JP2007071555A - Substrate having protein immobilized thereon and microreactor using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microreactor wherein a liquid sending system having a simple constitution and high accuracy is integrated for rapid processing and detection, capable of highly accurate detection, and a micro comprehensive analysis system. <P>SOLUTION: A protein and a pH-adjusting flocculation accelerator are immobilized on the surface of a substrate. The pH of protein solution is adjusted at an isoelectric point of the protein or its periphery by adding the pH-adjusting flocculation accelerator in the presence of the substrate, to thereby adsorb the protein on the substrate surface. The protein suitable therefor is streptoavidin or albumin. The pH-adjusting flocculation accelerator is preferably gluconic acid, phosphoric acid or glucono-δ-lactone. The substrate is used as a microreactor substrate or a suitable substrate for a passage element, and high sensitivity analysis of a trace sample becomes possible by being used properly for the microreactor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a substrate on which a protein is immobilized and a microreactor using the same, and particularly to a micro total analysis system including a bioreactor that can be suitably applied to a biological sample test.

In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. A system integrated on a microreactor has been developed. This is also referred to as μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chips, and biomicroreactor. In the medical examination / diagnosis field, environmental measurement field, and agricultural production field, Application is expected. Especially when complex processes, skilled techniques, and equipment operations are required, as seen in biological sample inspection, automated, high-speed and simplified microanalysis systems are costly and require samples. It can be said that not only the amount and the required time but also the benefits of enabling the analysis regardless of time and place are great (Non-Patent Document 1).

  In these analytical microreactors, importance is attached to the quantitativeness of analysis, the accuracy of analysis, and the economy. For that purpose, it is a challenge to establish a highly reliable liquid feeding system with a simple configuration, and a microfluidic control element having high accuracy and excellent reliability is required. The present inventors have already proposed a micropump system suitable for this (Patent Documents 1 and 2).

Since the sample filled in the microreactor is usually a very small amount, there is often a very small amount of the substance to be analyzed. In the microreactor, it is required to take technical measures so that even a minute amount can be analyzed with high sensitivity and high accuracy. In such a case, if a problem of non-specific adsorption occurs in the fine flow path, it obviously becomes an obstacle to the miniaturization of the analysis.
JP 2001-322099 JP 2004-108285 A "DNA microreactor technology and its application", "Protein Nucleic Acid Enzyme", Vol.43, No.13 (1998), Fumio Kimizuka, Yasunobu Kato, Kyoritsu Publishing Co., Ltd.

  The base material of the present invention has been made in view of the above-mentioned circumstances, and is provided with a base material that is versatile and suitable for high sensitivity of analysis, and is used as a substrate or a flow path element of a microreactor. An object of the present invention is to provide a microreactor and a micro total analysis system that enable rapid processing and highly accurate detection.

The base material of the present invention is a base material on which a protein and a pH-adjusting aggregation promoter are immobilized.
A suitable said protein is streptavidin or albumin.

The pH-adjustable aggregation accelerator is preferably gluconic acid, phosphoric acid or glucono-δ-lactone.
The substrate is preferably a solid film, an extruded plastic substrate, or a polymer bead.

The substrate is characterized by comprising at least polystyrene or polypropylene.
As one embodiment of the present invention, there is a trap base material in which streptavidin and a pH-adjustable aggregation promoter are immobilized on a surface, and a biotinylated substance is captured by forming a biotin-streptavidin complex. As another embodiment, a microreactor substrate characterized in that microchannels for flow paths are formed on a substrate, and proteins and pH-adjustable aggregation promoters are immobilized on at least the surface of the microgrooves.

  In the method of the present invention, in the presence of a substrate, the pH of the protein solution is adjusted to or near the isoelectric point of the protein by adding a pH-adjusting aggregation promoter. A method for immobilizing a protein on a substrate, wherein the protein is adsorbed and immobilized.

It is desirable that the protein is streptavidin or albumin and the pH-adjusting aggregation promoter is an acid.
The microreactor of the present invention is
A sample container into which at least a sample or control is injected in one chip;
A reagent storage section for storing reagents;
A flow path communicating with these accommodating portions;
A pump / connector that can be connected to a treatment / reaction unit for processing or reacting with a specimen, and a separate micropump for sending the liquid in the storage unit and the flow path, is provided upstream of each storage unit.
The substrate according to any one of claims 1 to 6 for trapping a biotinylated substance delivered from the treatment / reaction unit is disposed in a flow path downstream of the substrate, and the microreactor via a pump connection unit. The micropump is connected, and the sample contained in the sample containing portion is processed, separated, reacted, and detected.

Furthermore, a flow path through which a specimen, a control, or a reagent flows may be formed on the substrate according to claim 9.
A preferred microreactor embodiment is a microreactor for genetic testing, which is connected to a micropump via a pump connection part to the microreactor, and the sample contained in the specimen storage part or the DNA extracted from the specimen, and the reagent storage part The stored reagent is sent to the flow path, mixed in the flow path for amplification reaction, the reaction solution containing the amplified DNA and the denaturing liquid are mixed, and the amplified DNA is single-stranded. The treatment solution obtained by denaturation treatment is sent to a flow path in which the trap base material according to claim 6 is placed, and the amplified DNA is immobilized, and the amplified DNA and probe A microphone configured to mix and hybridize probe DNA sent from the DNA storage unit in the flow path and detect an amplification reaction based on the reaction product. A reactor.

The micro total analysis system of the present invention is
A device main body in which at least a micropump, a detection device, and a control device are integrated, and the microreactor according to any one of claims 10 to 12, which can be attached to the device main body, and the microreactor is attached to the device main body. The micropumps are connected to these pump connection parts, and the reaction is started by pushing the reagent from the reagent storage part to the flow path by supplying the driving liquid from each micropump. It is characterized by automatic detection.

The micropump includes a first flow path in which flow path resistance changes according to a differential pressure;
A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than the first flow path;
A pressurization chamber connected to the first flow path and the second flow path;
An actuator for changing the internal pressure of the pressurizing chamber;
It is desirable to provide a drive device for driving the actuator.

  In the base material of the present invention, the protein is efficiently adsorbed on the surface of the base material in the vicinity of the isoelectric point and is effectively immobilized. Such a substrate is suitable as a substrate or flow path element of a microreactor. By using the substrate of the present invention, a versatile microreactor that enables highly sensitive detection even with a small amount of specimen can be produced at low cost.

The microreactor and the micro total analysis system of the present invention are gene expression analysis, gene function analysis, single gene polymorphism analysis (SNP), drug screening, safety / toxicity inspection of drugs, agricultural chemicals or various chemical substances, medical clinical diagnosis It can be applied in fields such as food inspection, forensic medicine, chemistry, brewing, agriculture and forestry, fishery, livestock, and agricultural production.
Detailed Description of the Invention
In this specification, the “fluid” refers to a fluid that is sent out from a fluid storage unit or the like by a micropump and flows through a flow path in the microreactor, and may be a liquid, a fluid, a gas, or the like. Actually, the target fluid is often a liquid, and specifically includes various reagents, sample liquids, denaturant liquids, cleaning liquids, driving liquids, and the like. Note that the “flow path element” refers to a functional component installed in the microreactor. The “fine channel” is a minute groove-like channel formed on the microreactor substrate of the present invention. The “inspection chip” is an embodiment of the present microreactor. “Gene” refers to DNA or RNA carrying genetic information that expresses some function, but may also be simply referred to as DNA or RNA that is a chemical entity. “Biotinylated substance” refers to a substance to which biotin is bound. The target substance (analytical object) to be analyzed is sometimes referred to as “analyte”.

