are formed on the surface of a substrate 200 made of a light-transmitting material such as glass or plastic. Has been. Furthermore, a ligand fixing part 105 is formed in the microchannel 101. A ligand that specifically reacts with the specimen is immobilized on the ligand immobilization unit 105 by a known immobilization method such as physical adsorption or immobilization using a covalent bond with an amino group of the ligand. Here, the ligand means a chemical substance having a specific affinity for a substance to be analyzed, and is also referred to as an analyte substance.

  FIG. 14 is a diagram for explaining a specimen detection method using the microchannel device shown in FIG. As shown in FIG. 14, first, the liquid containing the specimen 120 and the liquid containing the labeled antibody 123 are mixed and reacted. Here, the labeled antibody 123 is formed by binding an optically detectable labeling substance 121 and an antibody 122 capable of binding to the specimen 120. The labeled antibody 123 and the specimen 120 react to form an immune complex 124 (reaction conjugate between the labeled antibody and the specimen).

  Next, the liquid mixture containing the immune complex 124 is injected from the injection hole 102 shown in FIG. 13 using an external pump, and is circulated through the microchannel. As shown in FIG. 14, the immune complex 124 and the ligand are immobilized. The antibody 125 immobilized on the part 105 is reacted. As a result, a complex 126 composed of the antibody 125 -the specimen 120 -the labeled antibody 123 is formed in the ligand fixing part 105.

  Thereafter, the labeling substance 121 of the immune complex 124 bound to the ligand fixing part 105 is subjected to a predetermined analytical instrument such as ultraviolet-visible spectroscopic analysis, fluorescence analysis, chemiluminescence analysis, or thermal lens analysis according to the type of the labeling substance. It is used to detect light absorption, fluorescence or luminescence of the labeling substance. As a result, the amount of the specimen 120 in the detection substance can be measured.

  According to the technique of Patent Document 1 described above, the reaction field (ligand fixing part) can be reduced, and the reaction surface area can be increased. Therefore, the reaction is required in addition to downsizing the device and simplifying the chip structure. Time can be significantly reduced.

  However, in Patent Document 1, one specimen is detected in one microchannel, but when a plurality of specimens are to be detected in one microchannel, a plurality of ligands are immobilized in one microchannel. There is a need.

  Here, if a micro spotter is used, a plurality of ligands can be immobilized in one channel. In this case, the ligand is immobilized before the microchips are bonded together. However, if the immobilization is performed before the microchip is bonded, it becomes difficult to fix the ligand because the thermocompression bonding and bonding with an organic solvent during bonding are adverse conditions for the immobilized ligand. , You will not be able to make accurate measurements. For this reason, it is necessary to immobilize the ligand after the bonding step of the analyzer or the like.

  Patent Documents 2 to 5 disclose a technique capable of immobilizing a ligand only at an arbitrary site in a microchannel without the need for the bonding step as described above after immobilization of the ligand. Specifically, by using the analysis apparatus or method disclosed in these patent documents, a plurality of ligands are introduced into the channel one by one and the immobilization is repeated. It is also considered that it can be immobilized on.

International Publication No. 2006/054689 Pamphlet (released on May 26, 2006) JP 2003-329682 A (published on November 19, 2003) International Publication No. 2004/088319 (published on October 14, 2004) JP 2004-301515 A (released on October 28, 2004) JP 2007-309725 A (published November 29, 2007)

  However, when a plurality of ligands are immobilized in one channel by the techniques of Patent Documents 2 to 5, the following problems arise.

  The first problem is a problem due to nonspecific adsorption of the ligand. Specifically, when immobilizing ligands one by one, it is necessary to introduce the ligand into the channel and perform immobilization using each method only at a specified position. However, since the ligand adsorbs nonspecifically at any site other than an arbitrary site in the channel, there is a possibility that a plurality of ligands may be immobilized at random other than the specified site. Accordingly, there is a problem that the amount of the specimen in the detection substance cannot be accurately measured.

  As a second problem, the methods according to Patent Documents 2 to 5 are very high compared to the concentration of the immobilized ligand solution (less than 10 μg / ml) used for detection on a widely used microtiter plate. Concentration is required. For example, Patent Document 2 describes HSA 500 μg / ml, Patent Document 3 describes 2.5 mg / ml, Patent Document 4 describes 10 μg / ml, and Patent Document 5 describes 1.25 mg / ml. Even if a high concentration of the immobilized ligand solution is added, only a part of the ligand is immobilized, and most of the immobilized ligand solution is not immobilized and becomes a waste liquid. In addition, the higher the concentration of the ligand solution, the more nonspecific adsorption in the microchannel increases. When non-specific adsorption increases, the ligand is non-specifically adsorbed in addition to any site in the channel, causing the above-described problems.

  The present invention has been made in view of the above problems, and its purpose is to suppress non-specific adsorption of a substance to be analyzed, and to immobilize only a specified site in a microchannel, thereby It is an object of the present invention to provide a method for immobilizing a specimen substance capable of accurately performing the above analysis and a microanalyzer using the immobilization method. In particular, when a plurality of different analyte substances are immobilized in a microchannel, a plurality of analyte substances can be reliably immobilized at a predetermined position with a single channel, and a plurality of analytes can be detected with a small amount of detection substance. It is an object of the present invention to provide a method for immobilizing a test substance capable of detecting urine and a microanalyzer using the immobilization method.

  An analyte substance immobilization method according to the present invention is an analyte substance immobilization method for immobilizing an analyte substance that can bind to an analyte in a microchannel in a microanalyzer, in order to solve the above-described problem. The microchannel is provided with an immobilizing electrode and a counter electrode, and a nonspecific adsorption preventing treatment agent is brought into contact with the microchannel so that the microchannel and the immobilizing electrode are nonspecific. A non-specific adsorption preventing step for performing an adsorption preventing treatment, the pH of the solution containing the analyte substance is adjusted to charge the analyte substance, and the pH-adjusted solution can react with the non-specific adsorption preventing treatment agent Mixing step of preparing a mixed solution by mixing a functional group and a cross-linking agent having a functional group capable of reacting with the analyte, and bringing the mixed solution into contact with the microchannel A current is allowed to flow between the immobilization electrode provided in the microchannel and the counter electrode, and an electric potential having a polarity opposite to that of the charged electric potential of the analyte is supplied to the immobilization electrode. A non-specific adsorption prevention treatment is applied to the immobilization electrode that has been subjected to a non-specific adsorption prevention treatment by applying a variable voltage to the immobilization electrode and applying activation energy to the immobilization electrode. It is characterized by including an immobilization step of immobilizing by crosslinking with an agent.

  According to the above-described configuration, before the analyte substance is electrically attracted to the electrode for immobilization by bringing the mixed liquid containing the analyte substance into contact with the microchannel, and the analyte substance is immobilized by the cross-linking agent. Since the non-specific adsorption preventing process is performed on the microchannel, the analyte substance can be prevented from being nonspecifically adsorbed in the microchannel. Since the analyte substance electrically attracted to the immobilizing electrode is bonded via the cross-linking agent, the analyte substance can be immobilized only at an arbitrary place. As a result, the sample can be accurately analyzed. In particular, by repeating the immobilization process of the present invention, it is possible to immobilize a plurality of different analyte substances at arbitrary locations in the microchannel. Thus, when a plurality of analyte substances are immobilized in the microchannel, a plurality of analyte substances can be reliably immobilized at a predetermined position in one channel, and a plurality of different specimens in the detection substance can be fixed. Can be analyzed more accurately.

  In the method for immobilizing an analyte according to the present invention, the crosslinking agent is a photocrosslinking agent, the functional group capable of reacting with the non-specific adsorption preventing treatment agent is a photoreactive substituent, and the analyte The functional group capable of reacting with a substance preferably has a photoreactive substituent, and the activation energy is preferably light energy.

