JP5521454B2 - Sample analysis chip, sample analysis apparatus and sample analysis method using the same - Google Patents

Description

  The present invention relates to a sample analysis chip and a sample analysis method used for detection and analysis of biochemical reactions.

  Conventionally, in the field of biochemical reactions such as DNA reaction and protein reaction, a technique called μ-TAS (Total Analysis System) or Lab-on-Chip is known as a reaction apparatus for processing a small amount of solution sample. . 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 technologies 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.

  Using this technique, multiple types of specimens can be processed simultaneously with the same reagent, and conversely, multiple types of specimens can be processed simultaneously, greatly reducing the time and effort required in the past. It is possible to reduce.

  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.

  In Patent Document 1, in a chip that sends liquid from a liquid reservoir to a well using centrifugal force, the flow path is deformed and sealed in order to make the well independent. Therefore, a mechanism for crushing the flow path is necessary, and automation is difficult. In addition, when the centrifugal liquid feeding is performed from the central liquid reservoir to the surrounding wells as in the conventional centrifugal liquid feeding chip, the liquid feeding amount to each well varies.

  In patent document 2, the dispersion | distribution in the liquid feeding amount to each well is solved by interweaving rotation + revolution of the centrifugation method. However, this method also requires a complicated mechanism and space for the tip to rotate and revolve.

  In Patent Document 3, an analysis medium in which a plurality of wells each having a liquid reservoir and a channel extending in the centrifugal direction are connected is disclosed. However, this document does not pay attention to the liquid distribution and the like. The fluid is controlled by pushing against the air clogged in the well. In this method, the liquid in the flow path between the liquid storage part and the liquid storage part is not supplied, and the amount of liquid supplied to each well varies greatly, resulting in a difference in the results for each reaction.

JP-T-2004-502164 Japanese Patent No. 3699721 JP 2008-83017 A

  In view of the above-described problems of the prior art, the present invention provides a sample analysis chip for delivering liquids to wells, in which the liquid delivery method is simple and there is no variation in the amount of liquid in each well. Is an issue.

The invention according to claim 1 of the present invention, which has been made to solve the above-mentioned problems, includes a plurality of wells in a substrate, a channel connected to each well, and a note for injecting a solution into the channel. A sample analysis chip having an inlet and rotating the substrate to distribute a solution to the well, wherein the flow path communicates with the inlet and is provided closer to the center of rotation than the well. One liquid reservoir portion communicates with the first liquid reservoir portion via the communication channel, and the second liquid reservoir portion corresponding to each well, and the second fluid reservoir portion and the branch flow channel that communicates with the well The sample analysis chip is characterized in that the first liquid reservoir and the second liquid reservoir are partitioned by independent islands .
According to a second aspect of the present invention, the second liquid reservoir portion communicates with an adjacent second liquid reservoir portion, and the communication passage is at a communication location with the adjacent second liquid reservoir portion. The sample analysis chip according to claim 1, wherein the sample analysis chip is in communication .
The invention according to claim 3 is the sample analysis chip according to claim 2, characterized in that each of the communication channels communicates with two adjacent second liquid reservoirs .
The invention according to claim 4 is any one of claims 1 to 3, wherein the first liquid reservoir, the second liquid reservoir, the well, the branch channel, and the communication passage communicate with each other on the same plane . The sample analysis chip described in 1.
The invention according to claim 5 is the sample analysis chip according to any one of claims 1 to 4, wherein the cross-sectional area of the communication flow path is larger than the cross-sectional area of the branch flow path. .
The invention according to claim 6 is the sample analysis chip according to any one of claims 1 to 5, wherein the branch channel is formed to be inclined with respect to the rotation center direction.
7. The sample analysis according to claim 1, wherein the well and the second liquid reservoir are arranged circumferentially with respect to the center of rotation. Chip.
According to an eighth aspect of the present invention, the sample analysis chip includes a first base material on which the well and the flow path are formed, and a second base material bonded to the base material. The sample analysis chip according to any one of the above.
The invention according to claim 9 is the sample analysis chip according to claim 8, wherein any one of the substrates is formed of a light transmissive material.
The invention according to claim 10 is the sample analysis chip according to claim 9, wherein the first base material is a light-transmitting resin material, and the second base material is a metal material.
According to an eleventh aspect of the present invention, there is provided a sample analysis comprising: means for installing and rotating the sample analysis chip according to any one of the first to tenth aspects; and a detection measurement means for detecting a reaction in the well. Device.
According to a twelfth aspect of the present invention, there is provided a step of injecting a solution into the first liquid reservoir of the sample analysis chip according to any one of the first to eleventh aspects, and rotating the sample chip at a first rotation speed. And filling the second liquid reservoir with the solution, and rotating the sample chip at a second rotational speed greater than the first rotational speed to distribute the solution to the wells. This is a sample analysis method.
The invention according to claim 13 is a gene analysis method characterized by using the sample analysis method according to claim 12.