Hereinafter, a substrate of the present invention, a microreactor using the same, and a micro total analysis system including the microreactor, a micropump, a control device, and a detection device will be described.
Base material The base material of the present invention is a base material on which a protein and a pH-adjusting aggregation promoter are immobilized. Such a substrate can take various forms, and is preferably used not only as an analytical tool such as an assay plate, bead, chip, tube, membrane, but also as a substrate or flow path element of the microreactor of the present invention. .

The said protein is suitably selected according to the objective which uses a base material as a member. In particular, a protein having an isoelectric point in the acidic region is preferable. When the substrate on which the protein is immobilized is used for selective trapping, separation, concentration, etc. of the substance, it is not particularly limited as long as it is a protein involved in binding / separation of biomolecules. For example, antigen, antibody, avidin, streptavidin and the like can be mentioned. Avidin and streptavidin which are widely used for various analyzes of biological materials are desirable. Streptavidin is particularly preferable. Streptavidin is a biotin affinity protein and forms a highly specific and strong bond (Kd = 10 ⁻¹⁵ M) with biotin. Therefore, it is suitable for selective capture of a biotinylated substance.

  The substrate can take various forms such as a solid film, an extruded plastic substrate, or polymer beads, sheets, tubes, and the like. In the stretched film, protein is thinly adsorbed in the surface direction, and there is a problem from the viewpoint of precise immobilization.

The base material is preferably a polymer resin, and examples of the base polymer include acrylic, vinyl, and styrene, and it is preferable that at least polystyrene or polypropylene be included. A styrene / acrylic copolymer may also be used.

  Polystyrene is hydrophobic and uses a property that has a strong tendency to adsorb proteins as will be described later, so that a biotin-binding protein (for example, avidin, streptavidin, etc.) is adsorbed to a downstream point on a fine channel. A path element, for example a detection site, is also formed. As an example of a specific embodiment, in a microreactor for genetic testing, streptavidin and a pH-adjustable aggregation promoter are immobilized on the surface, and biotin-labeled DNA (biotinylated DNA) is converted into a biotin-streptavidin complex. The substrate of the present invention is used as a flow path element for detecting DNA by forming and trapping.

  In order to efficiently and effectively immobilize the protein on such a substrate, it is desirable to carry out at or near the isoelectric point of the protein. This is because the solubility of the protein is minimized at the isoelectric point. This is because, as seen in isoelectric focusing and isoelectric precipitation, proteins generally have neutralized surface charge at and near their intrinsic isoelectric point and weak electrostatic repulsion. On the other hand, other interactions such as hydrophobic bonding are relatively strengthened. Therefore, proteins of the same type are easily aggregated, and the hydrophobic portion where the protein is exposed promotes the hydrophobic binding interaction with the substrate hydrophobic surface.

  In order to promote protein immobilization, a protein coagulant is used in the present invention. Various types of protein coagulants are known, mainly protein denaturing agents such as denaturing agents such as ethanol, acetone and DMSO, flocculants such as polyethylene glycol, and salts containing divalent metal ions. In the present invention, as a preferable coagulant, a pH-adjustable aggregation accelerator for transferring the pH of the protein solution to the isoelectric point or the vicinity thereof is used. Here, the “pH-adjustable aggregation promoter” means a coagulant capable of varying the solubility mainly by adjusting the pH of the protein solution. For such a pH-adjustable aggregation accelerator, an acid for adjusting the pH of the solution is generally used. The acid may be a mineral acid or an organic acid. For example, gluconic acid, glucono-delta-lactone, phosphoric acid, ascorbic acid, phytic acid, etc. are mentioned. Particularly preferred pH-adjustable aggregation promoters are gluconic acid, phosphoric acid or glucono-δ-lactone. In the solution, gluconic acid and glucono-δ-lactone are in an equilibrium state that varies with pH.

  In the method of the present invention, the method for immobilizing a protein on a substrate is carried out by adding a pH-adjusting aggregation promoter in the presence of a substrate that is a carrier for immobilization, and adjusting the pH of the solution containing the protein. It is characterized in that the protein is adsorbed and immobilized on the surface of the base material by adjusting the isoelectric point or the vicinity thereof. As a preferred immobilization mode in this case, the pH of the protein solution is rapidly increased to the vicinity of its isoelectric point by adding a pH-adjusting aggregation accelerator in the presence of a base material (so to be shocking) ) It is desirable to cause agglomeration and adhesion to the substrate surface accompanying a rapid decrease in protein solubility at the same time.

In particular, it is desirable that the protein is streptavidin and the pH-adjusting aggregation promoter is an acid. As the acid, gluconic acid, glucono-δ-lactone or phosphoric acid is preferred.
Another preferred embodiment of the substrate of the present invention in which the blocking substrate protein for microreactor and the pH-adjusting aggregation promoter are immobilized on the surface is a substrate for microreactor. That is, it is characterized in that a flow channel fine groove is formed on the substrate, and the pH-adjustable aggregation promoter and protein are immobilized on at least the surface of the fine groove as described above.

  In the analysis using the micro analysis system described above, mixing a plurality of fluids, for example, reagents and samples by circulating in the micro flow path of the micro reactor is incorporated as a necessary step almost without exception. In a microreactor, since a minute flow path and a flow path element are provided on a small plate, fluid flows smoothly when clogging occurs in such a minute space or air is mixed and bubbles are generated. It is not efficient to be divided, mixed and reacted. Furthermore, when analyzing a trace amount substance, it may adsorb | suck in the middle of a flow path and a loss may arise. A reagent solution containing an enzyme or the like also increases the amount of reagent required when the enzyme is adsorbed on the inner wall of the flow path.

  Therefore, the base material of the substrate that forms and processes the flow path such as the groove forming substrate is not easily deformed due to water absorption, and is hydrophobic and hydrophobic so that a small amount of sample liquid can be sent without loss in the middle. The plastic is preferred. Examples of such materials include resins such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polyethylene vinyl alcohol, polycarbonate, polymethylpentene, fluorocarbon, and saturated cyclic polyolefin. For the groove forming substrate, polystyrene resin is particularly preferable. This is because polystyrene is excellent in transparency, mechanical properties and moldability, and is easily finely processed, and is also easily subjected to the following blocking treatment.

The microreactor substrate of the present invention has flow channel microgrooves formed on the substrate, and proteins and pH-adjustable aggregation promoters are immobilized on at least the surface of the microgrooves (blocking treatment). As described above, the immobilization method involves adding a pH-adjusting aggregation promoter in the presence of a substrate that is an immobilization carrier, and adjusting the pH of the solution containing the protein to its isoelectric point or its By adjusting to the vicinity, the protein is adsorbed and immobilized on the surface of the substrate. Suitable proteins include albumin, casein, gelatin or heparin. One of them may be used, but two or more may be used in combination. Particularly preferably, albumin, especially bovine serum albumin (BSA) is inexpensive and readily available.

