JP2006121935A - Micro-reactor for inspecting biosubstance equipped with pretreatment means and waste liquid storage tank - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro-reactor and an inspection device each for inspecting a biosubstance that has a pretreatment means and enables multi-testing items to be tested on the biosubstance with high reliability. <P>SOLUTION: The biosubstance-inspecting device is constituted by separating chip components for individual testing specimens each loading reagents/solution-sending elements, and control/detection components as inspection main bodies. Since this system has a pretreatment part for concentrating the components to be inspected in the specimen, a substance can be concentrated even when the specimen is dilute, simultaneously, harmful matters hindering a reaction and foreign substances causing clogging in flow channels can be removed. In addition, this system has a flow channel structure that can analyze a positive control and a negative control at a time. Accordingly, grave problems in microanalysis and amplification reaction, for example, cross-contamination, carry-over contamination, or the like, scarcely occur. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bioreactor microreactor having a pretreatment means and a waste liquid reservoir, 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,
It is also called μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chips, biochip, and its application is expected in medical examination / diagnosis field, environmental measurement field, agricultural production field Has been. 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 where various tests such as clinical tests are performed, quantitativeness, accuracy of analysis, and the like are regarded as important in the measurement using these analysis chips that produce results quickly regardless of location. Since the analysis chip has severe restrictions in terms of its size and form, it is necessary 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 order to perform inspection on a chip, it is the greatest challenge required for a microreactor to minimize the amount of sample required and to reduce the amount of analysis that requires a small amount of reagent. However, depending on the sample, the concentration of the detection target (analyte) may be dilute. Since the amount of sample that can be introduced into the chip is also limited, such a sample amount does not fall within the measurable range. Therefore, a preliminary concentration or separation operation is required before introduction into the chip. Alternatively, it is necessary to mount a mechanism capable of detecting or quantifying a minute amount of reaction product with high sensitivity and easily. For this reason, in gene detection, an amplification reaction by a PCR (polymerase chain reaction) method is usually used. When a biological fluid such as blood is used as a sample, it cannot often be used as an analyte as it is, and usually it is often required to add some pretreatment.

  Furthermore, workers who routinely process a large number of measurement specimens, such as clinical specimens, are constantly exposed to the risk of infection with pathogenic bacteria such as viruses and bacteria. Furthermore, appropriate consideration is required for the specimen and waste liquid after measurement in order to prevent environmental pollution. For these reasons, it is desired that the chip as a microreactor be disposable, and it is necessary to overcome problems such as manufacturing costs. In order to reduce the cost of the microreactor, it is desired to adopt a multi-purpose type and a multi-item simultaneous measurement type.

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

  The present invention, which has been made in view of the above circumstances, proposes a microreactor for biological material inspection that enables high-sensitivity analysis using a chip having a pretreatment means and a waste liquid reservoir even for a very small amount of sample. In addition, the microreactor is of a disposable type in order to provide an analysis tool with a low risk of infection and contamination. A further object of the present invention is to provide a biological material testing device that incorporates a simple configuration and a high-accuracy liquid feeding system, and that enables highly accurate analysis for at least one item.

The present invention
It has a sample storage unit, a reagent storage unit, a sample pretreatment unit, a waste liquid storage unit, a micropump connection unit, and a fine channel,
The specimen liquid is sent from the specimen container to the specimen pretreatment section, and the specimen liquid is pretreated using the pretreatment means of the specimen pretreatment section, thereby concentrating the biological material to be measured, and As measured by flowing to the flow path constituting the reaction site, then the flow path constituting the detection site, provided downstream of the fine flow channel,
The bioreactor microreactor is characterized in that waste liquid generated as a result of concentration and measurement of a specimen is transferred to and confined in the waste liquid reservoir.

  The bioreactor microreactor is a microchannel in which the microchannel is branched into at least two or more, in the microchannel in which the sample liquid after concentration is branched by a micropump and a liquid feeding / dividing unit. The reagent or its mixed solution fed downstream and / or encapsulated is sent downstream of the microchannel branched by the micropump and the liquid feeding dividing means, and downstream of each of the branched microchannels, It is characterized by being able to measure simultaneously on multiple items and / or controls of specimens.

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.

The liquid feed dividing means includes
Branched fine flow path,
The pump pressure of the micropump that allows the liquid to pass by blocking the passage of the liquid until the liquid feeding pressure in the positive direction reaches a preset pressure and applying a liquid feeding pressure equal to or higher than the preset pressure. The liquid feed control unit that can control the passage of liquid by the above, and the backflow prevention unit that prevents the backflow of the liquid in the flow path, liquid feed in the branched flow path, determination of the liquid feed amount and each liquid It is desirable to control the mixing.

