JP2006149379A - Microreactor for testing biological substance and biological substance test device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological substance test device, particularly to provide a microreactor for testing biological substances enabling multi-item test in high reliability by changing a loaded probe as appropriate, and to provide a related device. <P>SOLUTION: The biological substance test device represents such a system constitution as to separately comprise reagents for each specimen, the microreactor loaded with a fluid transmission element, and a control/detective component. This device is high in fluid transmission accuracy because its flow path system including pumps and valves is simple in construction and air bubbles are seldom entrained and dead volume is small as well. Therefore, serious problems including cross contamination and carryover contamination in the case of microanalysis and amplification reaction seldom occur. The microreactor has a flow path construction enabling controls to also be analyzed simultaneously in order to exclude influence attributable to contamination and background level rise, etc. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bioreactor microreactor and a biomaterial test device including the microreactor.

  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. Systems integrated on a chip have been developed. This is also called μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chip, biochip, and is used in the medical examination / diagnosis field, environmental measurement field, and agricultural production field. Application is expected. In particular, as seen in genetic testing, when complicated processes, skilled procedures, and equipment operations are required, automated, accelerated, and simplified microanalysis systems are more cost effective The benefits of enabling analysis not only on time but also on time and place are enormous.

  In the field of various tests including clinical tests, importance is attached to the quantitativeness of analysis, the accuracy of analysis, and the economy of these analytical chips that produce results quickly regardless of location. Since the analysis chip is severely restricted by its size and form, it is a challenge to establish a highly reliable liquid delivery system with a simple configuration. Therefore, there is a need for a microfluidic control element that has high accuracy and excellent reliability. The present inventors have already proposed a micropump system suitable for this (Patent Documents 1 and 2).

  In addition, minimizing the amount of analysis that requires a very small amount of sample and requires a small amount of reagent is the greatest challenge required for a microreactor. In order to realize this, it is necessary to efficiently mix liquids (specimens and reagents) in the mixing channel or the reaction chamber. In addition, a mechanism capable of detecting or quantifying a minute amount of reaction product with high sensitivity and simply needs to be provided on the chip. In gene detection, amplification reaction by PCR (polymerase chain reaction) method is usually used, and its usefulness is widely recognized. However, since the PCR method can amplify a trace amount of genes present in a sample several hundred thousand to several million times or more, the effects of cross contamination and carry-over contamination become extremely serious. It has also been pointed out that reading errors frequently occur during DNA amplification. Even in immunoassays, the possibility of non-specific interactions needs to be excluded. Failure to consider these potential analytical pitfalls can lead to incorrect conclusions based on incorrect results. There is also a need to construct an analysis system that properly responds to microreactors.

  Furthermore, for chips that target a large number of samples to be measured, especially clinical samples that are at risk of contamination or infection, it is desirable to be disposable, and to overcome problems such as versatility and manufacturing costs. There is.

The microreactor that provides simple and quick inspection means as described above has raised specific problems that should still be solved in practice, and the solution is desired.
JP 2001-322099 A JP 2004-108285 A "DNA chip technology and its application", "Protein Nucleic Acid Enzyme", Vol. 43, No. 13 (1998), published by Fumio Kimizuka, Yasunobu Kato, Kyoritsu Publishing Co., Ltd.

  The bioreactor microreactor and the DNA test device including the same according to the present invention have been made in view of the above circumstances, and incorporate a simple configuration and a high-accuracy liquid feeding system, and at least one highly accurate analysis. An object of the present invention is to provide a microreactor that enables the above items.

  It is an object of the present invention to provide a biological material testing apparatus equipped with a disposal type microreactor and its function control, detection and processing means.

The present invention
A plurality of microchannels branched from microchannels for transporting liquid;
In each of the plurality of microchannels, a backflow prevention unit that prevents backflow of liquid flowing through the plurality of microchannels,
A liquid feed control unit that blocks the passage of the liquid until the pressure reaches a preset pressure, and allows the passage of the liquid above the preset pressure; and
A liquid feeding dividing means consisting of
The liquid fed from the fine channel is blocked by the liquid feeding control unit, and filled with a certain amount of the liquid defined between the backflow prevention unit and the liquid feeding control unit, A biological material testing microreactor characterized in that a predetermined amount of the liquid is divided into a plurality of fine channels.

Furthermore, the present invention provides
It has a specimen storage part, a reagent storage part, a control storage part, a micropump connection part, and a branched fine channel,
The encapsulated reagent or a mixed solution thereof is fed into the microchannel branched into at least two or more by the micropump and the liquid feeding and dividing means, and the flow constituting the reaction site is downstream of each branched microchannel. By flowing to the flow path and then to the flow path constituting the detection site,
It is also a microreactor for biological material testing that can simultaneously measure samples and controls.

The micropump preferably has a first flow path whose flow path resistance changes according to a differential pressure,
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber.

  In the microreactor for biological material inspection, a liquid supply control unit and a backflow prevention unit are provided in the fine channel, and the liquid in the flow channel branched by the micropump, the liquid supply control unit, and the backflow prevention unit is provided. It is characterized by controlling liquid feeding, quantification of liquid feeding amount and mixing of each liquid.

It is desirable that at least the flow path of the detection site is made of polystyrene.
The biotin affinity protein adsorbed to the detection site preferably binds to biotin labeled on the probe substance or biotin labeled on the 5 ′ end of the primer used in the gene amplification reaction.

The biotin affinity protein is preferably streptavidin.
The present invention also provides
The biological material bound to the biotin-containing probe at the reaction site of the biological material test microreactor and its microchannel is bound to the biotin affinity protein adsorbed to the detection site of the microchannel, and then the probe is colored. And a detection device that optically detects the biological material inspection device.

  The biological substance testing device includes a device main body in which a micropump and a temperature control device are integrated with the detection device, and a microreactor that can be attached to the device main body. By attaching the microreactor to the device main body, It is characterized by automatically performing the aforementioned binding of a substance, color development of a probe, and detection thereof.

  The biological material testing device of the present invention has a system configuration in which a chip component for each specimen, on which elements for reagents and a liquid feeding system are mounted, and a control / detection component as a testing device main body are separated. Furthermore, since the flow path system including the pump and the valve has a simple configuration, bubbles are difficult to enter and the dead volume is small, so that the liquid feeding accuracy is high. Therefore, serious problems such as cross-contamination and carry-over contamination are less likely to occur in microanalysis and amplification reactions. The microreactor of the present invention has a flow path configuration that can simultaneously analyze a positive control and a negative control in order to eliminate the influence of reaction inhibition, contamination, background increase and the like.

