JPWO2007055165A1 - Nucleic acid separation method, nucleic acid testing microreactor, nucleic acid testing system - Google Patents

Abstract

The present invention provides a microreactor for nucleic acid testing and a nucleic acid testing system that have pretreatment means and enable with reliability. The nucleic acid test system of the present invention has a system configuration in which a chip component for each sample, on which elements for reagents and a liquid feeding system are mounted, and a control / detection component as a test apparatus main body are separated. Since it has a sample pretreatment section that performs a pretreatment step for extracting and concentrating nucleic acids in a sample, the substance can be concentrated even in a diluted sample, and at the same time, harmful substances that interfere with the reaction, and the flow path Exclude foreign substances that cause clogging. Moreover, it has a flow channel configuration that can simultaneously analyze positive control and negative control. Therefore, serious problems such as cross-contamination and carry-over contamination are less likely to occur for microanalysis and amplification reactions.

Description

  The present invention relates to a nucleic acid separation method, a nucleic acid testing microreactor, and a nucleic acid testing system including the microreactor. More specifically, the present invention relates to a nucleic acid test microreactor having a pretreatment means to which a nucleic acid separation method is applied, and a nucleic acid test system 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 gene or nucleic acid to be detected 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. 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.

  As a method for extracting and separating nucleic acid from a biological sample, a chemical method or a physical method is used. As methods belonging to the latter, methods for extracting nucleic acids from cells by the action of vibrating beads (Patent Document 3) and methods for separating and concentrating by applying an electric field (Patent Documents 4 and 5) have been reported. There are many problems in applying these methods as they are to microreactors of ultrafine devices.

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 Special Table 2003-522521 JP 2004-217 JP WO02 / 23180 Publication

  The present invention, which has been made in view of the above circumstances, proposes a microreactor for nucleic acid testing that enables high-sensitivity analysis using a chip having a pretreatment means and a waste liquid reservoir even for a sample with a small amount or a low concentration. 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 nucleic acid test system that incorporates a simple configuration and a high-accuracy liquid feeding system and that enables high-accuracy analysis.

The method for separating nucleic acids of the present invention is a method for extracting and separating nucleic acids from cells,
(I) A cell-containing liquid is sequentially passed through a filter A having a pore size larger than the cell and a filter B having a pore size smaller than the cell to hold the cell on the supply port side of the filter B.
(Ii) The cells are taken out into the washing solution by flowing a washing solution from the outlet of the filter B opposite to the side holding the cells.
(Iii) Next, the washing liquid containing the cells is passed through a cell destruction unit that holds vibrating beads, thereby destroying the cells and obtaining a nucleic acid extract.
(Iv) introducing the nucleic acid extract from the cell disruption section through the flow path into the nucleic acid capture section where the electric field is applied in a direction crossing the flow path, and collecting the nucleic acid on the cathode side of the nucleic acid capture section;
(V) collecting the nucleic acid collected on the cathode side of the nucleic acid capture unit from the nucleic acid capture unit with a nucleic acid recovery solution;
It is characterized by that.

  The beads are vibrated by the action of an external actuator attached to the upper surface of the cell destruction part that holds them. This external actuator is preferably a piezoelectric PZT ceramic vibrator.

  It is desirable that the electric field is applied by a pair of external counter electrodes attached to the upper surface of the nucleic acid capturing unit or the contaminant capturing unit having a comb structure.

  In addition, when collecting the nucleic acid collected on the cathode side of the nucleic acid capturing unit, it is desirable to apply a voltage to the external counter electrode so as to reverse the original direction of the electric field.

  The cell filtration device of the present invention is a cell filtration device used for carrying out the steps (i) and (ii) in the nucleic acid separation method described above, wherein the filter A and the filter B are used as partition walls, respectively. A space that can be hermetically sealed is formed by the partition wall and the side wall of the apparatus, and a cleaning liquid discharge pipe is passed through the side wall surface of the apparatus. It has the means by which each hole in is opened and closed.

  The nucleic acid test microreactor of the present invention is released from a specimen receiving part that receives a liquid containing cells, a cell destruction part that destroys cells delivered from the specimen reception part, and cells destroyed in the cell destruction part. A sample pretreatment unit having a nucleic acid capture unit for capturing the nucleic acid to be captured, wherein the sample receiving unit, the cell destruction unit, and the nucleic acid capture unit are communicated with each other through a fine channel.

Furthermore, the microreactor for nucleic acid test of the present invention has a reagent storage unit that stores a reagent, a waste liquid storage unit, a micropump connection unit, and a fine channel that communicates each unit,
Obtained by reacting in the reaction section, the flow path constituting the reaction section where the sample liquid pretreated in the sample pretreatment section and the reagent contained in the reagent containing section react downstream of the fine flow path A flow path that constitutes a detection unit for detecting a reactant is provided.

The cell disruption unit has beads that are vibrated by an external actuator disposed in the cell disruption unit to destroy the cell,
The nucleic acid capturing unit captures a nucleic acid released from a cell destroyed by the cell destructing unit by an electric field applied to an external electrode disposed in the nucleic acid capturing unit.

The micropump connection 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;
An actuator for changing the pressure inside the pressurizing chamber;
It is connected to the micropump which has this.

  The waste liquid storage section is provided at the bottom of the microreactor, and is characterized in that it is a hollow chamber communicating with at least the end of the specimen pretreatment section and the detection section.

Moreover, the microreactor for nucleic acid test of the present invention is
Branched microchannels,
A liquid feed controller that blocks passage of liquid at a pressure lower than a preset pressure and allows passage of liquid at a pressure equal to or higher than a preset pressure; and
A backflow prevention unit for preventing backflow of liquid in the flow path;
Consisting of
It is characterized by having a liquid feed dividing means for controlling the liquid feed in the branched flow path and the quantification of the liquid feed amount.

  Furthermore, a device main body in which a micropump and a temperature control device are integrated with a detection device that optically detects nucleic acids extracted from cells, and the nucleic acid testing microreactor of the present invention that can be attached to the device main body. The present invention also includes a nucleic acid testing system characterized in that a nucleic acid is measured by mounting the microreactor on the apparatus main body.

