JP4660768B2 - Microanalyzer and method for analyzing microsample - Google Patents

Description

The present invention relates to a microanalyzer used for analysis and detection of a very small amount of sample and a method for analyzing a microsample, and more particularly to a microanalyzer for performing measurement using enzyme activity and a method for analyzing a microsample.

At present, in the field of chemical sensors, researches are being actively carried out to miniaturize and integrate analysis systems using micromachining technology and semiconductor technology such as photolithography. These are called Micro Total Analysis System (μTAS). The advantage of μTAS is that it can be measured anywhere without taking up space due to the small size of the device. These advantages of μTAS are exactly what is required in the current medical field, and the significance of research toward the realization of μTAS for medical use is very large.

By the way, as an example of a device capable of analyzing for a bedside diagnosis (POC (point of care) analysis) in which a measurement necessary for medical diagnosis is performed in the vicinity of a patient, a capillary is formed between a pair of flat plate members, 2. Description of the Related Art A chip-type analyzer (see, for example, Patent Document 1 and Patent Document 2) that analyzes a predetermined component in a mixed liquid by flowing a liquid reagent and a sample is known. In addition, as a device specialized in the examination of liver function, a biosensor array for GOT and GPT measurement in which a sample and a substrate are mixed on a chip and sensing is also known (see, for example, Non-Patent Document 1). This biosensor consists of a sample inlet, a meandering channel for mixing, and a sensor chip formed on a glass substrate. The sample and substrate are flown from the sample inlet, GOT and GPT are measured.

  As a biosensor capable of such GOT and GPT measurement, a micro flow path is formed on a substrate where a flow path of a Y-shaped substrate solution and a flow path of a sample solution are merged, and the micro flow path is merged An electrode system composed of a plurality of electrodes formed facing the section, and a predetermined enzyme arranged in the vicinity of one electrode in the electrode system to react with the substrate solution and the sample solution joined at the joining section There is also known a device that includes an enzyme supply means and calibrates the enzyme activity of the sample solution based on a current value generated in the electrode system (see, for example, Patent Document 3).

JP 2001-165939 A Japanese Patent Laid-Open No. 2000-2777 JP 2005-351882 A I.Moser, G. Jobst, P Svasek, M Varaham, G Urban "Rapid Liver Enzyme Assay with Miniaturized Liquid Handling System Comprising Thin Film Biosensor Array" Sensors and Actuators B, 1997, vol. 44, 377-380

  By the way, γ-GTP, GOT, and GPT, which are liver function test items, can be measured by detecting L-glutamic acid, which is the same reaction product, and these can be measured by using an enzyme sensor using glutamate oxidase. Three items of γ-GTP, GOT, and GPT can be detected by one analysis system. For more information,

  The reaction using L-glutamate oxidase is as follows, and is an enzyme that oxidatively deaminates L-glutamate, and hydrogen peroxide and ammonia are produced as reaction products. The L-glutamic acid concentration is measured by measuring the oxidation current value generated by the hydrogen peroxide, and the values of γ-GTP, GOT, and GPT can be detected from this measurement.

  The above-mentioned known techniques are designed to induce an enzyme reaction by forming a micro flow channel on each substrate or chip and flowing the sample solution and the substrate solution together in the flow channel. However, it is not easy to flow a sample solution or a substrate solution through a minute flow path, and a system for flowing a fluid by centrifugal force or a minute device for flowing a fluid such as a micropump or a microsyringe pump is arranged. If it is necessary to do so, or if the shape is such that the flow path is routed, the time until the enzyme reaction is developed becomes long, and as a result, the measurement itself takes time.

  In addition, when measuring in the form of a chip or the like, it is easier if the liquid to be supplied is only a sample liquid. For example, in the case of a liver function test for measuring the values of γ-GTP, GOT and GPT, When the substrate solution is also supplied to the chip, the wrong substrate solution may be added, so that the measurement data may be incorrect, which can be a problem.

  Therefore, in view of the above technical problems, the present invention greatly reduces the work of handling samples and substrates during measurement in a microanalyzer that performs various calibrations using enzyme activity and a method for analyzing microsamples. An object of the present invention is to provide a microanalyzer and a method for analyzing a microsample that can give high reliability to analysis data.

In order to solve the above technical problem, the microanalyzer of the present invention forms a microchannel connected to an injection opening into which a sample solution is injected on a substrate, and faces the microchannel. the enzyme to form an enzyme-carrying portion for carrying the porous carrier to dissolve the lyophilized substrate in the fine channel adjacent to the injection opening in the vicinity of the enzyme carrier part to unrealized the sample solution It is arranged to cause a measurable enzyme activity by flowing the sample solution through the microchannel through the injection opening, and to analyze the enzyme activity in the sample solution corresponding to the substrate.