  According to said structure, there exists the further effect that an analyte can be fix | immobilized by simple operation of irradiating the light which activates a photocrosslinking agent.

  In the method for immobilizing an analyte substance according to the present invention, it is preferable that at least a part of the immobilization electrode is covered with a synthetic polymer layer before the nonspecific adsorption preventing treatment.

  According to said structure, since the area | region which can be fix | immobilized on the electrode for fixation | immobilization is built in three dimensions, a to-be-analyzed substance can be fix | immobilized more densely. Thereby, there is an additional effect that the specimen can be detected with higher sensitivity.

  In the immobilization method of the analyte substance according to the present invention, it is preferable that the variable voltage applied between the immobilization electrode and the counter electrode is 1 V or more.

  According to said structure, the to-be-analyzed substance which exists between electrodes can be attracted to the said electrode for fixation more efficiently.

  In the method for immobilizing an analyte substance according to the present invention, the analyte substance concentration in the solution after pH adjustment is preferably 0.01 μg / ml or more and 10 μg / ml or less.

  According to said structure, since the density | concentration of analyte substance is low enough, there exists the further effect that the nonspecific adsorption | suction of analyte substance can be suppressed more.

  In order to solve the above problems, a microanalyzer according to the present invention is a microanalyzer provided with a microchannel, in which an analyte substance that can bind to a specimen is immobilized on the microchannel, Is provided with an immobilization electrode and a counter electrode, the immobilization electrode of the microchannel is coated with a non-specific adsorption prevention treatment agent, and the analyte is subjected to non-specific adsorption prevention treatment. The immobilized electrode is cross-linked with the non-specific adsorption preventing treatment agent by a crosslinking agent having a functional group capable of reacting with the non-specific adsorption preventing treatment agent and a functional group capable of reacting with the analyte. It is characterized by being fixed.

  According to the above configuration, the non-specific adsorption preventing layer formed by coating the non-specific adsorption preventing treatment agent is disposed on the immobilizing electrode, so that the analyte is adsorbed non-specifically. Can be prevented. As a result, the sample can be accurately analyzed. Further, according to the above configuration, the immobilization electrode is coated with the nonspecific adsorption preventing treatment agent, but the nonspecific adsorption preventing treatment agent and the analyte can be bound through the cross-linking agent. Can do.

  In the microanalyzer according to the present invention, the crosslinking agent is a photocrosslinking agent, and the functional group capable of reacting with the non-specific adsorption preventing treatment agent is a photoreactive substituent, and can react with the analyte. Such functional groups preferably have a photoreactive substituent.

  According to said structure, there exists the further effect that an analyte can be fix | immobilized by simple operation of irradiating the light which activates a photocrosslinking agent.

  In the microanalyzer according to the present invention, at least a part of the immobilization electrode is covered with a synthetic polymer layer, and the synthetic polymer layer is covered with the non-specific adsorption preventing treatment agent. It is preferable.

  According to the above configuration, since the synthetic polymer layer is disposed between the immobilization electrode and the nonspecific adsorption prevention layer, the immobilizable region is three-dimensionally formed on the immobilization electrode. As a result, the analyte substance can be immobilized at a higher density. Thereby, there is an additional effect that the specimen can be detected with higher sensitivity.

  In the microanalyzer according to the present invention, the microchannel is provided with two or more fixing electrodes, and a counter electrode is provided downstream or upstream of the two or more fixing electrodes. It is preferable that

  According to the above configuration, by providing two or more immobilization electrodes in the microchannel, it is possible to immobilize a plurality of different analytes, and a plurality of different analytes can be obtained in one microchannel. There is a further effect that it can be analyzed.

  The microanalyzer according to the present invention further comprises one injection hole for bringing the liquid into contact with the microchannel and one discharge hole for discharging the liquid, and the injection hole and the discharge hole are: It is preferable that they are connected by one microchannel.

  According to said structure, since the injection hole and the discharge hole are provided in the microchannel, there exists the further effect that injection | pouring (contact) and discharge | emission of a solution are easy.

  The microanalyzer according to the present invention further includes one injection hole for bringing the liquid into contact with the microchannel and two or more discharge holes for discharging the liquid, the one injection hole and the 2 The microchannel connecting two or more discharge holes has a structure in which a path from the one injection hole is branched at a branch point, and the microchannel branched in the microchannel downstream from the branch point. It is preferable that the immobilizing electrode is provided for each channel branch, and the counter electrode is provided in the microchannel upstream of the branch point.

  According to the above configuration, a sample of one detection substance is injected from the injection hole and branched at the branch point in the microchannel, and then detected on the immobilization electrode provided in each branch path. Thus, it is possible to analyze a plurality of properties (a plurality of specimens) with one detection substance sample. For example, it is very effective when detecting a reaction in which a substrate changes due to an enzyme substrate reaction.

  In the microanalyzer according to the present invention, it is preferable that a detection electrode for detecting an electrochemically active substance is provided on the downstream side of the immobilization electrode.

  According to the above configuration, when the specimen changes to an electrochemically active substance, the changed substance can be detected by the detection electrode on the downstream side thereof, and the specimen can be detected more quickly and easily. There is a further effect that detection can be performed. For example, it is very effective when the substrate is changed into an electrochemically active substance by an enzyme substrate reaction.

  In order to solve the above problems, a method for immobilizing an analyte according to the present invention is a microanalyzer that includes a microchannel, and an analyte that can bind to an analyte is immobilized on the microchannel, The microchannel is provided with an elongate immobilizable region along the microchannel, and at both ends of the microchannel, one injection hole for bringing the liquid into contact with the microchannel and the liquid are discharged. Each of which is provided with a reference electrode in the vicinity of the injection hole and a counter electrode in the vicinity of the discharge hole, or a reference electrode in the vicinity of the discharge hole. And a counter electrode is provided in the vicinity of the injection hole, and when immobilizing the analyte substance on the microchannel, non-specific adsorption prevention treatment In the immobilizable region that has been subjected to the non-specific adsorption prevention treatment by the liquid, the liquid containing the analyte and the photocrosslinking agent is injected from the injection hole and electrically attracted to the discharge hole. The mask has a functional group capable of reacting with the non-specific adsorption preventing treatment agent and a functional group capable of reacting with the analyte, by irradiating only a plurality of positions in the immobilizable region with light. When the photocrosslinking agent is activated, the analyte substance is cross-linked with the non-specific adsorption preventing treatment agent and immobilized at any of a plurality of positions in the immobilizable region.

  According to the above configuration, before the sample substance is electrically attracted to the discharge hole by bringing the liquid mixture containing the substance to be in contact with the inside of the microchannel, and the analyte substance is immobilized by the cross-linking agent, Since the nonspecific adsorption preventing process is performed on the channel, it is possible to prevent the analyte substance from being nonspecifically adsorbed in the microchannel. Since the analyte substance electrically attracted to the immobilizing electrode is bonded via the cross-linking agent, the analyte substance can be immobilized only at an arbitrary place. As a result, the sample can be accurately analyzed. In particular, it is possible to immobilize a plurality of different analyte substances at arbitrary locations in the microchannel by irradiating only a plurality of positions in the immobilizable region using a photomask. Become. Thus, when a plurality of analyte substances are immobilized in the microchannel, a plurality of analyte substances can be reliably immobilized at a predetermined position in one channel, and a plurality of different specimens in the detection substance can be fixed. Can be analyzed more accurately.

  In the microanalyzer according to the present invention, the nonspecific adsorption preventing treatment agent is preferably a protein, a synthetic polymer, or a mixed substance of a synthetic polymer and a protein.