  According to the sample analysis chip of the present invention, a simple, functional, safe and inexpensive reaction chip can be realized. Furthermore, a plurality of processes can be performed on one type of specimen.

  Further, according to the sample analysis chip of the present invention, when a solution such as a liquid sample is sent from the first liquid reservoir to the well by centrifugal force, the first second liquid reservoir is distributed to each well. Since the liquid volume is uniformly adjusted and then distributed to the wells, there is little variation from well to well.

Plan view and sectional view of one embodiment of sample analysis chip of the present invention The perspective view for description of the sample analysis chip of the present invention Sectional drawing for description of the sample analysis chip of the present invention Plan view for explaining the sample analysis chip of the present invention Plan view for explaining the sample analysis chip of the present invention Graph of detection measurement results in Examples Graph of negative control measurement results in Examples Graph of positive control measurement results in Examples

A sample analysis chip of the present invention will be described with reference to the drawings.
FIG. 1A is a plan view showing an embodiment of the sample analysis chip of the present invention. FIG. 1B is a cross-sectional view taken along the line II ′ of the sample analysis chip of FIG. The chip of the present invention has a plurality of wells 102 on a substrate 101 and a flow path for sending a solution, for example, a liquid sample (solution) to the wells.

  Since the sample analysis chip of the present invention distributes liquid to each well 102 by centrifugal force generated by rotating the chip, the disk shape has a point through which the rotation shaft penetrates (hereinafter, center point) at the center. However, there is no particular limitation as long as it is formed so as to be rotatable with respect to the rotating shaft that penetrates the chip. In the case of the disk shape, the well can be arranged so as to be concentric with the disk-shaped chip so that the center thereof is the rotation axis, and thus the space is efficient. Although it is important to apply centrifugal force evenly to distribute wells to the wells, the tip should have rotational symmetry about the center point except for the region of the solution inlet / outlet 107. It can be easily realized by designing. That is, assuming that there are N wells, centrifugal force can be applied evenly if N wells are provided. That is, in the sample analysis chip of the present invention, from the central point, the primary liquid reservoir 108, the communication channel 110 that connects the primary liquid reservoir and the secondary liquid reservoir 109, the secondary liquid reservoir, the secondary liquid reservoir, and the well are communicated. It is preferable to arrange the concentric circles in the order of the branch channel 105 and the well. Of course, this is not the case when the liquid distribution amount of each well is varied. Further, since wells and flow paths are arranged concentrically, all wells can be analyzed in one inspection region by rotating the substrate.

  The flow path has a flow path communicating with at least each well in order to send liquid to each well. For example, as shown in FIG. 1, a branch channel 105 communicating with each well communicates with a pond-shaped liquid reservoir 108 in the center, or a main channel provided at the center of the well and a flow of the main channel. A configuration in which communication passages are connected in order in the direction of the road can be mentioned, but the present invention is not limited to this, and a plurality of wells and a solution are distributed to each well inside (on the rotation center side described later). Any flow path (including a communication path, which will be described later, and a secondary liquid reservoir 109) is provided so as to communicate with the primary liquid reservoir 108.

  Unlike the branch channel 105, the liquid reservoir and the main channel are present at the stage where the solution is injected into the chip (the stage before the rotation). This is referred to as a reservoir 108. The primary liquid reservoir 108 has an inlet for injecting a solution, a discharge outlet for excess solution, and an air outlet (air hole) 107. A plurality of through holes such as discharge holes and air holes may be provided in the primary liquid reservoir. For example, in the case of a pond shape, an injection port can be formed at the center and an air vent hole can be arranged around the inlet. In the case of the main channel shape, an inlet can be provided at one end of the channel and an outlet can be provided at the other end.