  The nucleic acid test system of the present invention has a system configuration in which a chip component, which is a microreactor for each specimen, and a control / detection component, which is a main body of the apparatus, are mounted with elements for reagents and liquid feeding systems. 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 nucleic acid or bacteria when the concentration is low. According to the configuration of the microreactor for nucleic acid test of the present invention, it has a specimen pretreatment section that can extract nucleic acids and genes as analysis target substances from cells, and separate and concentrate them. 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 for nucleic acid testing of the present invention has a flow path configuration that can simultaneously analyze positive control and 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.

  Various gene testing methods using the nucleic acid testing system and nucleic acid testing microreactor of the present invention are far fewer than 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 small amount of sample, a small amount of labor, and a simple apparatus.

  The nucleic acid testing system of the present invention is used for various gene analysis, clinical testing / diagnosis, pharmaceutical screening, pharmaceuticals, agricultural chemicals or chemicals safety / toxicity testing, environmental analysis, food testing, forensic medicine, chemistry, brewing, fishery It can be used in livestock, agricultural production, agriculture and forestry.

FIG. 1 is a schematic view of a nucleic acid test system comprising a nucleic acid test microreactor and an apparatus main body. FIG. 2 is a schematic view of a microreactor for nucleic acid testing according to an embodiment of the present invention. The micropump belongs to a separate device from the nucleic acid testing microreactor. FIG. 3 is a schematic view of the cell filtration device of the present invention, with the upper figure showing a front view and the lower figure showing a cross-sectional view. FIG. 4 is a diagram showing a procedure for collecting cells using a cell filtration device. FIG. 5 shows a cross-sectional view of the pretreatment unit attached to the microreactor for nucleic acid testing of the present invention. The left compartment represents a cell disruption part, and the right part represents a nucleic acid capture part. FIG. 6 shows a piezo pump, FIG. 3A is a cross-sectional view showing an example of the pump, and FIG. 3B is a top view thereof. FIG. 3C is a cross-sectional view showing another example of the piezo pump. FIG. 7 is a diagram showing a configuration around the pump connection part of the microreactor for nucleic acid testing when the piezo pump is separated from the microreactor.

Explanation of symbols

1 Main body 2 Microreactor (inspection chip)
3 Peltier element 4 Heater 5 Photo diode 6 LED
10 Driving fluid tank 11 Micro pump (piezo pump)
DESCRIPTION OF SYMBOLS 12 Pump connection part 13 Liquid supply control part 15 Flow path 18 Reagent accommodating part 19 Sample 20 Sample receiving part 24 Drive liquid accommodating part 25 Sealing liquid accommodating part 26 Air vent flow path 31 Reagent 41 Upper substrate 42 Substrate 43 Vibration plate 44 Piezoelectric Element 45 Pressurizing chamber 46 First flow path 47 Second flow path 48 First liquid chamber 49 Second liquid chamber 71 Silicon substrate 72 Port 73 Port 81 Entrance side 82 Extraction port side 83 Cleaning liquid discharge pipe 84 Filter A
85 Filter B
86 Filtration space

  In general, biological samples such as blood and urine generally require sample pretreatment prior to analysis in order to remove unnecessary components (proteins, lipids, ionic substances, etc.) contained in the sample. . 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 sample itself is a complicated and time-consuming process, and the current situation is that 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. Such sample pretreatment can also be carried out safely on the same chip for analysis and detection, even if it is wholly or in its entirety, and if a sample that can be measured can be prepared quickly and automatically, such a mode can be used. The significance of making a chip is extremely large.

  Hereinafter, a nucleic acid test microreactor (hereinafter sometimes simply referred to as a microreactor) and a nucleic acid test system including the microreactor, a micropump, various control devices, and a detection device will be described. In the present specification, the “specimen” is a fluid containing the nucleic acid to be measured. “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. These and the polynucleotide are collectively referred to as “nucleic acid”. “Element” refers to a functional component installed in the microreactor. The “fine channel” is a channel formed in the microreactor of the present invention.

[Nucleic acid separation method]
The method for separating nucleic acids of the present invention is a method for extracting and separating nucleic acids from cells,
(I) A cell-containing liquid is sequentially passed through a filter A having a pore size larger than the cell and a filter B having a pore size smaller than the cell to hold the cell on the supply port side of the filter B.
(Ii) The cells are taken out into the washing solution by flowing a washing solution from the outlet of the filter B opposite to the side holding the cells.
(Iii) Next, the washing liquid containing the cells is passed through a cell destruction unit that holds vibrating beads, thereby destroying the cells and obtaining a nucleic acid extract.
(Iv) introducing the nucleic acid extract from the cell disruption section through the flow path into the nucleic acid capture section where the electric field is applied in a direction crossing the flow path, and collecting the nucleic acid on the cathode side of the nucleic acid capture section;
(V) collecting the nucleic acid collected on the cathode side of the nucleic acid capture unit from the nucleic acid capture unit with a nucleic acid recovery solution;
It is characterized by that.

  The filter mesh used in the nucleic acid separation method of the present invention considers the size of cells, blood cells, viruses, bacteria, and the like. The form of the filter, the thickness of the filter layer, etc. are also set appropriately according to the purpose. For example, insoluble substances and dust are first filtered and removed, and then two types of filters with different sizes are used in combination to perform a predetermined treatment. As the shape of the filter, there are a layered form, a resin layer, a hollow fiber aggregate form, and the like, which are arbitrarily selected.

  The filter A is a filter containing a target nucleic acid and having a pore size larger than that of the cells in the sample, and is a so-called “prefilter” for filtering out insoluble contaminants, dust, and the like. On the other hand, the filter B is a “sterile filter” having a pore size smaller than that of the cell, and the cell cannot pass through the filter B. The inside of the space isolated by the filter A and the filter B or the supply port side of the filter B (removal) Held on the opposite side of the mouth).