According to a preferred example of the present invention, an electrode is formed on the enzyme carrying part, and the enzyme is disposed on or near the surface of the electrode. The electrochemical reaction can be measured by the electrode, and for example, it is configured with two electrodes, more preferably three electrodes. In the case of the three-electrode system, an electrode system is formed by a working electrode, a reference electrode, and a counter electrode. For example, a thin metal film is formed in a required pattern on a plate-like member, and a part of the metal thin film is passed through a microchannel. It is possible to measure with current flowing. Various materials can be used as the metal material for forming the metal thin film, but materials such as platinum and silver can be used. Furthermore, for disposable applications, a carbon-based conductive paste is used. It is also possible to use an electrode. In the case of the three-electrode system, the enzyme supporting part can be formed in the vicinity of the working electrode.

  Any enzyme may be used as long as it produces a reaction product that can be measured with an electrode such as hydrogen peroxide. For example, an oxidoreductase such as glutamate oxidase may be used.

As a method of supporting such an enzyme, a method of immobilizing an enzyme in the vicinity of an electrode by a method comprising a carrier binding method, a crosslinking method, a comprehensive method or a combination thereof, or a permeable membrane or the like is formed so as to face a flow path. And a method of supplying an enzyme through the permeable membrane. The carrier binding method is a method of immobilizing an enzyme on a water-insoluble carrier, and a physical adsorption method, an ionic bond method, a covalent bond method, or the like is known depending on the binding mode. The cross-linking method is a method of fixing by cross-linking an enzyme using a reagent having two or more functional groups. In the cross-linking method, the enzyme can be easily fixed under mild conditions, so that the enzyme is hardly deactivated, and the stability of the enzyme can be increased by using an appropriate carrier. As an example, when L-glutamate oxidase is used as a target and L-glutamate oxidase is used as an enzyme, bovine serum albumin (BSA) is used as a polymer carrier, the enzyme is cross-linked using glutaraldehyde, and the membrane is formed. The enzyme carrier may be configured by adhering a molded product to the electrode. The inclusion method can be divided into a lattice type in which the enzyme is incorporated into a fine lattice of the polymer gel and a microcapsule type in which the enzyme is covered with a semipermeable polymer film. It can be used for enzyme immobilization.

  As the substrate, various materials such as glass, synthetic resin, ceramic, metal, wood, paper, and composite materials thereof can be used. A microchannel is formed in a required shape, and a sample solution is allowed to flow. It is only necessary to have a function of holding an electrode or the like in the microchannel formed in the substrate. According to an example of the present invention, a flow path pattern is formed of a photoresist on a glass substrate, and a flow path forming layer made of a transparent resin layer such as PDMS (polydimethylsiloxane) is formed on the glass substrate. It is also possible to form the flow path by removing the photoresist. The substrate itself may have a required rigidity or flexibility for the convenience of handling by the user. A microchannel having an arbitrary shape is formed on such a substrate. However, the microchannel may be formed by opening a channel forming layer around the enzyme carrying part. .

  In the microanalyzer according to the present invention, a substrate corresponding to the enzyme to be measured in the sample is lyophilized and disposed, and is dissolved by the sample solution to start the enzyme reaction. It is not supplied as a liquid. In lyophilization, the substrate solution is filled in a microchannel, and after freeze-up of the substrate solution in the microchannel, vacuum lyophilization can be performed. More preferably, the substrate is arranged so as to face the microchannel with a medium mixed with a protein such as bovine serum albumin and a cross-linking agent such as glutaraldehyde. When the substrate solution is processed using such a lyophilization method, the substrate solution becomes more porous, and the liquid can be dissolved and mixed faster and more uniformly by capillary action.

Moreover, various enzymes can be targeted as the measurement target enzyme of the microanalyzer of the present invention. The enzyme to be measured, the lyophilized substrate, and the supported enzyme are summarized as follows.For example, these substrates are selected and introduced into the microchannel, and the lyophilized one is arranged. When a sample solution such as blood is introduced, measurement in a very short time is realized.

