JP5441035B2 - Sample analyzer - Google Patents

Description

  The present invention relates to a sample analyzer that can analyze a combination of a large number of solid samples and liquid samples with a simple structure, and more specifically, a structure using a substrate made of a gas permeable material, a pressure adjusting mechanism, and a valve mechanism. The present invention relates to a sample analysis apparatus that can be analyzed with the above. As a preferred example, a reaction chamber in which a primer is pre-applied to a substrate made of a gas permeable material such as PDMS (polydimethylsiloxane) and dried, and a certain amount of solution is put into the reaction chamber using the gas permeability of PDMS. The present invention relates to a PCR chip for gene analysis provided with a mechanism for introduction.

  Polymerase chain reaction (hereinafter referred to as “PCR”) is one of biomolecule operations used for amplification of nucleic acids (eg, DNA, RNA). Because it is possible to obtain a large number of copies of nucleic acid fragments from the specificity to the sequence and a very small amount of sample, it is used for sequence analysis and genotype analysis in various fields such as biology, clinical diagnosis, forensic examination, etc. It has been.

  In recent years, development of technology for performing PCR on a chip of several centimeters has been actively performed by downsizing a conventional PCR device by using a micro electro mechanical system (MEMS) technology. It has become to. Compared to the conventional PCR method, this miniaturized PCR chip has a shorter analysis time, shorter heating / cooling cycle time, and requires a small amount of PCR solution. There is an advantage that a mechanism for pre-processing can be integrated. An important advantage is that a large number of minute PCR reactors fabricated in the chip can be used to amplify various sequences as targets from nucleic acids contained in a very small amount of sample. It is expected that a large number of target sequences contained in one minute sample can be efficiently detected by performing parallel processing of quantitative PCR using a channel on the chip. However, the development of this PCR chip has problems to be solved as described below.

(Complicated PCR chip)
In a PCR chip, it is necessary to measure and mix the primer and sample in the chip. In order to analyze m types of target sequences contained in n samples, the method of creating an mxn matrix in the chip and performing the corresponding PCR at the intersection can greatly save the labor of the experiment. It is effective because it can. However, to achieve this, it is necessary to weigh and mix the primer and sample sample at each intersection in a small chip. The conventional technique requires a large number of pipes, flow paths, microvalves, micromixers, and the like, and has a very complicated structure (for example, see Non-Patent Documents 1 to 4).

  Therefore, instead of integrating complicated pipes, microvalves, and pumps, there is also a report in which a PCR reagent is fed by capillary action using the characteristics of micro-sized flow paths and measured (for example, Non-Patent Document 5). ). In the method using the capillary phenomenon described in Non-Patent Document 5, a surfactant is mixed in the PCR reaction solution in order to minimize the contact angle between the flow path wall surface and the PCR solution and increase the liquid feeding efficiency. However, since the surfactant inhibits the PCR reaction, it is not suitable for amplifying a very small amount of genome.

(Measurement and bubble remaining problems)
Moreover, in the conventional PCR chip, bubbles often remain in the reactor. As factors for the generation of bubbles, (1) mixing of gas when the liquid sample is first introduced can be cited. That is, gas is mixed into the liquid sample due to small dust, dust, and protrusions in the flow path. This also occurs in straight channels. Further, (2) it is also generated when the dissolved gas in the liquid sample is vaporized during the temperature rise of the chip accompanying the PCR reaction step. This is because as the temperature rises, the solubility of the gas in the liquid decreases, and a gas that cannot be completely dissolved appears as a gas. Furthermore, (3) it also occurs when dissolved gas in the resin substrate (for example, PDMS) is vaporized and mixed into the liquid sample through the channel wall during the temperature rise of the chip.

  As described above, the generation of bubbles is confirmed not only during the introduction of the liquid sample but also during the PCR process, and the remaining bubbles significantly hinder measurement (quantitativeness) by PCR. Moreover, the determination of the presence or absence of a gene is not a big problem, but it is fatal when quantitativeness is strictly required as in the diagnosis of Down's syndrome. Bubbles repeat expansion and contraction during the temperature cycle, which causes additional measurement errors.

(Evaporation of sample solution)
In addition, in order to perform PCR using a very small amount of sample in a small reactor in the chip, it is indispensable to devise measures for preventing evaporation of the reaction solution. In particular, denaturation of DNA requires heating to a temperature close to one hundred degrees, so the solution evaporates quickly, and the sample may dry out and disappear completely under standard atmospheric pressure (eg, non-patented). Reference 6).