  The primary liquid reservoir 108 has a shape in which the side surface on the side having the connecting portion with the communication channel 110 has one peak with the adjacent connecting portion as a valley between the adjacent communication channels in the rotation center direction. Preferably there is. By adopting such a shape, when the chip is rotated, the solution naturally moves to each connecting portion by centrifugal force, thereby reducing liquid distribution variation. When the primary liquid reservoir has a channel shape, the volume of the channel determines the amount of solution between the valleys, which is particularly effective.

  The primary liquid reservoir 108 is connected to a communication channel 110 that communicates with the secondary liquid reservoir 109. When the tip is not rotated, the solution does not enter the secondary liquid reservoir 109. For this reason, an open end such as an air hole is not provided on the secondary liquid reservoir side of the communication channel 110, and the communication channel opening (channel cross-sectional area) that communicates the primary liquid reservoir 108 and the secondary liquid reservoir 109. However, the width and the cross-sectional area may be such that the solution does not pass through the solution.

  The secondary liquid reservoir 109 is connected to the well 102 via the branch channel 105. A feature of the secondary reservoir 109 is that it corresponds to each well 102. That is, the same number of secondary liquid reservoirs as the wells are formed, and each is connected one-to-one with the branch channel. By setting it as such a structure, the quantity of the solution distributed to each well is controllable with the capacity | capacitance of a secondary liquid reservoir part. For a reaction in which heat is strictly applied, it is desirable that the reaction system is uniform. Therefore, it is preferable that the reaction system does not remain in the branch channel when the solution is sent to the well. Therefore, it is desirable that the volume of the secondary liquid reservoir is equal to the well.

  Further, each secondary liquid reservoir 109 is partially communicated with an adjacent secondary liquid reservoir. In the embodiment of FIG. 1, the communication channel 110 is disposed between two adjacent secondary liquid reservoirs and coincides with the communication region. By adopting such a configuration, it is possible to adjust the amount of solution in the adjacent secondary liquid reservoirs at the stage where the solution is filled to the communication position at the same time as the liquid is distributed to each secondary liquid reservoir by centrifugation. The communication position is preferably provided at a position closest to the center point of the secondary liquid reservoir. It is possible to reduce the variation caused by the connection location when the liquid is fed to the well.

  Moreover, it is preferable that the bottom part (well side) of the secondary liquid reservoir 109 has a funnel shape with the branch channel 105 as a mouth in a plan view on the substrate. Moreover, it is preferable that the upper part (center point side) of the secondary liquid reservoir is formed to be inclined from the center point direction. Residual solution can be prevented by the centrifugal force during tip rotation.

  In the sample analysis chip of the present invention, the solution does not enter the well 102 in a state where the chip is not rotated and in the centrifugal force generated by the rotation of the chip at the stage of filling the secondary liquid reservoir 109. . That is, it is important that the pressure required to pass through the branch channel 105 connecting the well and the secondary liquid retaining portion is larger than the pressure required to pass through the communication passage 110. For this reason, as a means, for example, the relative pressure loss of the branch channel can be increased by making the channel cross-sectional area of the branch channel smaller than the channel cross-sectional area of the communication channel.

  Further, in the sample analysis chip of FIG. 1, the branch channel 105 is formed to be inclined from the center point direction. By forming the side passages in this way, the air in the well 102 moves along the inner side of the branch channel in the direction of the main channel when a centrifugal force is applied, while the solution moves along the outer side of the branch channel. Since it moves in the well direction, the solution can be smoothly moved into the well. As the angle to be inclined, the angle formed by the direction of the center point and the branch channel is preferably between 10 degrees and 80 degrees. If it is less than 10 degrees, the exhaust from the well and the entry of the solution into the well may interfere to prevent the solution from entering, and if it exceeds 80 degrees, the centrifugal force in the well direction of the branch channel is weak, May not move to the well.