The pretreatment means may be a filter, bead, or membrane that selectively adsorbs the biological material or bacteria or virus.
The filter that adsorbs the biological substance is preferably a filter that traps DNA.

The waste liquid storage part is a hollow chamber provided at the bottom of the microreactor and communicated with at least the specimen pretreatment part and the end of the detection site, and an extra specimen, a cleaning liquid generated in the specimen concentration step, a waste liquid, and It is characterized by being a waste liquid reservoir for storing waste liquid generated as a result of measurement of a specimen.

  Furthermore, a device body in which a micropump and a temperature control device are integrated together with a detection device that optically detects a biological material contained in a specimen, and a biological material testing microreactor that can be attached to the device body. Thus, the present invention also includes a biological material testing device that automatically measures a biological material by mounting the microreactor on the apparatus body.

  The biological material testing device of the present invention has a system configuration in which a chip component that is a microreactor for each specimen and a control / detection component that is a device main body, each of which is equipped with elements for reagents and a liquid delivery system. Therefore, cross-contamination and carry-over contamination are less likely to occur in trace analysis and amplification reactions.

  Since the capacity of the microreactor chip into which the sample can be introduced is extremely limited, it is difficult to capture the target substance or bacteria if the concentration is low. According to the configuration of the microreactor of the present invention, the sample pretreatment unit that can concentrate the analyte in the sample can be concentrated in the filter by flowing a large amount of sample liquid through the adsorption filter. . Therefore, the substance can be concentrated even in a dilute sample, and at the same time, harmful substances that interfere with the reaction and contaminants that clog the flow path can be excluded.

  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. Such a microreactor realizes highly reliable and highly accurate analysis even with a very small amount of specimen.

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 microreactor of the present invention can measure multiple items simultaneously and have versatility for multipurpose use.
Detailed Description of the Invention
Hereinafter, a bioreactor testing microreactor of the present invention and a bioreactor testing device including the microreactor and 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 also be simply referred to as DNA or RNA that is a chemical entity. “Element” refers to a functional component installed in the microreactor. The substance to be analyzed is sometimes called “analyte”. The “fine channel” is a channel formed in the microreactor of the present invention.
Outline of Microreactor and Biological Material Testing Device The bioreactor testing microreactor of the present invention has a specimen storage part, a reagent storage part, a specimen pretreatment part, a waste liquid storage part, a micropump connection part, and a fine channel,
The specimen liquid is sent from the specimen container to the specimen pretreatment section, and the specimen liquid is pretreated using the pretreatment means of the specimen pretreatment section, thereby concentrating the biological material to be measured, and As measured by flowing to the flow path constituting the reaction site, then the flow path constituting the detection site, provided downstream of the fine flow channel,
The microreactor is characterized in that the waste liquid generated as a result of the concentration and measurement of the specimen is transferred and confined to the waste liquid reservoir.

Further, the fine flow channel is a fine flow channel branched into at least two or more, and the sample liquid after concentration is fed to the downstream of the branched fine flow channel by a micropump and a liquid feed dividing unit, And / or the encapsulated reagent or a mixed solution thereof is sent to the downstream of the microchannel branched by the micropump and the liquid-feeding dividing means, and the multiple items of the specimen are respectively downstream of the branched microchannel, and It is characterized by being able to measure simultaneously on / or control.

  FIG. 1 is a schematic diagram of an embodiment of a biological material testing device (also referred to as “biological material testing device”) comprising a biological material testing microreactor detachable from the device body and the device body, and FIG. It is the schematic of the said microreactor in one Embodiment. In the various embodiments, the present invention can be arbitrarily modified and changed in accordance with the spirit of the present invention, and these 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.

  The microreactor 1 shown in FIGS. 1 and 2 is a single chip manufactured by appropriately combining one or more 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 described later, and can easily form a detection site on a fine channel. The fine channel is formed with a width of about 100 μm and a depth of about 100 μm, for example. Further, in such a microreactor, in order to optically detect a fluorescent substance or a product of a color reaction, the detection part covering at least the detection part of the microchannel on the surface of the microreactor is preferably a transparent member, preferably transparent It is necessary to be made of 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. Each of these is a flow path leading to the waste liquid storage section 23, and such a basic fine flow path is configured so that the reagent flows to the “analysis flow path” in the following. 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 to be analyzed, 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 device body 2 in which a micropump, a control device that controls the micropump, a temperature control device that controls temperature, a detection device, and the like are integrated, and a microreactor that can be attached to the device body 2 1 and. When the sample liquid is injected into the sample storage part of the microreactor 1 in which the reagent has been sealed in advance and the microreactor is attached to the main body 2 of the biological material testing device, a mechanical connection for operating the liquid feed pump, if necessary Control electrical connections are also made. Therefore, when the main body and the microreactor are joined, the flow path of the microreactor is also activated. Preferably, when analysis is started, the flow of specimens and reagents, gene amplification based on mixing, reaction such as binding of analyte and probe, detection of reactants and optical measurement are performed as a series of continuous steps. The measurement is automatically performed, the measurement data is stored in a file together with necessary conditions and recorded items, and the measurement of the biological material is automatically performed.