  When using a non-specific adsorption inhibitor for proteins, the protein can be obtained very easily and the non-specific adsorption prevention capability is high, thereby reducing costs and ensuring non-specific adsorption prevention more reliably. it can. In addition, when a non-specific adsorption preventing treatment agent for a synthetic polymer is used, there is an advantage that deterioration due to storage is very small as compared with protein. Moreover, when using the non-specific adsorption preventing treatment agent for the mixed substance of the synthetic polymer and protein, both the effects of the synthetic polymer and protein can be exhibited.

  In the microanalyzer according to the present invention, the synthetic polymer preferably has a function of a non-specific adsorption preventing treatment agent.

  Thereby, since the synthetic polymer functions as a non-specific adsorption preventing treatment agent, non-specific adsorption can be further prevented.

  In the microanalyzer according to the present invention, the protein is preferably bovine serum albumin, casein, or gelatin.

  These proteins are preferable from the viewpoint of availability.

  In the microanalyzer according to the present invention, the synthetic polymer is preferably electrically neutral. The synthetic polymer is preferably a synthetic polymer having a phosphorylcholine group.

  These synthetic polymers are preferable from the viewpoint that deterioration due to storage is very small compared to proteins. Furthermore, since the analyte substance immobilization field is three-dimensionally formed by the synthetic polymer, more analyte substances can be immobilized. As a result, an effect of increasing the detection sensitivity of the sample can be expected.

  In the microanalyzer according to the present invention, the cross-linking agent is 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimide ester or bis-[(4-azidosalicylamido) ethyl] disulfide. {Bis-[(4-Azidosalylamido) ethyl] disulphide} is preferable.

  These crosslinking agents are preferable from the viewpoint of availability.

  In the microanalyzer according to the present invention, the analyte is preferably an antibody or an artificial antibody.

  According to said structure, the antibody or the artificial antibody has the further effect that specificity with a test substance is very high, and it is excellent also in durability.

  In the microanalyzer according to the present invention, the artificial antibody is preferably a synthetic peptide, an aptamer, or a molecular imprinting polymer.

  According to said structure, there exists the further effect that a synthetic peptide, an aptamer, or a molecular imprinting polymer can be easily produced with a well-known technique.

  In the microanalyzer according to the present invention, the specimen is preferably a blood component.

  According to said structure, if the microanalyzer of this invention is used for a blood test, it will become possible to detect several samples (item) in a short time.

  In the microanalyzer according to the present invention, the blood component preferably contains at least one of adiponectin, leptin, resistin, and TNF-α.

  This can be used as an index of abnormal lipid metabolism.

  In the microanalyzer according to the present invention, it is preferable that a pump is connected to the injection hole.

  This makes it possible to immobilize more effectively because the analyte is affected not only by the attraction of electricity but also by the diffusion rate of the pump. Become.

  In the method for immobilizing an analyte according to the present invention, the microchannel includes an immobilization electrode and a counter electrode, and a nonspecific adsorption preventing treatment agent is brought into contact with the microchannel, so that the microchannel A non-specific adsorption preventing step for performing non-specific adsorption preventing treatment on the electrode for immobilization and the electrode for immobilization, adjusting the pH of the solution containing the analyte substance, charging the analyte substance, A mixed solution preparation step of preparing a mixed solution by mixing a functional group capable of reacting with the non-specific adsorption preventing treatment agent and a cross-linking agent having a functional group capable of reacting with the analyte, and the mixed solution A potential that is brought into contact with the microchannel, causes a current to flow between the immobilization electrode provided in the microchannel and the counter electrode, and the analyte is charged on the immobilization electrode; Applying a variable voltage that supplies a potential opposite in nature, and further applying activation energy to the immobilization electrode, the analyte substance is subjected to non-specific adsorption prevention treatment. The electrode includes an immobilization step of immobilizing the electrode by crosslinking with the non-specific adsorption preventing treatment agent.

  In the microanalyzer according to the present invention, the microchannel includes an immobilization electrode and a counter electrode, and the microchannel immobilization electrode is coated with a non-specific adsorption preventing treatment agent, The analyte substance has a functional group capable of reacting with the non-specific adsorption preventing treatment agent and a functional group capable of reacting with the analyte substance on the immobilizing electrode that has been subjected to non-specific adsorption preventing treatment. Thus, the non-specific adsorption preventing treatment agent is cross-linked and immobilized.

  Therefore, the immobilization method of the analyte substance and the microanalyzer using the immobilization method in the present invention suppress nonspecific adsorption of the analyte substance and immobilize it only at a specified site in the microchannel. Thus, there is an effect that the sample can be analyzed accurately.

  That is, according to the present invention, it is possible to independently detect a plurality of items from a minute sample without erroneous recognition.

It is a top view which shows the microanalyzer in one Embodiment of this invention. It is sectional drawing which shows the electrode for fixation of the microanalyzer in one Embodiment of this invention. It is sectional drawing which shows the electrode for fixation of the microanalysis apparatus in other embodiment of this invention. It is a top view which shows the microanalysis apparatus in other embodiment of this invention. It is a top view which shows the microanalyzer in other embodiment of this invention. It is a top view which shows the microanalyzer in other embodiment of this invention. It is a top view which shows the microanalyzer in other embodiment of this invention. It is a top view which shows the microanalyzer in other embodiment of this invention. 2 is a plan view showing a microanalyzer used in Example 1 and Comparative Examples 1 and 2. FIG. FIG. 3 is a diagram showing a fluorescence image of an immobilization electrode after immobilizing an analyte in Example 1. It is a figure which shows the fluorescence image of the electrode for fixation after immobilizing the analyte substance in the comparative example 1. It is a figure which shows the fluorescence image of the electrode for fixation after immobilizing the to-be-analyzed substance in the comparative example 2. It is the schematic which shows the structure of the microanalyzer in a prior art. It is the schematic which shows reaction in the immobilization part of the microanalyzer in a prior art.

  Hereinafter, embodiments of a method for immobilizing an analyte and a microanalyzer according to the present invention will be described with reference to the drawings.

[Embodiment 1]
Hereinafter, the first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a plan view of a microanalyzer according to Embodiment 1 of the present invention, and FIG. 2 is a cross-sectional view of an immobilizing electrode of the microanalyzer. FIG. 3 is a cross-sectional view of an immobilization electrode of another microanalyzer according to Embodiment 1 of the present invention.

<Main body of micro analyzer>
A microanalyzer (microchannel chip) according to a first embodiment of the present invention includes a substrate 100 as shown in FIG. 1 and a lid substrate (not shown) that adheres and seals the substrate 100. The substrate 100 and the lid substrate are bonded to each other. The substrate 100 is formed with the injection hole 1, the discharge hole 7, and the concave microchannel 2 that connects the injection hole 1 and the discharge hole 7.

  As the substrate 100, an insulating substrate can be used. As the insulating substrate, for example, a silicon substrate, a quartz substrate, an aluminum oxide substrate, a glass substrate, a plastic substrate, or the like having an insulating material such as an oxide film formed on the surface can be used. If chemiluminescence at the time of detection is used, for example, a plastic material having a small spontaneous fluorescence and transparent as disclosed in Japanese Patent Application Laid-Open No. 2003-149252 can also be used.

  The thickness of the substrate 100 is preferably about 0.1 to 5 mm. The depth of the microchannel 2 is preferably about 0.1 to 1000 μm, and the width is preferably about 0.1 to 1000 μm.

  The cross-sectional shape of the microchannel 2 can be rectangular or trapezoidal. Moreover, the bottom of the flow path may be round like a part of a circle.

  The injection hole 1 and the discharge hole 7 have a diameter of 10 μm or more. Further, the injection hole 1 and the discharge hole 7 are formed as through holes in either the substrate 100 or the lid substrate in order to inject and discharge the liquid.

  Examples of the method for forming the unevenness of the substrate 100 include a method by machining, a method by laser processing, an injection molding using a mold, a press molding, a method by casting, and the like as a direct processing method. Among them, a method using a mold is preferable because mass productivity is good and shape reproducibility is high.