  Since the well 102 is finally filled with the solution by the rotation of the chip, the well 102 is disposed on the outermost periphery of the flow path. However, this is a comparison with the above components, and does not prevent another component, for example, a waste chamber for discarding excess solution from being further provided on the outer periphery.

  The volume of the well 102 is preferably 1 μl or more and 100 μl or less. If it is smaller than 1 μl, the centrifugal force does not work sufficiently, and it is difficult to send the solution to the well. If it is larger than 100 μl, the mixing property of the reagent is lowered and the uniformity of the temperature in the well is lowered. May occur.

  Further, in order not to leave air in the well 102, it is preferable to connect to the flow path at a point closest to the center point of the well. That is, it is preferable that the branch channel 105 and the well be connected at a point closest to the center point on the well side.

  Next, the manufacturing method of the sample analysis chip of the present invention will be described.

FIG. 2 is a perspective view showing one embodiment of the structure of the sample analysis chip of the present invention.
The sample analysis chip of the present invention can be produced by bonding the second base material 402 to the first base material 401 in which wells and flow paths (including each liquid reservoir and the flow path) are formed. At least one of the first base material and the second base material has a supporting unit 405 for fixing to the chip rotating mechanism, for example, as a rotating means for rotating the chip by the chip rotating mechanism included in the sample analyzer. . In addition, at least one through hole for an inlet (INLET / OUTLET) that also serves as an inlet / outlet for air is formed in one of the first substrate and the second substrate. The through hole is made to coincide with a predetermined position as described above in the primary liquid reservoir 108 when the base material is bonded. In the following, for convenience of explanation, the base material side positioned on the surface to be measured when detecting and measuring the fluorescence reaction and the like is referred to as “upper side”, and the lower side is referred to as “lower side”.

  The base material is not particularly limited as long as it does not affect the sample, but good visible light transmission can be secured by using a resin material containing any of polypropylene, polycarbonate, and acrylic. . 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 is excellent in thermal conductivity and sealing performance. In addition, by making at least the well bottom of the upper substrate out of the substrates to be bonded transparent, detection and analysis of fluorescence and the like can be performed from the outside. In the present invention, “transparent” and “light transmissivity” mean that the total average transmittance in the wavelength region of the detection light is 70% or more. If a light-transmitting material is used in the visible light region (wavelength 350 to 780 nm), it is easy to visually recognize the sample state in the chip, but this is not restrictive.

  As a processing method of the base material for forming the well and the flow path, 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 first base material, good light transmittance, heat resistance, and strength can be ensured. Moreover, when the thickness of the 1st base material exists in the range of 50 micrometers-3 mm, favorable light transmittance, heat resistance, and intensity | strength can be ensured and the process of a recessed part can be performed reliably.

  Moreover, when the thickness of a 2nd base material exists in the range of 20 micrometers-300 micrometers, both the heat conductivity and sealing performance of a 2nd base material can be satisfied. When the thickness of the second base material is larger than 300 μm, the heat capacity becomes large and the thermal responsiveness may be lowered.

  FIG. 3 shows a cross-sectional view of the sample analysis chip of the present invention. The first base material 401 communicates with a solution inlet 403 penetrating the chip, grooves 108 and 109 serving as liquid reservoirs for the injection liquid to flow into the chip, and each well extending to the outer periphery of the chip. A groove 105 serving as a branch flow path and a depression 102 serving as a well in the outer peripheral portion of the chip are formed. The cross-sectional view of FIG. 3 schematically shows the path from the inlet to the well, and the shapes of the main flow path and the side path are not limited to this. In order to fill all the wells with the solution to be injected, the volume of the main channel needs to be larger than the sum of the volumes of each well. However, when the reagent 501 is fixed to the well, the amount of the liquid sample to be put into the reaction well is reduced by that amount, and therefore the volume of the flow channel into which it flows may be reduced accordingly. When performing detection measurement on the first substrate side for fluorescence reaction or measurement, it is preferable that the concave portion of the well has a smooth shape that does not scatter light.

  The reaction reagent 501 is fixed to the well 102 before the substrates are bonded. Different reagents can be used in each well. By fixing different reagents in each reaction well, a plurality of treatments can be performed on one specimen (sample). Further, a part of the reagent for actually performing the reaction may be fixed to each well, and the remaining reagent may be introduced together with the liquid sample.