The microreactor of the present invention has a configuration suitable for mass production including materials and components, and since probes and reagents used for detection can be easily obtained, it can be manufactured at low cost. The biological material testing device and the microreactor of the present invention can measure multiple items simultaneously, and have versatility for multiple purposes.
Detailed Description of the Invention
Hereinafter, a microreactor of the present invention and a biological material testing device including the microreactor, a micropump, various control devices, and a detection device will be described. In the present specification, “gene” refers to DNA or RNA carrying genetic information that expresses some function, but may be simply referred to as DNA or RNA that is a chemical entity. “Element” refers to a functional component installed in the microreactor. The “fine channel” is a channel formed in the microreactor of the present invention.
Overview of microreactor and biological material testing device
A plurality of microchannels branched from microchannels for transporting liquid;
In each of the plurality of microchannels, a backflow prevention unit that prevents backflow of liquid flowing through the plurality of microchannels,
A liquid feed control unit that blocks the passage of the liquid until the pressure reaches a preset pressure, and allows the passage of the liquid above the preset pressure; and
A liquid feed dividing means consisting of
The liquid fed from the fine channel is blocked by the liquid feeding control unit, and filled with a certain amount of the liquid defined between the backflow prevention unit and the liquid feeding control unit, A biological material testing microreactor characterized in that a predetermined amount of the liquid is divided into a plurality of fine channels.

The bioreactor microreactor of the present invention has a specimen storage section, a reagent storage section, a specimen pretreatment means, a micropump connection section, and a branched microchannel, and a specimen pretreated by the pretreatment means, The liquid is fed into the microchannel branched into at least two or more by the micropump and the liquid feeding dividing means, and the flow path constituting the reaction site and the flow constituting the detection site are downstream of each branched microchannel. It is a microreactor that can measure multiple items at the same time in sample analysis by flowing to the channel.

  Furthermore, the microreactor of the present invention has a specimen storage section, a reagent storage section, a control storage section, a micropump connection section, and a branched microchannel, and the enclosed reagent or a mixed solution thereof can be divided into a micropump and a liquid feed division. By feeding into the microchannel branched at least into two or more by means, and flowing downstream of each branched microchannel to the flow channel constituting the reaction site and then to the flow channel constituting the detection site, It is characterized by being able to measure samples and controls simultaneously.

  FIG. 1 is a schematic view of an embodiment of a biological material testing device (also referred to as “biological material testing apparatus”) comprising a microreactor for biological material testing of the present invention and an apparatus body, and FIG. 2 is an embodiment of the present invention. It is the schematic of the said microreactor in.

  The microreactor 1 shown in FIGS. 1 and 2 is composed of a single chip manufactured by appropriately combining members such as plastic resin, glass, silicon, and ceramics. Preferably, the fine flow path and the casing of the microreactor are formed of a plastic resin that is easy to process and inexpensive, and easy to dispose of by incineration. Among them, polystyrene resin is desirable because it has excellent moldability, has a strong tendency to adsorb streptavidin and the like as will be described later, and can easily form a detection site on a fine channel. In such a microreactor, in order to optically detect a fluorescent substance or a color reaction product, the detection part covering at least the detection part of the fine channel on the surface of the microreactor is transparent, preferably transparent. It must be plastic.

  FIG. 2 shows an example of a typical flow path configuration of the microreactor of the present invention. In the arrangement of the flow path and the liquid feeding element in FIG. 2, basically, there are three analysis flow paths (after branching into three flow paths) from the reagent storage section 18 at the most upstream part and the flow path 15 to the reagent division. It is a flow path to each waste liquid storage part 23, and it is comprised so that a reagent may flow into the fine flow path of such a basic skeleton to a "analysis flow path" below. The analysis channel on the left side is a channel for analyzing the sample, and corresponds to the analysis of one item in FIG. The central analysis channel is a positive control channel, and the right analysis channel is a negative control channel. In FIG. 2, there is one channel for sample analysis. However, for multi-item analysis, at least two or more analysis channels need to be formed for sample analysis. The number is limited not only by the number of items but also by the size of the chip and the number and arrangement of elements to be placed.

  The biological material testing device of the present invention includes a micropump, a control device that controls the micropump, a temperature control device that controls the reaction temperature, and a device main body 2 integrated with a detection device, and a microreactor that can be attached to the main device body 2 1 and. When the specimen is injected into the microreactor 1 in which the reagent is preliminarily sealed and the microreactor is attached to the apparatus main body 2, a mechanical connection for operating the liquid feeding pump and, if necessary, an electrical connection for control are also made. Binding of biological material to the probe, color development of the probe, and detection thereof are automatically performed.

The microreactor and biological material testing device of the present invention can be suitably used particularly for testing genes or nucleic acids. In that case, a mechanism for PCR amplification is mounted on the microreactor. In the following specification, a microreactor and a biological material testing device mainly used for genetic testing will be described in detail. However, it can be said that the basic configuration of biological substances other than genes, such as microreactors for analysis of proteins, enzymes, etc., is almost the same. Usually, the sample pretreatment unit 20a, reagents, probes, and the like may be changed. In this case, the arrangement and number of liquid feeding elements will change. A person skilled in the art can easily change the type of analysis by, for example, mounting elements necessary for an immunoassay method on a microreactor and making modifications including slight changes in flow path and specifications. it can.

  The microreactor chip for genetic testing includes a sample storage unit, a reagent storage unit, a probe DNA storage unit, a control storage unit, a flow path, a pump connection unit, a liquid feed control unit, a backflow prevention unit, a reagent quantitative unit, and a mixing unit. Each element is installed at a functionally appropriate position by a microfabrication technique. If necessary, a reverse transcriptase portion may be placed. The sample storage unit communicates with the sample injection unit and temporarily stores the sample and supplies the sample to the mixing unit. If necessary, actions such as blood cell separation and viscosity adjustment of the sample liquid may be performed. 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 They may be mixed so as to have a desired mixing ratio.

  By injecting a specimen such as blood into the specimen storage part of the microreactor and attaching the microreactor to the apparatus main body, the gene amplification reaction and the processing necessary for its detection are automatically performed in the chip, And it is configured so that genetic testing can be performed in a short time. In a preferred embodiment of the microreactor for genetic testing of the present invention, the necessary reagents are encapsulated in a predetermined amount in advance in the microreactor as a chip for detecting a sample DNA or RNA, a predetermined amplification reaction, and an amplification product. The microreactor is used for each specimen.

  On the other hand, a control system related to each control of liquid feeding, temperature, and reaction, a unit responsible for optical detection, data collection and processing constitutes a main body of the biological material testing device of the present invention together with a micropump and an optical device. This device body is commonly used for the specimen sample by mounting the chip on the device body. Therefore, even if there are a large number of samples, they can be processed efficiently and quickly. In the prior art, when analyzing or synthesizing different contents, it is necessary to configure a microfluidic device corresponding to the changed contents each time. In contrast, in the present invention, only the above-described detachable chip needs to be replaced. If it is necessary to change the control of each device element, the control program stored in the apparatus main body may be appropriately modified.