The detection device irradiates the detection site on the analysis flow path for each inspection item with measurement light from, for example, an LED, and transmits or reflects light by optical detection means such as a photodiode or a photomultiplier tube. Is a device for detecting As optical detection means, there are various optical devices having different principles, but an ultraviolet / visible spectrophotometer is desirable. It may be an apparatus incorporated in the inspection device, or may be connected as a separate apparatus at the time of use. Preferably, in the biological material testing device of the present invention, the detection device that optically detects the biological material contained in the specimen is incorporated and integrated with the liquid feeding means including the micropump and the temperature control device. It has a configuration.

  A unit responsible for control of liquid feeding, temperature, and reaction, optical detection, data collection and processing constitutes the main body of the biological material testing device of the present invention together with the micropump and the optical apparatus. This device body is commonly used for the specimen sample by mounting the chip on the device body. The reaction and detection such as amplification described above were installed in the biological material testing device as a program together with the micropump and temperature control and optical detection data processing as conditions set in advance with respect to the order of delivery, volume, timing, etc. Built into the software. In the conventional analysis chip, when performing different analysis or synthesis, it is necessary to configure a microfluidic device corresponding to the contents to be changed 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 element, the control program stored in the apparatus main body may be appropriately modified.

  In the biological material testing device of the present invention, since all components are miniaturized and are in a form that is convenient to carry, the place and time of use are not restricted, and workability and operability are good. 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.

  The microreactor and biological material testing device of the present invention can be suitably used particularly for testing genes or nucleic acids. In the following specification, these are described by taking the case of genetic testing as an example. In that case, a mechanism for PCR amplification is mounted on the microreactor. However, it can be said that the basic structure of biological materials other than genes is almost the same. Usually, the sample pretreatment unit, 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 reagents necessary for immunoassay on the microreactor and making modifications including slight changes to the flow path elements and specifications. can do. Biological substances other than genes mentioned here refer to various metabolites, hormones, proteins (including enzymes and antigens), and the like.

A preferred aspect of the microreactor for biological material testing of the present invention is:
Within one chip,
A specimen containing part into which a specimen or an analyte substance (for example, DNA) extracted from the specimen 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 connectable to a separate micropump for feeding the liquid in each storage portion and the flow path;
And a waste liquid storage part,
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,
While performing the above reaction and detection in the same manner for the positive control housed in the positive control housing section and the negative control housed in the negative control housing section,
Communicating with the specimen pretreatment section and the detection site through a through-hole, and is a hollow chamber provided at the bottom of the microreactor. Measurement of extra specimen, cleaning liquid, waste liquid, and specimen generated in the specimen concentration process As a result, it is characterized by having a waste liquid storage part which is a sealed waste liquid reservoir for storing the generated waste liquid and the like.

  In the microreactor of the present invention, each element such as each storage section, flow path, pump connection section, liquid feed control section, backflow prevention section, reagent quantitative section, mixing section, waste liquid storage section, etc. is placed at a functionally appropriate position. It is installed with fine processing technology. If necessary, the microreactor chip for genetic testing may be provided with a reverse transcriptase containing part.

After injecting a sample such as blood into the sample storage part of the microreactor and performing sample pretreatment in the chip, the microreactor is attached to the main body of the device, so that a predetermined reaction (for example, gene amplification in the case of genetic testing) Reaction) and processing necessary for the detection thereof are automatically performed, and the analysis of the analyte can be performed simultaneously and in a short time for many items. Alternatively, the sample pretreatment may be performed after the microreactor is mounted on the apparatus main body.
Sample container The sample container 20 of the microreactor of the present invention has a configuration as shown in FIGS. The sample storage unit 20 communicates with the sample injection unit and temporarily stores the sample and supplies the sample to the mixing unit. 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. Preferred sample pretreatment includes separation or concentration of the analyte (analyte), 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, only one sample storage unit 20 and one sample storage unit 17b are required as illustrated. 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 figure, particularly when the specimen pretreatment unit 20a is also installed, unnecessary liquid is discarded in the waste liquid storage unit 23.
It is more convenient to place the sample pretreatment unit 20a below the sample storage unit 20. Measurement item is 2
Therefore, when the sample liquid is divided so as to flow through the two analysis channels, the sample container 20, the sample port 19 and the like are arranged between the two analysis channels as shown in FIG. That's fine.