  As a method for fixing the reagent 501, for example, a liquid reagent is dropped onto the well portion of the first base material with a pipette or the like, and the first base material 401 is centrifuged at 2000 to 3000 rpm for about 5 minutes with a centrifuge, so that an appropriate amount is obtained. The liquid reagent can be fixed to the well by allowing the liquid surface to remain flat and drying it.

  Alternatively, wax 502 may be dropped after fixing the reagent to the well. Specifically, wax is melted on a hot plate and dropped using a pipette so as to cover the dried reagent. At this time, the wax solidifies in a few seconds. The wax has a role of fixing the reagent in the recess of the well.

  Examples of the method for bonding the substrates include a method in which a resin coating layer is provided as an adhesive layer on one substrate, and this is melted to bond both substrates. The resin coating layer is preferably provided on a metal material substrate having high thermal conductivity and melt bonded. As a material for the resin coating layer, a resin material such as PET, polyacetal, polyester, or polypropylene can be used.

  In this laminating method, a light-transmitting resin material suitable for fluorescence measurement is used for the first base material, and the second material has a high thermal conductivity and is provided with a resin coating layer for melting. It is preferable to use a metal material that can be easily bonded by bonding. Further, by forming a resin coating layer on the surface of the metal substrate, the chemical resistance of the metal substrate itself need not be considered when selecting the material.

  Further, when the resin coating layer is formed on the surface of the substrate, it is possible to perform fusion using a laser by forming an anchor layer as a base of the resin coating layer. Carbon black (light-absorbing material) that absorbs laser wavelength light is kneaded into the anchor layer, and the resin coating layer can be melt-bonded by generating heat when irradiated with laser light. Alternatively, instead of adding carbon black to the anchor layer, carbon black may be added to the resin coating layer, or the surface of the resin coating layer may be painted black. For example, the resin coating layer can be efficiently melted also by irradiating light of an infrared photodiode laser having a wavelength of about 900 nm. Laser welding, unlike thermal welding, does not require heating of the chip, so that the substrates can be bonded together with little effect on the chip and the reagent fixed to the chip.

  Next, a sample analysis method using the sample analysis chip of the present invention will be described.

  The sample analysis chip of the present invention can be used, for example, for detection and analysis of biochemical substances in a sample such as DNA or protein. A reagent is fixed to each well 102, and a liquid sample is distributed to each well. In this case, different reagents can be used for each well.

  Next, with respect to the sample analysis chip of the present invention in which the first base material 401 and the second base material 402 are bonded together, first, a solution such as a reagent is injected into the primary liquid reservoir 108 from the injection port 403 (107). To do. At this stage, only the primary liquid reservoir is filled with the solution and does not enter the secondary liquid reservoir 109 and the well 102 as described above. This is because there is air pressure from the well side due to the surface tension of the solution and the absence of air holes on the well side. The sample analyzer used for the sample analysis method may be provided with such a solution injection means.

  Next, the sample analyzer used for the sample analysis method has a chip rotation mechanism for rotating the sample analysis chip. A known general centrifugal device can be used for the tip rotating mechanism. A sample analysis chip is installed in the sample analyzer, and the chip is rotated by the rotation mechanism with the vertical direction of the chip as the rotation axis at the center point of the chip. The rotation speed requires a rotation speed at which the centrifugal force applied to the solution overcomes the aforementioned air pressure and surface tension and flows into the well. Although it depends on the shape of the chip, it is preferably about 500 rpm or more. If the rotation speed of the chip is less than about 500 rpm, the solution does not flow into the secondary liquid reservoir 109 and the liquid amount may not be constant. Further, the rotation speed of the chip needs to be a rotation speed that does not enter the well.

  As shown in FIG. 4, first, each secondary liquid reservoir 109 is filled with the solution injected into the primary liquid reservoir 108 by centrifugal force at the first rotational speed. Next, as shown in FIG. 5, the solution is moved from the secondary liquid reservoir 109 to the well 102 at a second rotation speed higher than the first rotation speed.