In order to avoid this evaporation problem, mineral oil having a boiling point higher than 100 ° C. and a density of 1.0 g / cm ³ or less is often used as an evaporation preventing film (for example, Patent Document 1, Non-Patent Document 7). , 8). The methods described in these documents are suitable for PCR using an open well reactor in which a sample is introduced by pipetting. However, in a PCR chip for gene analysis in which many elements are integrated, PCR is performed in a sealed reactor, so that it is difficult to prevent evaporation by the mineral oil method.

JP 2007-175006 A

BioMark Product, "Dynamic array for real-time qPCR", [online], Fluidigm, [October 28, 2009 search], Internet <URL: http://www.fluidigm.jp/biomark_qPCR.htm> Jian Liu, Carl Hansen, Stephen R. Quake; Solving the “world-to-chip” interface problem with amicrofluidic matrix; Ana. Chem, 75, 4718-4723 David S. Kong, Peter A. Carr, Lu Chen, Shuguang Zhang, Joseph M. Jacobson; Parallel gene synthesis in the microfluidic device; Nucleic Acids Research, 2007, Vol. 35, No. 8 Chunsun Zhang, Da Xing, Yuyuan Li; Micropumps, microvalves, and micromixers within PCR microfluidicchips: Advances and trends; Biotechnology Advances 25 (2007) 483-514 N. Ramalingam, Hao-BingLiu, Chang-Chun Dai, Yu Jiang, Hui Wang, Quighui Wang, Kam M Hui, Hai-QingGong; Real-time PCR array chip with capillary-driven sample loading and reactorsealing for point-of-care applications; Biomed Microdevices; Doi 10.1007 / s10544-009-9318-4 Chunsun Zhang, Da Xing; Miniaturized PCR chips for nucleic acid amplification and analysis: lastestadvances and future trends; Nucleic Acids Research, 2007, Vol.35, No 13 (4233-4237) Neuzil P, Zhang C.Y, Pipper J, Oh S, Zhuo L; Ultra fast miniaturized real-time PCR: 40 cycles inless than six minutes; Nucleic Acids Research, 2006, Vol. 35, No 13 (4233-4237) Xiang Q, Xu B, Fu R, LiD; Real time PCR on disposable PDMS chip with a miniaturized thermal cycler; Biomed.Microdevices, 7, 273-279.

  The present invention has been proposed in view of such conventional circumstances, and an object of the present invention is to provide a sample analyzer that eliminates the disadvantages (eg, complexity of the device structure) of conventional gene analyzers and the like. And That is, an object of the present invention is to provide a sample analyzer that can analyze a combination of a large number of samples and targets with a simple structure.

  In addition, the present invention provides a sample analysis device that eliminates other drawbacks (for example, residual bubbles and evaporation of sample solution) of a conventional gene analysis device and the like and can accurately measure the sample solution. Objective.

  Another object of the present invention is to provide a sample analysis apparatus that can analyze the reaction between a solid sample and a liquid sample with a simple structure, not limited to the field of gene analysis.

  If the inventors of the present application select a gas-permeable material for a substrate (or a part thereof) having a plurality of reaction chambers, the sample can be put into the reaction chamber without directly connecting the pressure increasing / decreasing mechanism and the reaction chamber. If a solution can be introduced, and a part of the sample solution (various primers in the PCR method) is applied (spotted) in advance inside the reaction chamber and dried, it is transferred from the storage chamber to each reaction chamber. If the sample solution can be introduced with a simple structure, and these points are combined, problems with conventional gene analyzers can be solved at once, and highly reliable gene analysis can be achieved. As a result, the present invention has been completed.

  The inventors of the present application have found that the above idea can be applied not only to gene analysis but also to analysis in which a solid sample and a liquid sample are reacted, and have completed the present invention.

That is, the sample analyzer according to the present invention is
At least one storage chamber capable of containing a liquid sample;
At least one reaction chamber containing a solid sample;
A liquid introduction channel communicating the storage chamber and the reaction chamber;
A discharge chamber provided in the vicinity of the reaction chamber;
And having
A wall containing a gas permeable material is provided between the reaction chamber and the discharge chamber,
Due to the pressure difference generated between the reaction chamber and the discharge chamber, the gas in the liquid introduction flow path and the reaction chamber and the gas in the liquid sample are sucked into the discharge chamber through the wall and the liquid sample from the storage chamber to the reaction chamber. Is introduced and filled ,
The reaction chamber has a cylindrical shape,
The discharge chamber forms a groove concentrically surrounding the reaction chamber,
The wall thickness is in the range of 10 μm to 500 μm,
The pressure of the discharge chamber, for introducing a liquid sample into the reaction chamber, is characterized in Rukoto is set to 0.2 to 0.9 atm.

  According to the sample analyzer of the present invention, the reaction between a solid sample and a liquid sample can be analyzed with a simple structure.