  When the material of the substrate 100 is silicon, glass, or the like, the pattern of the microchannel 2 on the substrate 100 can be formed by a conventionally proposed photolithography method or etching method.

<Immobilization electrode, counter electrode and reference electrode>
As shown in FIG. 1, a plurality of immobilization electrodes 3, 4, 5 for immobilizing a plurality of ligands are arranged in the microchannel 2. The immobilization electrodes 3, 4, and 5 are linearly arranged along the linear microchannel 2.

  As described above, the ligand (analyte substance) means a chemical substance having specific affinity for the substance to be analyzed.

  In the vicinity of the injection hole 1 on the upstream side of the fixing electrodes 3, 4, 5, the counter electrode 6 is arranged. Here, the upstream side or the downstream side corresponds to the flow from the injection hole 1 to the discharge hole 7.

  The microchannel 2 may be further provided with a reference electrode, and may be composed of three electrodes, an immobilization electrode, a counter electrode, and a reference electrode.

  Each electrode can be formed by a microfabrication technique using a conventional photolithography technique. As the conductive material of the electrode, for example, gold, platinum, silver, chromium, titanium, iridium, copper, or carbon can be used.

  The widths of the fixing electrodes 3, 4, 5 and the counter electrode 6 are in the range of 1 μm to 3000 μm, and more preferably set equal to the channel width. The shape of each electrode may be a polygon including a rectangle as shown in FIG. 1 or may be a circle.

  The fixing electrodes 3, 4, and 5 are arranged in the channel such that the distance between the counter electrode 6 and the fixing electrode 3 is the same as the distance between the fixing electrodes 3 and 4 and the fixing electrode 4. It is preferable that the distance between the electrode 5 and the immobilization electrode 5 is 10 times or more. The counter electrode 6 may be disposed near the injection hole 1 as shown in FIG. 1 or may be disposed near the discharge hole 7.

  The distance between the counter electrode 6 and the immobilizing electrode 3 is at least 10 times the distance between the immobilizing electrode 3 and the immobilizing electrode 4 and the distance between the immobilizing electrode 4 and the immobilizing electrode 5. According to the configuration, the following effects can be obtained. That is, when a liquid containing an analyte is put into the microchannel 2 and a voltage is applied between each of the immobilizing electrodes 3, 4, and 5 and the counter electrode 6, the ligand between the electrodes to which the voltage is applied is It migrates and is concentrated on the electrode. However, since the interval between any of the fixing electrodes 3, 4, 5 and the counter electrode 6 is sufficiently larger than the interval between the fixing electrodes 3, 4, 5, the fixing electrode 3, The amount of the analyte substance that is concentrated and fixed to 4, 5 is less uneven in each of the immobilizing electrodes 3, 4, 5. In other words, when immobilizing a plurality of analyte substances, non-uniformity in the amount of analyte substances to be immobilized can be reduced.

<Non-specific adsorption prevention treatment agent>
In the analyte immobilization method and the microanalyzer according to the present invention, before flowing the liquid containing the analyte into the microchannel 2, the microchannel 2 is contacted with a non-specific adsorption preventing treatment agent, and the microchannel 2 is brought into contact with the microchannel 2. A non-specific adsorption preventing treatment agent layer 12 (see FIG. 2) is provided. Here, the “non-specific adsorption preventing treatment agent” refers to an agent that prevents non-specific adsorption.

  Examples of the non-specific adsorption preventing treatment agent include proteins, synthetic polymers, and mixed substances of synthetic polymers and proteins.

  When using a non-specific adsorption inhibitor for proteins, the protein can be obtained very easily and the non-specific adsorption prevention capability is high, thereby reducing costs and ensuring non-specific adsorption prevention more reliably. it can. In addition, when a non-specific adsorption preventing treatment agent for a synthetic polymer is used, there is an effect that deterioration due to storage is very small compared to protein. When using a mixed material of a synthetic polymer and protein, both effects can be achieved.

  Examples of the protein include bovine serum albumin (BSA), casein, and gelatin.

  As the synthetic polymer, a commercially available non-specific adsorption preventing treatment agent such as an electrically neutral polymer or a polymer having a phosphorylcholine group can be used.

<Ligand (analyte substance)>
As the ligand in the ligand solution, an antibody or an artificial antibody is preferable. This is because an antibody or an artificial antibody has very high specificity with a specimen and is excellent in durability. Artificial antibodies can be synthetic peptides, aptamers, or molecular imprinting polymers. Synthetic peptide production, aptamer production, and molecular imprinting production techniques can be easily produced by known techniques.

  The ligand solution is adjusted to a pH at which the ligand is charged either positively or negatively from the pKa or isoelectric point. Here, the pH of the ligand solution is preferably adjusted to 3 to 6 when acidic and 8 to 11 when alkaline. The final concentration of the ligand solution after pH adjustment is preferably adjusted to 0.01 μg / ml or more and 10 μg / ml or less. This is because when it is less than 0.01 μg / ml, a sufficient amount cannot be immobilized on the immobilized electrode even when immobilization is performed by the immobilization method according to this patent. This is also not preferable because the ligand may be adsorbed nonspecifically to the chemical adsorption inhibitor.

<Crosslinking agent>
As a crosslinking agent, the photocrosslinking agent described in the said patent document 4 is preferable. In the first embodiment, a photocrosslinking agent is used. As the photocrosslinking agent, a substance having two or more photoreactive substituents or a substance having a photoreactive substituent and an active substance substituent can be used.

  The photo-crosslinking agent is activated by reacting the photoreactive substituent with the non-specific adsorption preventing agent adsorbed on the surface of the microchannel, and reacting the photoreactive substituent or active substance substituent with the ligand. It plays a role of forming a bridging bond between the substance and the non-specific adsorption preventing treatment agent.

  The photoreactive substituent is not particularly limited as long as it reacts with and binds to the non-specific adsorption preventing agent by photoreaction, and examples thereof include an azide group and a benzophenone group, and an azide group is particularly preferable. The active substance substituent may be any substance that reacts with a ligand to form a bond. For example, when the ligand is a protein or an enzyme, it is preferably a substituent having reactivity with an amino group, a carboxy group, a thiol group, or the like that they have. Examples of such a substituent include an amino group, a carboxy group, an activated ester of a carboxylic acid, an isothiocyanate group, a thiol group, and a sulfonyl chloride. Examples of such a photocrosslinking agent include a compound having an azide group and an active ester of a carboxylic acid, a compound having an azide group and a carboxy group, a compound having an azide group and an azide group, and the like. Specifically, for example, 4-azido-2,3,5,6-tetrafluorobenzoic acid, 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimide ester (hereinafter referred to as “ATFB-”). SE ”), Bis- (4-Azidosalylamido) ethyl] disulphide and the like are preferable, and ATFB-SE is particularly preferable from the viewpoints of reactivity and availability.

  Moreover, as a crosslinking agent, other crosslinking agents, such as a thermal crosslinking agent, can be used. The functional group possessed by the crosslinking agent is not limited to the above-mentioned photoreactive substituent or active substance substituent, but a functional group capable of reacting with a non-specific adsorption preventing treatment agent and a ligand (analyte substance). It is sufficient that the ligand and the non-specific adsorption preventing treatment agent can be crosslinked with a crosslinking agent by applying activation energy such as heat.

<Immobilization method>
Hereinafter, the ligand immobilization method in Embodiment 1 will be described.

  A non-specific adsorption preventing treatment agent is brought into contact with the microchannel 2 of FIG. 1 to form a non-specific adsorption preventing treatment agent layer 12 (see FIG. 2) on the microchannel 2, and then the pH-adjusted ligand solution and photocrosslinking The liquid mixture with the agent is brought into contact with an amount sufficient to fill the microchannel 2 through the injection hole 1.