  The filter A having a large hole size filters coarse particles such as salt crystals, cell debris, hair, and tissue. Therefore, the average hole size is selected to be small enough to filter out such coarse material from the sample fluid and large enough to allow the target cells and viruses containing nucleic acids to pass through. Is done. In general, the size of the hole should be in the range of about 2-25 μm, and preferably about 5 μm.

  The filter B having a small hole size captures cells and viruses in the sample fluid. The average hole size is selected according to the average size of cells or viruses containing the target nucleic acid. For example, the thickness is 0.1 to 5 μm, preferably 0.5 to 2 μm.

  The filter may be a commercially available filter, such as a membrane filter such as cellulose acetate, cellulose nitrate mixed ester, polytetrafluoroethylene, polyethersulfone, nylon, polyvinylidene difluoride, or glass fiber. Examples of the housing material include polyvinyl carbonate, polyethylene, polypropylene, and glass.

  The cells can be taken out into the washing liquid by pouring the washing liquid into the space from the takeout port on the side opposite to the side of the filter B holding the cells. Next, the washing liquid in which the cells are dispersed is passed through the flow path to the cell destruction section that holds the vibrating beads. Thereby, the cell is destroyed by colliding with the vibrating bead, and its contents are released into the liquid. The nucleic acid extract containing the released nucleic acid or the like is further passed through a nucleic acid capturing part to which an electric field is applied in a direction crossing the flow path. Nucleic acids that are negatively charged linear molecules have a tendency to migrate and collect on the anode side. Nucleic acid collected on the cathode side of the nucleic acid capture unit can be separated from the nucleic acid capture unit with a nucleic acid recovery solution.

  The washing liquid, the extraction liquid, and the recovery liquid are not particularly limited, but are aqueous liquids such as various salt solutions, buffer solutions, and physiological saline.

  Cells can be easily collected by the method for separating nucleic acids of the present invention, and nucleic acids can be extracted from the cells and separated in a concentrated form. The nucleic acid separation method of the present invention is easily carried out in the following sample filtration apparatus and specimen pretreatment unit installed on a microreactor.

[Cell filtration device]
The cell filtration device of the present invention is a cell filtration device used for carrying out the steps (i) and (ii) in the nucleic acid separation method of the present invention, and has the following configuration.

  The filter A and the filter B are arranged as partition walls on the inlet side 81 and the outlet side 82 of the device, respectively, and a space (filtering space) 86 that can be sealed by these partition walls and the device side wall is formed on the side wall surface. A cleaning liquid discharge pipe 83 is provided, and each hole in the inlet, the outlet, and the discharge pipe has means that can be arbitrarily opened and closed (FIG. 3).

  The space 86 is a filtrate reservoir, but is preferably cylindrical, for example a syringe type device is convenient. Transparent plastics such as polyethylene, polypropylene, and polycarbonate are suitable materials because they are easy to handle and easy to produce, but are not particularly limited thereto.

  Furthermore, the inlet for introducing the sample liquid containing cells, the outlet for discarding the filtrate, and the hole in the cleaning liquid discharge pipe for collecting the cleaning liquid have means that can be opened and closed. Open and close those holes as needed. A preferable example of such means may be a detachable screw cap, a plug stopper, or a three-way cock.

  The size of the filtration device of the present invention, particularly the size of the space 86, is appropriately set depending on the type and concentration of the sample. In general, if the volume is 1 to 50 ml, it is easy to handle and operate.

  By using this cell filtration device, step (i) and step (ii) in the method for separating nucleic acids of the present invention can be simply carried out as follows (FIG. 4).

  An inlet side 81 of the cell filtration device of the present invention is placed in a sample solution in a sample container (such as a blood collection tube, a urine cup, a vial, or a sample bottle) that contains the collected sample liquid. The sample liquid is sucked from the opposite outlet side 82 and introduced into the filtration space (filtrate reservoir) 86 through the filter A84. When a sample solution of the required amount is collected, the cell filtration device is taken out from the sample container. Further, suction is continued, and the filtrate in the filtration space is filtered through the next filter B85, and the filtrate is discarded from the outlet side 82. As a result, the portion remaining in the filtration space includes cells that cannot pass through the pores of the filter B85.

  The suction may be performed by an appropriate pressure reducing means such as a water flow pump. However, if it is not necessary to increase the amount of the sample, a disposable syringe, a vacuum blood collection tube, etc. are inserted into the hole on the outlet side. It may be attached to and aspirated. In the case of a vacuum blood collection tube, suction is automatically performed after mounting. The filtrate that has passed through the filter B is convenient because it is accommodated in a disposable syringe or a vacuum blood collection tube.

  Next, a washing liquid is fed into the space from the outlet side 82, and the liquid containing cells is washed. Unnecessary cleaning liquid is removed by suction from the outlet side 82. Repeat this further if necessary.

  Next, the hole on the inlet side 81 is closed, the hole of the cleaning liquid discharge pipe 83 that has been closed until then is opened, and the cleaning liquid sent from the outlet side is pressurized by sending air from the outlet side 82 and pressurizing it. Remove from open cleaning fluid drain. The washing solution contains cells.

  The washing solution containing the cells is directly injected into the sample receiving part of the microreactor for nucleic acid analysis of the present invention. For this reason, it is preferable that the cleaning liquid discharge tube and the sample receiving portion are joined in a liquid-tight manner. The position, size, and the like of the washing liquid discharge pipe provided on the side wall of the cell filtration device are designed to be optimal in the above relationship with the microreactor that is a chip. For convenience, a needle tip may be attached to the washing liquid discharge tube, and the needle may be injected through the sample insertion opening made of silicon rubber in the sample receiving portion of the microreactor.

  Advantages of using the cell filtration device of the present invention as a separate device from the microreactor for nucleic acid analysis include the following.

  First, due to restrictions on the dimensions of the microreactor, the configuration, size, and the like of the cell filtration device according to various circumstances of the specimen can be designed separately from the microreactor by using a separate device. As apparent from the above configuration, the cell filtration device of the present invention has a very simple configuration.

  It is not necessary to install a filter in the microchannel of the microreactor, and it is possible to avoid troubles such as an increase in channel resistance due to clogging of the filter and the occurrence of turbulence.