  According to the present invention, it is possible to provide a sample analysis apparatus (for example, a PCR chip) that eliminates the drawbacks of conventional apparatuses, particularly in the field of gene analysis. Specifically, a PCR chip capable of analyzing a combination of a large number of samples and targets with a simple structure can be provided. In addition, it is possible to provide a PCR chip that eliminates problems such as residual bubbles and evaporation of the sample solution of a conventional genetic analyzer. Furthermore, in addition to the absence of residual bubbles and the like, the sample chamber can be filled to the full volume of the reaction chamber (ie, quantitative), so a PCR chip capable of PCR processing using a precisely weighed solution can be provided. Can be provided.

It is the figure which showed the invention concept (main structure) of the sample analyzer of this invention. It is the figure which showed the sample analyzer containing m types of liquid samples and n types of solid samples. It is the figure which showed the disassembled perspective view of the PCR chip for gene analysis which is one Example of this invention. Example 1 It is the figure which showed the decomposition | disassembly top view of the PCR chip | tip of this invention. It is the figure which showed the reaction chamber and discharge chamber which were provided in the PCR chip | tip of this invention. It is the figure which showed the sample solution which flows in into the reaction chamber of this invention. It is the figure which showed the valve mechanism provided in the PCR chip | tip of this invention. It is the graph which showed the relationship between wall thickness D of the wall which demarcates a reaction chamber and a discharge chamber, and sample solution introduction time. It is the figure which showed the cross-section of the PCR chip | tip which is another Example of this invention. It is the figure which showed the mode of the PCR chip | tip before and after a thermal cycle test. It is the figure which expanded and showed the reaction chamber of the PCR chip | tip before and after a thermal cycle test. It is the figure which showed the mode of the PCR chip after PCR execution. It is the fluorescence image which showed the reaction chamber of the PCR chip after PCR execution.

Hereinafter, although the present invention is explained based on an embodiment shown in a drawing, the present invention is not limited to the following concrete embodiment at all.

  FIG. 1 is a diagram showing an inventive concept (main structure) of a sample analyzer 1 of the present invention. The sample analysis apparatus 1 uses a sample composed of a liquid part (liquid sample) 2 and a solid part (solid sample) 3 and evaluates the chemical reaction by bringing both parts 2 and 3 into contact with each other. Here, the liquid sample 2 is, for example, a sample (template DNA, polymerase, dNTP, etc.) obtained by excluding those that specify the analysis site (primer, TaqMan (registered trademark) probe, etc.) from the PCR adjustment solution, genetic testing Sample, RNA test sample and the like, but are not necessarily limited thereto. On the other hand, the solid sample 3 includes, but is not necessarily limited to, one that specifies the analysis site as described above, for example, a primer serving as a DNA target, a TaqMan probe, a substrate-specific enzyme, and an antibody.

The sample analyzer 1 includes at least one storage chamber 32 (one in FIG. 1) capable of accommodating the liquid sample 2 and at least one reaction chamber (one in FIG. 1) in which the solid sample 3 is accommodated. ), A liquid introduction flow path 34 communicating the storage chamber 32 and the reaction chamber 35, and a discharge chamber 36 provided in the vicinity of the reaction chamber 35. The sample analyzing apparatus 1 further includes a wall W _p is provided that includes a gas permeable material between the reaction chamber 35 and the discharge chamber 36. The sample analyzer 1 then discharges the gas in the liquid introduction flow path 34 and the reaction chamber 35 and the gas in the liquid sample 2 through the wall W _p due to the pressure difference generated between the reaction chamber 35 and the discharge chamber 36. The liquid sample 2 is introduced into and filled in the reaction chamber 35 from the storage chamber 32 while being sucked into the chamber 36.

Moreover, the sample analyzer 1 of the present invention may further include a pressure difference generating mechanism that generates a pressure difference between the reaction chamber 35 and the discharge chamber 36. This pressure difference generating mechanism may be a pressure reducing mechanism that reduces the pressure in the discharge chamber 36 or a pressure mechanism that pressurizes the pressure in the reaction chamber 35. In the following embodiments and examples, the case where the pressure difference generating mechanism is a pressure reducing mechanism is described. As shown in the figure, the decompression mechanism includes, for example, a discharge port 33 and a discharge flow path 37 connected to the discharge port 33 and the discharge chamber 36, and the discharge chamber 36 is discharged from the discharge port 33 by a micro pump or the like (not shown). The discharge chamber 36 is decompressed by discharging the gas inside. Decompression mechanism, by reducing the pressure of the discharge chamber 36, the gas in the reaction chamber 35 through the wall W _p is aspirated and introduced and filled with the liquid sample 2 into the reaction chamber 35 from the storage chamber 32.