By using such a substrate, among the fine channels through which the fluid of the microreactor circulates, at least the fine channel through which the specimen flows or the inner wall of the fine channel through which the reagent containing the protein flows is coated with protein. Nonspecific binding or adsorption of biological material to the inner wall of the flow path is prevented. In the microreactor, a substrate on which the above-mentioned streptavidin or the like is immobilized together with such a substrate may be used at an appropriate site depending on the purpose.
Microreactor and micro total analysis system The microreactor and micro total analysis system of the present invention will be described.

The micro total analysis system of the present invention is
It consists of a device body in which at least a micropump, a detection device, and a control device are integrated, and the following microreactor that can be attached to the device body.
The apparatus main body is disposed integrally with at least the base main body and within the base main body,
A micropump unit including a microreactor connection having a channel opening for communicating with the microreactor and a plurality of micropumps;
A detection device for detection and processing;
A control device for controlling at least the temperature, the function of the micropump unit and the function of the detection device;
With
By attaching a microreactor to the main body of the apparatus and in a state where the pump connection part of the microreactor and the microreactor connection part of the micropump unit are in close contact with each other, the micropumps are connected to these pump connection parts. By supplying a driving liquid from the reagent container, the reagent is pushed out from the reagent container to the flow path to start the reaction, and the process, separation, reaction or detection is automatically performed.

The microreactor is
A sample container into which at least a sample or control is injected in one chip;
A reagent storage section for storing reagents;
A flow path communicating with these accommodating portions;
A pump / connector that can be connected to a treatment / reaction unit for processing or reacting with a specimen, and a separate micropump for sending the liquid in the storage unit and the flow path, is provided upstream of each storage unit.
The substrate according to any one of claims 1 to 6 for trapping a biotinylated substance delivered from the treatment / reaction unit is disposed in a flow path downstream of the substrate, and the microreactor via a pump connection unit. The micropump is connected, and the sample contained in the sample containing portion is processed, separated, reacted, and detected.

The microreactor in this case is an analysis microreactor also called an analysis chip or a test chip. Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a conceptual diagram showing a configuration in an embodiment of a micro comprehensive analysis system of the present invention. In this embodiment, as shown in the figure, as a test chip (microreactor) 2 and a device that accommodates the microreactor, a heating / cooling unit (Peltier element 3, heater 4) for reaction, a liquid-feeding micropump 11, A micropump unit having a driving liquid tank 10 and a microreactor connection part, a control device (not shown) related to each control of liquid feeding, temperature and reaction, an optical detection system (LED6, photodiode 5, etc.), data The system main body 1 is provided with a detection processing device (not shown) for collecting (measuring) and processing.

  The inspection chip 2 is made of, for example, resin, glass, silicon, ceramics, and the like, and a microchannel having a micro-order size of about 10 μm to several hundred μm in width and height is formed by a microfabrication technique. It is. The vertical and horizontal sizes are usually several tens mm and the height is about several mm.

  In the test chip 2, the liquids in the storage units such as the various reagent storage units and the sample storage unit are communicated with each of the storage units by the pump connection unit 12 having a channel opening for communicating with the micropump. Liquid is fed by the micropump 11.

The components other than the inspection chip 2 are desirably configured as a system apparatus main body 1 in which these are integrated, and the inspection chip 2 is configured to be attached to and detached from the apparatus main body. Although the micropump 11 can be provided on the inspection chip 2, normally, a plurality of micropumps are incorporated in the apparatus main body. A micropump unit including the plurality of micropumps and a microreactor connection portion having a channel opening for communicating with the microreactor is arranged in the base body of the system body of the present invention. As shown in the figure, the inspection chip 2 is mounted on the apparatus main body, and the pump connection part of the inspection chip 2 is connected to the port of the microreactor connection part in the micro pump unit of the apparatus main body by overlapping the surfaces. Yes.

  The device of the electric control system that controls the micropump 11 sets a target value of the flow rate and supplies a drive voltage corresponding to the target value to the micropump. A control apparatus responsible for such control may also be incorporated into the apparatus main body of the system of the present invention and controlled to operate when the pump connection portion of the microreactor is connected to the microreactor connection section of the micropump unit of the apparatus main body.

  A control device for controlling at least the function of the micropump unit and the function of the detection processing device is incorporated in the main body of the system of the present invention. The control device may further control the system comprehensively including temperature management, measurement data recording and processing, and the like. The control device in this case has various conditions set in advance with respect to the liquid delivery sequence, capacity, timing, etc., as well as the micropump and temperature control, as the contents of the program.

  A series of analysis steps of pretreatment, reaction, and detection of a specimen as a measurement sample is performed with the microreactor attached to the system device body 1 in which the micropump, the detection processing device, and the control device are integrated. Done. The analysis may be started after injecting the sample into the attached microreactor or after attaching the microreactor injected with the sample to the apparatus main body. Predetermined reactions and optical measurements based on sample and reagent delivery, pretreatment, and mixing are automatically performed as a series of continuous processes, and the measurement data is stored in a file along with the necessary conditions and recorded items. Desirable form is desirable.

  Although not particularly limited as an optical system detection means, for example, a technique such as visible spectroscopy or fluorescence photometry is applied, and an LED, a photomultiplier, a CCD camera, etc. are appropriately installed in the system apparatus body as a component of the detection processing apparatus. Is done.

-Microreactor The microreactor according to the present invention injects a specimen such as blood into a specimen injecting section of the microreactor, thereby causing a predetermined reaction in the microreactor, for example, a synthesis reaction, a chemical reaction, an antigen-antibody reaction, a gene amplification reaction, and a reaction / detection. It is possible to automatically perform other processing (pretreatment, separation, concentration, etc.) necessary for the inspection so that multiple items can be inspected simultaneously and in a short time.

  The microreactor according to the present invention controls a microreactor in which a micropump is connected to a microreactor in which a flow path is formed, a micropump previously connected to the microreactor, and a micropump, a valve and the like together with the microreactor. An embodiment having a control system is also included. In addition, it may be a mode in which a microreactor is attached to a separate apparatus main body equipped with a micropump and the like to perform reaction and detection, and this apparatus main body is a control system related to each control of liquid feeding, temperature, and reaction. It consists of units that are in charge of optical detection, data collection and processing. In genetic testing applications, the apparatus main body is commonly used for specimen samples by attaching a microreactor to the apparatus body, so that even if there are a large number of specimens, they can be processed efficiently and quickly.

  In the above microreactor, all components are miniaturized and are in a form that is convenient to carry. Therefore, the microreactor is not restricted by the place and time of use, and has good workability and operability.