  Next, a voltage is applied between the fixing electrode to be fixed (here, the fixing electrode 5) and the counter electrode 6. At this time, a variable voltage is applied so that a constant current flows. The value of the constant current is 100 μA or less in absolute value. The range is preferably 1 μA or more and 100 μA or less, and more preferably 1 μA or more and 10 μA or less.

  Here, the voltage applied to the immobilizing electrode 5 needs to be given a potential opposite to the polarity of the charged ligand. By applying such a voltage to the immobilization electrode 5, the charged ligand is attracted to the immobilization electrode 5 and concentrated.

  At the same time as the voltage is applied, light energy is applied to the fixing electrode 5. As a result, the photocrosslinking agent in the mixed solution is activated, and the ligand attracted and concentrated on the immobilizing electrode 5 is cross-linked by the non-specific adsorption preventing treatment agent and the photocrosslinking agent (using a thermal cross-linking agent). If you do, you will give thermal energy). Since the photocrosslinking agent is not activated in the portion to which no light energy is applied, it is not crosslinked with the non-specific adsorption preventing treatment agent. After immobilization, the inside of the microchannel is washed with a washing solution such as a buffer solution. As a result, the ligand is immobilized only at an arbitrary site (on the immobilization electrode 5) to which light energy is applied on the immobilization electrode 5.

  According to the above-described immobilization method, since non-specific adsorption prevention treatment is performed in advance on the microchannel, the ligand is not immobilized in the microchannel 2 other than the portion where the light energy of the immobilization electrode 5 is not applied. .

  By repeating the same operation as the immobilization electrode 5 on the immobilization electrode 4 and the immobilization electrode 3, each ligand is not mixed, and there is no nonspecific adsorption in the microchannel other than the prescribed site. It is possible to immobilize on the immobilizing electrode 4 and the immobilizing electrode 3. FIG. 2 is a cross-sectional view showing the immobilization electrode of the microanalyzer after such immobilization. As shown in FIG. 2, a non-specific adsorption preventing treatment agent is applied on the immobilizing electrode 13 to form a non-specific adsorption preventing treatment agent layer 12, and the photocrosslinking agent 11 and the ligand 10 are non-specifically adsorbed. The prevention treatment agent layer 12 is cross-linked and has a structure in which the ligand 10 is immobilized.

  Hereinafter, another configuration of the ligand immobilization method according to the first embodiment will be described.

  In the microanalyzer according to Embodiment 1 of the present invention, as shown in FIG. 3, before applying the nonspecific adsorption preventing treatment agent, a synthetic polymer is coated (coated) on the immobilizing electrode 29. In the microchannel, the synthetic polymer layer 28 can be disposed so as to cover at least a part of the immobilizing electrode 29.

  By performing such a process, it is possible to construct a three-dimensional region that can be immobilized on the immobilizing electrode 29. When immobilizing the ligand on the immobilizing electrode having the same area, the amount of immobilization can be ensured twice or more when the polymer is coated as compared to the case where the polymer is not coated. Thus, when a microchannel chip having a ligand immobilized at a high density is used, the detection sensitivity of the specimen can be improved.

  Furthermore, this synthetic polymer may have a function of a non-specific adsorption preventing treatment agent. In this case, non-specific adsorption can be further prevented. Further, the synthetic polymer layer and the non-specific adsorption preventing treatment agent layer can be formed as one layer, and the structure can be further simplified.

<Detection substance and sample>
An analysis target of the microanalyzer of the present invention is a specimen in a detection substance. Examples of the detection substance include blood or a solution containing blood.

  Examples of the specimen include blood components. The blood component can include any of adiponectin, leptin, resistin, and TNF (Tumor Necrosis Factor) -α. That is, when the detection substance is blood, any one or more of adiponectin, leptin, resistin, and TNF-α can be detected in one sample of blood using the microanalyzer of the present invention. it can.

[Embodiment 2]
FIG. 4 shows the structure of the microanalyzer according to the second embodiment of the present invention. As shown in FIG. 4, the microanalyzer (microchannel chip) according to the second embodiment includes a substrate 100 and a lid substrate (not shown) that adheres and seals the substrate 100. The substrate 100 and the lid substrate are bonded to each other. The substrate 100 is formed with an injection hole 30, a discharge hole 36, and a microchannel 31 that connects the injection hole 30 and the discharge hole 36. Further, as in the first embodiment (see FIG. 1), three fixing electrodes 32, 33, and 34 are arranged in the microchannel 31. Further, the counter electrode 35 is arranged in the vicinity of the injection hole 30 as in the first embodiment.

  Similarly to the first embodiment, after the non-specific adsorption preventing treatment agent is brought into contact with the microchannel 31, the mixed solution of the ligand solution and the photocrosslinking agent after pH adjustment is transferred from the injection hole 30 into the microchannel 31. Contact.

  The difference between the second embodiment and the first embodiment is that a counter electrode 37 is also disposed in the vicinity of the discharge hole 36.

  In the first embodiment, when a voltage is applied between the immobilization electrode and the counter electrode in the microchannel 31, the ligand existing between the electrodes is attracted to the immobilization electrode and concentrated. Therefore, in the configuration of FIG. 1 in the first embodiment, it is difficult to attract all the ligands in the microchannel 2 to the immobilization electrodes 3, 4, and 5. Further, the distance between the immobilizing electrode 3 and the counter electrode 6, the distance between the immobilizing electrode 4 and the counter electrode 6, and the distance between the immobilizing electrode 5 and the counter electrode 6 are different, so that they are attracted. The amount of ligands is inevitably different.

  On the other hand, in the second embodiment, since the counter electrode 37 is also arranged in the vicinity of the discharge hole 36, the counter electrode 35 and the counter electrode 37 and the fixing electrode (here, the fixing electrode) to be fixed. The voltage can be simultaneously applied to the electrode 33 for use. As a result, the ligand present in the microchannel 31 can be simultaneously attracted from both the opposing electrode 35 and the opposing electrode 37 to the immobilizing electrode 33 from both sides.

  Therefore, a larger amount of the ligand present in the microchannel 31 can be attracted to the immobilizing electrode 33, and the ligand is attracted from both sides of the immobilizing electrode 33, so that the amount of the attracted ligand is made uniform. It becomes possible to do.

  In the second embodiment, the method for adjusting the ligand solution, the electro-attractive immobilization method including the photocrosslinking agent and the non-specific adsorption preventing treatment agent are the same as those in the first embodiment.

[Embodiment 3]
FIG. 5 shows the structure of the microanalyzer according to the third embodiment of the present invention. As shown in FIG. 5, the microanalyzer according to the third embodiment includes a substrate 100 and a lid substrate (not shown) that adheres and seals the substrate 100. The substrate 100 and the lid substrate are bonded together. In the substrate 100, the injection hole 40, the discharge hole 44, the discharge hole 47, the discharge hole 50, and the microchannel 41 that connects the injection hole 40 and the discharge holes 44, 47, 50 are formed.

  The microchannel 41 has a structure in which the path of the microchannel from the injection hole 40 is branched into a plurality (three in FIG. 5) as many as the number of fixing electrodes, and from one branch point as shown in FIG. A branched structure may be used, or a structure branched from different points.

  The immobilization electrode 42 and the detection electrode portion 43, the immobilization electrode 45 and the detection electrode portion 46 are provided at three branch paths in the microchannel 41 branched corresponding to the discharge holes 44, 47 and 50. , And an immobilization electrode 48 and a detection electrode portion 49 are disposed, and the counter electrode 51 is disposed in the vicinity of the injection hole 40.

  Each of the detection electrode portions 43, 46, 49 may be composed of two electrodes, that is, a working electrode for detecting an electrochemically active substance and a counter electrode thereof, or a counter electrode, a working electrode, and a reference electrode. You may comprise from 3 poles. Here, examples of the electrochemically active substance include ferrocene, paraaminophenol (PAP), and potassium ferricyanide.