(A) Measurement enzyme: gamma glutamyl transpeptidase (γ-GTP)
Lyophilized substrate: L-γ-glutamyl-L-glutamic acid and glycyl-glycine Carrying enzyme: glutamate oxidase (b) Measuring enzyme: glutamic oxaloacetec transaminase (GOT)
Lyophilized substrate: L-aspartic acid and α-ketoglutaric acid Supported enzyme: glutamate oxidase (c) Measuring enzyme: glutamic pyruvic transaminase (GPT)
Lyophilized substrate: L-alanine and α-ketoglutarate Carrying enzyme: Glutamate oxidase (d) Measuring enzyme: Leucine aminopeptidase Lyophilizing substrate: Leucylglutamate Carrying enzyme: Glutamate oxidase (e) Measuring enzyme: Glutaminase Lyophilized substrate: Glutamine Supported enzyme: glutamate oxidase

  For example, gamma-glutamyl transpeptidase (γ-GTP), glutamic oxaloacetec transaminase (GOT) and glutamic pyruvic transaminase (GPT), which are liver function test items, are all the same reaction product L -It becomes measurable by detecting glutamic acid, and if glutamate oxidase is used as the enzyme to be supported, these three items of γ-GTP, GOT, and GPT can be detected by one analysis system. In this case, the enzyme fixed at or near the working electrode may be made to face the microchannel with a medium in which bovine serum albumin (BSA) is mixed with L-glutamate oxidase and glutaraldehyde, for example.

In addition, the microsample analysis method of the present invention forms a microchannel connected to an injection opening into which a sample solution is injected, and forms an enzyme carrying section for carrying an enzyme so as to face the microchannel. a step, a step of placing a porous carrier that dissolves the lyophilized substrate in the fine channel adjacent to the injection opening in the vicinity of the enzyme carrier part to unrealized the sample solution, the injection opening And supplying the sample solution from the microchannel to the freeze-dried substrate, and analyzing the enzyme activity of the sample solution with respect to the substrate.

  Furthermore, the microanalyzer according to the present invention forms a microchannel on a substrate, forms an enzyme A supporting portion for supporting enzyme A so as to face the microchannel, and the microchannel in the vicinity of the enzyme supporting portion. The enzyme B that has been freeze-dried is placed in the flow path, and the substrate corresponding to the enzyme B in the sample liquid is analyzed by flowing the sample liquid through the micro flow path.

According to a preferred example of the present invention, an electrode is formed on the enzyme A carrying portion, and the enzyme A is disposed on or near the surface of the electrode. The electrochemical reaction can be measured by the electrode, and for example, it is configured with two electrodes, more preferably three electrodes. In the case of the three-electrode system, an electrode system is formed by a working electrode, a reference electrode, and a counter electrode. It is possible to measure with current flowing. Various materials can be used as the metal material for forming the metal thin film, but materials such as platinum and silver can be used. Furthermore, for disposable applications, a carbon-based conductive paste is used. It is also possible to use an electrode. In the case of the three-electrode system, the enzyme A carrying part can be formed in the vicinity of the working electrode.

  The enzyme A to be supported may be any enzyme that produces a reaction product that can be measured with an electrode such as hydrogen peroxide. For example, an oxidoreductase such as glutamate oxidase, cholesterol oxidase, glycerophosphate oxidase, and sarcosine oxidase. Can be used.

As the method for supporting enzyme A, as described above, a method of immobilizing enzyme A in the vicinity of the electrode by a method comprising a carrier binding method, a crosslinking method, a comprehensive method, or a combination thereof, or a permeable membrane or the like is exposed to the flow path. And a method of supplying enzyme A through the permeable membrane.

  Similarly to the above, the substrate can be made of various materials such as glass, synthetic resin, ceramic, metal, wood, paper, and composite materials thereof. It is only necessary to have a function of holding an electrode or the like in a microchannel formed on the substrate by flowing a liquid.

  In the microanalyzer of the present invention, the enzyme B is lyophilized and disposed, and has a structure in which the enzyme reaction starts by being dissolved by the sample solution. As the enzyme B to be arranged, any enzyme may be used as long as it induces a substrate of the enzyme A. Specifically, hydrolyzing enzymes such as lipoprotein lipase, corsterol esterase, glycerol role kinase, creatininase, creatinase, glutaminase, One or a plurality of dehydrogenases such as glutamate dehydrogenase or phosphorylase may be used. When measuring ammonia or urea, glutamate dehydrogenase is enzyme B, and α-ketoglutarate, which is one of its substrates, needs to be lyophilized together with enzyme B.

  Such freeze-drying of the enzyme B can be performed by filling the enzyme solution into a microchannel and freezing the enzyme B solution in the microchannel, followed by vacuum freeze-drying. More preferably, the enzyme B is arranged so as to face the microchannel with a medium mixed with a protein such as bovine serum albumin and a cross-linking agent such as glutaraldehyde. In general, when the enzyme solution is treated using such a freeze-drying method, the enzyme solution becomes more porous, and the liquid can be dissolved and mixed faster and more uniformly by capillary action. Furthermore, in order to prevent a decrease in the stability of enzyme B due to lyophilization, a new enzyme stabilizer such as gamma-poly-L-glutamic acid is added to the enzyme B solution from 0.001% to 0.1%, preferably 0.01%. Add to a certain degree and freeze-dry.