In addition, the part that injects the specimen on the upper surface of the specimen storage part (specimen injection part) is formed with a stopper made of an elastic material such as rubber to prevent leakage, infection and contamination to the outside and to ensure sealing performance. It is desirable that it is 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.
-Specimen The specimen to be measured in the present invention is an analyte-containing sample derived from a living body. There is no particular limitation on the sample itself, but for example, most samples derived from living organisms such as whole blood, plasma, serum, buffy coat, urine, stool, saliva, sputum are applicable. In the case of genetic testing, a gene, DNA, or RNA is an analyte as a nucleic acid that serves as a template for an amplification reaction. The specimen may be prepared or isolated from a sample that may contain such nucleic acid. Therefore, in addition to the above-mentioned samples, cell cultures; nucleic acid-containing samples such as viruses, bacteria, molds, yeasts, plants, animals; samples that may contain or contain microorganisms, and other nucleic acids All possible samples are targeted. A method for preparing a gene, DNA or RNA from such a sample is not particularly limited, and conventional techniques can be used. DNA can be separated and purified from a sample by phenol / chloroform extraction and ethanol precipitation according to a conventional method. The processing of specimens containing biological substances other than genes is also performed as necessary by applying conventional techniques.

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, a blood sample of about 2 to 3 μL is simply injected into a chip having a length and width of several centimeters. 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 specimen can be obtained, and there are few restrictions on the specimen surface, and a small amount of reagents are inevitably required, thereby reducing the examination cost.
Specimen pretreatment section Generally, biological samples such as blood and urine generally require sample pretreatment prior to analysis in order to remove unnecessary components (proteins, ionic substances, etc.) contained in the sample. It is. Even in a microreactor, the on-board analysis system can provide excellent performance only when the sample is in an ideal state, such as a standard product. In many cases, the success or failure of the analysis will affect the success or failure of the analysis. However, the pretreatment of the specimen itself is a complicated process, and at the present time, technology and experience are required for proper implementation. In particular, samples from clinical sites always contain the risk of infection and contamination with viruses and bacteria. Pretreatment of such specimens can also be performed safely on the same chip that performs analysis and detection, and if a specimen that can be measured can be quickly and automatically prepared, it is very significant to make a chip in this manner. .

The specimen pretreatment unit 20a is a part that concentrates, separates, lyses, etc. the substance to be analyzed (analyte). In the case of a blood sample, the specimen pretreatment unit 20a may be responsible for separating blood cells or lymphocytes if necessary. Good. The pretreatment method is usually on a case-by-case basis depending on the type of sample, the type and concentration of the target substance, the presence or absence of interfering substances, and the like. Therefore, the microreactor of the present invention is provided with a pretreatment unit 20a for performing pretreatment as necessary from the viewpoint of the specimen and analysis. In the specimen processing liquid storage unit 20b communicating with this, a lysis reagent, a hemolysis reagent, an extraction liquid, a denaturing liquid, a washing liquid, an elution liquid, and the like are sealed according to the contents of the pretreatment.
-Pretreatment of specimens The pretreatment of specimens varies depending on the type and properties of the sample, or the type and concentration of the substance to be analyzed. For example, in order to prevent clogging of the fine channel, it is necessary to remove insoluble impurities. It is desirable to remove in advance substances that inhibit the reaction for analysis and substances that react together. It is also desirable to concentrate and separate the target substance in advance prior to detection. Depending on the specimen, the substance concentration to be detected is extremely dilute. In such a case, the amount of sample that can be introduced into the microreactor (a few μL for a chip of several centimeters in a few μL) is limited, and as such, it does not fall within the measurable range. Therefore, a preliminary concentration or separation operation of the target substance is required.

  The pretreatment method varies depending on the type of specimen and the analytical method used. Usually, pretreatment for a biological sample frequently used is cell destruction (lysis or hemolysis treatment) or solubilization, extraction, deproteinization, and concentration. Adsorption / desorption, washing, dialysis (desalting), filtration, hydrolysis or derivatization are performed. Further, when the sample liquid is viscous, dilution may be performed as necessary for adjusting the viscosity or the surface tension so that the fine flow path in the microreactor is smoothly fed as a laminar flow. .