In the microreactor used in the system of the present invention, each flow path element or structure is functionally appropriate so as to be used as a test chip 2 for chemical analysis, various tests, sample processing / separation, chemical synthesis, and the like. It is arranged at a position by microfabrication technology (FIG. 2). The test chip 2 is provided with a plurality of reagent storage units for storing each reagent in addition to a sample storage unit for storing a sample as a fluid storage unit, and in this reagent storage unit, reagents used for a predetermined reaction, A cleaning solution, a denaturing solution, etc. are accommodated (FIG. 2). This is because it is desirable that the reagents are stored in advance so that the inspection can be performed quickly regardless of the place or time. Reagents and the like incorporated in the microreactor are sealed on the surface of the reagent storage portion in order to prevent evaporation, leakage, mixing of bubbles, contamination, denaturation, and the like.

  The preferred structure in the microreactor of the present invention is that a basic substrate consisting of a groove forming substrate and a covering substrate is used, and a pump connection portion, a valve base portion and a liquid reservoir portion (reagent storage portion, specimen storage portion, cleaning liquid storage portion) Etc., each of the storage units, the waste liquid storage unit), the liquid feeding control unit, the backflow prevention unit, the reagent quantification unit, the mixing unit, the waste liquid storage unit, etc.) It is formed on the groove forming substrate. It is desirable that the structure portion, the flow path, and the detection portion of the groove forming substrate are covered with a coated substrate, and at least the detection portion is covered with a light-transmitting coated substrate.

  In a preferred test mode using the microreactor of the present invention, necessary reagents are sealed in a predetermined amount in advance, and the microreactor is used for each specimen. For example, a microreactor for genetic testing is used as a unit that performs amplification reaction of sample DNA (including DNA synthesized by reverse transcription reaction from RNA extracted from the sample) and reagents for DNA amplification reaction, and detection of amplification products. Is done. That is, a microreactor suitable for genetic testing is configured as follows.

A micropump is connected to the microreactor via a pump connection unit, and the sample contained in the sample storage unit or the DNA extracted from the sample and the reagent stored in the reagent storage unit are fed to the flow channel, The reaction solution containing the amplified DNA is mixed with the denaturing solution, and the treatment solution obtained by denaturing the amplified DNA into a single strand is used for the trap. The solution is sent to the channel where the substrate is placed to immobilize the amplified DNA, and the amplified DNA is mixed with the probe DNA sent from the probe DNA storage section in the channel to increase the DNA. Hybridization is performed, and amplification reaction is detected based on the reaction product.

  The basic structure of the microreactor is usually manufactured by appropriately combining one or more molding materials, but various molding materials can be used as the molding material, and are used according to individual material characteristics. For example, fluorocarbon resins such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, polycarbonate, polytetrafluoroethylene, polysiloxane polymers such as polydimethylsiloxane, polyolefins such as polymethyl methacrylate, polyvinyl alcohol, and ethylene-vinyl alcohol copolymer Polymers, polyester polymers such as polyethylene terephthalate and polybutylene terephthalate, polyamide polymers such as 6-nylon and 6,6-nylon, cyclic cycloolefin resins, polyarylate resins, cellulose polymers such as cellulose acetate and nitrocellulose, Various inorganic glass, silicon, ceramics, metal, etc. are mentioned. Of these, polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, polyvinyl chloride, polycarbonate, polysiloxane polymers such as polydimethylsiloxane, silicon, and glass are preferable. However, the present invention is not limited to these examples.

As described above, a wide variety of materials can be used, but it is desired to satisfy processability, chemical resistance, heat resistance, and low cost. Since there is no single excellent material that can meet all of these requirements, it is necessary to appropriately select a microreactor material in consideration of the structure, application, detection method, and the like of the microreactor. For example, silicon that can make use of processing techniques such as photolithography and etching cultivated in semiconductor manufacturing technology has the disadvantage of being opaque and expensive. Glass, which is a transparent heat-resistant material, has a problem that biological substances are adsorbed non-specifically and workability is not always good. Therefore, a microreactor in which a plurality of materials are appropriately combined is also manufactured. In addition, it is desirable that the microreactor intended for a large number of measurement specimens, particularly clinical specimens at risk of contamination or infection, should be disposable, and should have versatility and mass productivity.

  From the above viewpoint, in the microreactor used in the system of the present invention, the flow path, the flow path element, and the housing can be mass-produced in order to make the microreactor a disposable type that is easy to manufacture. It is made of plastic resin that can be easily disposed of by incineration. The plastic resin used preferably has good properties such as processability, non-water absorption, chemical resistance, heat resistance, and low cost. If a plastic having these material characteristics is used as much as possible, the types of members constituting the microreactor are reduced, and the manufacturing process is not complicated. On the other hand, an integrated microreactor manufactured by combining a plurality of substrates on which various flow path elements are arranged by subjecting the substrate to complicated microfabrication causes system complexity, reduced accuracy, and increased manufacturing costs. Cheap.

When heating near 100 ° C. is necessary for the convenience of analysis, it is necessary to change to a resin having excellent heat resistance, such as polycarbonate, polyimide, polyetherimide, polybenzimidazole, polyetheretherketone and the like.

  In order to advance the reaction for detecting the analyte, a predetermined location or reaction site of the microreactor channel is often heated to a desired temperature. The temperature for locally heating in the heating region is usually up to about 100 ° C. On the other hand, it may be necessary to cool specimens and reagents that become unstable at high temperatures. It is desirable to select a material with an appropriate thermal conductivity in view of such local temperature increases and decreases within the microreactor. Examples of such a material include a resin material and a glass material. By forming these regions with a material having low thermal conductivity, heat conduction in the surface direction is suppressed, and only the heating region is selectively selected. Can be heated.

  In order to optically detect a fluorescent substance or a product of a color reaction, it is necessary that at least the detection portion covering the detection portion of the fine channel on the surface of the microreactor is a light transmissive member. . Therefore, a transparent material, alkali glass, quartz glass, and transparent plastics can be used for the light-transmitting coated substrate. The coated substrate is bonded to the groove forming substrate as a transparent plate so as to cover at least the structure portion, the fine flow path, and the detection portion on the groove forming substrate. Such a coated substrate may cover the entire top surface of the microreactor.

When flexibility is required in addition to transparency, materials such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, and polydimethylsiloxane are preferred.
-Fine flow path The flow path on the inspection chip as the microreactor is formed on the substrate according to the flow path arrangement designed in advance according to the purpose (FIG. 2). The flow path through which the fluid flows is, for example, a micro flow path having a micrometer order width formed to have a width of several tens to several hundreds μm, preferably 10 to 200 μm, and a depth of about 5 to 500 μm, preferably 10 to 300 μm. If the channel width is less than 50 μm, the channel resistance increases, which is inconvenient for fluid delivery and detection. The advantage of the microscale space is diminished in the flow path exceeding 500 μm in width. The forming method is based on a conventional fine processing technique. Typically, transfer of a fine structure using a photosensitive resin by a photolithography technique is suitable, and unnecessary portions are removed, necessary portions are added, and shapes are transferred using the transfer structure. A pattern that models the constituent elements of the microreactor is produced by photolithography, and this pattern is transferred and molded on a resin. Therefore, the material of the basic substrate forming the microchannel of the microreactor is preferably a plastic that can accurately transfer a submicron structure and has good mechanical properties. Polystyrene, polydimethylsiloxane and the like are excellent in shape transferability. If necessary, processing by injection molding, extrusion molding or the like may be used.