Here, the gas permeable material which can be used in wall W _p, for example, polydimethylsiloxane (PDMS), an acrylic polymer, silicone rubber, various elastomers, and the like. Here, PDMS is suitable for detection by an optical analysis method because it is a material having excellent transparency, and is suitable for biological sample analysis because of its biocompatibility. Since it has self-adsorbing properties, no special bonding process is required, it has excellent shape transferability, and it is also inexpensive, making the PCR chip structure and manufacturing method simpler and simpler at a lower cost. Is possible. Moreover, since it is excellent in heat resistance, various surface modifications are possible. Further, the reaction chamber 35, the discharge chamber 36, and the wall _Wp may be provided on one substrate 31, and the substrate 31 itself may be made of a gas permeable material (see FIGS. 3 and 4).

  1 shows only one storage chamber 32 and one reaction chamber 35 for convenience of explaining the concept of the present invention, but as shown in FIG. 2, a plurality (m in FIG. 2) of storage chambers 32 A plurality (n in FIG. 2) of reaction chambers 35 are preferably installed in the sample analyzer 1, and the liquid introduction flow path 34 is branched from one storage chamber 32 to the plurality of reaction chambers 35. It is preferable to provide the path 34a.

  With this configuration, a solid sample (for example, n types of target primers) 3 is applied to n reaction chambers 35 one by one, and m types of liquid samples (including cells and nucleic acids) are applied. 1 type of solution 2 is stored in the storage chamber 32 one by one, the combination of the m types × n types of liquid samples 2 and the solid samples 3 and the evaluation of the reaction can be performed in a single sample analysis apparatus 1 with a simple flow. This can be achieved with a road structure.

  For example, when analysis is performed using a solution containing cells in the liquid sample 2, a cell culture chamber may be provided on the liquid introduction channel 34 that communicates between the storage chamber 32 and the reaction chamber 35. The number of cells corresponding to the solid sample 3 applied to all the reaction chambers 35 can be cultured, and the liquid sample 2 can be introduced into each of the reaction chambers 35.

  The liquid introduction channel 34 is preferably provided with a valve mechanism 5 for opening and closing the channel, and as shown in FIG. 2, the valve mechanism 5 is preferably provided on each branch channel 34a. By adopting such a configuration, it is possible to eliminate the risk that the liquid sample 2 once introduced into the reaction chamber 35 flows back to or flows out of the storage chamber 32 or the adjacent reaction chamber 35. In addition, it is possible to ensure the independence of the reaction in each reaction chamber 35 in the thermal cycle necessary for PCR analysis.

Example 1
Next, a PCR chip for gene analysis will be described as an example of the sample analysis apparatus 1 of the present invention actually produced. FIG. 3 shows an exploded perspective view of the PCR chip 1. As illustrated, the PCR chip 1 includes at least one valve control layer 10, at least one channel layer 30, and a thin film layer 20 sandwiched therebetween. Hereinafter, the configuration and function of each layer will be described in detail.

(Configuration of sample analyzer)
FIG. 4A shows a plan view of the valve control layer 10. The valve control layer 10 includes, for example, a gas amount adjusting port 12, a plurality (five in FIG. 4A) of valve openings 14, and a gas on a flat substrate 11 made of polydimethylsiloxane (PDMS). A gas amount adjusting flow path 13 that communicates the amount adjusting port 12 and each valve opening 14 is provided.

FIG. 4B shows a plan view of the flow path layer 30. The flow path layer 30 is, for example, a storage chamber 32 in which the liquid sample 2 is temporarily stored on a flat substrate 31 made of polydimethylsiloxane (PDMS) (or a liquid sample supply connected to the storage chamber 32). Port), liquid introduction flow paths 34, a plurality (five in FIG. 4B) of reaction chambers 35, a plurality (a number corresponding to the number of reaction chambers 35, that is, five) of discharge chambers 36, A discharge channel 37 and a discharge port 33 are provided. The liquid introduction channel 34 is branched into a plurality of (five in FIG. 4B) branch channels 34a so that the liquid sample 2 is supplied from the storage chamber 32 to the reaction chamber 35. The reaction chamber 35 is thrust per Riniaru cavity branch channel 34a, to the portion of the inner surface of the cavity, the primer 3 to be targeted of the liquid sample 2 are pre spots are dried (In other words, the solid sample 3 is applied). As through the wall W _p that is part of the PDMS substrate 31 surrounding the reaction chamber 35, discharge chamber 36 is respectively provided on the substrate 31. The discharge chamber 36 is connected to a discharge flow path 37a, and the gas acquired in the discharge chamber 36 is discharged from the discharge port 33 through the discharge flow paths 37a and 37.