When the specimen is blood, it is desirable to fractionate into blood cells and other plasma (or serum). When analyzing blood cells, it is necessary to lyse in advance.
When the measurement target is a protein, it is preferable that the protein is separated from the coexisting protein through a resin layer, a filter or the like, and concentrated. Using a filter or the like to which a specific antibody is bound can efficiently adsorb the target protein, which is efficient.

When the target substance is a gene, it is effective as a pretreatment to remove protein or pass through a filter that selectively adsorbs only DNA. Furthermore, in the case of RNA, it is converted to cDNA using an appropriate reverse transcriptase and then used for analysis.

When detecting the genes of microorganisms such as viruses or fungi (bacteria, fungi), as described above, the microreactor chip has a very limited capacity to introduce the sample, so if the concentration of the target bacteria in the sample is low It is virtually impossible to capture this. Therefore, a large amount of sample liquid is passed through the adsorption filter and concentrated in the filter. Even considering the capacity of the waste liquid storage section and the adsorption capacity of the filter described later, it is probably about 100% of the capacity of the specimen storage section.
It is also possible to pour a sample solution of double to 1000 times volume into the filter. In such a case, a sample liquid exceeding the capacity in the sample storage unit may be continuously introduced from the sample injection unit through the sample storage unit. Such a pretreatment method is different from the conventional method of applying a certain amount of sample to a predetermined position of the analysis chip and measuring the sample at a time, and is originally performed as a stage before application to the chip. This is characterized in that the pre-processing is performed on the same chip. As a preferred embodiment, a lysis reagent is allowed to flow into the specimen storage portion to perform lysis treatment, and a treatment solution for the lysed bacteria is sent to the specimen pretreatment section. Since the sample pretreatment unit is equipped with a filter for adsorbing DNA, the sample treatment solution is passed through the filter to temporarily adsorb DNA in the treatment solution. Thereafter, the DNA adsorbed through the washing treatment is eluted by flowing an eluent and sent downstream of the fine channel.

  Alternatively, a target microbial cell may be used to trap the target bacterium, and a lysis reagent may be flowed to expose the gene, and the solution may be sent downstream of the fine channel. Thereafter, a lysis reagent may be flowed to generate a gene and flow downstream of the microchannel.

  As described above, since various processes are assumed as the pretreatment of the specimen as described above, a necessary process is selected as a pretreatment process based on the specimen and the measurement method, and members, apparatuses, and the like necessary for that purpose are selected. Is preferably incorporated into the specimen pretreatment unit 20a. The specimen pretreatment unit 20a has any shape, structure, etc., and is filled with pretreatment means such as the filter and beads. Furthermore, the sample pretreatment unit supplies the sample from the sample storage unit 20 and the processing liquid used for the preprocessing from the reagent storage unit 18 or the sample processing liquid storage unit 20b (may be provided separately from the reagent storage unit 18). While receiving, it communicates with the flow path from the reagent storage to the reaction site downstream of the fine flow path. In addition, since the waste liquid storage part (waste liquid storage) 23 is provided in the bottom face part of a chip | tip, the valve | bulb arrange | positioned in the boundary with a pre-processing part can be opened, and an unnecessary sample liquid and washing | cleaning liquid can be thrown away there.

  Whether the sample pretreatment is performed before the microreactor is mounted on the biological material testing device body or whether the sample pretreatment is performed after mounting depends on the properties of the sample and the content of the pretreatment. The procedure should be set individually. In addition, it is desirable that the specimen pretreatment is performed in a batch process for the same target sample, and then divided into analysis channels corresponding to individual test items.

Pretreatment means In the microreactor of the present invention, the pretreatment means is not particularly limited in form as a carrier that selectively adsorbs biological material or bacteria or viruses, but specifically, a filter, a bead, a gel or a membrane. It is characterized by being. Depending on the purpose, it may be a combination of a plurality of filters or the above carriers. From the viewpoint of availability, ease of use, and the like, a filter is preferable, and pretreatment means in which these are laminated is preferable.

Examples of the filter that adsorbs the biological substance include a filter that traps DNA. The filter that traps DNA may be, for example, a filter that specifically adsorbs DNA molecules under certain conditions. As an example, “porous membrane” of “Automatic nucleic acid extraction system QuickGene-800” of Fuji Photo Film Co., Ltd. can be used. Or what packed the substance which can selectively adsorb | suck DNA in the column shape may be used.

Alternatively, a case where a specific substance or functional group bound to DNA specifically interacts with the filter is also included. For example, a filter that binds a substance that intercalates into the inside of double-stranded DNA, for example, an intercalator such as an acridine dye or ethidium bromide may be used.