  In the genetic testing device of the present invention, all components are miniaturized and are in a form that is convenient to carry. Therefore, the genetic testing device is excellent in workability and operability without being restricted by the place and time of use. Since it can be measured quickly regardless of location and time, it can be used for emergency medical care or for personal use in home medical care. Since a large number of micropump units used for liquid feeding are incorporated in the apparatus main body, the chip can be used as a disposable type.

  As described above, the outline of the bioreactor test microreactor and the biomaterial test device of the present invention in the case of the genetic test has been described, but the present invention is not limited to various modifications in accordance with the spirit of the present invention in various embodiments. Modifications are possible and are included in the present invention. That is, the structure, configuration, arrangement, shape, dimensions, material, method, method, etc. of the microreactor and the inspection apparatus of the present invention may be variously modified as long as they meet the spirit of the present invention. it can. Therefore, the microreactor and test device for gene testing will be mainly described below.

Preferred embodiment of the bioreactor microreactor The preferred embodiment of the biomaterial test microreactor of the present invention is:
In one chip,
A specimen storage part into which a specimen or a biological material extracted from the specimen (for example, DNA) is injected;
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 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,
A micropump is connected to the chip via a pump connection unit, a sample contained in the sample storage unit or a biological material extracted from the sample (for example, DNA or other biological material), and a reagent stored in the reagent storage unit Are mixed in the reaction site of the fine channel, for example, the site of gene amplification reaction (in the case of protein, antigen-antibody reaction, etc.) and reacted, and then the detection unit in the downstream channel The treatment solution obtained by treating the reaction solution and the probe accommodated in the probe accommodating portion are fed, mixed in the flow path and combined (or hybridized) with the probe. Detect substances,
Similarly, the positive control housed in the positive control container and the negative control housed in the negative control container are reacted in the flow path with the reagent housed in the reagent container and then housed in the probe container. It is characterized in that the reaction is detected based on the reaction product by binding (or hybridization in the case of gene analysis) to the probe in the flow path.

Reagent Division and Sample Division / Micropump and Pump Connection Unit In this embodiment, the sample storage unit 20, the reagent storage unit 18, the positive control storage unit 21h, and the negative control storage unit 21i are each contained in the content liquid of these storage units. Is provided. The micropump 11 is connected to the upstream side of the reagent storage unit 18, and supplies the driving liquid to the reagent storage unit side by the micropump 11, thereby pushing out the reagent to the flow path and feeding the reagent. The micropump unit is incorporated in an apparatus main body (biological substance testing device) separate from the microreactor, and is connected to the microreactor from the pump connection unit 12 by mounting the microreactor on the apparatus main body.

In this embodiment, a piezo pump is used as the micro pump. That is,
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber. Details thereof are described in Patent Documents 1 and 2 described above. -Liquid feeding and dividing means In the present invention, when analyzing multiple items for one specimen and when analyzing positive control and negative control at the same time, the reagents and specimen are divided into two or more, and each analysis is performed. It is necessary to send liquid to the flow path. The liquid feed dividing means is provided for that purpose. Specifically, as shown in FIG. 2 and FIG. 3, the liquid feed dividing means is composed of a branched fine flow path, a liquid feed control unit 13, and a backflow prevention unit 16.

The liquid feeding control unit 13 blocks the passage of the liquid until the liquid feeding pressure in the forward direction reaches a predetermined pressure, and allows the liquid to pass by applying a liquid feeding pressure equal to or higher than the predetermined pressure. The backflow prevention unit 16 for preventing the backflow of the liquid in the flow path is a check valve in which the valve body closes the flow path opening due to the backflow pressure, or the valve body is pressed against the flow path opening by the valve body deforming means. And an active valve for closing the opening.

The microreactor of the microreactor of the present invention is branched by the micropump, a liquid feeding control unit that can control the passage of liquid by the pump pressure, and a backflow prevention unit that prevents the backflow of the liquid in the channel. The liquid feeding in the flow path, the quantification of the liquid feeding amount, and the mixing of each liquid are controlled. Therefore, the reagents and the sample are divided at an appropriate ratio by the action of the liquid feeding and dividing means and the micropump 11. That is, at a pressure lower than the pressure at which the liquid supply control unit 13 is opened, all of the liquid supply control units 13 in each of the branched microchannels are in a blocked state, and the liquid flow control unit 13 of each microchannel is backflowed. A predetermined amount (predetermined amount) of liquid feeding is filled between the prevention units 16. Therefore, according to the liquid feeding and dividing means of the present invention, it is possible to divide a predetermined amount of liquid into each fine channel.
In addition, by supplying the liquid from the flow path communicating with the downstream portion of the backflow prevention unit 16, it is possible to send, quantitate, and mix a predetermined amount of the divided liquid.
Sample storage unit The sample storage unit 20 of the microreactor of the present invention has a configuration as shown in FIGS. The sample injected into the sample storage unit 20 is connected to the micropump 11 and the pump connection unit 12, and is sent to the sample pretreatment unit 20a by these actions. In the sample pretreatment unit 20a, pretreatment is performed with the treatment liquid sent from the sample treatment liquid storage unit 20b. Such a specimen pretreatment unit 20a is placed as necessary. Specific examples of the sample pretreatment include separation or concentration of a substance to be analyzed, deproteinization, and the like. Therefore, the specimen pretreatment unit 20a may include a separation filter, an adsorption resin, beads, and the like.

  Next, if necessary, the pretreated specimen is divided into two or more microanalytical channels for specimen analysis by the above-described liquid sending and dividing means, and sent to the downstream analysis flow path that communicates. The divided specimens enter and merge from the sample port 19 shown in FIG. 4 into a fine channel through which reagents flow. In this case, in order to divide the liquid feed so that the specimen flows through three or more analysis flow paths and to merge with the reagents, it is necessary to make the height of the port from which the specimen flows out and the analysis flow path to be joined different from each other. Occurs. The reason for this positional relationship is as follows.