  Insoluble impurities can be removed in advance, and the cells can be easily separated from the sample solution, and a washing solution containing only the target cells can be injected into the sample receiving portion of the microreactor in an appropriate amount.

  In the cell filtration device of the present invention, the number of cells in the washing liquid in which the cells are suspended can be easily adjusted. In reality, there are many samples in which the nucleic acid to be detected, particularly the genes of microorganisms such as viruses or fungi (bacteria, fungi), are present at very low concentrations. For example, in the detection of infectious diseases, Gram-negative bacteria are present in less than 10 copies per milliliter of blood, Cryptosporidium is usually only a few copies per gallon of drinking water, and anthrax is less than 100 copies per milliliter of water. appear. As described above, in the microreactor chip, the capacity for introducing the specimen is extremely limited. Therefore, if the concentration of the target bacteria in the sample solution is low, it is virtually impossible to capture the target bacteria. Therefore, it is extremely useful that a large amount of sample liquid can be passed through filter A and concentrated in filter B.

  By using the cell filtration device of the present invention, cells can be easily and cleanly separated without being contaminated by bacteria, viruses, etc., and the specimen can be transferred to the microreactor for nucleic acid testing of the present invention.

  If there is no particular risk of contamination, a commercially available disposable syringe and syringe filter can be used to perform the same treatment as described above, although it is somewhat complicated. Specifically, a commercially available syringe filter corresponding to filter A (a small volume filtration filter of the type attached to the syringe) is first attached to a plastic syringe of an appropriate volume, and the sample liquid is placed in the syringe by placing it in the sample liquid. To suck. Next, the filter A is removed, and instead, a commercially available syringe filter corresponding to the filter B is attached as the second filter, and the sample liquid in the syringe is filtered through the filter by pushing the plunger. Thereafter, with the second filter attached, the cleaning liquid is sucked and discharged, and if necessary, the cleaning operation is repeated. Since the cells remain in the syringe, after the washing operation, the second filter may be removed, and a washing solution containing the cells may be transferred to the microreactor by attaching a needle tip instead.

[Outline of microreactor for nucleic acid test and nucleic acid test system]
FIG. 1 is a schematic view of an embodiment of a nucleic acid test system comprising a nucleic acid test microreactor detachable from the apparatus main body and the apparatus main body. FIG. 2 is a schematic view of a microreactor for nucleic acid testing in one embodiment of the present invention. 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, as for the whole or a part of the nucleic acid test microreactor and nucleic acid test system of the present invention, various structures, configurations, arrangements, shapes, dimensions, materials, systems, methods, etc. are applicable as long as they meet the gist of the present invention. Can be.

  The nucleic acid testing microreactor 2 shown in FIGS. 1 and 2 is a single chip produced 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 will be described later, and can easily form a detection portion 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 for nucleic acid test of the present invention.

  The nucleic acid test system of the present invention includes a device body 1 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 2 that can be attached to the device body 1 It consists of. When the sample liquid is injected into the sample receiving part of the microreactor 2 in which the reagent is preliminarily sealed and the microreactor 2 is attached to the main body 1 of the nucleic acid test system, a mechanical connection for operating the liquid feeding pump, if necessary Control electrical connections are also made. Therefore, when the apparatus main body 1 and the microreactor 2 are joined, the flow path of the microreactor 2 is also activated. Preferably, when analysis is started, a series of continuous steps are performed, such as feeding samples and reagents, amplification of nucleic acids based on mixing, reactions such as binding of nucleic acids and probes, detection of reactants and optical measurement. The measurement data is stored automatically in a file together with necessary conditions and recorded items, and nucleic acid measurement is automatically performed.

  The detection device irradiates the detection unit 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. A device incorporated in the detection device may be used, or a separate device may be connected in use. Preferably, in the nucleic acid test system of the present invention, a detection device for optically detecting a nucleic acid contained in a sample is incorporated in the device main body 1 together with the liquid feeding means including the micropump and the temperature control device. It has become a structure.

  A control system relating to each control of liquid feeding, temperature, and reaction, optical detection, data collection, and processing constitute a main body of the nucleic acid test system of the present invention together with a micropump and an optical device. The apparatus main body 1 is commonly used for a specimen sample by attaching a nucleic acid testing microreactor 2 to the apparatus main body 1. The above-mentioned reactions such as amplification and detection are software installed in the nucleic acid test system as a program together with micropump and temperature control, optical detection data processing as conditions set in advance for the order of feeding, volume, timing, etc. It is built in. 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. Unlike this, in the present invention, only the removable microreactor for nucleic acid testing needs to be replaced. If it is necessary to change the control of each element, the control program stored in the apparatus main body 1 may be appropriately modified.

  In the nucleic acid test system of the present invention, all components are miniaturized and are in a form that is convenient to carry. Therefore, the nucleic acid test system is not restricted by the place and time of use, and has good workability and operability. 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 on the apparatus main body 1 side, the microreactor for nucleic acid testing can be used as a disposable type.

  The microreactor and nucleic acid test system for nucleic acid testing of the present invention can be suitably used particularly for testing genes or nucleic acids. In that case, a mechanism for DNA amplification is mounted on the microreactor for nucleic acid testing. However, it can be said that the basic configuration of nucleic acids other than genes is almost the same. For example, a reverse transcriptase storage unit may be installed. 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.

[Microreactor for nucleic acid testing]
The microreactor for nucleic acid test of the present invention has a sample receiving part, a sample pretreatment part, a reagent storage part, a waste liquid storage part, a micropump connection part, and a fine channel. A specimen receiving part that receives a washing liquid containing cells, a specimen pretreatment part, a cell destruction part, and a nucleic acid capturing part are provided from the cell filtration device through the washing liquid discharge pipe, and each part is communicated with a fine channel. . A cleaning liquid containing cells and viruses in the specimen receiving section is sent as a specimen liquid to the specimen pretreatment section through a fine channel, and the specimen liquid is pretreated using the pretreatment means of the specimen pretreatment section. To extract, separate and concentrate the nucleic acid to be measured, and flow the substance to the flow path constituting the reaction part and then the flow path constituting the detection part provided downstream of the fine flow path and measure The nucleic acid testing microreactor is characterized in that the waste liquid produced as a result of the concentration and measurement of nucleic acid is transferred and confined to the waste liquid reservoir.