-Microreactor in which the flow path is blocked In a fine space, the viscosity of the fluid rises due to the influence of capillary force and flow path resistance. In such a situation, the material of the channel also affects the channel resistance of the fluid flowing through the fine channel. Therefore, the flow path wall that is hydrophobic rather than hydrophilic has less interaction with the aqueous fluid, so that the flow path resistance is not increased. In addition, it is convenient for controlling the fluid motion such as stopping or relaxing the fluid flow. Therefore, if a water-repellent plastic resin is used for the substrate on which the fine channel is formed, the water-repellent coating is not particularly required in the channel.

  On the other hand, when a hydrophobic plastic resin is used, enzymes, antibody proteins, etc. in the reagent are adsorbed on the bottom surface or side surface of the flow path, and a significant loss occurs before reaching the reaction site. Alternatively, the contaminant protein derived from the specimen may be adsorbed and remain in the fine channel to disturb the laminar flow, or the channel may be partially narrowed. The more serious problem is that, especially in the case of a small amount of sample liquid, in the case of a dilute sample liquid containing only a trace amount of target substance as an analyte, the loss of such target substance in the flow path is This greatly affects the accuracy and sensitivity of the analysis. In addition, nucleic acids, cells, etc. can also cause nonspecific adsorption. Such sample loss is due to non-specific adsorption onto the plastic constituting the flow path.

  In order to prevent non-specific adsorption of biomolecules such as proteins and DNA to the inner wall of the flow path, the inner surface of the micro flow path is preliminarily made of a substance that is easily adsorbed on the flow path wall and does not affect the reaction. May be coated. Here, “blocking” is to prevent reaction between the two by generally blocking the reactive group from the reactant such as a reagent. In the present invention, the target substance or the target substance is particularly preferred. To inhibit non-specific binding or adsorption to a solid surface including a channel wall. The treatment performed so as to develop the action is a blocking treatment, and the composition containing the above substance to be used is called a blocking agent.

For example, nonspecific binding or adsorption of proteins to plastics depends on the type and concentration, and the degree of adsorption varies depending on the relative degree of hydrophobicity in each protein's higher order structure. By performing a blocking treatment on the plastic part before use, it is possible to increase the sensitivity of analysis of a diluted sample solution. Although the blocking agent may be uniformly coated on the plastic inner wall, it is not necessarily required. This is because the target substance or the like does not have to be non-specifically bound or adsorbed even if it is not uniform.

  A suitable blocking agent of the present invention is a protein and is not particularly limited. However, it is also necessary not to cause an interaction or reaction with a reagent, particularly a reaction reagent brought into contact with the target substance for detection.

In the blocking treatment, a solution containing a protein adsorbed on the inner wall of the fine channel is used as a blocking agent, and more specifically, an aqueous solution of albumin, casein, gelatin, or heparin. One of them may be used, but two or more may be used in combination. Particularly preferably,
Albumin, especially bovine serum albumin (BSA) is inexpensive and readily available. In such blocking treatment, the pH-adjustable aggregation promoter is added to a solution containing 0.1 to 10% by weight, preferably 0.5 to 5% by weight, more preferably 1 to 3% by weight of protein in the flow path of the microreactor substrate. Then, by adjusting the pH to or near the isoelectric point of the protein, the protein and the pH-adjustable aggregation promoter are adsorbed on the substrate surface.
If the protein concentration is less than 0.1% by weight, the blocking effect will not be achieved, and 10% by weight.
If it exceeds, the part that is not adsorbed will come out as wasted part.

  When the channel is filled and allowed to stand for a certain period of time, for example, 5 minutes to 2 hours, preferably 10 minutes to 1 hour, the protein is adsorbed and covered by the inner wall of the fine channel. Thereafter, a cleaning liquid is flowed to remove excess blocking agent, bubbles, dust, and the like from the fine channel as necessary. For the convenience of storage until the microreactor is used, it is desirable to dry the blocking agent applied to the flow path. After drying, the protein of the blocking agent remains on the inner wall, and shows a masking action on the adsorptive substance in the fluid during use.

  The flow path inner wall may be attached in any form such as bonding, adsorption, and adhesion. The solvent of the solution for blocking treatment is not particularly limited, but water that has little influence on the surface properties of the inner wall of the flow path and that does not excessively extend the solution is suitable.

  It is not always necessary for the flow path to be blocked to be applied to all the fine flow paths of the microreactor. However, it is desirable that the flow path through which the reagent solution containing at least the protein, the sample solution, or the sample treatment solution pass is subjected to a blocking treatment.

  By the blocking treatment, BSA or the like exists on the surface of the inner wall of the fine channel, and biological substances such as proteins in the fluid cannot be bonded. Therefore, non-specific adsorption of proteins or the like in the flow path is prevented, the fluid in the flow path flows smoothly, and the fluid is mixed and divided smoothly. Moreover, generation | occurrence | production of a bubble is suppressed or air becomes easy to escape from a flow path.

-Micropump unit A system main body 1 of the present invention is a micropump unit including a base body and a microreactor connecting portion disposed in the base body and having a channel opening for communicating with the microreactor, and a plurality of micropumps. As a structural unit.

  The micropump 11 may be incorporated in the apparatus main body 1 as a microreactor-like pump unit in which a plurality of micropumps are formed by a photolithography technique or the like. As the micro pump, various types such as a check valve type pump in which a check valve is provided in an inflow / outflow hole of a valve chamber provided with an actuator can be used, but a piezo pump is preferably used. FIG. 3A is a cross-sectional view showing an example of a piezo pump, and FIG. 3B is a top view thereof. In this micropump, a substrate 42 on which a first liquid chamber 48, a first flow path 46, a pressurizing chamber 45, a second flow path 47, and a second liquid chamber 49 are formed and laminated on the substrate 42. Upper substrate 41, diaphragm 43 laminated on upper substrate 41, piezoelectric element 44 laminated on the side of diaphragm 43 facing pressure chamber 45, and drive unit for driving piezoelectric element 44 ( (Not shown). The drive unit and two electrodes on the surface of the piezoelectric element 44 are connected by wiring such as a flexible cable, and a voltage having a specific waveform is applied to the piezoelectric element 44 by the drive circuit of the drive unit through the connection. It has become.

  In this example, a photosensitive glass substrate having a thickness of 500 μm is used as the substrate 42, and etching is performed until the depth reaches 100 μm, whereby the first liquid chamber 48, the first flow path 46, the pressurizing chamber 45, the second A flow path 47 and a second liquid chamber 49 are formed. The first channel 46 has a width of 25 μm and a length of 20 μm. The second flow path 47 has a width of 25 μm and a length of 150 μm.

  By stacking the upper substrate 41, which is a glass substrate, on the substrate 42, the upper surfaces of the first liquid chamber 48, the first flow path 46, the second liquid chamber 49, and the second flow path 47 are formed. The portion of the upper substrate 41 that corresponds to the upper surface of the pressurizing chamber 45 is processed by etching or the like and penetrates.

  A vibration plate 43 made of thin glass having a thickness of 50 μm is laminated on the upper surface of the upper substrate 41, and a piezoelectric element 44 made of, for example, lead zirconate titanate (PZT) ceramic having a thickness of 50 μm is laminated thereon. ing.