  The elements such as the sample storage unit 20 and the sample pretreatment unit 20a shown in FIG. 4 are preferably provided on the analysis channel (left fine channel) for sample analysis as shown in FIG. It is arranged at a position downstream of the portion 18. That is, in FIG. 2, when the number of sample measurement items is one, as shown in the figure, only one sample storage unit 20 and one sample storage unit 17b are required. On the other hand, when there are two or more measurement items, it is necessary to divide the sample according to the number of measurement items as described above and join the respective analysis channels. It is arranged at an appropriate position (not necessarily directly above) on the analysis channel. The positional relationship is also shown in FIGS. 5 and 6, for example. In order to divide the sample liquid so that the sample flows through three or more analysis flow paths and join the reagents with three or more measurement items, before the sample and the reagent join, the port 19 The flow path through which the specimen from the flow path must cross the flow path through which the reagent flows and the liquid flow in both the flow paths do not merge. As shown in the drawing, particularly when the sample pretreatment unit 20 a is also installed, it is more convenient to place the sample pretreatment unit 20 a below the sample storage unit 20 in order to discard unnecessary liquid in the waste liquid storage unit 23. . When there are two measurement items and the sample liquid is divided so as to flow through the two analysis channels, the sample container 20 and the sample port 19 are interposed between the two analysis channels as shown in FIG. And so on.

In each flow path on the upstream side of the merging section for merging the sample solution containing the biological material that is the reaction site measurement target and the reagent (mixed solution), the sample storage section that stores the sample, and the reagent liquid And a reagent storage section to be stored, and a pump connection section is provided on the upstream side of each of these storage sections,
By connecting the micropumps to these pump connection parts, and supplying the driving liquid from each micropump, the solution of the specimen and the reagent in each storage part are pushed out and merged so that a gene amplification reaction or antigen It is configured to initiate a reaction required for analysis such as an antibody reaction. The mode of such a reactive site is not particularly limited, and various forms and modes are conceivable. Basically, at least two kinds of liquids containing the reaction reagent are sent together by a micropump and joined together,
A fine channel provided in advance from the merging portion and in which each of the liquids is diffusively mixed;
A liquid reservoir portion that is provided first from the downstream end of the fine flow path, and has a space wider than the fine flow path, and stores a mixed liquid that is diffusely mixed in the fine flow path to perform a reaction; It is preferable to provide.

-Sample In the case of genetic testing, the sample to be measured in the present invention is a gene, DNA or RNA as a nucleic acid to be a template for an amplification reaction. It may be prepared or isolated from a sample that may contain such nucleic acid. A method for preparing a gene, DNA or RNA from such a sample is not particularly limited, and conventional techniques can be used. There are no particular restrictions on the sample itself, but most biological samples such as whole blood, serum, buffy coat, urine, feces, saliva, sputum; cell cultures; viruses, bacteria, molds, yeasts, plants, animals, etc. Samples that may contain nucleic acids; samples that may contain or contain microorganisms, and other samples that may contain nucleic acids.

  DNA can be separated and purified from a sample by phenol / chloroform extraction and ethanol precipitation according to a conventional method. In the case of RNA, analysis may be performed after converting to cDNA using an appropriate reverse transcriptase. Reverse transcriptase is readily available.

  The microreactor of the present invention requires a very small amount of specimen as compared with the case of manual work using a conventional apparatus. For example, in the case of a gene, the DNA is 0.001 to 100 ng. For this reason, the microreactor according to the present invention includes a case where only a very small amount of sample can be obtained, and there are few restrictions on the specimen surface, and the amount of reagents is inevitably small, and the test cost is reduced. The sample is introduced from the injection part of the “sample storage part”.

The processing of specimens containing biological substances other than genes is also performed as necessary by applying conventional techniques.
-Gene amplification method The amplification method is not limited in the microreactor of the present invention. For example, the DNA amplification technique can use a PCR amplification method that is actively used in various fields. Various conditions for carrying out the amplification technique have been examined in detail and described in various documents including improvements. In PCR amplification, it is necessary to manage the temperature by raising and lowering between three temperatures. However, a channel device that enables temperature control suitable for a microchip has already been proposed by the present inventors (Japanese Patent Application Laid-Open No. 2004-2005). -108285). This device system may be applied to the amplification channel of the chip 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, so DNA amplification is performed in a much shorter time than conventional methods that are performed manually in microtubes, microvials, etc. be able to.

Recently developed ICAN (Isomeric chimera primed nucleic acid) that does not require complicated temperature control like PCR reaction
acid amplification) has a feature that DNA amplification can be carried out in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Japanese Patent No. 3433929). Therefore, the ICAN method is a suitable amplification technique because the microreactor of the present invention requires simple temperature control. By hand, the method, which takes 1 hour, is 10 to 20 minutes, preferably 15 minutes to complete the analysis in the bioreactor of the present invention.

The DNA amplification reaction may be another PCR improvement method or PCR modification method, and the microreactor of the present invention is flexible enough to cope with any change in the design of the flow path. When using any DNA amplification reaction, details of the technique are disclosed, and those skilled in the art can easily introduce them.
-Reagents When analyzing a biological substance in a specimen, reagents necessary for 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. Next, the reagents necessary for genetic testing are described.
(I) Primers PCR primers are two kinds of oligonucleotides complementary to both ends of a DNA strand at a specific site to be amplified. A dedicated application has already been developed for the design, and those skilled in the art can easily create the design using a DNA synthesizer, chemical synthesis, or the like. The primer for the ICAN method is a chimeric primer of DNA and RNA, but the preparation method thereof has already been technically established (Japanese Patent No. 3343929). It is important to use the most appropriate primer design and selection to determine the success and efficiency of the amplification reaction.

Further, when biotin is bound as a label to the 5 ′ end of the primer, the DNA of the amplification product can be immobilized on the substrate through binding to streptavidin on the substrate, and can be used for quantification of the amplification product. Examples of other primer labeling substances include digoxigenin and various fluorescent dyes.
(Ii) Reagents for amplification reaction Reagents including enzymes used for the amplification reaction can be easily obtained for both PCR and ICAN methods.

  Reagents for PCR include at least 2'-deoxynucleoside 5'-triphosphate, Taq DNA polymerase, Vent DNA polymerase, or Pfu DNA polymerase.

Reagents in the ICAN method include at least 2′-deoxynucleoside 5′-triphosphate, a chimeric primer that can specifically hybridize to a gene to be detected, a DNA polymerase having strand displacement activity, and an RNase of endonuclease.
(Iii) Control The internal control is used as an internal standard substance for monitoring or quantifying amplification of the target nucleic acid (DNA, RNA). The internal control sequence can be amplified in the same manner as the sample because it has sequences that can hybridize to the same primer as the sample primer on both sides of the sequence different from the sample. The sequence of the positive control is a specific sequence for detecting the sample, and the portion where the primer hybridizes and the sequence between them are the same as the sample. Nucleic acids (DNA, RNA) used for control may be those described in known technical literature. The negative control includes all reagents other than nucleic acids (DNA, RNA) and is used for checking for the presence of contamination and for background correction.
(Iv) Reverse Transcription Reagent In the case of RNA samples, there are reverse transcriptase, reverse transcription primer and the like for synthesizing cDNA from RNA, which are also commercially available.