  Furthermore, in addition to each storage unit, flow path, and pump connection unit, each element such as a liquid feed control unit, backflow prevention unit, reagent quantitative unit, and mixing unit is installed at a functionally appropriate position by microfabrication technology. Yes.

[Sample pretreatment unit]
-Pretreatment As a pretreatment of the sample, it is desirable to separate and concentrate the target nucleic acid prior to amplification and detection. Depending on the sample, the nucleic acid concentration to be detected is extremely dilute. In such a case, the amount of sample solution that can be introduced into the microreactor (several μL for a chip of several centimeters) is limited, and as such, it does not fall within the measurable range. Therefore, it is necessary to concentrate or separate the target substance. Furthermore, in the case of RNA, it is converted to cDNA using an appropriate reverse transcriptase and then used for analysis.

  Such a pre-processing operation is an operation different from a method in which a predetermined amount of sample is applied to a predetermined position of a conventional analysis chip at a time and the sample is measured. The present invention is characterized in that there are two stages: a pre-process before the application to the nucleic acid test microreactor and a pre-process performed on the nucleic acid test micro-reactor thereafter.

  The specimen pretreatment unit is a part that preliminarily performs extraction, separation, concentration, and the like of the nucleic acid to be analyzed by cell destruction (bacterial or hemolysis). The shape, structure, etc. are arbitrary, and pretreatment means such as vibrating beads, an electric field by an external electrode, etc. are applied. In the reagent storage unit communicating with the pretreatment unit, an extraction liquid, a cleaning liquid, a recovery liquid, and the like are sealed as necessary.

The specimen pretreatment unit is composed of at least two independent portions in the flow path, a cell destruction unit, and a nucleic acid capturing unit.
-Cell destruction part The specimen containing cells and viruses is sent from the specimen receiving part to the cell destruction part of the specimen pretreatment part through the fine channel. In the cell destruction part, nucleic acids are extracted by physically destroying cells and viruses. The cell destruction part provided on the fine flow path downstream from the specimen receiving part is a space wider than the flow path before and after it, and spreads up and down. Inside the space, the required number of beads can be added. (FIG. 5). Therefore, it should be thick enough to provide space for the beads to move or to rupture cells or viruses. The size of the beads needs to be larger than the diameter of the fine channel so as to remain in the space, and is, for example, in the range of 50 to 600 μm, preferably 100 to 400 μm. The average diameter of the beads should be selected according to the cells, bacteria, and viruses to be ruptured by the beads, and it is not necessary to make the sizes uniform as long as they remain in the space inside the cell destruction portion. The beads may be porous or non-porous, and the shape is not particularly limited. The material of the beads is not particularly limited, and suitable beads for rupturing cells or viruses include zirconia, borosilicate glass, lime glass, silica, and polystyrene beads. In particular, a material that does not adsorb nucleic acids is preferred.

  Beads that remove unwanted substances (eg, proteins and peptides) or chemicals (eg, salts, metal ions, detergents) that interfere with DNA amplification from the sample may be accommodated. For example, ion exchange beads for removing proteins are known, and beads having a metal ion chelator such as iminodiacetic acid remove metal ions from biological samples.

  The beads are vibrated by an external actuator attached to the cell destruction part. A piezoelectric PZT (lead zirconate titanate) ceramic vibrator is preferable as such an actuator. Cells that come into contact with or collide with beads vibrated by the PZT vibrator are destroyed in the membrane structure, and intracellular substances are released out of the cells. Intracellular substances include the target nucleic acid. By such treatment, bacterial cells and viruses are lysed very rapidly, and as a result, the nucleic acid comes out in the liquid and a nucleic acid extract is obtained. It is done. The vibration intensity of the beads may be set as appropriate according to the type and nature of the cells in the specimen.

  Conventional methods of lysis and cell destruction are carried out by physical or chemical methods. The method for extracting and separating nucleic acids in the microreactor for nucleic acid testing of the present invention is free from the problems and inconveniences seen in the conventional methods described below, and can separate nucleic acids that are smoothly amplified.

  In chemical methods, a lysis agent (for example, a surfactant, an enzyme, or an organic compound) is used to destroy cells and dissociate nucleic acids. Thereafter, treatment of the extract with a chaotropic salt is required to denature contaminating proteins (enzymes used) and potentially interfering proteins. As a further disadvantage, the chemical agent inhibits subsequent DNA amplification, and a denaturing agent such as a surfactant or ethanol may change the water repellency of the water-repellent valve in the microchannel. This necessitates a step of separating the target nucleic acid from the chemical agent, which results in a reduction in the amount of nucleic acid as well as complexity. Moreover, in the method using an enzyme, it takes time including the time and effort of incubation.

  Although physical methods for destroying cells do not require chemicals, physical methods also have drawbacks. The heat method, which is a typical physical method, causes protein denaturation, and the protein adheres to the dissociated nucleic acid to prevent subsequent DNA amplification. Another method, freeze-thawing, may be insufficiently disrupted by certain spores and viruses. There is also known a method of passing a small-diameter hole under high pressure, but such a system is large and expensive, and has many limitations. It is also known that nucleic acids can be separated by exposing cells to ultrasonic vibrations. In a typical example, an ultrasonic probe is directly placed in a liquid containing cells to rupture the cells. Since the probe is in direct contact with the sample solution, bubbling due to cross contamination and cavitation leads to a dangerous and complicated situation.