The substance to be measured in the microanalyzer of the present invention may be any enzyme B substrate that produces a substrate for enzyme A or a substrate for enzyme B that is finally led to a substrate for enzyme A by a plurality of enzymes B. A variety of substrates can be targeted. For example, by using the enzymes A and B as shown below, measurement of the substrate to be measured can be realized in a very short time when a sample solution such as blood of a subject is introduced.

(I) Measurement substrate: cholesterol Enzyme A: Corsterol oxidase Enzyme B: Lipoprotein lipase and cholesterol sterase (ii) Measurement substrate: Neutral fat Enzyme A: Glycerophosphate oxidase Enzyme B: Lipoprotein lipase and / or glycerol kinase (iii) ) Measurement substrate: creatinine enzyme A: sarcosine oxidase enzyme B: creatininase and / or creatinase

  Furthermore, the method for analyzing a micro sample according to the present invention comprises a step of forming an enzyme A carrying part for forming an enzyme A so as to face the minute channel, and the vicinity of the enzyme A carrying part. Placing the freeze-dried enzyme B in the microchannel, and supplying a sample solution containing a substrate corresponding to the enzyme B from the microchannel to the dried enzyme B, It is characterized by analyzing a substrate in a sample.

  In the microanalyzer of the present invention, a freeze-dried substrate or enzyme is disposed in a microchannel that is a very narrow area, so that when a sample solution is introduced therein, it is quickly mixed. As a result, the start-up of the enzyme reaction is fast, and a synergistic effect between the capillary phenomenon in the microchannel and the capillary phenomenon due to the lyophilized substrate or the porous material of the enzyme material can be obtained. According to such a measuring method, calibration by an enzyme reaction can be performed in a time of about 2 or 3 minutes, and for example, a quick measurement result can be provided at a medical site.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. In the microanalyzer 10 of this embodiment, an electrode group is formed on a small fan-shaped flat glass substrate 11, and a polyimide insulating layer 12 in which a microchannel 26 is formed and a PDMS (polydimethylsiloxane) substrate 13 are laminated. In particular, it is shorter than gamma-glutamyl transpeptidase (γ-GTP), glutamic oxaloacetec transaminase (GOT), and glutamic pyruvic transaminase (GPT), which are test items for liver function. Calibration based on enzyme activity over time is possible.

  As will be described later, the glass substrate 11 is formed by dividing a glass wafer having a diameter of 3 inches and a thickness of 500 μm so that the radial direction is a dividing line, and an electrode group is formed on the glass substrate 11. The working electrode 16, the reference electrode 18, and the counter electrode 19 constituting the structure are formed in a linearly dotted manner in a region in the flow path. The working electrode 16, the reference electrode 18, and the counter electrode 19 are made of a metal thin film formed on the glass electrode substrate 11 by sputtering, and the working electrode 16 and the reference electrode 18 are made of a chromium / platinum-based metal thin film. The counter electrode 19 has a structure in which a silver / silver chloride layer 34 is laminated.

The working electrode 16 is disposed at a position closest to the injection opening 24 that serves as an introduction portion of the sample liquid into the microchannel 26, and the surface of the glass substrate 11 is connected to the working electrode 16 through a wire that is led out in an L shape. The working electrode pad 21 is connected to the ammeter at the working electrode pad 21. The reference electrode 18 is formed immediately downstream of the working electrode 16, and the surface of the glass substrate 11 is extended to the reference electrode pad portion 23 through a wiring drawn out in an L shape from the reference electrode 18. The pad unit 23 is connected to an ammeter. The counter electrode 19 is formed further downstream of the reference electrode 18, and extends to the counter electrode pad portion 22 through the wiring that is substantially linearly drawn from the counter electrode 19. The counter electrode pad portion 22 Connected to ammeter.

  On the surface of the working electrode 16, an enzyme-carrying film 17 is formed in which L-glutamate oxidase, which is cross-linked and fixed using glutaraldehyde, is formed in a film shape and the enzyme is supported so as to face the microchannel 26. Yes. Specifically, for example, 0.1 mg of L-glutamate oxidase is mixed with 10% by weight of bovine serum albumin as a matrix, and 10% by weight of glutaraldehyde is mixed and crosslinked by 2 μL (liter) of each. Therefore, when L-glutamic acid passes around the working electrode 16, the reaction of the above formula 2 occurs, and L-glutamic acid oxidase acts on the L-glutamic acid to produce hydrogen peroxide, which is peroxidized. A current corresponding to the hydrogen concentration flows.