A predetermined amount of each of these amplification substrates (2′-deoxynucleoside 5′-triphosphate), gene amplification reagents, and the like is enclosed in advance in the reagent storage section of one microreactor. Therefore, the microreactor of the present invention does not need to be filled with a necessary amount of reagent each time it is used, and is ready for immediate use.
Detection Site In the bioreactor microreactor of the present invention, a detection site for detecting a biological material, for example, an amplified gene, is provided downstream of the reaction site in the fine channel. At least the detection part of the microreactor is transparent, preferably transparent plastic, to allow optical measurements. Furthermore, the biotin-affinity protein adsorbed at the detection site on the fine channel binds to biotin labeled on the probe substance or biotin labeled on the 5 ′ end of the primer used in the gene amplification reaction. Thereby, the probe labeled with biotin or the amplified gene is trapped at the detection site. It is desirable that at least the fine channel of the detection site is made of polystyrene.

  As a method for detecting the amplified DNA of the target gene or other biological material, generally, a detection method such as a visible spectroscopy side light method, a fluorescence measurement method, or a luminescence luminescence method is used. Furthermore, techniques such as an electrochemical method, surface plasmon resonance, and quartz crystal microbalance are also included.

A method for detecting the separated biological material or the amplified DNA of the target gene is not particularly limited, but a preferred embodiment is basically performed in the following steps. That is, using the microreactor,
(1a) A sample or a DNA extracted from a sample, or a cDNA synthesized by reverse transcription reaction from RNA extracted from a sample or a sample, and a primer modified with biotin at the 5 ′ position, and a fine channel downstream from these containing parts To liquid. A step of amplifying a gene in a microchannel of a reaction site, a step of mixing an amplification reaction solution containing a gene amplified in a microchannel and a denaturing solution, and denaturing the amplified gene into a single strand A step of feeding a treatment solution obtained by denaturing the amplified gene into a single strand to a detection site in a microchannel to which a biotin-affinity protein (preferably streptavidin) is adsorbed, and trapping the amplified gene Then, a probe DNA whose end is fluorescently labeled with FITC (fluorescein isothiocyanate) is allowed to flow through the detection site in the microchannel where the amplified gene is trapped, and this is hybridized with the immobilized gene. (A previously hybridized gene amplified with fluorescently labeled probe DNA may be trapped at the detection site.)
(1b) An antibody against an antigen present in the specimen, preferably a reagent containing a monoclonal antibody, is mixed with the specimen. 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 is modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow into the microchannel, and the FITC hybridized to the FITC of the antigen-antibody reaction immobilized thereby or to the gene The gold colloid is adsorbed on the modified probe.
(3) The concentration of the gold colloid in the fine channel is optically measured.

Examples of the biotin affinity protein include avidin, streptavidin, and extraavidin (R). These avidins have four biotin binding sites, preferably streptavidin because it has a higher degree of specificity. The present inventors have clarified suitable conditions for adsorbing the protein derived from Streptomyces avidinii in the microchannel. When immobilizing streptavidin in the microchannel formed in the polystyrene substrate, no special chemical treatment is required. Simply dissolve the biotin affinity protein in SSC buffer or physiological saline to prepare a solution with a concentration of 10-35 μg / mL, preferably 20-30 μg / mL, which is downstream of the amplification reaction site made of polystyrene. It is only necessary to apply onto a microchannel and adsorb the biotin-affinity protein onto the channel. By immobilizing streptavidin in this way, a detection site for trapping the amplified gene can be provided very simply. In order to increase the amount of streptavidin adsorbed, the surface area of the detection site may be increased by providing fine irregularities, such as filaments, in the polystyrene adsorbing portion. The biotin-affinity protein adsorbed on the detection site in this way binds to biotin labeled on the probe substance or biotin labeled on the 5 ′ end of the primer used in the gene amplification reaction.

  The probe is to be bound to a biological substance. When the measurement target is a protein, an antibody in which FITC, which is a fluorescent label for detection, is bound together with the biotin corresponds to the probe. In addition, fluorescently labeled oligodeoxynucleotides are preferably used as probe DNA for genetic testing. As the DNA base sequence, a sequence that is complementary to a part of the base sequence of the gene to be detected is selected. By appropriately selecting the base sequence of the probe DNA, it is possible to detect with high sensitivity without being affected by coexisting DNA and background, which specifically binds to the target gene.

  As a fluorescent dye for labeling the probe, a known fluorescent dye can be used. For example, fluorescent substances such as general FITC, RITC (rhodamine isothiocyanate), NBD, Cy3, and Cy5 are shown. In particular, FITC is desirable because anti-FITC antibodies such as gold colloid anti-FITC anti-mouse IgG are available. . The probe DNA may be labeled with steroid hapten digoxigenin (DIG) instead of the fluorescent dye. In this case, an anti-DIG-alkaline phosphatase labeled antibody is used as an alternative to the anti-FITC antibody.

  It is also possible to measure the fluorescence of the fluorescent dye FITC. However, it is necessary to consider light fading of the fluorescent dye, background noise, and the like. For this reason, a method capable of finally measuring with high sensitivity by visible light is preferable.

  The biological material testing device of the present invention utilizes optical detection of gold colloid using gold colloid anti-FITC anti-mouse IgG. Alternatively, the probe may be labeled with horseradish peroxidase (HRP) instead of the fluorescent dye. A coloring reaction catalyzed by this enzyme can also be used. As typical coloring substances for that purpose, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), p-phenylenediamine (OPD), 5-aminosalicylic acid ( 5AS), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (4C1N), 4-aminoantipyrine, o-dianisidine and the like are known.

In addition to peroxidase, enzyme / coloring systems such as alkaline phosphatase and galactosidase can also be used.
The reason why such visible light absorption analysis is superior is that the instrument is more versatile than fluorescence photometry, and there are few interference factors and data processing is easy. Preferably, an optical detection device therefor is integrated with a liquid feeding means including the micropump and a temperature control device for controlling the reaction temperature of each reaction in the flow path of the microreactor, and has an integrated configuration. Detection is performed using the biological material testing device of the present invention.

Preferably, the method includes a step of feeding a cleaning liquid into the flow path where streptavidin is adsorbed as necessary between the respective steps. As such a cleaning solution, for example, various buffer solutions, salt aqueous solutions, organic solvents and the like are suitable. In the above process, the denaturing solution is gene DN.
A reagent for making A a single chain, such as sodium hydroxide and potassium hydroxide.
In the analysis of biological material for control, a negative control is usually added to the analysis and performed in parallel with the analysis of the specimen. This is because it is essential for correction of contamination, for example, color development and fluorescence of substances mixed in reagents and the like. Furthermore, in order to increase the reliability of the analysis results, it is also necessary to add positive controls. This is useful for detection of interfering factors in reagents to be added, appropriateness of set conditions, and verification of non-specific interactions. Similarly, it is often necessary to add an internal control, which is particularly useful for quantitative analysis.