  After dispensing the solution sample, oil that does not inhibit the reaction of the sample / reagent may be dispensed to each well in the same process. Oil injection can prevent evaporation of the liquid during the reaction. It is necessary to use oil having a lighter specific gravity than the previously dispensed solution. This is because when the tip is rotated and dispensed by centrifugal force, it serves as a stopper for each well on the side path side. The type of oil is not particularly limited as long as it does not inhibit the sample / reagent reaction, but mineral oil or silicone oil can be preferably used.

  When the wax 502 is used for fixing the reagent, the sample analyzer may be provided with a temperature control means using a heater or a Peltier element made of a heating wire or the like. By heating the chip above the melting point of the wax, the wax can be melted and the reagent and the solution (sample) can be mixed in the well. The temperature control means can also be used for reaction control of reagents such as PCR reaction.

  Thereafter, the reagent and the sample are mixed in the well, and the reaction state can be analyzed by a technique such as fluorescence detection. The sample analyzer has a detection measurement means for performing measurement at the position of the well on the upper side of the substrate of the sample analysis chip. A predetermined well can be measured by rotating the chip by a rotating mechanism. In the sample analysis chip of the present invention, it is possible to perform optical measurement from the outside of the chip by making the upper side of the substrate transparent.

  As described above, by providing a mechanism that acts on the sample analysis chip in each step, a sample analysis apparatus that saves space and facilitates sample analysis can be provided.

  Next, an example of the sample analysis method of the present invention will be described.

  Examples of gene analysis include detection of somatic mutation and detection of germ cell mutation. Since the type of protein to be expressed differs depending on the genotype, for example, it causes a difference in the function of the metabolic enzyme of the drug, resulting in individual differences in the optimal dose of the drug and the likelihood of side effects. By utilizing this in the medical field and examining the “genotype” of each patient, custom-made medical care can be performed.

-Detection of SNPs About 0.1% of the human genome has a unique nucleotide sequence difference, which is called SNP (Single Nucleotide Polymorphism), and is one of germline mutations. As one of the SNP identification methods, for example, a PCR-PHFA (PCR-Preferred Modulation Formation Assay) method using fluorescence is used. The PCR-PHFA method includes a PCR process for amplifying a detection mutation site and a competitive strand displacement reaction process using an amplified fragment and a corresponding probe. According to this method, mutation is detected by the difference in luminescence of the fluorescent reagent, but by using the sample analysis chip of the present invention, since there is little liquid distribution variation in each well, accurate SNPs detection can be performed. In addition, the sample analysis chip of the present invention can also be used for the Invader method (registered trademark), the Taqman PCR method and the like as SNP detection methods other than those described above.

Hereinafter, an analysis example using the PCR-PHFA method for SNPs involved in side effects on warfarin (an anticoagulant, used as a drug for heart disease and hypertension) will be described using the present invention.

A sample nucleic acid obtained from blood or the like is purified to obtain a solution sample. The sample nucleic acid is amplified before or after the injection into the sample analysis chip of the present invention. Note that SNPs in VKORC1 and CYP2C9 are often discussed for detection of SNPs involved in warfarin, and CYP2C9 * 2 and CYP2C9 * 3 are famous. Gene fragments containing these SNPs from the specimen are amplified by multiplex PCR.

  In the detection method described above, two detection wells are required to determine one SNP. Therefore, it is preferable to use a sample analysis chip in which 10 or more wells are formed for each specimen sample. A reagent for SNP detection for performing competitive strand displacement reaction is fixed.

  The sample in which the nucleic acid is amplified by the PCR is filled in each well. Each well is temperature-controlled, and mutation is detected by the difference in luminescence of the fluorescent reagent mixed in the reagent. If only one of the two wells is positive for one SNP, it can be determined to be homozygous, and if two are positive, it can be determined to be heterozygous.

• Detection of K-ras gene mutations Most of the mutations characteristic of cancer cells and those that are resistant to molecular target drugs are somatic mutations. In the case of germ cell mutations (SNP, etc.), a common mutation is seen in all cells, whereas in somatic mutations, cells are mutated only in the mutated cells, and cells that are not mutated (usually normal cells) ) Shows no mutation.