  A polyimide insulating layer 12 is formed on the glass substrate 11 so as to electrically insulate the wirings from the working electrode 16, the reference electrode 18 and the counter electrode 19 formed on the glass substrate 11 from each other. The polyimide insulating layer 12 has an outer shape that follows the fan shape of the glass substrate 11, and the injection opening 24 and the discharge opening 25 are formed in the minute flow channel 26 facing the working electrode 16, the reference electrode 18, and the counter electrode 19. The portion of the polyimide insulating layer 12 that has been cut out functions as the microchannel 26.

  In the microanalyzer 10 of the present embodiment having such a configuration on the glass substrate 11 side, the PDMS substrate 13 functioning as a flow path forming layer is disposed with the substrate material portion 15 lyophilized in the micro flow path 26. It is pasted together. The PDMS substrate 13 is formed on a template using a thick film photoresist, and by forming a mold of a micro flow channel 26, an injection opening 24 and a discharge opening 25 in the template, Reflecting these types, the microchannel 26, the injection opening 24, and the discharge opening 25 are formed. An example of the shape of the microchannel 26 is a channel extending in a simple rectangular shape, and the length, height, and width are, for example, 15 mm, 800 μm, and 1 mm, respectively. The injection opening 24 has a substantially cylindrical shape opened in a circular shape having a diameter slightly larger than the width of the microchannel 26, and a silicone rubber member can be provided in this portion in order to easily introduce the sample liquid.

The lyophilized substrate material portion 15 is disposed in the microchannel 26 near the portion where the injection opening 24 is connected to the microchannel 26. An example of the method of forming the substrate material part 15 by lyophilization will be described. First, a solution containing an appropriate amount of substrate is prepared, mixed using the substrate solution, bovine serum albumin solution, and glutaraldehyde, and glass Immediately injecting into the microchannel 26 of the PDMS substrate 13 to which the substrate 11 is not bonded, and fixing in the microchannel 26 by crosslinking. After such immobilization for several minutes, the PDMS substrate 13 is placed in a refrigerator and kept at -25 ° C. for about 1 hour to further advance the crosslinking reaction, followed by vacuum lyophilization for about 30 minutes. The substrate material part 15 can be obtained by freeze-drying.

  In this embodiment, the example in which the substrate solution is poured into the microchannel 26 of the PDMS substrate 13 and vacuum freeze-dried has been described. However, the present invention is not limited to this, and the substrate solution is in a state of being freeze-dried in vacuum. A structure in which fragments are arranged may be used. A structure in which a substrate solution is freeze-dried in a flow path such as a fine straw and fixed and then cut and mounted for each chip. Any other structure may be used as long as it is a structure in which a fragment in a state where the substrate solution is finally lyophilized in vacuum is disposed in the vicinity of the electrode.

  The substrate solution to be lyophilized can be replaced by an enzyme whose activity is to be measured. For example, as shown in Chemical Formula 1 above, when gamma-glutamyl transpeptidase (γ-GTP) is measured, a substrate containing L-γ-glutamyl-L-glutamic acid and glycyl-glycine can be used. In the case of measuring glutamic oxaloacetec transaminase (GOT), a substrate containing L-aspartic acid and α-ketoglutaric acid can be used, and in the case of measuring glutamic pilvic transaminase (GPT), A substrate containing L-alanine and α-ketoglutarate can be used.

In the present embodiment, the microanalyzer 10 designed to be suitable for the liver function test item is provided. However, the microanalyzer according to the present invention is not limited to the liver function test, and other enzyme activities. The present invention can be applied to a sensor that uses an antibody, and further can be applied to a sensor using an antibody / antigen reaction. For example, in order to obtain a microchannel device that can be detected by the ELISA method, a freeze-dried substrate is placed in advance in the microchannel, the antibody is immobilized on the inner wall of the microchannel, and then the antigen and enzyme label By sequentially supplying the antibodies, discrimination by the ELISA method is also realized. In addition, as an application of a sensor capable of detecting lifestyle-related diseases, the microanalyzer according to the present invention can be applied to a method for calibrating uric acid levels using uricase. A single biosensor chip that can also perform value testing is possible.

  As described above, the PDMS substrate 13 having the substrate material portion 15 obtained by pouring the substrate solution into the microchannel 26 and lyophilized in vacuum, the working electrode 16, the counter electrode 18 and the reference electrode 19 protected by the polyimide insulating layer 12 are provided. The microanalyzer 10 is created by bonding the formed glass substrate 11 together. It is also possible to form the polyimide insulating layer 12 on the back surface side of the PDMS substrate 13 and bond it thereafter.