The filter mesh takes into account the size of viruses and bacteria. The form of the filter, the thickness of the filter layer, etc. are appropriately set according to the purpose. For example, two or more types of filters with different sizes may be used in combination in order to filter off insoluble substances and dust first, and then perform a predetermined treatment. The shape of the filter is arbitrary, such as a layered form, a form filled with particles, a resin layer, and a collective form of hollow fibers.

In addition to the filter, it may be a bead that adsorbs viruses or bacteria, a gel such as agarose, or a membrane.
Waste liquid storage part Waste liquid storage part 23 is a hollow chamber provided at the bottom of the microreactor, an extra sample,
This is a sealed waste liquid reservoir that contains all of the cleaning liquid and waste liquid generated in the specimen separation / concentration process, and the waste liquid generated as a result of the reaction and measurement of the specimen. When the analyte pretreatment unit 20a performs the analyte concentration step as a pretreatment, it is necessary to treat the waste liquid that accompanies the concentration and washing. It is not bothered to automatically store such waste liquid inside rather than discharging it outside the microreactor for processing. Preferably, the waste liquid storage unit 23 is provided immediately below the valve unit 64 of the sample pretreatment unit 20a of the microreactor, and at least communicates with the sample pretreatment unit, the reaction unit site of the flow path, the end of the detection site, and the like. What is necessary is just the cavity in which the through-hole which connects with each site | part which produces a waste liquid with the sealing structure which has a capacity | capacitance is formed. The waste liquid storage unit 23 may be a single hollow chamber or may be in the form of a multi-compartment cavity divided into a plurality of compartments. The volume, shape, etc. are not particularly limited, but it is necessary to take into consideration the strength of the chip itself, space limitations associated with the arrangement of other elements, and the like. For example, the capacity of the waste liquid storage part is several hundreds μL to 2 mL when it is provided on the most bottom part of a chip of several cm square. In addition, a valve that opens and closes at an appropriate place in the through hole may be provided as necessary so that the waste liquid in the waste liquid storage unit does not enter other parts of the microreactor or leak outside. .

In particular, in a specimen having a low concentration such as analyte, a large amount of specimen liquid is flowed in the specimen pretreatment section 20a to trap analyte, bacteria, viruses, etc. on the adsorption filter. Waste liquid is generated. Such concentration pretreatment requires a large waste liquid reservoir. In order to obtain a large waste liquid reservoir, almost the entire bottom portion of the chip may be a waste liquid storage part composed of one or a plurality of hollow structures.

  The installation and structure of the waste liquid reservoir further provide the following advantages to the user. Workers who analyze clinical samples are constantly exposed to the risk of infection from the sample, such as bacteria or viruses. Not only handling of specimens and pretreatment thereof, but also treatment of washing liquid during measurement, waste liquid after measurement and the like are complicated, and there is still a risk of infection and the possibility of environmental pollution. In the microreactor of the present invention, the waste liquid storage part 23 is provided at the bottom of the microreactor, and the above-described unnecessary specimens, waste liquids and the like all have a flow path configuration that is finally thrown into the waste liquid reservoir. The waste liquid storage unit 23 is completely sealed inside the microreactor, and the disposable microreactor is stored in a hazard box provided in an examination room or the like as it is after use and is incinerated.