(Production method of PCR chip)
An example of a method for producing the PCR chip 1 having the above configuration will be described. First, SU8-2100 (manufactured by MICRO CHEM) is spin coated, soft baked, exposed, and the like on a silicon substrate to create a mold corresponding to the desired shape of each layer. Next, when PDMS is poured into a mold and baked under conditions of 75 ° C. and 90 minutes, the valve control layer 10 and the flow path layer 30 are completed. Since PDMS is self-adsorbing, the PCR chip 1 can be completed by combining the layers 10, 20, and 30 made of PDMS. Since the bonding force between the valve control layer 10 and the thin film layer 20 is weak when simply brought into physical contact with each other, the surface wettability is improved, and O ₂ can be chemically modified. It is preferable to further add a plasma treatment to improve the bonding force.

(Liquid sample introduction method utilizing reaction chamber structure and gas permeability)
Next, as an example, a specific structure of the reaction chamber 35 in the PCR chip 1 and its peripheral part will be described with reference to FIG. 5A shows a perspective view of the reaction chamber 35 and its peripheral part, and FIG. 5B shows a plan view of the reaction chamber 35 and its peripheral part.

The illustrated reaction chamber 35 is provided on the substrate 31 and has a cylindrical shape with a bottom. The branch flow path 34 a for transporting the liquid sample 2 to each reaction chamber 35 has a groove shape having a rectangular cross section and extends toward the center of the cylinder of the reaction chamber 35. The discharge chamber 36 is provided outside the reaction chamber 35 so as to concentrically surround the cylindrical reaction chamber 35 and has a groove shape having a rectangular cross section. Between the reaction chamber 35 and the discharge chamber 36 is partitioned by a wall W _p having a wall thickness D. The discharge flow path 37 a connected to the discharge chamber 36 is a groove having a rectangular cross section, and extends away from the reaction chamber 35.

  The upper side of each element (the reaction chamber 35, the discharge chamber 36, the branch flow path 34a, etc.) formed on the substrate 31 of the flow path layer 30 described in the present embodiment is covered with the thin film layer 20 and sealed. .

The discharge chamber 36 is normally filled with a gas (for example, air). At the time of analysis, a micropump (not shown) connected to the discharge port 33 sucks air in the discharge chamber 36, thereby reducing the pressure of the discharge chamber 36. Is done. Then, since the wall W _p is gas permeable, the gas (for example, air) in the reaction chamber 35 and the branch flow path 34a is passed through the wall W _p , the discharge chamber 36, the discharge flow path 37a, and the discharge port 33. It is discharged outside the PCR chip 1. Thereby, the liquid sample 2 is introduced into the reaction chamber 35 in which the primer 3 is preliminarily applied to a part of the inner surface, bubbles in the liquid sample 2 are removed, and only the liquid sample 2 is filled in the reaction chamber 35. Thus, a very small amount of the liquid sample 2 weighed more precisely can be introduced into the reaction chamber 35. FIG. 6 is an image showing a state in which the liquid sample 2 stained with methyl green is introduced and filled into the actual reaction chamber 35.

8, the wall thickness D (unit: mm) of the wall W _p between the reaction chamber 35 and the discharge chamber 36 and the time until the liquid sample 2 is completely introduced into the reaction chamber 35 (in seconds) FIG. Here, the differential pressure ΔP caused to the wall _{W p} was set at about 0.5 atmospheres. As can be seen from this figure, the larger the wall thickness D, the longer the introduction time. When D = 0.6 mm, it takes about 1400 seconds (about 23 minutes). If the value of the differential pressure ΔP is increased, the introduction time is reduced (the plot line shown in the figure decreases overall), and if the value of the differential pressure ΔP is reduced, the introduction time is further increased (the plot line shown is overall. Going up). In addition, since the suction pressure of the micropump normally used for the PCR chip 1 at present is 0.2 to 0.9 atm, the wall thickness D is preferably set in the range of 10 μm to 500 μm. If it is less than 10 μm, it is easily broken by reduced pressure.

  The preferred diameter φ of the cylindrical reaction chamber 35 of Example 1 is preferably in the range of φ = 20 μm to 5 mm. When the diameter φ is less than the lower limit, the influence of the evaporation of the liquid sample 2 becomes significant, and signals such as fluorescence are not sufficiently detected. When the diameter φ is larger than the upper limit, a large number of reaction chambers 35 are included in the microfluidic PCR chip. Can no longer be installed.