Another method for cell disruption is disclosed by Murphy et al. In US Pat. No. 5,374,522. According to this method, a suspension of cells is placed in a container with small beads. The container is placed in the ultrasonic bath until the cells rupture and release the cellular components. However, the distribution of ultrasonic waves in the tank is not uniform, so an engineer must locate a high energy area in the tank and place the container in this area. The uneven distribution of the ultrasonic energy produces inconsistent results. In addition, the ultrasonic bath does not concentrate energy in the container, so it often takes several minutes to complete the cell rupture.
-Nucleic acid capture | acquisition part A nucleic acid capture | acquisition part is located in the downstream of a cell destruction part, and may adjoin substantially. A pair of external electrodes are attached oppositely to the outer surface of the chip corresponding to the upper and lower sides of the trapping part and the foreign matter trapping part facing each other (FIG. 5). An electric field is applied in the crossing direction. In the present invention, an electric field is generated in a direction crossing the flow path and applied to the cell component in association with performing electrophoresis auxiliary for nucleic acid separation. Since the direction crossing the flow path, for example, the width direction or height direction of the flow path is extremely smaller than the length direction of the flow path, the applied voltage required to generate the desired electric field can be small. Since the channel width is up to about 200 μm, the voltage applied between the channel widths is about 40 to 60V. When electrophoresis is performed as a driving force for biomolecules, it can be seen that it is sufficient to apply a very low voltage as compared with the necessity of applying a voltage of several kV. Note that the application of a voltage to the electrode and the action of the PZT vibrator of the cell destruction unit are electrically controlled from the apparatus body while the microreactor is mounted on the apparatus body.

  The pair of opposing external electrodes may be formed by depositing an electrode such as platinum on the upper surface of the nucleic acid capturing unit or on the channel side wall. Alternatively, these electrodes and a structure having a comb structure can be manufactured by an etching technique as disclosed in, for example, Japanese Patent Application Laid-Open No. 2002-233792.

  A nucleic acid extract containing nucleic acid is sent from the cell disruption section to the nucleic acid capture section through the fine channel. When the nucleic acid extract is passed through the nucleic acid capturing part to which an electric field is applied in the direction crossing the flow path, the nucleic acid having a negative charge moves toward the anode (anode), and the comb corresponding to the anode It will be connected to the mold structure or will be gathered around it. On the other hand, the positively charged impurities are the opposite surface corresponding to the cathode (cathode), that is, the surface of the flat portion facing the concave comb structure in FIG. Named). The liquid containing other cell components passes through the capturing part as it is and flows downstream. This liquid may be sent to the waste liquid reservoir at an appropriate downstream position of the fine channel. Aims to prevent clogging of microchannels by removing bacterial cells, cell membrane residues, and contaminants, as well as to remove substances that inhibit reactions for analysis and react with them in advance. It is desirable that the waste liquid storage unit be disposed at a position immediately after the capturing unit. The sample pretreatment unit receives a sample from the sample receiving unit 20 from the reagent storage unit 18 (which may be provided separately from the other reagent storage units), and receives the processing liquid used for the pretreatment from the reagent storage unit 18. It communicates with the flow path to the reaction section downstream of the fine flow path. Since the bottom surface of the chip is provided with a waste liquid storage part (waste liquid reservoir), for example, a valve disposed at the boundary with the pretreatment part may be opened to discard unnecessary specimen pretreatment waste liquid and cleaning liquid there. it can.

  The nucleic acid capturing part preferably has a comb structure as a microstructure. Within a relatively narrow range of the trap, the nucleic acid is attracted by electrophoresis under an applied electric field, and the nucleic acid is gathered densely away from the fluid flow. The fluid is pumped out, but the nucleic acid is taken into the comb-shaped region so that it does not flow with the fluid. Furthermore, by taking a comb-shaped structure, it does not occupy the entire cross section of the flow path, and nothing is arranged on the other side of the flow path, so it does not hinder the flow of fluid, and continuously There is also an advantage that the nucleic acid is efficiently separated and recovered from other components.

  Instead of the comb structure, the structure of the nucleic acid capturing part is disclosed in Japanese Patent Application Laid-Open No. 2004-217. A method of trapping and releasing near a narrowed portion may be used.

  Nucleic acids collected in the concave nucleic acid capture unit on the anode side can be separated from the nucleic acid capture unit with a nucleic acid recovery solution. At that time, when the deflecting means applies a voltage to the counter electrode so as to generate an electric field in the opposite direction to the nucleic acid to release the nucleic acid from the capturing part, the nucleic acid is separated and recovered more efficiently. Specifically, when the electric field is turned off, the impurities trapped on the cathode-side flat surface (contaminant trapping portion) are also released, but such impurities tend to flow out before the nucleic acid in the recess. For this reason, when changing the direction of the electric field, it is desirable to make adjustments so that the electric field is weaker than the trapped voltage. Nucleic acids can be concentrated by such treatment. Next, the sample pretreated as described above is divided into two or more fine flow channels for sample analysis by the following liquid sending / dividing means, if necessary, and sent to the downstream analysis flow channel communicating therewith. Is done. The divided specimens enter from the sample port into a fine flow path through which reagents flow and merge.

[Sample receiving part]
The sample receiving part 20 of the microreactor for nucleic acid testing of the present invention has the following configuration. The sample receiving unit 20 communicates with the sample injection unit to temporarily store the sample (that is, the above-described washing liquid containing cells or viruses) and supply the sample to the mixing unit. The cell-containing liquid injected into the sample receiving unit 20 is connected to the micropump 11 and the pump connection unit 12, and is sent to the sample pretreatment unit by these actions.

Further, a portion (sample injection portion) for injecting the sample on the upper surface of the sample receiving portion 20 has a stopper made of an elastic material such as a rubbery material in order to prevent leakage, infection and contamination to the outside and to ensure sealing performance. Preferably, it is formed or 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 not particularly limited to a biological sample itself, but most biological specimens such as whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum, etc. Is applicable. In the case of a genetic test, a gene, DNA, or RNA is an analysis target 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.

  The microreactor for nucleic acid test of the present invention requires a very small amount of sample compared to the case of manual test. 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 for nucleic acid test of the present invention includes few cases from the sample surface, including the case where only a very small amount of sample can be obtained, and inevitably requires a small amount of reagents, thereby reducing the test cost.