By providing such a waste liquid storage part, after injecting clinical specimens into the microreactor, there is no possibility of consistent contact with specimens at all during and after examination, even after disposal. Ensuring safety from infection. Moreover, disposal can be easily performed without fear of contamination. In addition, there are advantages such as weight reduction of the chip and reduction of manufacturing cost.
Reagent storage part In the bioreactor microreactor of the present invention, a predetermined amount of necessary reagents are sealed in advance in a reagent storage part 18 in the 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. Further, the necessary cleaning liquid is appropriately stored in the cleaning liquid storage portion 21d.
-Reagents 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.
Reagent division and sample division / micropump and pump connection section In this embodiment, the contents of the container are respectively contained in the sample container 20, the reagent container 18, the positive control container 21h, and the negative control container 21i. A micropump 11 for feeding liquid 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. The details are described in Patent Documents 1 and 2 above.
Liquid feeding and dividing means In the present invention, when analyzing multiple items for one sample and simultaneously analyzing a positive control and a negative control, the reagents and the sample are divided into two or more, and each analysis flow is divided. Need to be sent to the road. 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 constituted by 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. Liquid feeding in the flow path, quantification of the liquid feeding amount, and mixing of each liquid are controlled. By the action of the liquid feeding and dividing means and the micropump 11, the reagents and the specimen are divided at an appropriate ratio.
A specimen storage section in which the specimen is stored in each flow path upstream of a merging section that joins a solution of a specimen containing a biological substance (analyte) that is a reaction site measurement target and a reagent (mixed solution); And a reagent container for accommodating the reagent liquid, and a pump connection part is provided upstream of each of the container parts. The micro pump is connected to the pump connection part and driven from each micro pump. By supplying the solution, the solution of the specimen and the reagent in each container are pushed out and merged to start a reaction necessary for analysis such as gene amplification reaction, analyte trap or antigen-antibody reaction. It is configured. 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. 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.
-Gene amplification method In the genetic test | inspection which uses the microreactor of this invention, an amplification method is not limited. 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. 2004-2000). -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. 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 amplification) method as an improvement of PCR is performed under any constant temperature at 50 to 65 ° C.
A Amplification can be performed in a short time (Japanese Patent No. 3433929). Therefore, ICAN
In the microreactor of the present invention, the method is a suitable amplification technique because simple temperature control is sufficient. By hand, the method, which takes 1 hour, ends in 10-20 minutes, preferably 15 minutes, in the bioreactor of the present invention.
Detection Site In the microreactor of the present invention, a detection site for detecting an analyte, for example, an amplified gene, is provided on the downstream side of the reaction site of the fine channel. At least the detection part is made of a transparent material, preferably a transparent plastic, in order to enable optical measurement. Furthermore, a biotin-affinity protein (avidin, streptavidin, extraavidin (R), preferably streptavidin) adsorbed to a detection site on the fine channel is used for biotin labeled on the probe substance or gene amplification reaction. It specifically binds to biotin labeled at the 5 ′ end of the primer. Thereby, the probe labeled with biotin or the amplified gene is trapped at the detection site.

A method for detecting the separated analyte or the amplified DNA of the target gene is not particularly limited. However, as a preferred embodiment, the method is basically performed in the following steps. That is, using the microreactor,
(1a) A micro-channel downstream from a container containing a sample or DNA extracted from a sample or cDNA synthesized from a sample or RNA extracted by reverse transcription and a biotin-modified primer at the 5 ′ position 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 The processed solution obtained by denaturing the amplified gene into a single strand is sent to the detection site in the microchannel adsorbed with the biotin affinity protein, and the amplified gene is passed through the step of trapping the amplified gene. A probe DNA whose end is fluorescently labeled with FITC (fluorescein isothiocyanate) is flowed to the detection site in the trapped microchannel, and this is hybridized to the immobilized gene. (A previously hybridized amplification gene and fluorescently labeled probe DNA may be trapped at the detection site.) (1b) Specific antibodies against analytes such as antigens, metabolites, and hormones 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 has been modified with an anti-FITC antibody that specifically binds to FITC flows in the fine channel, and this hybridizes to the FITC of the immobilized analyte / antibody reaction product or to the gene. The gold colloid is adsorbed to the FITC-modified probe.
(3) The concentration of the gold colloid in the fine channel is optically measured.

  When immobilizing streptavidin in the microchannel formed in the polystyrene substrate, no special chemical treatment is required. It is only necessary to apply the biotin affinity protein onto a fine channel downstream of the amplification reaction site and adsorb the biotin affinity protein on the channel. The probe is bound to an analyte, and when the measurement target is a protein analyte, a specific 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.

  Known fluorescent substances such as FITC, RITC, NBD, Cy3, and Cy5 can be used as the fluorescent dye for labeling the probe. In particular, FITC is desirable due to the availability of anti-FITC antibodies such as gold colloid anti-FITC anti-mouse IgG. Instead of the fluorescent dye, digoxigenin (DIG) may be labeled on the probe DNA. In this case, an anti-DIG-alkaline phosphatase labeled antibody is used as an alternative to the anti-FITC antibody.

Although it is possible to measure the fluorescence of the fluorescent dye FITC, it is necessary to consider light fading of the fluorescent dye, background noise, and the like. A system that can finally measure with high sensitivity by visible light is preferable. Absorption analysis of visible light is superior because the instrument is more versatile than fluorescence photometry, and there are few interference factors and data processing is easy. Instead of using optical detection of colloidal gold using colloidal gold anti-FITC anti-mouse IgG, the probe may be labeled with horseradish peroxidase (HRP) instead of the fluorescent dye. For the detection, a coloring reaction catalyzed by the enzyme can also be used. As typical coloring substances for this purpose, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), p-phenylenediamine (OPD), and the like are known. . In addition, enzyme / coloring systems such as alkaline phosphatase and galactosidase can also be used.
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 trace 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.