  By the way, in order to promote introduction of the liquid sample 2, as shown in FIG. 4B, a branch channel 34b having one end connected to the liquid introduction channel 34, and a branch having the other end connected to the branch channel 34b. The chamber 34c, the discharge chamber 36b surrounding the branch chamber 34c, and the discharge flow path 37b communicating the discharge chamber 36b and the discharge flow path 37 may be further provided. As a result, the liquid sample 2 can be pulled more quickly and more uniformly to the respective inlets of the branch flow paths 34 a connected to the reaction chamber 35.

(Configuration and operation of valve mechanism)
Next, an example of the valve mechanism 5 of the PCR chip 1 of the present invention will be specifically described. The valve mechanism 5 achieves a desired function by the structure provided in the valve control layer 10, the thin film layer 20, and the flow path layer 30 (see FIGS. 3, 4A, and 7). . As shown in FIG. 7, for example, a valve opening 14 that forms a cubic space is formed in the valve control layer 10, and a thin film layer 20 and a branch channel 34 a are laid below the valve opening 14. . The branch channel on 34a, the corresponding positions of the valve opening 14, an area is smaller than the valve opening 14 solid cubic body-shaped projections 34 _v are provided.

For example, before PCR analysis, after introduction / filling of the liquid sample 2 into the reaction chamber 35, or at the time of PCR thermal cycle, the flow rate adjustment means (not shown) passes through the gas amount adjustment port 12 and the gas amount adjustment flow path 13. Gas (for example, air) flows into the valve opening 14 and the valve opening 14 covered with the thin film layer 20 is pressurized. Thus, as shown in FIG. 7 (b), the thin film layer 20 is fixed on the upper surface of the protrusion 34 _v deflect the flow channel layer 30 side, and thus, to close the branch passage 34a at the position of the protrusion 34 _v become.

  On the other hand, when the liquid sample 2 is introduced into the reaction chamber 35, the gas in the valve opening 14 is passed through the gas amount adjusting flow path 13 and the gas amount adjusting port 12 by a flow rate adjusting means (not shown). The valve opening 14 discharged from 1 and covered with the thin film layer 20 is decompressed. As a result, as shown in FIG. 7A, the thin film layer 20 is bent to the valve control layer 10 side and fixed on the inner upper surface of the valve opening 14, and as a result, the branch flow path 34a is opened.

  For example, if the valve control layer 10, the thin film layer 20, and the flow path layer 30 are made of a substrate having the same plane as shown in FIG. 4, the valve opening 14 and the branch flow path 34 can be easily aligned. The valve mechanism 5 can be formed (see FIG. 4C).

  Although the method for introducing the liquid sample 2 into the reaction chamber 35 using gas permeability has been described with a specific configuration, the shape and structure of elements such as the reaction chamber 35, the discharge chamber 36, the valve opening 14 and the like have been described. Is not limited to the above examples. For example, the discharge chamber 36 is not limited to a groove shape that concentrically surrounds the reaction chamber 35, and may be a space that is vertically separated from the reaction chamber 35 by a predetermined distance.

(Example 2)
Next, another embodiment of the present invention will be described. FIG. 9A shows a cross-sectional structure of the PCR chip 1 of Example 1, and FIG. 9B shows a cross-sectional structure of the PCR chip 1 of Example 2. The configuration is the same as that of the first embodiment except that the valve control layer 10 and the flow path layer 30 are each divided into two layers and the gas barrier layers 40a, 40b, 40c, 40d, 40e, and 40f are inserted between the layers. Therefore, the description of other configurations is omitted.

Each layer 10, 20, 30 of the first embodiment is made of PDMS as described above and allows gas to pass therethrough. For example, the gas compressed by the valve mechanism 5 passes through the substrate 11 and the thin film layer 20, and branches. 34a, when bubbles are mixed into the liquid sample 2 flowing through the reaction chamber 35 or the like, or when the liquid sample 2 evaporates during the PCR thermal cycle, the gas leaks from the upper and lower substrates 11 and 31 before being introduced into the reaction chamber 35. May end up. Accordingly, any one of the gas barrier layers 40 (40a, 40b, 40c, 40d, 40e, and 40f in the figure) is laid at a position between the respective layers as shown. Examples of the material of the gas barrier layer 40 include parylene, PVA, unsaturated polyester, HDPE, and glass. As shown by reference numeral 40f in FIG. 9, when the gas barrier layer 40 is provided outside the PCR chip 1, it is preferable to use hard glass. In addition, since PVA is a soft material, when using PVA as the gas barrier layer 40, it is preferable to arrange | position between the board | substrates 11a and 11b of the valve control layer 10, or between the board | substrates 31a and 31b of the flow-path layer 30 (code | symbol of a figure). 40a, 40e). When the gas barrier layer 40 is bonded to the other layers 10, 20, and 30, it is preferable to perform O ₂ plasma treatment.