In other words, when many of the samples are normal cells and some mutant cells are included, it is necessary to detect a small number of mutant genes present in many normal genes. The difference is that it makes detection of genetic mutations in somatic cells more difficult.

  The K-ras gene is a gene whose molecular target drug has been shown to be ineffective in most patient groups when mutations are present in cancer cells. Hope to detect this gene simply, quickly, inexpensively and with high accuracy It is being done.

An example of analysis of the K-ras gene by the PCR-PHFA method is described below.

A reagent containing a probe nucleic acid is fixed to the well for detecting a genetic mutation. Since detection of K-ras gene has 13 types of mutations in the wild type, the sample analysis chip of the present invention in which at least 14 wells are formed is used, and reagents corresponding to each of the wells are immobilized. Is preferred.

  Collect cancer cells such as colorectal cancer, purify the sample nucleic acid, and use it as a solution sample. The sample nucleic acid is amplified before or after the injection into the sample analysis chip of the present invention.

  The sample in which the nucleic acid is amplified by the PCR is filled in each well. A well can be temperature-controlled and a variation | mutation can be detected by the luminescence difference of the fluorescence reagent mixed in the said reagent.

  The following experiment was conducted in order to investigate the accuracy of the liquid distribution variation of the sample of the sample analysis chip of the present invention.

  As the first base material 401 of the sample analysis chip of the present invention, polypropylene resin is used and has a disk-like outer shape as shown in FIG. 4, and the primary liquid reservoir 108 and the secondary liquid reservoir 109 are connected on concentric circles. The communication channel 110, the secondary liquid reservoir, the branch channel 105 connecting the secondary liquid reservoir and the well 102, and the chip having the well are formed by machining the acrylic resin with an end mill from φ6 mm to φ0.4 mm. did. The base material is formed with a primary liquid reservoir, 24 wells and a secondary liquid reservoir, and a corresponding flow path. The internal volume of each well was 12 μl, the volume of each secondary liquid reservoir corresponding to this was designed to be 12 μl, and the volume of the primary liquid reservoir was designed to be 12 × 24 μl.

  As the second base material 402 to be bonded to the polypropylene base material, an aluminum sheet base material coated with a polypropylene resin as a resin coating layer was used. A resin coating layer having a thickness of about 0.07 mm was used. The resin coating layer has a melting point of around 120 degrees, and is coated on the aluminum base so that it melts when heat is applied to the aluminum side.

  The polypropylene base material and the aluminum base material were overlapped, and heat of 130 degrees or more was applied to the aluminum base material side, whereby the resin coating layer was melted and the polypropylene base material and the aluminum base material were welded.

  Further, as a comparative example, a sample analysis chip that does not have a secondary liquid reservoir but directly communicates with the primary liquid reservoir 108 and the branch channel 105 that communicates with the well 102 is obtained by using an end mill with acrylic resin of φ6 mm to φ0.4 mm. Machine cutting was performed, and the base materials were bonded together in the same manner as in the example.

  A solution sample of an aqueous solution moderately colored with bromophenol blue was fed to both the chip of this example and the comparative example with a pipette. In each chip, the solution sample was fed only to the primary liquid reservoir.

  After feeding, both tips were rotated about the tip center. As a means for applying centrifugal force to the chip, a simple centrifuge device using a desktop small centrifuge used for separation of reagents in chemical and biological reactions was prepared and used. The rotational speed during centrifugation was measured and adjusted with a rotational speed measuring device.

  First, when both chips were rotated at a rotation speed of 1000 rpm, in the chip of the example, the solution did not enter the well side, and each secondary liquid reservoir 109 was filled with a volume that seems to be almost equal. It was. On the other hand, in the chip of the comparative example, the solution did not enter the well side and remained in the primary liquid reservoir.

  Next, when both chips were rotated at a rotation speed of 4000 rpm, a solution sample was fed to each well of the chip. In the chip of the example, each well was completely filled with the solution sample, and further a part of the branch channel was filled. In the chip of the comparative example, the sample solution in some wells was insufficient and not completely filled.