  Simultaneously performing positive control and internal control is particularly important in gene amplification by PCR and antigen-antibody reaction. This is because it is particularly necessary to check that the PCR reaction and the antigen-antibody reaction are correctly occurring. For example, when any problem occurs, it is optimal for verifying whether it originates from set conditions, reagents, operation, analysis system, or sample. In particular, the PCR method can amplify a very small amount of genes present in a specimen several hundreds of thousands to several million times, so that the influence of contamination such as cross-contamination is extremely serious.

  These control settings, useful for determining false positives and false negatives, follow conventional analytical technique practice. According to the flow channel configuration of the microreactor of the present invention, the analysis can be performed simultaneously in the analysis flow channel separate from the specimen under the same reagent and the same conditions.

The reaction and detection such as amplification described above are incorporated into the biological substance testing device software as a program together with the micropump, temperature control, and optical detection data processing as conditions set in advance with respect to the order of delivery, volume, timing, etc. It is included. When the biological substance testing device body and the microreactor detachable from the apparatus body are joined, the flow path of the microreactor is also activated. The analysis is preferably started automatically, and reaction such as sample and reagent delivery, gene amplification based on mixing, detection of reactants and optical measurement are automatically performed as a series of continuous steps. Data is stored in a file along with necessary conditions and recorded items.
Examination by Microreactor In the biological material testing device of the present invention and its microreactor, two gene testing aspects are mainly formed from the gene amplification technique and the hybridization technique adopted as detection methods. First, by using a primer having a sequence specific to a specific gene as a primer used in a gene amplification reaction, by measuring the presence or absence of amplification or amplification efficiency, the DNA derived from the gene in the sample It can be used to determine whether the gene is the same or different. In particular, it is effective for rapidly identifying or determining a causative virus or bacterium of an infectious disease from a gene. When performed as allele-specific PCR using allele-specific oligonucleotides as PCR oligomers, even slight mutations between alleles on homologous chromosomes can be detected. Since this microreactor also supports simultaneous measurement of multiple items, prepare multiple primers with different base sequences as appropriate for use in genetic testing. For example, identify mutant strains between bacteria and viruses of the same species. It is also preferably used for identification.

  Furthermore, the detection accuracy is increased by designing the nucleotide sequence of the probe DNA that hybridizes to the amplified gene DNA so as to be complementary to the target gene. Alternatively, the present invention can be applied to detection of gene mutations using a mismatch with a synthetic probe in hybridization as an index.

Alternatively, determination of genetic predisposition indicating susceptibility to specific diseases, gene mutations involved in side effects on drugs, coding regions, and mutations in the promoter region of regulatory genes are also detected by genetic testing using the microreactor of the present invention be able to. In that case, a primer having a nucleic acid sequence containing a mutation portion is used. The gene mutation means a mutation in the nucleotide base of the gene. Furthermore, analysis of gene polymorphism by using the test apparatus of the present invention is useful for identification of disease susceptibility genes.

  Various genetic testing methods using the testing device of the present invention are much more accurate than conventional nucleic acid sequence analysis, restriction enzyme analysis, and nucleic acid hybridization analysis, with much smaller sample amount, less labor, and a simple device. It is clear from the configuration of the apparatus and the analysis principle.

  In addition to the above-described genetic test, simultaneous measurement of multiple items in the microreactor of the present invention can be realized for multi-item analysis of antigens, hormones, metabolites, etc. for clinical specimens, for example, by designing probes and detection methods used. it can.

  The bioreactor test microreactor and biomaterial test apparatus of the present invention include gene expression analysis, gene function analysis, single gene polymorphism analysis (SNP), clinical testing / diagnosis, pharmaceutical screening, pharmaceuticals, agricultural chemicals, or various chemical substances. It can be used in the fields of safety / toxicity inspection, environmental analysis, food inspection, forensic medicine, chemistry, brewing, fishery, livestock, agricultural production, agriculture and forestry.

  Hereinafter, genetic testing will be further described by way of example with reference to the drawings shown as examples of preferred embodiments of the present invention. The present invention is not limited to such embodiments and examples.

  The microreactor consisting of a single resin chip shown in FIG. 2 automatically performs a gene amplification reaction by the ICAN method and its detection in the chip by injecting a gene sample extracted from blood or sputum, It is configured so that multiple genes can be diagnosed simultaneously. For example, an amplification reaction and its detection can be performed by attaching a chip to the apparatus main body 2 of FIG. 1 simply by dropping a blood sample of about 2 to 3 μL onto a chip having a length and width of several centimeters. .

The sample injected into the sample storage unit 20 in FIG. 2 and a reagent used in a gene amplification reaction previously enclosed in the reagent storage units 18a to 18c (a biotin-modified chimeric primer that specifically hybridizes to a gene to be detected, A DNA polymerase having strand displacement activity and an endonuclease are fed to a flow path communicating with each housing portion by a micropump (not shown) incorporated in the apparatus main body of FIG. The sample and the reagent are mixed in the flow path via the and the amplification reaction is performed. The fine channel is formed with a width of about 100 μm and a depth of about 100 μm, for example. The DNA amplified in this manner is detected by optically measuring the concentration at which the gold colloid is bound. Specifically, it is detected by an optical detection device (not shown) incorporated in the apparatus main body 2 of FIG. For example, measurement light is irradiated from a LED or the like to a detection site on the analysis flow path for each inspection item, and transmitted light or reflected light is detected by a light detection means such as a photodiode, a CCD camera, or a photomultiplier tube. Thus, the amplified DNA (gene) labeled through the hybridized probe DNA can be detected.

In the present embodiment, the microreactor is configured in the following manner in order to perform highly accurate and quick reliable genetic testing with a single chip.
First, all the controls are integrated in one chip, and the internal control, the positive control, and the negative control are enclosed in the microreactor in advance. Reagents are divided by the operation of the microreactor, and predetermined steps for the amplification reaction and detection of these controls are performed simultaneously with the amplification reaction and detection operation of the specimen. As a result, a highly reliable genetic test can be performed quickly.

  Secondly, at each predetermined position of the flow path, the liquid is blocked from passing until the liquid supply pressure in the positive direction reaches a preset pressure. In order to allow the passage of the liquid, a liquid feed control unit that controls the passage of the liquid by the pump pressure of the micropump and a backflow prevention unit that prevents the backflow of the liquid in the flow path are provided. Liquid feeding in the flow path is controlled by the functions of the micropump, the liquid feeding control unit, and the backflow prevention unit. That is, the reagent and the specimen are divided during the liquid feeding, and the reagent and the like can be quantitatively fed with high accuracy, and a plurality of reagents introduced from the branch channel can be mixed quickly.

