JP2014070991A - Biochemical reaction chip for analyzing plural samples, and analysis device thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a low cost sample analysis chip capable of performing simultaneous processing of a plurality of biochemical substances by a simple liquid feed method.SOLUTION: Disclosed is a sample analysis chip whose center of a circle does not exist on a base material when arranged in concentric circles on a device processing stage in spite of the fact that the base material is a right-left asymmetrical non-fan shape along a biochemical reaction processing structure, and a device for processing the sample analysis chip is provided. Processing is capable of being efficiently performed in a space saving manner by arrangement of the sample analysis chip in concentric circles.

Description

  The present invention relates to a biochemical reaction processing chip and an analysis method thereof.

  Conventionally, in the field of biochemical reactions such as DNA reaction and protein reaction, as a reaction apparatus for processing a small amount of sample solution, a technique called μ-TAS (Total Analysis System) or Lab-on-Chip is known. Yes. This is a single chip or cartridge provided with a plurality of reaction chambers (hereinafter referred to as wells) and channels, and can analyze a plurality of specimens or perform a plurality of reactions. These techniques have been considered to have various merits by reducing the amount of chemicals handled by downsizing the chip and cartridge.

  The benefits include, for example, the fact that the amount of strong acids and strong alkaline chemicals that have been used in the past has been reduced to a much lower level, and the impact on the human body and the environment will be significantly reduced. For example, the amount of reagents consumed can be reduced so that the cost of analysis and reaction can be reduced.

  In order to perform biochemical reactions most efficiently using chips and cartridges, different types of chemicals, specimens, and enzymes are placed in multiple wells, and one or more reagents that react with these chemicals, specimens, and enzymes are used. It is necessary to pour into several wells from several main conduits to produce different reactions.

  By using this method, multiple types of specimens can be processed simultaneously with the same reagent, and conversely, multiple types of specimens can be processed simultaneously. This saves time, effort, and cost. It can be greatly reduced.

In addition, if the sample or reagent used for the reaction is a dangerous substance or an infectious biological sample, it is preferable to keep it in this chip as much as possible, and it will leak out of the chip by evaporation or volatilization. Things must also be prevented.

  When using this type of technique, a technique for feeding an equal amount of sample to a plurality of reaction fields and a technique for preventing the contents of each well from being mixed are important. The following is mentioned as a prior art about the chip | tip which performs liquid feeding to such a well.

  Japanese Patent Application Laid-Open No. H10-228561 shows a method of sending a liquid to a process chamber using centrifugal force.

  Patent Document 2 shows a sample fraction type centrifugal liquid feeding system having a sample loading region.

Patent Document 3 shows a centrifugal liquid feeding system in which structures are radially formed.

Japanese Patent No. 4181406 Japanese Patent No. 4499720 JP-T-2004-529333

  However, Patent Document 1 shows a method of sending a liquid to a process chamber using centrifugal force. In this method, after introducing the liquid into the process chamber, each flow path leading to the process chamber is physically set. It needs to be crushed and closed, and is not suitable for automation or downsizing, and requires complicated work by human hands. Even if the other end of the loading port is opened, a method for discarding excess liquid is necessary, which is not preferable for dangerous chemicals or infectious biological samples.

  Patent Document 2 shows a sample fraction-type centrifugal liquid feeding system having a sample loading region. However, since both ends of the channel are designed for sample loading, evaporation of sample and reagent such as heat treatment is performed. In order to prevent this, it is necessary to put a lid, and even in the examples, it is necessary to apply a film by a technique. Moreover, since the sample fraction structure is radial, the area of the base material cannot be effectively used, and the size is increased.

  Further, Patent Document 3 shows a centrifugal liquid feeding system in which structures are radially formed. However, unnecessary sample solution is released from an open waste port, and biochemical reaction is performed. When performing, there is a possibility that the processing sample before and after may cause contamination, and it is impossible to use dangerous chemicals or processing using an infectious biological sample.