(Example 3)
Except for adding the evaporation inhibitor 4 to the liquid sample 2, the configuration is the same as that of the first embodiment. Examples of the evaporation inhibitor 4 include glycerol, ethylene glycol, sucrose, and trehalose. In the thermal cycle in PCR, there is a step of heating to around 95 ° C., and at this time, the liquid sample 2 evaporates, and the amount of the liquid sample 2 essential for PCR detection may not be obtained. However, by adding the evaporation inhibitor 4 as described above to the liquid sample 2, it becomes possible to raise the boiling point of the liquid sample 2 by several degrees Celsius, and a sufficient amount of the liquid sample 2 can be obtained even after the thermal cycle is completed. It can remain in the reaction chamber 35.

Example 4
The gas barrier layer 40 of Example 2 is added to the apparatus configuration of Example 1, and the evaporation inhibitor 4 of Example 3 is added to the liquid sample 2.

(Thermal cycle test results)
FIG. 10 shows the results of a thermal cycle test using the PCR chip 1 of Example 1. FIG. 10A shows an image of the reaction chamber 35 of the PCR chip 1 before the thermal cycle test. For the liquid sample 2, uncolored water was used. As is apparent from this figure, it can be seen that the liquid sample 2 is completely filled in the reaction chamber 35, respectively. FIG. 10B shows an image of the reaction chamber 35 after the thermal cycle test. Here, the thermal cycle test includes step 1 in which the PCR chip 1 is held at 95 ° C. for 10 minutes, step 2 in which the PCR chip 1 is held at 95 ° C. for 15 seconds and then held at 65 ° C. for 60 seconds, and 4 ° C. This is a test consisting of step 3 maintained in step 3, and repeating step 2 30 times. From the image of FIG. 10B, it can be confirmed that although the liquid sample 2 remains in the reaction chamber 35, the amount of the liquid sample 2 is almost half.

  FIG. 11 shows the results of a thermal cycle test using the PCR chip 1 of Example 4. FIGS. 11A and 11B show images of the reaction chamber 35 of the PCR chip 1 before and after the thermal cycle test. The content of the thermal cycle test is the same as the test content of Example 1 described above. The liquid sample 2 was water colored with methyl green. As is clear from comparison of these figures, the liquid sample 2 was not evaporated even after the thermal cycle test was completed, and bubbles were not confirmed.

(PCR test results)
FIG. 12 shows an image of the reaction chamber 35 after the PCR test using the PCR chip 1 of Example 2. As a liquid sample 2, 10 × Buffer (5 μl), 2 mM each (M = mol / l) dNTP (5 μl), 25 mM MgCl ₂ (7 μl), 5 U / μl (U = units) DNA polymerase (Ampli) A solution containing Taq Gold (registered trademark) (1.25 μl), 10 ng / μl human gene DNA (1 μl), and water (30.75 μl) was prepared and supplied to the PCR chip 1. In the reaction chamber 35 of the PCR chip 1, a solution containing 3 μM forward primer, 3 μM reverse primer, and 2 μM TaqMan probe (Beta Actin Probe) is spotted in advance as the solid sample 3. (See Table 1 below for details on each component of the liquid sample 2 according to Example 2). This PCR chip 1 was subjected to the following PCR treatment. Specifically, the PCR process includes step 1 of holding at 95 ° C. for 10 minutes, step 2 of holding the PCR chip 1 at 95 ° C. for 15 seconds, then holding 60 ° C. for 60 seconds, and step of maintaining at 25 ° C. This test consists of 3 and repeats step 2 30 times. As is apparent from the image of FIG. 12, it can be seen that the liquid sample 2 remains in the reaction chamber 35 after the PCR process, but is somewhat evaporated during the PCR process.

FIG. 13 shows a fluorescence image of the reaction chamber 35 after performing a PCR test using the PCR chip 1 according to Example 4. As the liquid sample 2 according to Example 4, the liquid sample 2 in FIG. 13B has almost the same components as the liquid sample 2 described in Example 2, but the liquid sample 2 according to Example 2 In place of the water (30.75 μl) added in step 1, a solution containing glycerol (that is, the evaporation inhibitor 4) and water (water: 15 μl, glycerol: 15.75 μl, 30.75 μl in total) was added. The only difference is that On the other hand, the liquid sample 2 in FIG. 13A removes the human gene DNA (1 μl) contained in the liquid sample 2 according to Example 2 (see Table 1) for comparison with FIG. 13B. The removed amount was also replaced with water (thus, in comparison with the water in Example 2, in FIG. 13A, water: 16 μl, glycerol: 15.75 μl, 31.75 μl in total). The PCR process is the same as that described in FIG. 13A and 13B, it can be seen that the reaction chamber 35 is filled with the liquid sample 2 in its entire volume even after the PCR process. In FIG. 13A, the liquid sample 2 does not emit fluorescence, but in FIG. 13B, the liquid sample 2 emits fluorescence. This is because PCR was not performed in the reaction chamber 35 shown in FIG. 13 (a) because no human gene DNA containing the beta actin gene was present, and the reaction chamber 35 shown in FIG. The human gene DNA containing the beta-actin gene exists, a PCR reaction occurs, a specific gene region is amplified, and as a result, the reaction of the fluorescent substance appears in the fluorescence image by the Taqman probe. Thereby, it turns out that the presence or absence of a specific gene arrangement | sequence can be analyzed using the sample analyzer 1 of this invention.