  Next, with reference to FIG. 2, FIG. 3, operation | movement of the microchannel apparatus 10 of this embodiment is demonstrated. For example, in the case of measuring for gamma glutamyl transpeptidase (γ-GTP), L-γ-glutamyl-L-glutamic acid and glycyl-glycine are arranged in the microchannel 26 of the PDMS substrate 13. The substrate material portion 15 is obtained by freeze-drying the substrate. On the other hand, the film formed on the surface of the working electrode 16 faces the microchannel 26 with L-glutamate oxidase immobilized by crosslinking with glutaraldehyde. It is the enzyme carrying | support film | membrane 17 formed in this.

  In this state, the sample liquid 30 is introduced into the microchannel 26 from the injection opening 24 opened in the PDMS substrate 13, and the sample liquid 30 enters the cylindrical injection opening 24 as shown in FIG. Accumulate. Then, the sample solution 30 reaches the surface of the adjacent lyophilized substrate material portion 15, and the sample solution 30 further rapidly moves the porous surface such as cotton of the substrate material portion 15 to the downstream side by capillary action. It will go on. The downstream flow of the sample solution 30 is automatically generated by capillary action, and in particular, a downstream flow is formed without requiring a mechanism such as a micropump or a microsyringe pump. .

  The downstream flow of the sample solution 30 guided to the lyophilized substrate material part 15 reaches the enzyme-supporting film 17 fixed on the surface of the working electrode 16 as shown in FIG. By dissolving the substrate material part 15 in which the sample solution 30 is lyophilized, gamma glutamyl transpeptidase (γ-GTP), which is an enzyme to be calibrated, contained in the sample solution 30 is converted into L-γ-glutamyl-L-. It becomes active by the substrate of glutamic acid and glycyl-glycine to produce L-glutamic acid. The generated L-glutamic acid immediately reaches the enzyme-supporting film 17 fixed on the surface of the working electrode 16, and the reaction between the L-glutamic acid oxidase fixed here and the generated L-glutamic acid. As a result, hydrogen peroxide is generated, and a change in current value due to the hydrogen peroxide is obtained as a sensor output.

  The working electrode 16, the counter electrode 19 and the reference electrode 18 of the microanalyzer according to the present embodiment as described above can be measured by being connected to a potentiostat, and the working electrode pad portion 21 is connected via an ammeter. The reference electrode pad portion 23 is connected to the potentiostat.

  In the microanalyzer 10 of this embodiment having such a structure, since the lyophilized substrate material portion 15 is disposed in the microchannel 26 that is a very narrow area, the sample solution 30 is placed there. When introduced, the sample solution 10 is quickly mixed by utilizing the capillary phenomenon due to the substrate material portion, and the rise of the enzyme reaction is quick. When the sample liquid 30 is supplied to the microchannel 26 by arranging the freeze-dried substrate material portion 15 in the microchannel 26, the capillary phenomenon in the microchannel 26 and the freeze-dried substrate A synergistic effect with the capillary phenomenon due to the porosity of the material part 15 can be obtained. In particular, according to an electrochemical measurement method, calibration by an enzymatic reaction can be performed in a time of less than 1 minute. A quick measurement result can be used.

  Next, the assembly method of the microanalyzer 10 of the present embodiment will be further described with reference to FIGS. 4 and 5. As shown in FIG. 4, the microanalyzer 10 of the present embodiment has a disk-shaped glass wafer. The electrode groups as described above are sequentially arranged on 31 and the glass wafers 31 on which the electrode groups are formed are divided into fan shapes, whereby individual glass substrates 11 can be formed. Each PDMS substrate 13 can be formed by forming the PDMS substrate 32 so as to have a plurality of micro flow path patterns 33 and dividing the PDMS substrate 32 in a fan shape. Each of the fan-shaped division processes can be performed after the disk-shaped PDMS substrate 32 and the disk-shaped glass wafer 31 are bonded to each other, and the disk-shaped PDMS disk substrate 32 and the disk-shaped glass wafer 31 are respectively divided into fan-shaped sections. And you may make it stick together, respectively.

  FIG. 5 is an exploded perspective view of the microanalyzer 10 of the present embodiment. In particular, a flow path pattern is also formed in the polyimide insulating layer 12 functioning as an interlayer insulating film, and the pattern is a disc-shaped PDMS disk substrate 32. It is the same as the plurality of micro flow path patterns 33 formed in the above, and processing using the same mask may be used.