Next, an amplification reaction using the microreactor of this embodiment and a detection operation thereof will be described in association with main components of the microreactor.
Reagent storage unit The microreactor 1 is provided with a plurality of reagent storage units 18 for storing each reagent, and is used for a gene amplification reaction, a denaturing solution for denaturing the amplified gene, and hybridizing with the amplified gene. The probe DNA to be stored is accommodated.

  It is desirable that a reagent is stored in the reagent storage unit 18 in advance so that the inspection can be performed quickly regardless of the place and time. Reagents contained in the chip are sealed on the surface of the reagent storage portion to prevent evaporation, leakage, mixing of bubbles, contamination, denaturation, and the like. Further, when the microreactor is stored, the reagent is sealed with a sealing material in order to prevent the reagent from leaking into the fine flow path from the reagent container and reacting with the reagent. When a reagent is stored in advance in the microreactor, it is desirable to store the microreactor in a refrigerator for the stability of the reagent. This sealant is solidified or gelled under refrigerated conditions in which the microreactor is stored before use, and melts into a fluid state at room temperature during use. It is preferable to seal the reagent in the reagent container by filling a sealant between the reagent and the flow path 15 communicating with the reagent container 18. Note that air may be interposed between the sealant and the reagent, but it is preferable that the amount of air interposed is sufficiently small (relative to the reagent amount) from the viewpoint of quantitative liquid feeding.

As such a sealant, a plastic material that is hardly soluble in water can be used, and an oil or fat having a solubility in water of 1% or less is preferable. In addition, you may fill with a sealing agent similarly between each accommodating part which accommodates positive control and negative control, and the flow path connected to this.
-Reagent fixed_quantity | quantitative_assay part A reagent can be quantitatively sent using said liquid feeding control part and a backflow prevention part. As a configuration of the reagent quantification unit, a predetermined amount of reagent is filled in the channel (reagent filling channel 15a) between the backflow prevention unit 16 and the liquid feeding control unit 13a. Further, a branch channel that branches from the reagent filling channel 15a and communicates with the micropump 11 that feeds the driving liquid is provided. In addition, by providing the large-volume storage portion 17a in the reagent filling channel 15a, variation in fixed quantity is reduced.

The reagent is mixed by mixing two reagents through a Y-shaped channel. In this case, even if each reagent is fed simultaneously, the mixing ratio is not stable at the beginning of the liquid. Therefore, it is desirable to cut off the leading portion so that the mixed solution is fed to the next step after the mixing ratio is stabilized.
-Reaction site Reagents such as biotin-modified chimeric primer that specifically hybridizes to the gene to be detected, DNA polymerase having strand displacement activity, and endonuclease are shown in FIG.
The piezo pump 11 housed in the main body of the apparatus separate from the microreactor is connected to the upstream side of each reagent storage unit 18a, 18b, 18c by a pump connection unit 12, and each reagent storage unit The reagent is fed from these to the downstream flow path 15a by these pumps.

  The flow path 15a, the flow path to the next process branched from the flow path 15a, and the liquid feeding control units 13a and 13b cut off the tip of the mixed liquid of the reagent fed from each reagent storage unit, and the mixed state is A flow path is formed so that the reagent mixture is sent to the next step after stabilization. In each reagent storage unit, a total of more than 7.5 μL of reagent is stored, and a total of 7.5 μL of the reagent mixed solution with the tip cut off is divided into three flow paths 15 b, 15 c, 2.5 μL, The liquid is fed to 15d. The flow path 15b reacts with the analyte, to the detection system 22 (FIGS. 2 and 3), the flow path 15c reacts with the positive control, to the detection system 22 (FIGS. 2 and 3), and the flow path 15d corresponds to the negative control. Reaction to the detection system 22 (FIGS. 2 and 3).

  The mixed reagent sent to the flow path 15b is filled in the storage part 17a of FIG. A reagent filling flow path is formed between the check valve 16 on the upstream side of the reservoir 17a and the liquid supply control unit 13a on the downstream side, and the branch flow path communicates with the pump 11 that supplies the driving liquid. The above-described reagent quantification unit is configured together with the liquid feeding control unit 13b provided in the above.

  A sample extracted from blood or sputum is injected from the sample storage unit 20 of FIG. 2, and the storage unit 17b is filled with the sample in a fixed amount (2.5 μL) by the same mechanism as the reagent determination unit described above, and the subsequent flow path A fixed amount of liquid is fed to The specimen and reagent mixed solution filled in each reservoir 17 are sent to the flow channel 15e (volume: 5 μL) via the Y-shaped flow channel, and mixing and ICAN reaction are performed in the flow channel 15e. Here, the liquid supply of the specimen and the reagent alternately drives each pump 11 and alternately introduces the specimen and the reagent mixed solution into the flow path 15e in a circular shape so that the specimen and the reagent are quickly diffused and mixed. Like to do.

The amplification reaction is stopped by feeding 5 μL of the reaction liquid and 1 μL of the reaction stop liquid stored in the stop liquid storage portion 21a to the 6 μL flow path 15f and mixing them. Next, the denatured solution (1 μL) accommodated in the denatured solution accommodating portion 21b and the mixed solution (0.5 μL) of the reaction solution and the stop solution are fed to the 1.5 g volume channel 15g and mixed. The amplified gene is denatured into a single strand. Next, the hybridization buffer (2.5 μL) accommodated in the hybridization buffer accommodating portion 21 c and the denatured treatment solution (1.5 μL) are fed to the channel 15 h having a volume of 4 μL and mixed.
Detection site Next, 2 μL of this treatment solution is sent to each of the detection sites 22a and 22b in which streptazibine is adsorbed in the flow channel, and the amplified gene is immobilized in the flow channel. Into the flow path 22a in which the amplified gene is immobilized, a single pump 11 is used to wash the cleaning solution accommodated in the accommodating portions 21d, 21f, and 21e, the probe DNA solution whose end is fluorescently labeled with FITC, and the anti-FITC antibody The labeled colloidal gold solution is fed in the order shown in FIG. Similarly, the flow path 22b in which the amplified gene is immobilized is labeled with the washing solution, the internal control probe DNA solution, and the anti-FITC antibody accommodated in the accommodating portions 21d, 21g, and 21e by the single pump 11. The colloidal gold solution is fed in the order shown in FIG. Probe DNA is hybridized to the immobilized single-stranded amplified gene. Note that the necessary cleaning liquid is appropriately stored in the cleaning liquid storage portion 21d.

  By feeding the colloidal gold solution, the colloidal gold is bound to the immobilized amplified gene via the probe DNA FITC and immobilized. The presence or absence of amplification or amplification efficiency is measured by optically detecting the immobilized gold colloid.