  The present invention has been made in view of the above-described circumstances, and its object is to provide a simple liquid feeding method with little variation in the amount of liquid in each well, and a plurality of chemical reactions and enzyme reactions by heating and cooling or heating. It is possible to provide a low-cost sample analysis chip that can be performed simultaneously on a plurality of samples.

In order to solve the above problems, the present invention proposes the following means.
The sample analysis chip of the present invention includes a plurality of biochemical processing structures on a base material, and the biochemical processing structure has a mechanism for feeding a sample liquid by rotation of the base material. The processing structure has at least a plurality of reaction wells, a main channel for supplying a sample to the reaction well, a side channel for communicating the main channel and the reaction well, and a sample feeding unit for supplying the sample to the main channel And a surplus liquid reservoir for holding the surplus sample, and an air vent passage that is open to the atmosphere above the surplus liquid reservoir, and the main flow path is disposed closer to the center of rotation than the reaction well. It is characterized in that the biochemical treatment structure is arranged concentrically on the substrate.

Further, it is preferable that the distance from the center of the rotation is short in the order of the surplus liquid storage part, the sample liquid supply part, the main flow path, and the reaction well.

Moreover, it is preferable that the said excess liquid storage part is gathered and formed in the rotation center part.

Further, the reaction well is arranged on the outermost peripheral portion of the base material, whereby a fluorescent sample, a dye or the like can be measured from the side surface of the base material. The outermost peripheral part means that there is no structure obstructing the measurement from the reaction well to the side surface of the substrate.

The reaction well is preferably arranged within 3 mm from the outer periphery.

  The main flow path preferably has a wave-like peak near the rotation center and a valley far from the rotation center, and the flow path width is relatively small at the peak and relatively large at the valley.

  Further, the corrugated shape of the main channel does not need to be a symmetrical shape, and the sample analysis chip of the present invention can be made small by effectively using the surface area of the substrate by forming it into an inclined mountain shape. It becomes possible.

  The wells are preferably arranged concentrically on the substrate.

  Further, it is preferable that the biochemical treatment structure is arranged concentrically on the base material, and the air release end of the sample liquid feeding part is also arranged concentrically.

  In the sample analysis chip of the present invention, it is preferable that a support for rotating the substrate and feeding the sample solution by centrifugal force is provided on the substrate.

  Moreover, it is preferable to have the support part for the said centrifugation in the outer peripheral part of the said base material.

  Moreover, it is preferable that the sample analysis chip of the present invention includes a first base material in which the well and the main channel are formed on one side, and a second base material bonded to the first base material.

  Moreover, it is preferable that both the sample liquid feeding part and the air vent passage are open to the atmosphere on the surface opposite to the surface on which the main flow path is formed, and open upward to the atmosphere. “Done” means that the groove is formed with the groove shape of the base material facing downward, and there is an air vent upward.

  Moreover, it is preferable that the area of the cross section orthogonal to the flow direction of the sample solution in the surplus liquid storage part is larger than the area of the cross section orthogonal to the flow direction of the sample solution in the main channel.

  Further, the volume of the surplus liquid reservoir is preferably larger than the volume of the main channel, but the effect of the present invention can be utilized if a capacity for holding the surplus sample solution is obtained.

  Further, the volume of the excess liquid storage part is larger than the volume of the first buffer part, and the sum of the volume of the first buffer part and the volume of the excess liquid storage part is larger than the volume of the main flow path. Although it is preferable, it is not limited to this by the reason mentioned above.

  Further, it is a wall shape surrounding the other end of the sample liquid feeding section and the other end of the air vent passage, and after the sample solution is injected into the flow path, the other end of the sample liquid feed section and the air vent path It is preferable to have a lip portion that can be deformed by an apparatus or a jig and that can keep the flow path in a sealed state for the purpose of closing the other end.

  Moreover, it is preferable that the said side path is inclined with respect to the straight line which connects the well to which the said side path was connected among the said several wells, and the rotation center of the said base material.

  Moreover, it is preferable that at least any one of said 1st base material and said 2nd base material is formed with the light transmissive material.