  In the microanalyzer 10 of the present embodiment, the substrate shape has been described as a fan shape. However, the substrate shape is not limited to this shape, and may be various shapes such as other disk shapes, rectangular shapes, cylindrical shapes, prismatic shapes, and the like. Is possible. Also, the wiring pattern of the electrode wiring is arbitrary, and the pad portion can be provided not only on one side but also on both ends and both sides, for example.

  Next, the advantages of the present embodiment will be described based on experimental examples conducted by the inventors with reference to FIGS. First, it was verified that the substrate material part by freeze-drying was superior to those fixed by ordinary drying and those using a conventional Y-shaped channel. FIG. 6 is a diagram comparing the combinations of these three drying methods and the channel structure. As shown in FIG. 6, the example (a) in which the substrate material portion by freeze-drying is formed in the microchannel has the fastest rise of the curve, and then the substrate dried at room temperature is formed in the microchannel. In Example (b), the rise was fast and the slowest was (c) in which the sample solution and the substrate solution were passed through the Y-shaped channel.

  In particular, in the example in which the substrate material portion by lyophilization is formed in a microchannel, a curve rises in about 10 seconds or less and is stable in about 4 or 5 minutes. In this experiment, it was also found that the concentrations of bovine serum albumin and glutaraldehyde were important. When the concentration of bovine serum albumin was 10% by weight or more and the concentration of glutaraldehyde was 2% by weight or more, the support layer It was also found that the swelled and became sticky and the reaction became dull.

  Next, an experiment was conducted on the size of the microchannel. Here, an experiment was performed by forming a microchannel having a size as shown in Table 1 below.

  Example (a) in which a substrate material portion formed by freeze drying is formed in a micro channel by forming channels of such sizes, and an example in which a substrate dried at room temperature is formed in a micro channel ( b) Data was obtained for each of the examples (c) in which the sample solution and the substrate solution were passed through the Y-shaped channel. (A) of FIG. 7 is the result of examining the flow paths in Table 1 for an example in which the substrate material portion by lyophilization is formed in a micro flow path. However, it can be seen that it shows a quick response curve. FIG. 7 (b) shows data taken for an example (b) in which a substrate dried at room temperature is formed in a microchannel, and the substrate material portion formed by freeze drying is formed in the microchannel. The same tendency as in Example (a) is obtained, but the rise of the curve is dull, and in Example (c) where the sample solution and the substrate solution are passed through the Y-shaped channel, the rise is further dull. I found out.

  Next, experiments were also conducted on the viscosity of the microchannel and the sample solution. In this example, glutamic oxaloacetec transaminase (GOT) is contained in the sample aqueous solution, and GOT is contained in the human serum sample. Example (a) formed in a channel, Example (b) in which a substrate dried at room temperature is formed in a microchannel, Example (c) in which a sample solution and a substrate solution are passed through a Y-shaped channel We examined each of the above.

  FIG. 8 shows the relationship between the microchannel and the viscosity of the sample liquid. In the example (a) in which the substrate material part is formed in the microchannel by freeze-drying, different results are obtained between the example in which GOT is contained in the sample aqueous solution and the example in which GOT is contained in the human serum sample. The sample included in the sample had a slower rise of the curve than the example included in the sample aqueous solution, and a phenomenon that the capillary phenomenon seems to be somewhat disturbed in terms of viscosity was observed. This is in good contrast to the example (b) in which the substrate dried at room temperature is formed in the microchannel, and the difference in the example (b) is clear, but overall It can also be seen that the example (a) in which the substrate material part by freeze-drying is formed in the microchannel has a higher reaction rate. In addition, in the example (c) in which the sample solution and the substrate solution are flowed through the Y-shaped channel, the overall rise is gentle, and it is also shown that the higher the viscosity, the slower the reaction.

  Next, experiments were also conducted on electrode active interference substances such as L-ascorbic acid, uric acid, and acetaminophen. FIG. 9 is a diagram showing the experimental results, using the microanalyzer of the present embodiment, only the interference substance, a mixture of glutamic oxaloacetec transaminase (GOT) 45 U / L and the interference substance, GOT The reaction curves were examined for 3 types of those with only 45 U / L. As a result, in the case of only the interference substance, the current value became a steady value, and the data showed very little time change. On the other hand, when an enzyme to be reacted is included, a reaction curve is surely drawn, and it has been found that even if some interference substances are included, the sensor functions sufficiently.

  FIG. 10 and FIG. 11 show glutamic oxaloacetec transaminase (GOT) at each sample solution concentration (channel (a) in FIG. 10 and FIG. 11), Results obtained by investigating the reactions for pyruvic transaminase (GPT) (FIGS. 10 and 11 (b)) and gamma-glutamyl transpeptidase (γ-GTP) (FIGS. 10 and 11 (c)), respectively. It is. From these FIG. 10, it can be seen that a calibration curve corresponding to the concentration is shown for each of GOT, GPT, and γ-GTP. From FIG. 11 created from the data of FIG. Therefore, it was confirmed that a sensor with high sensitivity can be expected by using the microanalyzer of this embodiment.