  As described above, according to the sample analyzer of the present invention, the reaction between the solid sample and the liquid sample can be analyzed with a simple structure.

  According to the present invention, it is possible to provide a sample analysis apparatus (for example, a PCR chip) that eliminates the drawbacks of conventional apparatuses, particularly in the field of gene analysis. Specifically, a PCR chip capable of analyzing a combination of a large number of samples and targets with a simple structure can be provided. In addition, it is possible to provide a PCR chip that eliminates problems such as residual bubbles and evaporation of the sample solution of a conventional genetic analyzer. Furthermore, in addition to the absence of residual bubbles and the like, the sample chamber can be filled to the full volume of the reaction chamber (ie, quantitative), so a PCR chip capable of PCR processing using a precisely weighed solution can be provided. Can be provided.

  INDUSTRIAL APPLICABILITY The present invention can be used in gene analysis research and clinical diagnostic fields using a microfluidic chip, such as a PCR chip, in addition to analysis of various chemical reactions in which a solid sample and a liquid sample are reacted. In particular, application to fetal diagnosis using nucleated red blood cells is expected.

1 Sample analyzer (gene analyzer, PCR chip)
2 Liquid sample 3 Solid sample (primer)
4 Evaporation inhibitor 5 Valve mechanism 10 Valve control layer 11 Substrate 12 Gas amount adjusting port 13 Gas amount adjusting channel 14 Valve opening 20 Thin film layer 30 Channel layer 31 Substrate 32 Storage chamber 33 Discharge port 34 Liquid introduction channel 34a Branch passage 34 _v branched flow projections path 35 reaction chamber 36 exhaust chamber 37 discharge channel 40 (40a, 40b, 40c, 40d, 40e, 40f) gas barrier layer W _p wall

Claims (8)

At least one storage chamber capable of containing a liquid sample;
At least one reaction chamber containing a solid sample;
A liquid introduction channel communicating the storage chamber and the reaction chamber;
A discharge chamber provided in the vicinity of the reaction chamber;
And having
A wall containing a gas permeable material is provided between the reaction chamber and the discharge chamber,
Due to the pressure difference generated between the reaction chamber and the discharge chamber, the gas in the liquid introduction channel and the reaction chamber and the gas in the liquid sample are sucked into the discharge chamber through the wall, and The liquid sample is introduced and filled from the storage chamber to the reaction chamber ,
The reaction chamber has a cylindrical shape,
The discharge chamber has a groove concentrically surrounding the reaction chamber;
The wall thickness is in the range of 10 μm to 500 μm,
The pressure of the discharge chamber, for introducing the liquid sample into the reaction chamber, the sample analyzing apparatus according to claim Rukoto is set to 0.2 to 0.9 atm.   The pressure difference generating mechanism for generating a pressure difference between the reaction chamber and the discharge chamber is further provided, and the pressure difference generating mechanism is a pressure reducing mechanism for decreasing the pressure of the discharge chamber. 2. The sample analysis apparatus according to 1.   The sample analysis apparatus according to claim 1, wherein the liquid introduction flow path includes a branch flow path that branches from one storage chamber to a plurality of the reaction chambers.   The sample analysis apparatus according to claim 1, wherein the liquid introduction channel includes a valve mechanism that opens and closes the channel.   The sample analysis apparatus according to claim 1, wherein an evaporation inhibitor is added to the liquid sample.   The sample analysis apparatus according to claim 1, wherein a gas barrier layer is provided in the vicinity of the liquid introduction channel and the reaction chamber, and no gas barrier layer is provided on the wall. The sample analysis apparatus according to claim 1, wherein the gas permeable material is polydimethylsiloxane. The liquid sample includes a nucleic acid solution,
The solid sample includes a primer pre-applied to the reaction chamber and dried,
The sample analysis apparatus according to claim 1, wherein the sample analysis apparatus is used for PCR analysis.