  Due to the drive voltage from the drive unit, the piezoelectric element 44 and the vibration plate 43 attached thereto vibrate, whereby the volume of the pressurizing chamber 45 increases or decreases. The first flow path 46 and the second flow path 47 have the same width and depth, and the length of the second flow path is longer than that of the first flow path. When the differential pressure increases, turbulent flow is generated so as to create a vortex in the flow path, and the flow path resistance increases. On the other hand, in the second flow path 47, the flow path width is long, so that even if the differential pressure increases, it tends to become a laminar flow, and the rate of change in flow path resistance with respect to the change in differential pressure is smaller than in the first flow path.

  For example, the volume of the pressurizing chamber 45 is decreased while applying a large differential pressure by quickly displacing the vibration plate 43 inwardly of the pressurizing chamber 45 by the driving voltage for the piezoelectric element 44, and then outward from the pressurizing chamber 45. When the volume of the pressurizing chamber 45 is increased while slowly displacing the diaphragm 43 to give a small differential pressure, the fluid is fed in the direction B in FIG. Conversely, the diaphragm 43 is quickly displaced outwardly from the pressurizing chamber 45 to increase the volume of the pressurizing chamber 45 while applying a large differential pressure, and then the diaphragm 43 is slowly displaced inward from the pressurizing chamber 45. If the volume of the pressurizing chamber 45 is reduced while giving a small differential pressure, the fluid is fed in the direction A in FIG.

  It should be noted that the difference in flow rate resistance change ratio with respect to the change in differential pressure between the first flow channel and the second flow channel is not necessarily due to the difference in the length of the flow channel, but is based on other geometric differences. It may be.

  According to the piezo pump configured as described above, it is possible to control the liquid feeding direction and the liquid feeding speed of a desired fluid by changing the driving voltage and frequency of the pump. Although not shown in FIGS. 3A and 3B, the first liquid chamber 48 is provided with a port 72 connected to the driving liquid tank 10. It plays the role and receives the driving fluid from the driving fluid tank 10 at the port 72. The second liquid chamber 49 forms a flow path of the micropump unit, and a port 73 of the microreactor connection part is provided at the end of the flow path, and is connected to the “pump connection part” 12 of the microreactor.

  FIG. 3 (c) shows another example of this pump. In this example, the pump is composed of a silicon substrate 71, a piezoelectric element 44, and a flexible wiring (not shown). The silicon substrate 71 is obtained by processing a silicon wafer into a predetermined shape by a photolithography technique, and by etching, a pressurizing chamber 45, a diaphragm 43, a first channel 46, a first liquid chamber 48, a second channel 47, A second liquid chamber 49 is formed. The first liquid chamber 48 is provided with a port 72, and the second liquid chamber 49 is provided with a port 73 of a microreactor connection portion. For example, when this piezo pump is separated from the inspection chip 2 of FIG. Communicates with the pump connection portion 12 of the inspection chip 2 through the port 73. For example, the substrate can be connected to the inspection chip 2 by superimposing the substrate 74 with the ports 72 and 73 perforated and the vicinity of the pump connection portion of the microreactor up and down. Further, as described above, a plurality of pumps can be formed on one silicon substrate. In this case, it is desirable that the driving liquid tank 10 is connected to the port opposite to the port connected to the inspection chip 2. When there are a plurality of pumps, their ports may be connected to a common drive fluid tank.

  The relationship between the micropump and the system of the present invention shown in FIG. 1 will be described below. In the example of FIG. 1, the micropump belongs to the system main body as a device different from the inspection chip 2 and communicates with the driving liquid tank. When the micropump is bonded to each other in a predetermined form, the micropump has a microreactor connection port 73 in the micropump unit and a channel opening in the microreactor for communicating with the micropump. The pump connection section 12 having the above connection is connected to the microreactor flow path.

  FIG. 4 shows a configuration around the pump connection portion of the inspection chip 2 when a piezo pump as a micro pump is separated from the inspection chip 2 of FIG. In this figure, a flow path downstream from the pump connection portion 12 communicating from the fluid pumping port of the micro pump to the flow path of the micro reactor is on the micro reactor. (A) shows the configuration of the pump unit for feeding the driving liquid, and (b) shows the configuration of the pump unit for feeding the reagent. Here, reference numeral 24 denotes a drive liquid container, which corresponds to the drive liquid tank of FIG. The driving fluid may be either oil-based such as mineral oil or water-based. Reference numeral 25 denotes a sealing liquid container for sealing the reagent stored in advance. This sealing liquid is intended to prevent the reagent from reacting due to leakage into the fine flow path, and is solidified or gelled under refrigerated conditions where the microreactor is stored before use. When in use, when it is at a temperature suitable for inspection, it melts into a fluid state. The sealing liquid may be filled in the fine flow path, or may be filled in a reservoir provided for the sealing liquid.

  As another aspect, the micropump itself can be incorporated on the microreactor. In particular, this mode can be adopted in the case where the flow path on the microreactor is relatively simple and the purpose or use is based on the premise of repeated use, for example, a microreactor / microreactor for chemical synthesis reaction.

Embodiment of Microreactor The inspection chip 2 is, for example, a rectangular plate with a size of 50 × 76 × 2 mm, and is preferably made of a polystyrene-based resin that has excellent transparency, mechanical properties, and moldability and is easy to be finely processed. .

  The inspection chip 2 is patterned with fine flow paths for analysis or chemical synthesis. As an example of the dimension and shape of the fine channel, it is a groove having a rectangular cross section having a width of about 100 μm and a depth of about 100 μm. In the microreactor, a pump connection unit, a fine channel, a specimen storage unit, a reagent storage unit, a liquid feeding control unit, a reaction unit, a detection unit, and the like are provided, and are communicated with each other through a channel. Further, in order to increase the accuracy of liquid feeding, it is desirable to provide a backflow prevention unit, a quantitative liquid feeding mechanism, and the like. Various configurations and materials other than those described above can be employed.

-Aspect of analysisThe micro total analysis system of the present invention comprises:
After mounting the microreactor in the base body in a state where the pump connection part of the microreactor and the microreactor connection part of the micropump unit are in liquid tight contact with each other,
An analyte (target substance) contained in a sample liquid or a processing liquid obtained by processing the sample in a flow path;
The reagent stored in the reagent storage unit,
Liquid is sent to the flow path that constitutes the reaction site and merged,
After reacting these, the obtained reaction product substance or its treated substance is
It is desirable to send the liquid to the flow path constituting the detection site and automatically perform the detection by the detection processing device.

Typically, as shown in FIG. 5, each reagent 31 accommodated in a plurality of reagent accommodating portions 18 (18a, 18b, 18c) located at the most upstream portion is provided in the flow path 15 on the downstream side from the reagent accommodating portion. And the mixed reagent is sent to the downstream analysis channel. In the analysis channel, the specimen 19 and the mixed reagent are merged and mixed from the Y-shaped channel or the like, the reaction is started by temperature rise or the like, and the reaction is detected at a detection site provided downstream of the channel 15. .