It is a figure which shows the upper surface and cross section of an example of the microanalysis apparatus of this invention. It is sectional drawing of an example of the microanalysis apparatus of this invention, Comprising: It is a figure which shows the place which inject | poured the sample liquid. It is sectional drawing of an example of the microanalysis apparatus of this invention, Comprising: It is a figure which shows the place which the substrate material part melt | dissolved with the sample solution. It is a perspective view which shows the disk shaped board | substrate used when assembling an example of the microanalysis apparatus of this invention. It is a disassembled perspective view of an example of the microanalyzer of the present invention. It is a figure for contrasting and explaining an example of the microanalyzer of the present invention, and is a figure showing time change of an enzyme reaction according to a difference in a drying method of a channel and a substrate. It is a figure for contrasting and explaining an example of the microanalysis device of the present invention, and forms a channel of each size, and an example (a) in which a substrate material part by freeze drying is formed in a microchannel, Changes in time of enzyme reaction for each of the example (b) in which the substrate is dried at room temperature in the microchannel and the example (c) in which the sample solution and the substrate solution are passed through the Y-shaped channel FIG. It is a figure for contrasting and explaining an example of the microanalysis device of the present invention, Comprising: Example (a) in which substrate material part is formed in microchannel by changing the viscosity of sample and freeze-drying, substrate at room temperature The figure which showed the time change of the enzyme reaction about each of the example (b) which dried what was formed in the microchannel, and the example (c) which flowed the sample solution and the substrate solution in the Y-shaped channel It is. It is the figure which verified the influence of the interference substance about an example of the microanalysis apparatus of this invention, Comprising: It is the figure which showed the time change of the enzyme reaction. It is the figure which showed the time change of the enzyme reaction in each sample liquid density | concentration about an example of the microanalyzer of this invention, (a) is GOT, (b) is GPT, (c) is a figure of (gamma) -GTP. . It is a figure of the inclination of the reaction curve obtained based on the result of FIG. 10 about an example of the microanalyzer of this invention, (a) is GOT, (b) is GPT, (c) is a figure of (gamma) -GTP. .

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Microanalyzer 11 Glass substrate 12 Polyimide insulating layer 13 PDMS substrate 16 Working electrode 17 Enzyme carrying film 18 Reference electrode 19 Counter electrode 21 Working electrode pad portion 22 Counter electrode pad portion 23 Reference electrode pad portion 24 Injection opening portion 25 Discharge opening portion 26 Minute Channel 30 Sample solution 31 Glass wafer 32 PDMS disk substrate 33 Microchannel pattern

Claims (6)

On the substrate, a micro flow channel connected to the injection opening into which the sample liquid is injected is formed, an enzyme carrying unit for carrying the enzyme so as to face the micro flow channel is formed, and the vicinity of the enzyme carrying unit is formed. the lyophilized substrate in the fine channel adjacent to the injection opening is disposed a porous carrier which is soluble in unrealized said sample liquid, the sample liquid to the fine channel through said injection opening A microanalyzer that generates measurable enzyme activity by flowing and analyzes the enzyme activity in the sample solution corresponding to the substrate.   2. The microanalyzer according to claim 1, wherein an electrode is formed on the enzyme carrying portion, and the enzyme is disposed on or near the surface of the electrode.   The flow path forming layer is formed on the substrate, and the micro flow path is formed by opening the flow path forming layer around the enzyme supporting part on the substrate. Micro analyzer.   The enzyme to be supported is an oxidoreductase, and any one of gamma glutamyl transpeptidase (γ-GTP), glutamic oxaloacetic transaminase (GOT), or glutamic pilvic transaminase (GPT) in the sample solution. The microanalyzer according to claim 1, wherein   The microanalyzer according to claim 1, wherein the substrate is disposed so as to face the microchannel with a medium in which a protein and a crosslinking agent are mixed. Forming a microchannel connected to an injection opening into which a sample solution is injected and forming an enzyme-carrying part for supporting an enzyme so as to face the microchannel, and the injection in the vicinity of the enzyme-carrying part a step of lyophilized substrate in the fine channel adjacent to the opening is arranged a porous carrier which is soluble in unrealized the sample solution, the said freeze-dried from the fine channel through said injection opening And supplying the sample solution to the substrate, and analyzing the enzyme activity of the sample solution with respect to the substrate.