  Moreover, it is preferable that said 1st base material is a transparent resin material, and said 2nd base material is a metal material.

  The sample analysis method of the present invention is a sample analysis method using the sample analysis chip of the present invention, the step of injecting the sample solution into the main channel, and rotating the base material to each of the sample solutions. And a step of distributing the liquid to the well.

  Moreover, it is preferable to have the process of distributing mineral oil to each said well after the process of distributing the said sample solution to the said well.

  The sample processing method of the present invention includes a step of injecting the sample into the main channel, a step of rotating the base material to distribute the solution to the wells, and a step of measuring the wells. This is a sample processing method.

  The gene analysis method of the present invention is a gene analysis method characterized by using the sample analysis method of the present invention.

The sample analysis chip of the present invention can efficiently process a plurality of samples simultaneously or sequentially, and can produce a small and low-cost analysis chip.

  The sample analysis chip of the present invention is simple in liquid feeding method, has little variation in the amount of liquid in each well, can suitably perform chemical reaction and enzyme reaction by heating and cooling and heating, and is low in cost.

  Moreover, according to the sample analysis method and gene analysis method of the present invention, analysis with high analysis accuracy and reproducibility can be performed.

  In addition, according to the sample analysis method and gene analysis method of the present invention, it is possible to prevent contamination between samples during analysis.

  In addition, according to the sample analysis method and the gene analysis method of the present invention, dangerous chemicals and infectious biological samples can be safely processed and analyzed.

It is a top view of the 1st surface which shows the sample analysis chip of one embodiment of the present invention. It is a top view of the 2nd surface which shows the sample analysis chip of one embodiment of the present invention. It is a perspective view of the 1st surface side of the sample analysis chip. It is a perspective view of the second surface side of the sample analysis chip. It is a top view of the 1st surface which shows the sample analysis chip of one embodiment of the present invention. It is a top view of the 2nd surface which shows the sample analysis chip of one embodiment of the present invention. It is a perspective view of the 1st surface side of the sample analysis chip. It is a perspective view of the second surface side of the sample analysis chip. It is a block diagram which shows an example of the analysis method of the sample analysis chip | tip in this invention. It is a schematic diagram which shows an example of the analysis method in this invention. It is a block diagram which shows an example of the analysis method of the sample analysis chip | tip in this invention. It is a schematic diagram which shows an example of the analysis method in this invention. It is a simplified sectional view of one embodiment of a sample analysis chip in the present invention.

  A sample analysis chip according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a plan view of a first surface showing a uniform state of the sample analysis chip of the present embodiment. FIG. 2 is a plan view of a second surface showing a uniform state of the sample analysis chip of the present embodiment.

Next, the manufacturing method of the sample analysis chip of the present invention will be described.
First, as shown in FIG. 1, a base material (first base material) 101 having a reaction well 104 and a flow path (including a main flow path 106 and a side path 105) is formed by molding using a molding die.

  The base material 101 has a groove-like shape including a well 104, a main channel 106, a side channel 105, a sample liquid feeding unit 102, an air vent channel 103, and an excess liquid storage unit 107 after manufacturing on one surface in the thickness direction. Has been. Here, the sample liquid feeding section 102 and the air vent passage 103 are also opened on the other surface in the thickness direction of the substrate 101. Further, a lip portion (not shown) having a wall shape surrounding the sample liquid feeding portion 102 and the air vent passage 103 may be integrally formed on the other surface.

  Moreover, when the thickness of the base material 101 exists in the range of 100 micrometers-3000 micrometers, both the heat conductivity of the base material 101 and sealing performance can be satisfied suitably. When the thickness of the base material 101 is larger than 3000 μm, the heat capacity becomes large and the thermal responsiveness may be lowered.