  As a preferred embodiment of the micro total analysis system of the present invention, a biological sample inspection apparatus is presented. In particular, the system of the present invention can be suitably used for testing genes or nucleic acids (DNA, RNA). In this case, the fine flow path of the test chip 2 is configured to be suitable for PCR amplification as shown in FIG. 6 or FIG. 7, but the basic flow path configuration is substantially the same for biological materials other than genetic testing. It can be said that Usually, the specimen pretreatment unit, reagents, and probes may be changed. In this case, the arrangement and number of liquid feeding elements will change. A person skilled in the art can easily select the type of analysis by mounting the reagents necessary for the immunoassay method, for example, on the test chip 2 and making modifications including slight changes to the flow path elements and specifications. Can be changed. Biological substances other than genes mentioned here refer to analytes such as various metabolites, hormones, proteins (including enzymes and antigens).

-Biological sample inspection In a preferred embodiment of the inspection chip 2, in one microreactor,
A sample container into which a sample or an analyte extracted from the sample (eg, DNA, RNA, gene) is injected;
A sample pretreatment unit for performing pretreatment of the sample;
A reagent container for storing reagents used for probe binding reaction, detection reaction (including gene amplification reaction or antigen-antibody reaction);
A positive control accommodating part for accommodating a positive control;
A negative control accommodating portion for accommodating a negative control;
A probe accommodating portion that accommodates a probe (for example, a probe that hybridizes to a gene to be detected amplified by a gene amplification reaction);
A fine flow path communicating with each of these accommodating portions,
A pump connection portion that can be connected to each of the accommodating portions and a separate micropump for feeding the liquid in the flow path is provided (FIGS. 6 and 7).

  A micropump is connected to the test chip 2 via a pump connection unit 12, a sample 19 stored in the sample storage unit 20 or a biological material extracted from the sample (for example, DNA or other biological material), a reagent After the reagent 31 stored in the storage unit 18 is fed to the flow path 15 and mixed and reacted at the reaction site of the fine flow path, for example, the site of gene amplification reaction (in the case of protein, antigen-antibody reaction, etc.) Then, the processing solution obtained by processing this reaction solution and the probe stored in the probe storage unit are sent to the detection unit in the downstream channel, mixed in the channel, and combined with the probe (or hybridization) The biological material is detected based on the reaction product (FIGS. 6 and 7).

  In addition, the above reaction and detection are performed in the same manner for the positive control housed in the positive control housing section and the negative control housed in the negative control.

The sample storage unit 20 in the test chip 2 communicates with the sample injection unit, and temporarily stores the sample and supplies the sample to the mixing unit. Whether the specimen injection part that injects the specimen from the upper surface of the specimen storage part 20 is formed with a stopper made of an elastic material such as a rubber-like material in order to prevent leakage, infection and contamination to the outside and to ensure sealing performance. Alternatively, it is desirable to be covered with a resin such as polydimethylsiloxane (PDMS) or a reinforced film. For example, a specimen in a syringe is injected with a needle that has been pierced with a stopper made of the rubber material or a needle that has passed through a pore with a lid. In the former case, it is preferable that the needle hole is immediately closed when the needle is pulled out. Alternatively, another specimen injection mechanism may be installed.

  The sample 19 injected into the sample storage unit 20 is mixed with, for example, the sample 19 and the treatment liquid in the sample pretreatment unit provided in advance in the flow path 15 before mixing with the reagent 31 as necessary. By preprocessing. Such a specimen pretreatment unit may include a separation filter, an adsorption resin, beads, and the like. Preferred sample pretreatments include analyte separation or concentration, deproteinization, and the like. For example, lysis treatment and DNA extraction treatment are performed using a lysis agent such as a 1% SDS mixed solution. In this process, DNA is released from the inside of the cell and adsorbed on the membrane surface of the bead or filter.

  A predetermined amount of necessary reagents 31 are sealed in the reagent storage unit 18 of the test chip 2 in advance. Therefore, it is not necessary to fill the necessary amount of the reagent 31 each time it is used, and it can be used immediately. When analyzing a biological substance in a specimen, reagents necessary for the measurement are generally known. For example, when analyzing an antigen present in a specimen, a reagent containing an antibody against it, preferably a monoclonal antibody, is used. The antibody is preferably labeled with biotin and FITC.

  Reagents for gene testing may include various reagents used for gene amplification, probes used for detection, and coloring reagent, as well as pretreatment reagents used for the sample pretreatment if necessary. The cleaning liquid storage unit appropriately stores the required cleaning liquid (for example, 21d). By supplying the driving liquid from the micropump, the specimen liquid and the reagent liquid are pushed out from each container and merged to start a reaction required for analysis such as gene amplification reaction, analyte trap or antigen-antibody reaction. The

  The mixing of the reagent and the reagent and the mixing of the sample and the reagent may be performed at a desired ratio in a single mixing part, or a plurality of merging parts are provided by dividing one or both, and finally You may mix so that it may become a desired mixing ratio.

  The mode of such a reactive site is not particularly limited, and various forms and modes are conceivable. As an example, a micro flow channel in which each liquid is diffused and mixed is provided in advance of a joining portion (flow channel branching point) that joins two or more liquids containing reagents, and from the downstream end of the micro flow channel. The reaction is carried out in a liquid reservoir provided in advance and having a space wider than the fine channel.

  As a DNA amplification method, a PCR amplification method described in various documents including improvements and actively used in various fields can be used. In the PCR amplification method, it is necessary to manage the temperature by raising and lowering between three temperatures. However, a flow channel device that enables temperature control suitable for a microreactor has already been proposed by the present inventors (Japanese Patent Laid-Open No. 2004-2004). -108285). This device system may be applied to the amplification flow path of the microreactor of the present invention. As a result, the heat cycle can be switched at high speed, and the micro flow path is a micro reaction cell with a small heat capacity. Therefore, DNA amplification can be performed in a much shorter time than the conventional method that is performed manually.

Recently developed ICAN (Isothermal chimera primer initiated nucleic acid
The amplification method is a suitable amplification technique even in the system of the present invention because DNA amplification can be performed in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Japanese Patent No. 3343929). By hand, the method, which takes one hour, ends in 10-20 minutes, preferably 15 minutes, in the system of the present invention.

A detection site for detecting an analyte, for example, an amplified gene, is provided downstream of the reaction site in the fine flow path of the test chip 2. At least the detection part
In order to enable optical measurement, it is made of a transparent material, preferably a transparent plastic.

  Furthermore, biotin-affinity protein (for example, streptavidin) immobilized on the detection site on the microchannel is biotin labeled on the probe substance or biotin labeled on the 5 ′ end of the primer used in the gene amplification reaction. Bind specifically. Thereby, the probe labeled with biotin or the amplified gene is trapped at the detection site. Such a detection site can be configured by arranging the trap base material.

A method for detecting the separated analyte or the amplified DNA of the target gene is not particularly limited, but as a preferred embodiment, it is basically performed in the following steps.
(1a) A fine channel downstream from these containing portions of a sample or DNA extracted from the sample, or cDNA synthesized by reverse transcription from RNA extracted from the sample or sample, and a primer modified with biotin at the 5 ′ position To liquid.