  In the third embodiment, the method for adjusting the ligand solution, the electro-attractive immobilization method including the photocrosslinking agent and the non-specific adsorption preventing treatment agent are the same as those in the first embodiment.

  The branched structure according to the third embodiment of the present invention is very effective when an analyte is detected electrochemically. When the specimen is detected electrochemically, the process is such that the signal captured by the ligand in the specimen is converted into an electrochemical signal and detected by the detection electrode section.

  For example, when detecting a reaction in which a substrate changes due to an enzyme substrate reaction, after capturing the sample, the enzyme is labeled with a secondary antibody reaction, and then an electrochemically active substance is generated by the enzyme substrate reaction to detect it. The concentration is detected by the electrode section for use.

  On the other hand, when the immobilization electrode and the detection electrode section are arranged in the linear microchannel as shown in FIGS. 1 and 4, the substance having electrochemical activity generated by the reaction on the upstream side is downstream. This causes a problem of misrecognition by other detection electrode portions on the side. However, as shown in FIG. 5, by arranging the immobilization electrode and the detection electrode part one by one in the microchannel after branching, detection can be performed independently by the detection electrode part.

[Embodiment 4]
FIG. 6 shows the structure of the microanalyzer according to the fourth embodiment of the present invention. As shown in FIG. 6, the microanalyzer according to the fourth embodiment includes a substrate 100 and a lid substrate (not shown) that adheres and seals the substrate 100. The substrate 100 and the lid substrate are bonded together. The substrate 100 is formed with the injection hole 60, the discharge hole 64, the discharge hole 68, the discharge hole 72, and one microchannel 61 that connects the injection hole 60 and the discharge holes 64, 68, 72.

  The microchannel 61 has a structure in which the path of the microchannel from the injection hole 60 is branched into a plurality (three in FIG. 6) of the number of electrodes for immobilization, and from one branch point as shown in FIG. A branched structure may be used, or a structure branched from different points.

  Immobilization electrode 62 and detection electrode portion 63, immobilization electrode 66 and detection electrode portion 67 are arranged in three branch paths in microchannel 61 branched corresponding to each of discharge holes 64, 68 and 72. , And the immobilization electrode 70 and the detection electrode portion 71 are disposed, and the counter electrode 74 is disposed in the vicinity of the injection hole 60. These structures are the same as those of the third embodiment shown in FIG.

  The fourth embodiment differs from the third embodiment in that a counter electrode 65 is disposed near the discharge hole 64, a counter electrode 69 is disposed near the discharge hole 68, and a counter electrode 73 is disposed near the discharge hole 72. That is.

  According to this structure, it is possible to attract more ligands to the immobilization electrode and immobilize them by using four counter electrodes for one immobilization electrode when immobilizing.

  The method for preparing the ligand solution and the method for electrically attracting and immobilizing the photocrosslinking agent and the non-specific adsorption preventing treatment agent are the same as in the first embodiment.

[Embodiment 5]
FIG. 7 shows the structure of the microanalyzer according to the fifth embodiment of the present invention. As shown in FIG. 7, the microanalyzer according to the fifth embodiment includes a substrate 100 and a lid substrate (not shown) that adheres and seals the substrate 100. The substrate 100 and the lid substrate are bonded together. The substrate 100 is formed with an injection hole 80, a discharge hole 84, and one microchannel 82 that connects the injection hole 80 and the discharge hole 84.

  A ligand reference electrode 85 is disposed in the vicinity of the discharge hole 84, and a counter electrode 81 is disposed in the vicinity of the injection hole 80. Here, conversely, the counter electrode 81 may be disposed near the discharge hole 84, and the ligand reference electrode 85 may be disposed near the injection hole 80.

  In the fifth embodiment, a long (long) immobilizable region 83 along the microchannel 82 is provided in the microchannel between the injection hole 80 and the discharge hole 84. With this structure, in the method according to Embodiment 5, a plurality of ligands can be immobilized using only a pair of electrodes.

  That is, when the ligand is electrically attracted from the mixed solution of the ligand and the photocrosslinking agent, in Embodiments 1 to 4, photoimmobilization is performed after attracting the immobilization electrode. Since the immobilizable region 83 along the microchannel 82 is provided, light is irradiated only to an arbitrary point in the immobilizable region 83 using a photomask in the immobilizable region 83 that is being electrically attracted. To do.

  As a result, the photocrosslinking agent is activated when light is irradiated, and the ligand and the non-specific adsorption preventing treatment agent are cross-linked on the spot. By repeating this, a plurality of ligands can be immobilized at an arbitrary location of the microchannel 82 with only a pair of electrodes.

  The ligand solution adjustment method, photocrosslinking agent, non-specific adsorption preventing treatment agent and the like are the same as those in the first embodiment.

[Embodiment 6]
FIG. 8 shows the structure of the microanalyzer according to the sixth embodiment of the present invention. As shown in FIG. 8, the microanalyzer according to the sixth embodiment has a structure in which a pump 90 is installed so as to be connected to a microchannel 92 of the microanalyzer 93. The pump 90 can be connected to, for example, an injection hole or a discharge hole. In the present embodiment, the pump 90 is connected to the injection hole.

  The pump 90 and the injection hole are connected by a tube 91. The tube 91 is substantially unnecessary, but is installed if necessary for connection.

  As the microanalyzer 93, the microanalyzer of Embodiments 1 to 5 can be used. If immobilization is carried out while pumping the solution, the ligand can be immobilized more effectively because not only the attractive force of electricity alone but also the diffusion speed of the pump is increased.

  The ligand solution adjustment method, photocrosslinking agent, non-specific adsorption preventing treatment agent and the like are the same as those in the first embodiment.

  Next, effects of the structure of the present invention will be described with reference to Example 1 and Comparative Examples 1 and 2.

  In Example 1 and Comparative Examples 1 and 2, the microchannel chip shown in FIG. 9 was produced. The structure of the microchannel of the microanalyzer in Example 1 and Comparative Examples 1 and 2 was prepared using PDMS (Polydimethylsiloxane, manufactured by Toray Dow Corning). Both the fixing electrodes 203 and 204 and the counter electrode 205 were formed on a quartz substrate by a photolithographic method, and titanium and then gold were sputtered to produce a gold electrode.

  The microchannel 202 was manufactured with a width of 500 μm and a height of 50 μm. The fixing electrodes 203 and 204 were 200 μm × 500 μm, and the counter electrode 205 was 600 μm × 500 μm. A PDMS channel substrate and an electrode substrate were bonded together to produce a microchannel structure. The inside of the microchannel was subjected to non-specific adsorption prevention treatment with MPC (Methacryloyloxyethylphosphorylcholine) polymer (Daiichi Kikai Co., Ltd.) containing BSA and phosphorylcholine groups.

Example 1
Adiponectin antibody (manufactured by R & D System) modified with Hilyte 555 (manufactured by Dojindo) and resistin antibody modified with FITC (Fluoresceinisothiocyanate, manufactured by Dojindo) are each adjusted to pH 3.5, and the final concentration is adjusted to 1 μg / ml. did. Each antibody solution was mixed with a photocrosslinking agent ATFB-SE (manufactured by Thermo Fisher Scientific) so that the molar ratio of the photocrosslinking agent to the antibody was 500. The FITC-modified resistin antibody mixed solution was brought into contact through the injection hole 201, and a voltage was applied so that the current became −4 μA while irradiating light to the immobilizing electrode 203, thereby performing electro-induced light immobilization. Next, it was sufficiently washed with PBS (phosphate buffer saline, manufactured by Wako Pure Chemical Industries, Ltd.) containing Tween 20 (manufactured by Kishida Chemical Co., Ltd.). Further, the Hilite 555-modified adiponectin antibody mixed solution was brought into contact through the injection hole 201, and voltage was applied so that the current became −4 μA while irradiating the immobilizing electrode 204 with light, thereby carrying out electric induced light immobilization. Finally, the plate was thoroughly washed with PBS containing Tween 20.