[Waste liquid reservoir]
The waste liquid storage part is a hollow chamber provided at the bottom of the microreactor, and contains all of the excess specimen, the cleaning liquid produced in the specimen separation / concentration process, the waste liquid, and the waste liquid generated as a result of the reaction and measurement of the specimen. It is a sealed waste liquid reservoir. In the specimen pretreatment section, when an analyte concentration step is performed as a pretreatment, it is necessary to treat a waste liquid that accompanies 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 part is provided immediately below the valve part of the specimen pretreatment part of the microreactor, and communicates with at least the specimen pretreatment part, the reaction part of the flow path, the end part of the detection part, and the like and has a necessary capacity. What is necessary is just the cavity in which the through-hole which leads to each part which produces sealing structure, ie, a waste liquid, is formed. The waste liquid reservoir 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. .

  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. The microreactor for nucleic acid testing of the present invention has a waste liquid reservoir 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 discarded into the waste liquid reservoir. This waste liquid storage part is completely sealed inside the microreactor, and the disposable microreactor for nucleic acid testing according to the present invention is stored in a hazard box provided in a testing room or the like after use and incinerated. The

  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 unit]
In the microreactor for nucleic acid testing of the present invention, necessary reagents (including a cleaning solution) are sealed in a predetermined amount in a reagent storage unit 18 in the microreactor in advance. Therefore, the nucleic acid test 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 use. When analyzing nucleic acids in a specimen, necessary reagents are known, and examples include various reagents for amplification, probes used for detection, and coloring reagents.

[Micro pump and pump connection]
In the present embodiment, a micropump 11 is provided for each of the sample receiving unit 20, the reagent storage unit 18, and, if necessary, the control storage unit, to feed the storage unit contents. 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 (nucleic acid test apparatus) separate from the microreactor, and is connected to the microreactor from the pump connection portion 12 by mounting the microreactor on the apparatus main body (see FIG. 7).

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 (FIG. 6), the details of which are described in Patent Documents 1 and 2.

[Reaction part]
A sample receiving part for storing the sample and a reagent for storing the reagent liquid in each flow channel upstream of the joining part that joins the sample liquid containing the nucleic acid to be measured and the reagent (mixed liquid) And a pump connection portion provided upstream of each of the storage portions. The micropumps are connected to the pump connection portions, and the driving liquid is supplied from the micropumps to supply the driving liquid. The reaction solution necessary for gene amplification analysis is started by extruding the sample solution and the reagent in the container and joining them together. 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 reaction part is not particularly limited, and various forms and modes are conceivable. Basically, at least two kinds of liquids including a reagent and a sample liquid are fed 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.
-DNA amplification method In the genetic test using the microreactor for nucleic acid test of the present invention, the 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 the PCR amplification method, 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. 2005-318867). 2004-108285). This device system may be applied to the amplification flow path of the microreactor for nucleic acid testing 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.

  An ICAN (Isothermal chimera primer initiated nucleic acid amplification) method recently developed as an improvement of the PCR amplification method has a feature that DNA amplification can be performed in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Patent No. 3433929). No. publication). Therefore, the ICAN method is a suitable amplification technique because the temperature control in the microreactor for nucleic acid testing of the present invention requires simple temperature control. In the manual process, this method, which takes 1 hour, is completed in 10 to 20 minutes, preferably 15 minutes, in the microreactor for nucleic acid test of the present invention.

[Detection unit]
In the microreactor for nucleic acid testing of the present invention, a detection unit for detecting the amplified nucleic acid is provided on the downstream side of the reaction unit 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, the biotin-affinity protein (avidin, streptavidin) adsorbed on the detection part on the microchannel is composed of biotin labeled on the probe substance or biotin labeled on the 5 ′ end of the primer used in the gene amplification reaction. Bind specifically. As a result, the probe labeled with biotin or the amplified nucleic acid is trapped by the detection unit.

A method for detecting the separated amplified nucleic acid is not particularly limited, but a preferred embodiment is basically performed in the following steps. That is, using the microreactor for nucleic acid testing of the present invention,
(1) A sample or a DNA extracted from a sample, or a cDNA synthesized from a sample or RNA extracted from a sample or a sample by reverse transcription, and a biotin-modified primer at the 5 ′ position, and a microchannel downstream from these containing parts To liquid. A step of amplifying nucleic acid in the microchannel of the reaction section, a step of mixing the amplification reaction solution containing the amplified nucleic acid and the denaturing solution in the microchannel, and denaturing the amplified DNA into a single strand, amplification The treated solution obtained by denaturing the resulting DNA into a single strand is sent to the detection unit in the microchannel to which the biotin-affinity protein is adsorbed, and the amplified nucleic acid is trapped through the step of trapping the amplified gene. A probe DNA whose end is fluorescently labeled with FITC (fluorescein isothiocyanate) is allowed to flow through the detection section in the microchannel, and this is hybridized to the immobilized gene. (A previously hybridized amplification gene and fluorescently labeled probe DNA may be trapped by the detection unit.)
(2) A gold colloid solution whose surface is modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow through the fine channel, and thereby the gold colloid is adsorbed to the FITC-modified probe hybridized with DNA.
(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 on the fine channel downstream of the amplification reaction part and to adsorb the biotin-affinity protein on the channel. As a probe DNA for genetic testing, fluorescently labeled oligodeoxynucleotides are preferably used. 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.

  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.

A preferred embodiment of the microreactor for nucleic acid testing of the present invention is:
Within one chip,
A sample receiving part into which a sample or a nucleic acid extracted from the sample is injected;
A sample pretreatment unit for performing pretreatment of the sample,
A reagent storage section for storing reagents used in probe binding reactions, detection reactions (including gene amplification reactions, etc.);
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, and the nucleic acid extracted from the sample stored in the sample receiving unit and the reagent stored in the reagent storage unit are fed to the channel, After mixing and reacting in the reaction part, for example, the amplification reaction part, the treatment liquid treated with the reaction liquid and the probe accommodated in the probe accommodating part are sent to the detection part in the downstream flow path, Mix in the channel and bind (or hybridize) with the probe, detect the nucleic acid based on this reaction product,
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,
The sample pretreatment unit and the detection unit communicate with each other through a through-hole, and is a hollow chamber provided at the bottom of the microreactor. Measurement of extra sample, cleaning liquid, waste liquid, and sample generated in the sample concentration step 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 present invention, when the positive control and the negative control are simultaneously analyzed for one sample, it is necessary to divide the reagents and the sample into two or more and send them to the respective analysis channels. The liquid feed dividing means is provided for that purpose. The liquid feed dividing means is composed of a branched fine channel, a liquid feed control unit 13 and a backflow prevention unit.