As described above, the genetic test has been described as the main example with reference to the drawings shown as an example of a preferred embodiment of the present invention, but the present invention is not limited to such an embodiment and example.
Examination by microreactor The biological material test device of the present invention and various genetic test methods using the microreactor are far more in comparison with conventional nucleic acid sequence analysis, restriction enzyme analysis, and nucleic acid hybridization analysis due to the configuration and analysis principle of the apparatus. It is clear that highly accurate results can be obtained with a very small amount of sample, a small amount of labor, and a simple apparatus.

  From the gene amplification technique and the hybridization technique employed as detection methods in the present invention, two aspects of genetic testing are mainly formed. 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 gene-derived DNA in the sample is 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. 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 mutated portion is used. The gene mutation means a mutation in the nucleotide base of the gene. Furthermore, gene polymorphism analysis by using the test apparatus of the present invention is useful for identification of disease susceptibility genes and analysis of drug-metabolizing enzyme genes.

  In addition to the genetic test described above, the multi-item simultaneous measurement in the microreactor of the present invention can also be used for the multi-item analysis of antigens, hormones, various metabolites, etc. for clinical specimens, for example, by designing detection means such as probes used and detection methods. Can be realized. Specific test items include glucose, bilirubin, albumin, amylase, lipase, cholinesterase, alkaline phosphatase, γ-GTP for liver function tests, AST, ALT, LDH, and urea nitrogen (BUN) for renal function tests Analyzes such as total cholesterol, HDL cholesterol, LDL cholesterol, free cholesterol, triglycerides for cardiovascular examination, such as creatinine and uric acid.

  The biological material testing device of the present invention includes various gene analyzes, clinical examinations / diagnosis, pharmaceutical screening, pharmaceuticals, agricultural chemicals or safety / toxicity testing of various chemicals, environmental analysis, food testing, forensic medicine, chemistry, brewing, It can be used in fisheries, livestock, agricultural production, agriculture and forestry.

FIG. 1 is a schematic view of a biological material testing device including a microreactor and an apparatus main body. FIG. 2 is a schematic view 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. 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 showing 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. Further, the specimen processing liquid storage unit 20b is not shown in this figure. 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 (8)

It has a sample storage unit, a reagent storage unit, a sample pretreatment unit, a waste liquid storage unit, a micropump connection unit, and a fine channel,
The specimen liquid is sent from the specimen container to the specimen pretreatment section, and the specimen liquid is pretreated using the pretreatment means of the specimen pretreatment section, thereby concentrating the biological material to be measured, and As measured by flowing to the flow path constituting the reaction site, then the flow path constituting the detection site, provided downstream of the fine flow channel,
A bioreactor testing microreactor characterized in that a waste liquid generated as a result of concentration and measurement of a specimen is transferred and confined to the waste liquid reservoir.   The fine flow channel is a fine flow channel branched into at least two or more, and is fed to the downstream of the branched fine flow channel by the micropump and the liquid feed dividing means, and / or Alternatively, the encapsulated reagent or the mixed solution thereof is fed downstream of the microchannel branched by the micropump and the liquid feeding / dividing means, and the multiple items of the specimen are respectively downstream of the branched microchannels, and / or The bioreactor microreactor according to claim 1, wherein control can be performed 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;
3. 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 liquid feeding and dividing means is
Branched fine flow path,
The pump pressure of the micropump that allows the liquid to pass by blocking the passage of the liquid until the liquid feeding pressure in the positive direction reaches a preset pressure and applying a liquid feeding pressure equal to or higher than the preset pressure. The liquid feed control unit that can control the passage of liquid by the above, and the backflow prevention unit that prevents the backflow of the liquid in the flow path, liquid feed in the branched flow path, determination of the liquid feed amount and each liquid The bioreactor microreactor according to any one of claims 1 to 3, characterized in that the mixing is controlled.   The bioreactor microreactor according to any one of claims 1 to 4, wherein the pretreatment means is a filter, a bead, or a membrane that selectively adsorbs the biological material or bacteria or viruses. 6. The bioreactor microreactor according to claim 5, wherein the filter that adsorbs the biological material is a filter that traps DNA.   The waste liquid storage part is a hollow chamber provided at the bottom of the microreactor and communicated with at least the specimen pretreatment part and the end of the detection site, and an extra specimen, a cleaning liquid generated in the specimen concentration step, a waste liquid, and The bioreactor microreactor according to any one of claims 1 to 6, wherein the microreactor is a waste liquid reservoir for storing waste liquid generated as a result of measurement of a specimen. A device body in which a micropump and a temperature control device are integrated together with a detection device that optically detects a biological material contained in a specimen, and a biological material testing microreactor that can be attached to the device body, A biological material testing device, wherein a biological material is automatically measured by mounting the microreactor on an apparatus main body.

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