(Comparative Example 1)
The Hyte 555 modified adiponectin antibody and the FITC modified resistin antibody were each adjusted to pH 3.5, and the final concentration was adjusted to 1 μg / ml. The photocrosslinking agent ATFB-SE was not mixed in each antibody solution. The FITC-modified resistin antibody solution was brought into contact from the injection hole 201, and a voltage was applied so that the current became −4 μA while irradiating the immobilizing electrode 203 with light, thereby performing electro-induced light immobilization. Next, the plate was thoroughly washed with PBS containing Tween 20. Furthermore, a Hilyte 555-modified adiponectin antibody solution was brought into contact with the injection hole 201, and a voltage was applied so that the current became −4 μA while irradiating the immobilization electrode 204 with light, thereby performing electro-induced light immobilization. Finally, the plate was thoroughly washed with PBS containing Tween 20.

(Comparative Example 2)
The Hyte 555 modified adiponectin antibody and the FITC modified resistin antibody were each adjusted to pH 3.5, and the final concentration was adjusted to 1 μg / ml. Each antibody solution was mixed with the photocrosslinking agent ATFB-SE so that the molar ratio of the photocrosslinking agent to the antibody was 500. The FITC-modified resistin antibody mixed solution was brought into contact through the injection hole 201, and the electric attraction light immobilization was performed without applying a voltage while irradiating the immobilization electrode 203 with light. Next, the plate was thoroughly washed with PBS containing Tween 20. Furthermore, a Hilyte 555-modified adiponectin antibody mixed solution was brought into contact with the injection hole 201, and the electric attraction light immobilization was performed without applying voltage while irradiating the immobilization electrode 204 with light. Finally, the plate was thoroughly washed with PBS containing Tween 20.

(Comparison between Example 1 and Comparative Examples 1 and 2)
Each immobilization electrode 204 was observed with fluorescence. Since fluorescence is modified in the antibody, if fluorescence is observed, it indicates that the antibody is immobilized in the microchannel.

  In the case of Example 1, the fluorescence was clearly observed only at the site irradiated with light on the immobilizing electrode (FIG. 10). In the case of Comparative Example 1 in which no photocrosslinking agent was added, almost no fluorescence was observed (FIG. 11). In the case of Comparative Example 2 where no voltage was applied, almost no fluorescence was observed (FIG. 12).

  Thus, it can be seen that the ligand can be reliably immobilized on the immobilization electrode by inserting a photocrosslinking agent and applying a voltage in the structure of the microanalyzer of the present invention.

  The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments can be appropriately combined. Such embodiments are also included in the technical scope of the present invention.

  The embodiments and examples disclosed this time are examples in all respects and are not considered to be restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

The present invention described above can be paraphrased as follows. That is,
(1) A method for immobilizing an analyte substance of a microanalyzer that detects multiple items simultaneously, wherein the analyte substance is adjusted to a pH having a charging property, and the solution has a photocrosslinking group having a photoreactive substituent. The solution is injected into a microchannel that has been subjected to non-specific adsorption prevention treatment before immobilizing the analyte and immobilizing the analyte, and the immobilization electrode installed in the microchannel and its counter electrode A variable voltage is applied so that a constant current flows between them, and the analyte substance is immobilized by a photoreaction by irradiating the immobilizing electrode with light that gives activation energy. A method for immobilizing an analyte.

  (2) In the method for immobilizing an analyte substance described in (1) above, at least a part of the immobilization electrode is made of a polymer before performing nonspecific adsorption prevention treatment in the microchannel of the microanalyzer. Method for immobilizing an analyte material that coats the substrate.

  (3) In the method for immobilizing a specimen substance described in (1) or (2) above, the voltage applied between the immobilization electrode installed in the microchannel and the counter electrode is 1 V or more. A method for immobilizing an analyte.

  (4) The method for immobilizing an analyte substance according to (1) or (2) above, wherein the final concentration of the analyte substance is 10 μg / ml or less.

  (5) A microanalyzer that detects multiple items simultaneously, in which a non-specific adsorption preventing treatment agent is coated on an immobilizing electrode, and the analyte is non-specific by a photocrosslinking agent having a photoreactive substituent. A microanalyzer characterized by a cross-linked structure between the adsorption prevention treatment.

  (6) In the microanalyzer described in (5) above, at least a part of the immobilization electrode is coated with a polymer, a non-specific adsorption preventing treatment agent is further coated thereon, and the analyte is further irradiated with light. A microanalyzer characterized by a structure in which a non-specific adsorption preventing treatment is crosslinked by a photocrosslinking agent having a reactive substituent.

  (7) The microanalysis apparatus according to (5) or (6), wherein two or more immobilization electrodes are provided in the microchannel, and (one) counter electrode is provided downstream or upstream thereof. apparatus.

  (8) In the microanalyzer described in (7) above, the microchannel has a structure composed of one microchannel connecting one injection hole for injecting a solution and one discharge hole for discharging the solution. Features a micro-analyzer.

  (9) In the microanalyzer described in (7) above, the microchannel has a structure branched in the middle to connect to one injection hole for injecting the solution and two or more discharge holes for discharging the solution, A microanalyzer in which an immobilization electrode is branched in a microchannel downstream from a branch point, and one counter electrode is provided in a microchannel upstream from the branch point.

  (10) The microanalyzer according to the above (9), wherein a detection electrode for detecting an electrochemically active substance is provided downstream of the immobilization electrode.

  (11) The microanalyzer according to any one of (5) to (10) above, wherein the non-specific adsorption preventing treatment agent is a protein.

  (12) The microanalyzer according to any one of (5) to (10) above, wherein the non-specific adsorption preventing treatment agent is a synthetic polymer.

  (13) The microanalyzer according to any one of (5) to (10), wherein the non-specific adsorption preventing treatment agent is a mixed substance of a synthetic polymer and a protein.

  (14) The microanalyzer according to (11) or (13) above, wherein the protein is bovine serum albumin, casein, and / or gelatin.

  (15) The microanalyzer according to the above (12) or (13), wherein the synthetic polymer is electrically neutral.

  (16) The microanalyzer according to (15), wherein the non-specific adsorption preventing treatment agent is a synthetic polymer having a phosphorylcholine group.

  (17) In the microanalyzer according to any one of (5) to (16) above, the photocrosslinking agent having a photoreactive group is 4-azido-2,3,5,6-tetrafluoro. Microanalyzer, which is benzoic acid succinimide ester, Bis- (4-Azidosalylamido) ethyl] disulphide.

  (18) The microanalyzer according to any one of (5) to (17) above, wherein the analyte is an antibody or an artificial antibody.

  (19) The microanalyzer according to the above (18), wherein the artificial antibody is a synthetic peptide, aptamer, or molecular imprinting polymer.

  (20) The microanalyzer according to any one of (5) to (19) above, wherein the detection substance is a blood component.

  (21) The microanalyzer according to (20), wherein the blood component includes any one of adiponectin, leptin, resistin, and TNF-α.

  (22) The microanalyzer according to any one of (5) to (21), wherein a pump is connected to the injection hole.

  According to the analyte substance immobilization method and the microanalyzer of the present invention, a plurality of rapid and highly sensitive measurements can be simultaneously performed with a small amount of sample. Accordingly, the present invention provides a microchannel chip using μ-TAS technology, such as a microchannel chip used to detect a minute chemical substance, a chemical microdevice such as a microreactor, and a biosensor such as an allergen sensor. It can be applied to fields that use.