The flow paths 15c and 15d in FIG. 3 communicate with the positive control reaction, the detection system, and the negative control reaction and the detection system. By sending a reagent mixture to these, the above-described sample reaction and detection system are connected. In the same manner as in the above, after amplification reaction in the flow path with the reagent, it is hybridized with the probe DNA accommodated in the probe DNA accommodating portion, and the amplification reaction is detected based on this reaction product.

Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited in any way by such examples.
Reagents used : Streptavidin; manufactured by Nacalai Tesque.
Biotinylated gold colloid; Albumin-Biotin Gold Labeled 20 nm (Sigma, product number A4417)
This was diluted 50 times with the following 5 × SSC and used.
Buffer solution (filtered and sterilized with a 0.2 μm filter after preparation)
5 × SSC; 750 mM sodium chloride and 75 mM trisodium citrate saline; 0.9% sodium chloride 50 mM Tris-HCl; Tris is 2-amino-2-hydroxymethyl-1,3-propandiol.

Pure water
A light-emitting diode having a detection maximum wavelength of 520 to 530 nm and a photodiode were opposed to each other, and a measurement part of the sample was placed between them, and the output of the photodiode was measured. That is, when the value of the time nothing between the light emitting diode and the photodiode I _0, the numerical value of the silicon-based non adsorbed I _b, a numerical value obtained by reacting a gold colloid was I _g, adsorption The strength is
log (I ₀ / (I ₀ + (I _g −I _b ))).
Procedure A silicone rubber with a hole having a diameter of 4 mm is attached to a polystyrene sheet. Each hole is filled with 12 μL of a streptavidin solution of various concentrations (9 types in the range of 10-50 μg / mL) prepared with various buffers (Tris buffer, SSC buffer, hybrid buffer, physiological saline). To do. The silicone rubber hole was closed with a silicone rubber lid and allowed to stand at room temperature for 1 hour. The streptavidin solution was extracted and the wells were washed 3 times with various buffers. Next, 2 μL of biotin-labeled gold colloid was filled in the silicone hole. The biotin-labeled gold colloid was extracted and the holes were washed three times with various buffer solutions. The silicone rubber was removed and the polystyrene sheet was dried.

Then, the optical density of the hole part and other part of a polystyrene sheet was measured, and the adsorption strength of the gold colloid part was calculated according to the above formula. The obtained results are shown in Table 1.
The optimal concentration of streptavidin is 25 μg / mL. As a buffer solution to be used, physiological saline>SSC>Tris> pure water is preferable in this order.

FIG. 1 is a schematic view of a biological material testing device including a microreactor and an apparatus main body. FIG. 2-1 is a schematic diagram of a bioreactor microreactor according to an embodiment of the present invention. The micropump 11 belongs to a device separate from the present microreactor. FIG. 2-2 is a diagram illustrating a configuration of a part that performs reaction and detection between a sample and a reagent that communicate with each other through the flow path illustrated in FIG. The liquid feeding control unit 13 is omitted. The micropump 11 belongs to a device separate from the present microreactor. FIG. 3 is a diagram showing the configuration of the reagent mixing section and reagent division of the microreactor in one embodiment of the present invention. FIG. 4 is a diagram illustrating the sample storage unit 20, the sample preprocessing unit 20a, and the sample division. FIG. 5 is a cross-sectional view of a part of the microreactor. The positional relationship of the flow path from the reagent storage unit, the sample pretreatment unit 20a in FIG. It should be noted that an element indicated by a dotted line is not located at the same cross-section as a solid line element. FIG. 6 shows an example of arrangement positions of the sample storage unit 20 and the sample port 19 when the number of measurement items is two.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microreactor 2 Apparatus main body 11 Micropump 12 Pump connection part 13 Liquid supply control part 15 Flow path 16 Backflow prevention part 17a Reagent storage part 17b Sample storage part 18 Reagent storage part 19 Sample port 20 Sample storage part 20a Sample pretreatment part 20b Sample Treatment liquid storage part 21a Stop / stop liquid storage part 21b Denaturation liquid storage part 21c Hybridization buffer storage part 21d Washing liquid storage part 21e Gold colloid storage part 21f Probe DNA storage part 21g Internal control probe DNA storage part 21h Positive control storage part 21i Negative control accommodating part 22 Strept adivine adsorbing part 23 Waste liquid storage part 64 Valve part

Claims (9)

A plurality of microchannels branched from microchannels for transporting liquid;
In each of the plurality of microchannels, a backflow prevention unit that prevents backflow of liquid flowing through the plurality of microchannels,
A liquid feed control unit that blocks the passage of the liquid until the pressure reaches a preset pressure, and allows the passage of the liquid above the preset pressure; and
A liquid feed dividing means consisting of
The liquid fed from the fine channel is blocked by the liquid feeding control unit, and filled with a certain amount of the liquid defined between the backflow prevention unit and the liquid feeding control unit, A bioreactor microreactor characterized by dividing a predetermined amount of the liquid into a plurality of fine channels. It has a specimen storage part, a reagent storage part, a control storage part, a micropump connection part, and a branched fine channel,
The encapsulated reagent or a mixed solution thereof is fed into the microchannel branched into at least two or more by the micropump and the liquid feeding and dividing means, and the flow constituting the reaction site is downstream of each branched microchannel. By flowing to the flow path and then to the flow path constituting the detection site,
2. The bioreactor microreactor according to claim 1, wherein a sample and a control can be measured simultaneously. The micropump is
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
The bioreactor microreactor according to claim 1, wherein the bioreactor microreactor is a piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber.   The fine flow path is provided with a liquid feed control unit and a backflow prevention unit, and by the micropump, the liquid feed control unit, and the backflow prevention unit, liquid feeding in the branched flow path, quantification of the liquid feeding amount, and 4. The bioreactor microreactor according to claim 1, wherein mixing of each liquid is controlled.   The bioreactor microreactor according to any one of claims 1 to 4, wherein at least the flow path of the detection site is made of polystyrene.   2. The biotin-affinity protein adsorbed on the detection site binds to biotin labeled on a probe substance or biotin labeled on the 5 ′ end of a primer used in a gene amplification reaction. The bioreactor microreactor according to any one of -5.   The bioreactor microreactor according to any one of claims 1 to 6, wherein the biotin affinity protein is streptavidin. The biological material bound to the biotin-containing probe is bound to the biotin-affinity protein adsorbed to the detection site of the microchannel at the reaction site of the bioreactor microreactor and its microchannel, and then the probe is colored And a detection apparatus that optically detects the biological material inspection device.   A device main body in which a micropump and a temperature control device are integrated with the detection device, and a microreactor that can be attached to the device main body. By attaching the microreactor to the device main body, the binding of the biological material and the probe are performed. The biological material testing device according to claim 8, wherein color development and detection thereof are automatically performed.

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