  The step of amplifying the gene in the microchannel of the reaction site, the amplification reaction solution containing the gene amplified in the microchannel and the denaturing solution are mixed, and the amplified gene is made into a single strand by denaturation treatment. This is hybridized with a probe DNA fluorescently labeled with FITC (fluorescein isothiocyanate) at the end.

Next, the solution is sent to the detection site in the microchannel to which the biotin-affinity protein is adsorbed, and the amplified gene is trapped in the detection site in the microchannel (the probe labeled with the fluorescence after trapping the amplified gene at the detection site) It may be hybridized with DNA.)
(1b) A reagent containing an antibody, preferably a monoclonal antibody, specific to an analyte such as an antigen, metabolite or hormone present in the sample is mixed with the sample. In that case, the antibody is labeled with biotin and FITC. Therefore, the product obtained by the antigen-antibody reaction has biotin and FITC. This is sent to a detection site in a microchannel to which a biotin affinity protein (preferably streptavidin) is adsorbed, and is immobilized on the detection site through the binding of biotin affinity protein and biotin.
(2) A colloidal gold solution whose surface has been modified with an anti-FITC antibody that specifically binds to FITC flows through the fine channel, and this hybridizes to the immobilized FITC of the analyte / antibody reaction product or to the gene. The gold colloid is adsorbed to the FITC-modified probe.
(3) Optically measure the concentration of colloidal gold in the fine channel.

  Although the embodiments of the microreactor of the present invention have been described above, the present invention is not limited to these embodiments, and can be arbitrarily modified and changed in accordance with the spirit of the present invention. Included in the invention. In other words, the whole or a part of the microreactor of the present invention can have various structures, configurations, arrangements, shapes, dimensions, materials, methods, methods and the like as long as they meet the gist of the present invention.

FIG. 1 shows a micro total analysis system including a microreactor and an apparatus main body, and shows a schematic configuration as one embodiment of the micro total analysis system. FIG. 2 is a schematic diagram of the inspection chip (microreactor) of FIG. FIG. 3 shows a piezo pump, FIG. 3 (a) is a sectional view showing an example of the pump, and FIG. 3 (b) is a top view thereof. FIG. 3C is a cross-sectional view showing another example of the piezo pump. FIG. 4 is a diagram showing a configuration around the pump connection portion of the chip when the piezo pump is separated from the chip. FIG. 5 is a partially enlarged view showing a reagent container in the test chip. This figure shows an example of a flow path configuration in which fluids are merged and mixed. FIG. 6 shows one mode of the fine channel system on the DNA testing test chip. FIG. 7 shows an aspect in which the fine channel system is different from that in FIG.

Explanation of symbols

1 Device Main Body 2 Inspection Chip 3 Peltier 4 Heater 5 Photodiode 6 LED
10 Drive fluid tank 11 Micro pump (piezo pump)
12 Pump connection unit 13 Liquid supply control unit 15 Fine channel 16 Backflow prevention unit 17a Reagent storage unit 17b Sample storage unit 18 Reagent storage unit 19 Sample 20 Sample storage unit 21a Stop liquid storage unit 21b Denatured liquid storage unit 21c Hybridization buffer storage Section 21d Cleaning liquid storage section 21e Colloidal gold storage section 21f Probe DNA storage section 21g Internal control probe DNA storage section 21h Positive control storage section 21i Negative control storage section 21j Buffer storage section 22 Detection section 23 Waste liquid storage section 24 Drive liquid storage section 25 Sealing liquid container 26 Air venting channel 31 Reagent 41 Upper substrate 42 Substrate 43 Vibration plate 44 Piezoelectric element 45 Pressurizing chamber 46 First channel 47 Second channel 48 First liquid chamber 49 Second liquid chamber 71 Silicon Board 72 Port 73 Port

Claims (14)

  A substrate on which a protein and a pH-adjusting aggregation promoter are immobilized.   The base material according to claim 1, wherein the protein is streptavidin or albumin.   The base material according to claim 1, wherein the pH-adjustable aggregation accelerator is gluconic acid, phosphoric acid or glucono-δ-lactone.   The substrate of claim 1, wherein the substrate is a solid film, an extruded plastic substrate, or a polymer bead.   The base material according to any one of claims 1 to 4, wherein the base material comprises at least polystyrene or polypropylene.   Streptavidin and a pH-adjustable aggregation promoter are immobilized on the surface, and the biotinylated substance is a trap base material that forms and captures a biotin-streptavidin complex. The base material in any one of 5.   In the presence of the substrate, the pH of the protein solution is adjusted to the isoelectric point of the protein or in the vicinity thereof by adding a pH-adjusting aggregation promoter, thereby adsorbing the protein on the surface of the substrate. A method for immobilizing a protein on a substrate, comprising immobilizing the protein.   The immobilization method according to claim 7, wherein the protein is streptavidin or albumin, and the pH-adjusting aggregation promoter is an acid.   A microreactor substrate, characterized in that microchannels for flow paths are formed on a substrate, and a protein and a pH-adjustable aggregation promoter are immobilized at least on the surface of the microgrooves. A sample container into which at least a sample or control is injected in one chip;
A reagent storage section for storing reagents;
A flow path communicating with these accommodating portions;
A pump / connector that can be connected to a treatment / reaction unit for processing or reacting with a specimen, and a separate micropump for sending the liquid in the storage unit and the flow path, is provided upstream of each storage unit.
The substrate according to any one of claims 1 to 6 for trapping a biotinylated substance delivered from the treatment / reaction unit is disposed in a flow path downstream of the substrate, and the microreactor via a pump connection unit. The microreactor is configured to perform processing, separation, reaction, and detection of the sample stored in the sample storage unit by connecting the micropump.   The microreactor according to claim 10, further comprising a flow path through which a specimen, a control, or reagents circulates on the substrate according to claim 9. The microreactor is a genetic testing microreactor,
A micropump is connected to the microreactor via a pump connection unit, and the sample contained in the sample storage unit or the DNA extracted from the sample and the reagent stored in the reagent storage unit are fed to the flow channel, And a reaction solution containing the amplified DNA and a denaturing solution, and a treatment solution obtained by denaturing the amplified DNA into a single strand. The solution is sent to the flow path in which the trap base material described in the above is placed to immobilize the amplified DNA, and the amplified DNA and the probe DNA sent from the probe DNA storage section are flown into the flow path. The microreactor according to claim 10 or 11, wherein the microreactor is mixed and hybridized in order to detect an amplification reaction based on the reaction product.   A device main body in which at least a micropump, a detection device and a control device are integrated, and the microreactor according to any one of claims 10 to 12, which can be attached to the device main body, and the microreactor is attached to the device main body. The micropumps are connected to these pump connection parts, and the reaction is started by pushing the reagent from the reagent storage part to the flow path by supplying the driving liquid from each micropump. A micro total analysis system characterized by automatic detection. The micropump includes a first flow path in which flow path resistance changes according to a differential pressure;
A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than the first flow path;
A pressurization chamber connected to the first flow path and the second flow path;
An actuator for changing the internal pressure of the pressurizing chamber;
The micro comprehensive analysis system according to claim 13, further comprising: a driving device that drives the actuator.

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