  The liquid feeding control unit 13 blocks the passage of the liquid until the liquid feeding pressure in the forward direction (usually, the direction in which the fluid is pushed out by the pump, that is, the downstream direction) reaches a predetermined pressure. Addition allows the passage of liquid. Further, the backflow prevention part 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.

In the microchannel of the microreactor for nucleic acid testing of the present invention, the micropump, a liquid feed 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, the quantification of the liquid feeding amount, and the mixing of each liquid in the branched flow path 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.
・ Measurement of control In the analysis of nucleic acid, a negative control is usually added to the analysis in parallel with the analysis of the sample. 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. This is because it is particularly necessary to check that the PCR reaction is occurring correctly. For example, when any problem occurs, it is optimal for verifying whether it originates from set conditions, reagents, operation, analysis system, or sample. Since the PCR method can amplify a very small amount of gene present in a sample several hundred thousand to several million times or more, 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 genetic test has been described above 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.

Claims (13)

A method for extracting and separating nucleic acids from cells,
(I) A cell-containing liquid is sequentially passed through a filter A having a pore size larger than the cell and a filter B having a pore size smaller than the cell to hold the cell on the supply port side of the filter B.
(Ii) The cells are taken out into the washing solution by flowing a washing solution from the outlet of the filter B opposite to the side holding the cells.
(Iii) Next, the washing liquid containing the cells is passed through a cell destruction unit that holds vibrating beads, thereby destroying the cells and obtaining a nucleic acid extract.
(Iv) introducing the nucleic acid extract from the cell disruption section through the flow path into the nucleic acid capture section where the electric field is applied in a direction crossing the flow path, and collecting the nucleic acid on the cathode side of the nucleic acid capture section;
(V) collecting the nucleic acid collected on the cathode side of the nucleic acid capture unit from the nucleic acid capture unit with a nucleic acid recovery solution;
A method for separating nucleic acids.   2. The method for separating nucleic acids according to claim 1, wherein the beads are vibrated by the action of an external actuator attached to the upper surface of the cell destruction part that holds them.   3. The method for separating nucleic acids according to claim 2, wherein the external actuator is a piezoelectric PZT ceramic vibrator.   2. The nucleic acid according to claim 1, wherein the electric field is applied by a pair of external counter electrodes attached to an upper surface of the nucleic acid capturing unit or the contaminant capturing unit having a comb structure. Separation method.   5. The voltage according to claim 4, wherein a voltage is applied to the external counter electrode so as to reverse the original direction of the electric field when the nucleic acid collected on the cathode side of the nucleic acid capturing unit is recovered. A method for separating nucleic acids. A cell filtration device used for carrying out steps (i) and (ii) in the method for separating nucleic acids according to claim 1, comprising:
The filter A and the filter B are arranged as partition walls on the inlet side and the outlet side of the apparatus, respectively, and a space that can be sealed is formed by these partition walls and the apparatus side wall, and a cleaning liquid discharge pipe is formed on the side wall surface. A cell filtration device characterized in that it has a means for opening and closing each hole in the inlet, outlet and outlet tube.   A sample receiving part for receiving a liquid containing cells, a cell destroying part for destroying cells delivered from the sample receiving part, and a nucleic acid capturing part for capturing nucleic acids released from the cells destroyed in the cell destroying part A nucleic acid test microreactor comprising: a sample pretreatment unit, wherein the sample receiving unit, the cell disrupting unit, and the nucleic acid capturing unit communicate with each other through a fine channel. Furthermore, it has a reagent storage unit for storing the reagent, a waste liquid storage unit, a micropump connection unit and a fine channel that communicates each part,
Obtained by reacting in the reaction section, the flow path constituting the reaction section where the sample liquid pretreated in the sample pretreatment section and the reagent contained in the reagent containing section react downstream of the fine flow path The microreactor for nucleic acid testing according to claim 7, further comprising a flow path that constitutes a detection unit that detects a reactant. The cell disruption unit has beads that are vibrated by an external actuator disposed in the cell disruption unit to destroy the cell,
The nucleic acid capture unit captures a nucleic acid released from a cell destroyed by the cell destruction unit by an electric field applied to an external electrode disposed in the nucleic acid capture unit. Item 9. A microreactor for nucleic acid testing according to item 7 or item 8. The micropump connection 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;
An actuator for changing the pressure inside the pressurizing chamber;
The microreactor for nucleic acid test according to claim 8 or 9, characterized in that the microreactor is connected to a micropump having:   The waste liquid storage part is provided in the bottom of the microreactor, and is a hollow chamber communicating with at least the end part of the specimen pretreatment part and the detection part. A microreactor for nucleic acid testing according to claim 1. Branched microchannels,
A liquid feed controller that blocks passage of liquid at a pressure lower than a preset pressure and allows passage of liquid at a pressure equal to or higher than a preset pressure; and
A backflow prevention unit for preventing backflow of liquid in the flow path;
Consisting of
The microreactor for nucleic acid test according to any one of claims 8 to 11, further comprising a liquid feeding dividing means for controlling liquid feeding and quantification of the liquid feeding amount in the branched flow path. .   A device main body in which a micropump and a temperature control device are integrated with a detection device that optically detects nucleic acids extracted from cells, and the device main body according to claims 7 to 12, which can be attached to the device main body. A nucleic acid test system comprising the microreactor for nucleic acid test according to any one of the above, wherein the nucleic acid is measured by mounting the microreactor on the apparatus body.