  As a material for the base material 101, a resin having optical transparency can be suitably used. Further, when optical analysis (fluorescence measurement, colorimetric measurement, etc.) is performed on the solution in the well 104, it is preferable that the substrate 101 has high transparency. For example, the material of the base material 101 is not particularly limited as long as it does not affect the sample. However, if a resin material containing any of polypropylene, polycarbonate, and acrylic is used, good visible light transmission is ensured. can do. As polypropylene, homopolypropylene or a random copolymer of polypropylene and polyethylene can be used. Moreover, as an acryl, the copolymer of monomers, such as polymethyl methacrylate or methyl methacrylate, and other methacrylic acid ester, acrylic acid ester, styrene, can be used. Moreover, when using these resin materials, the heat resistance and intensity | strength of a chip | tip can also be ensured. Examples of the material other than the resin material include aluminum, copper, silver, nickel, brass, and gold. When a metal material is used, in addition, it has excellent thermal conductivity and wear resistance.

  In addition, by making the bottom part of the well 104 of the base material 101 or the wall part of the outer peripheral direction transparent, detection / analysis of fluorescence etc. can be performed from the outside. In the present invention, “transparent” and “light-transmitting” mean that the total average transmittance in the visible light region (wavelength 350 to 780 nm) when formed is 70% or more.

  As a processing method of the substrate 101, in the case of a resin material, various resin molding methods such as injection molding and vacuum molding, mechanical cutting, and the like can be used. In the case of a metal material, it can be formed by grinding or etching using a thick base material, or pressing or drawing a thin metal sheet.

  In addition, when a resin material including any of polypropylene, polycarbonate, and acrylic is used as the base material 101, good light transmittance, heat resistance, and strength can be ensured.

  5, 6, 7, and 8 show another embodiment of the sample analysis chip of the present invention.

  In another embodiment of the present invention, the reaction well 104 is not circular, and the end of the side passage 105 is used as it is as a reaction well.

  Three main flow paths 106 are arranged in the base material, and each has a sample introduction part 102, an air vent passage 103, and an excess liquid storage part 107, and three samples can be analyzed in one base material.

  FIG. 9 shows an embodiment for processing the sample analysis chip of the present invention.

In one analysis mode of the present invention, the upper and lower portions of the well portion 104 are sandwiched by the temperature adjustment member 902, and the fluorescence wavelength generated by the reaction can be read by the photometric device 901.
In FIG. 9, the temperature adjustment member 902 is on the front and back of the chip 1000, but either one may be used.

FIG. 10 is a schematic diagram of a photometric method of an analysis form of the present invention. By forming the outer peripheral shape of the sample analysis chip of the present invention into a corrugated shape, excitation into the well and emission surface from the well are formed. It can be secured greatly.

FIG. 11 is a perspective view of another embodiment for processing the sample analysis chip of the present invention.

FIG. 12 is a schematic plan view of another embodiment for processing the sample analysis chip of the present invention. The photometric device 1001 is scanned along the arrow to acquire electromagnetic waves including fluorescence from within the well, and the temperature adjustment member 1002 has a shape along the outer periphery of the sample analysis chip. Heating and rapid cooling can be performed.

In one analysis mode of the present invention, the photometric device 1001 is circularly arranged along the sequence of the reaction well 104 while the second surface and side surfaces of the well portion 104 are temperature-controlled by the temperature adjustment member 1002 along the outer peripheral shape of the sample analysis chip. It is possible to acquire the fluorescence wavelength and the like by scanning in the circumferential orbit.

  FIG. 13 is a simplified cross-sectional view of one embodiment of the present invention, and is a schematic view with the second surface up from the sample introduction part 102 to the air vent passage 103 through the main flow path.

The sample liquid supply port 102 may have a cone shape as shown in FIG. 13B when liquid is supplied by a member for supplying liquid, for example, by a pipette.

Further, it is desirable that the surplus liquid reservoir 107 has a sufficiently large cross-sectional area with respect to the main channel 106 and also extends in the height direction.

It is possible to prevent the solution from flowing upward by making a lip portion 501 for covering the sample liquid feeding portion 102 and the air hole 103, and minimize the movement of the internal liquid during the reaction. However, it is not necessary to prepare a mechanism that serves as a lid as a processing device or a separate member, or when a solution other than the reaction solution does not cause a problem.