1, 30, 40, 60, 80, 102, 201 Injection hole 2, 31, 41, 61, 82, 92, 101, 202 Microchannel 3, 4, 5, 13, 29, 32, 33, 34, 42, 45, 48, 62, 66, 70, 203, 204 Immobilization electrode 6, 35, 37, 51, 65, 69, 73, 74, 81, 205 Counter electrode 7, 36, 44, 47, 50, 64, 68, 72, 84, 104, 206 Discharge hole 10, 25 Ligand (analyte substance)
11, 26 Photocrosslinking agent (crosslinking agent)
12, 27 Non-specific adsorption preventing treatment agent layer 28 Synthetic polymer layer 43, 46, 49, 63, 67, 71 Detection electrode part 83 Immobilizable region 85 Ligand attracting electrode 90 Pump 91 Tube 93 Microanalyzer 100 Substrate 103 Pool part 105 Ligand fixing part 120 Sample substance 121 Labeled substance 122, 125 Antibody 123 Labeled antibody 124 Immune complex 126 Complex

Claims (26)

A method of immobilizing an analyte substance that immobilizes an analyte substance that can bind to an analyte in a microchannel in a microanalyzer,
The microchannel is provided with an immobilization electrode and a counter electrode,
A non-specific adsorption preventing step of bringing a non-specific adsorption preventing treatment agent into contact with the micro channel and performing non-specific adsorption preventing treatment on the microchannel and the immobilization electrode;
The pH of the solution containing the analyte is adjusted to charge the analyte, and the pH-adjusted solution can react with the functional group capable of reacting with the non-specific adsorption preventing treatment agent and the analyte A mixed liquid preparation step of mixing a crosslinking agent having a functional group to prepare a mixed liquid, and
The mixed solution is brought into contact with the microchannel, a current is passed between the immobilization electrode and the counter electrode provided in the microchannel, and the analyte substance is charged on the immobilization electrode. Applying a variable voltage that supplies a potential opposite in polarity to the potential, further applying activation energy to the immobilization electrode, and immobilizing the analyte substance on the non-specific adsorption prevention treatment A method for immobilizing an analyte, comprising an immobilization step of immobilizing the immobilization electrode by crosslinking with the non-specific adsorption preventing treatment agent.   The crosslinking agent is a photocrosslinking agent, the functional group capable of reacting with the non-specific adsorption preventing treatment agent is a photoreactive substituent, and the functional group capable of reacting with the analyte is photoreactive substitution. 2. The method for immobilizing an analyte according to claim 1, wherein the method includes the group and the activation energy is light energy.   3. The method for immobilizing an analyte according to claim 1, wherein at least a part of the immobilization electrode is covered with a synthetic polymer layer before the nonspecific adsorption prevention treatment.   The method for immobilizing an analyte according to any one of claims 1 to 3, wherein a variable voltage applied between the immobilization electrode and the counter electrode is 1 V or more.   The analyte according to any one of claims 1 to 4, wherein the concentration of the analyte in the pH-adjusted solution is in the range of 0.01 µg / ml to 10 µg / ml. Immobilization method of substance. A microanalyzer comprising a microchannel and an analyte substance that can bind to a specimen immobilized on the microchannel,
The microchannel is provided with an immobilization electrode and a counter electrode,
The electrode for immobilizing the microchannel is coated with a non-specific adsorption preventing treatment agent,
The analyte substance has a functional group capable of reacting with the non-specific adsorption preventing treatment agent and a functional group capable of reacting with the analyte substance on the immobilizing electrode that has been subjected to non-specific adsorption preventing treatment. The microanalyzer is characterized by being cross-linked and immobilized with the non-specific adsorption preventing treatment agent.   The crosslinking agent is a photocrosslinking agent, the functional group capable of reacting with the non-specific adsorption preventing treatment agent is a photoreactive substituent, and the functional group capable of reacting with the analyte is photoreactive. The microanalyzer according to claim 6, which has a substituent. At least a part of the immobilizing electrode is covered with a synthetic polymer layer,
8. The microanalyzer according to claim 6, wherein the synthetic polymer layer is coated with the non-specific adsorption preventing treatment agent. The microchannel is provided with two or more immobilization electrodes,
The microanalyzer according to any one of claims 6 to 8, wherein a counter electrode is provided on the downstream side or the upstream side of the two or more immobilization electrodes. And further comprising one injection hole for bringing the liquid into contact with the microchannel and one discharge hole for discharging the liquid,
The microanalyzer according to any one of claims 6 to 9, wherein the injection hole and the discharge hole are connected by one microchannel. And further comprising one injection hole for bringing the liquid into contact with the microchannel and two or more discharge holes for discharging the liquid,
The microchannel connecting the one injection hole and the two or more discharge holes has a structure in which a path from the one injection hole branches at a branch point;
In the microchannel downstream from the branch point, the immobilization electrode is provided for each branch path of the branched microchannel,
The microanalyzer according to any one of claims 6 to 9, wherein the counter electrode is provided in the microchannel upstream of the branch point.   The microanalysis according to any one of claims 6 to 11, wherein a detection electrode for detecting an electrochemically active substance is provided downstream of the immobilization electrode. apparatus. A microanalyzer comprising a microchannel and an analyte substance that can bind to a specimen immobilized on the microchannel,
The microchannel is provided with an elongate immobilizable region along the microchannel, and at both ends of the microchannel, one injection hole for bringing the liquid into contact with the microchannel and the liquid are discharged. Each has one discharge hole,
A reference electrode is provided in the vicinity of the injection hole and a counter electrode is provided in the vicinity of the discharge hole, or a reference electrode is provided in the vicinity of the discharge hole and the counter electrode is in the vicinity of the injection hole Is provided,
When immobilizing the analyte substance on the microchannel, a liquid containing the analyte substance and a photocrosslinking agent is provided in the immobilizable region that has been subjected to the nonspecific adsorption prevention treatment with the nonspecific adsorption prevention treatment agent. While injecting from the injection hole and electrically attracting to the discharge hole, a photomask is used to irradiate light to a plurality of positions of the immobilizable region to react with the non-specific adsorption preventing treatment agent. The immobilizable region is formed by crosslinking the analyte substance with the non-specific adsorption preventing treatment agent by activating the photocrosslinking agent having a functional group capable of reacting with the analyte substance. A microanalyzer fixed at a plurality of positions.   14. The microanalyzer according to claim 13, wherein the non-specific adsorption preventing treatment agent is a protein.   The microanalyzer according to claim 13 or 14, wherein the non-specific adsorption preventing treatment agent is a synthetic polymer.   The microanalyzer according to claim 15, wherein the synthetic polymer has a function of a non-specific adsorption preventing treatment agent.   The microanalyzer according to claim 13 or 14, wherein the non-specific adsorption preventing treatment agent is a mixed substance of a synthetic polymer and a protein.   The microanalyzer according to claim 14 or 17, wherein the protein is bovine serum albumin, casein, or gelatin.   The microanalyzer according to claim 15, wherein the synthetic polymer is electrically neutral.   The microanalyzer according to claim 19, wherein the non-specific adsorption preventing treatment agent is a synthetic polymer having a phosphorylcholine group.   The photocrosslinking agent is 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimide ester or bis-[(4-azidosalicylamido) ethyl] disulfide. Item 21. The microanalyzer according to any one of Items 13 to 20.   The microanalyzer according to any one of claims 13 to 21, wherein the analyte is an antibody or an artificial antibody.   23. The microanalyzer according to claim 22, wherein the artificial antibody is a synthetic peptide, aptamer, or molecular imprinting polymer.   The microanalyzer according to any one of claims 13 to 23, wherein the specimen is a blood component.   The microanalyzer according to claim 24, wherein the blood component contains at least one of adiponectin, leptin, resistin, and TNF-α.   The microanalyzer according to any one of claims 13 to 25, wherein a pump is connected to the injection hole.