  The reaction chip of the present invention can be used for detection and analysis of biochemical substances in samples such as nucleic acids. In particular, it can be used for detection of mutations in SNPs and methods for detecting mutations in genes such as cancer, germ cells and somatic cells. Further, it can be used as a container or a reaction container for mixing a plurality of solutions.

DESCRIPTION OF SYMBOLS 101 ... Base material 102 ... Sample liquid feeding part 103 ... Air vent passage 104 ... Well 105 ... Side passage 106 ... Main flow path 107 ... Excess liquid storage part

Claims (19)

  Provided with a plurality of biochemical treatment structures on a substrate, the biochemical treatment structure has a mechanism for feeding a sample liquid by rotation of the substrate, and the one biochemical treatment structure has at least a plurality of reactions. A well, a main channel for supplying the sample to the reaction well, a side channel for communicating the main channel and the reaction well, a sample liquid feeding unit for supplying the sample to the main channel, and for holding an excess sample A surplus liquid reservoir, and an air vent passage that is open to the atmosphere above the surplus liquid reservoir, wherein the main channel is disposed closer to the center of rotation than the reaction well. A sample analysis chip, characterized in that the treatment structures are arranged concentrically on the substrate. 2. The sample analysis chip according to claim 1, wherein a distance from the center of the rotation is short in the order of an excess liquid storage part, a sample liquid supply part, a main flow path, and a reaction well. 2. The sample analysis chip according to claim 1, wherein the surplus liquid storage part is formed by being gathered at a rotation center part. 3. 2. The sample analysis chip according to claim 1, wherein the reaction well is arranged on the outermost peripheral portion of the base material, whereby a fluorescent sample, a dye, or the like can be measured from the side surface of the base material. The sample analysis chip according to claim 1, wherein the reaction well is disposed within 3 mm from the outer periphery.   The main flow path has a peak part close to the rotation center in a corrugated form and a valley part far from the rotation center, and the flow path width is relatively small at the peak part and large at the valley part, The sample analysis chip according to claim 1.   The sample analysis chip according to claim 1, wherein the corrugated shape of the main channel is formed in an inclined mountain shape.   The sample analysis chip according to claim 1, wherein the wells are arranged concentrically on the substrate.   2. The sample analysis chip according to claim 1, wherein the biochemical treatment structure is concentrically arranged on a base material, and an open end of the sample liquid feeding unit is also concentrically arranged.   2. The sample analysis according to claim 1, wherein the sample analysis chip is provided with a support for rotating the substrate and feeding the sample solution by centrifugal force. Chip.   The sample analysis chip according to claim 1, further comprising a supporting portion for the centrifugation on an outer peripheral portion of the base material.   The sample analysis chip has a first base material in which the well and the main flow path are formed on one side, and a second base material bonded to the first base material. The sample analysis chip described.   2. The sample according to claim 1, wherein both the sample liquid feeding section and the air vent passage are open to the atmosphere on a surface opposite to a surface on which the main flow path is formed. Analysis chip.   The area of the cross section orthogonal to the flow direction of the sample solution in the surplus liquid storage part is larger than the area of the cross section orthogonal to the flow direction of the sample solution in the main flow path. Sample analysis chip.   A wall shape surrounding the other end of the sample liquid feeding portion and the other end of the air vent passage, and having a lip portion that is elastically deformed and can keep the flow path sealed. Item 8. The sample analysis chip according to Item 1.   The said side path is inclined with respect to the straight line which connects the well to which the said side path was connected among these wells, and the rotation center of the said base material, It was characterized by the above-mentioned. Sample analysis chip.   The sample analysis chip according to claim 1, wherein at least one of the first base material and the second base material is formed of a light transmissive material.   The sample analysis chip according to claim 1, wherein the first base material is a light-transmitting resin material, and the second base material is a metal material.   A processing method using the sample analysis chip according to any one of claims 1 to 18, wherein the sample is injected into the main channel, and the substrate is rotated to put the solution into the wells. A sample processing method comprising a step of liquid distribution and a step of photometrically measuring each of the wells.

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