  In order to examine the amount of variation in the liquid distribution, the amount of the solution sample filled in each well was measured with a micropipette. The amount of the solution sample filled in each well of the sample analysis chip of the example was 13.3 μl to It was 14.9 μl, and in the comparative example, it was 10.3 μl to 17.5 μl. From this, it was confirmed that in the sample analysis chip of the present invention, the distribution variation was greatly improved by the secondary liquid reservoir.

  Next, as an example of the sample analysis chip of the present invention and an analysis method using the sample analysis chip, an example of a SNPs analysis chip will be shown.

  Similarly to Example 1, a sample analysis chip of the present invention as shown in FIG. 4 was produced. Before the first base material 401 and the second base material 402 having the flow paths are bonded together, an invader reaction probe reagent, an enzyme such as a DNA polymerase and a chestnut base are dropped into the well 102 with a pipette and dried and fixed. . Thereafter, the first base material and the second base material were bonded together to complete the sample analysis chip of the present invention according to Example 2. Other chip manufacturing processes are the same as those in the first embodiment.

  A buffer solution containing the purified genome was pipetted as a solution sample into the chip produced in the above process, and filled into the primary liquid reservoir 108.

  Each reagent was used in the amounts shown in Table 1 below.

  After the feeding, the tip was rotated around the tip center at a rotation speed of 1000 rpm, and the secondary liquid reservoir 109 was filled with the solution sample. Further, a solution sample was distributed to each well at 4000 rpm.

  Subsequently, when the mineral oil described in the reagent list (Table 1) that does not inhibit the reaction was fed in the same manner, the sample solution filled the well, and the remaining solution filled about half of the side path. Completely filled the branch flow path and further filled a part of the secondary liquid reservoir 109.

  In this example, an invader reaction probe was immobilized as a reaction reagent in 22 wells. Further, in order to determine the success or failure of the reaction result, a negative control was set at one place as a confirmation of the presence or absence of contamination, and a reaction test was performed on one chip.

  35 cycles of 95 ° C. and 68 ° C. are alternately applied to a sample analysis chip in which the reaction vessel is independent of oil, and the sample genome is amplified by PCR reaction. Subsequently, the temperature is adjusted at 63 ° C. for 30 minutes, thereby causing a fluorescence detection reaction in the well by an enzyme reaction.

  At this time, since the polypropylene substrate side of the chip was transparent, fluorescence detection was performed from the outside through the polypropylene substrate. In this example, the fluorescence reaction was measured by a fluorescence detection apparatus combining a photomultiplier tube and an optical fiber.

  6 and 7 are graphs of the analysis results of SNPs by the fluorescence reaction detected by this example. The vertical axis of each graph is the intensity of detected light and indicates the intensity of fluorescence. The horizontal axis is the time axis.

  FIG. 6 shows the result of one well in which the reaction was performed, and it was confirmed that a fluorescence detection reaction was caused by the reagents mixed within a predetermined time.

  Since FIG. 7 is a well in which reagents are not fixed in advance, no fluorescence reaction was detected. This confirmed that there was no contamination from both sides.

  FIG. 8 shows detection data obtained by mixing the reagent and the sample at an optimum ratio in a general manner using a polypropylene tube (positive control). Comparing FIG. 6 and FIG. 8, the reaction in the chip according to this example shown in FIG. 6 is almost the same as the reaction in FIG. it can. Thus, it can be seen that a desired amount of sample can be accurately distributed.

  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, since SNP mutations can be detected, it can be used in 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.

101 ... Substrate 102 ... Well 105 ... Branch channel 107 ... Inlet / Outlet 108 ... Primary liquid reservoir 109 ... Secondary liquid reservoir 110 ... Communication channel
120 ... island-like portion 401 ... first base material 402 ... second base material 403 ... inlet / outlet (through hole)
405 ... Supporting part 501 ... Fixing reagents 502 ... Wax

Claims (13)

A sample analysis chip that has a plurality of wells on a base material, a flow channel connected to each well, and an inlet for injecting a solution into the flow channel, and rotates the base material to distribute the solution to the wells. There,
The flow path communicates with the inlet, and a first liquid reservoir provided on the rotation center side from the well; and
Communicating with the first reservoir through the communication channel, a second reservoir corresponding to each well,
A branch channel connecting the second liquid reservoir and the well ,
The sample analysis chip, wherein the first liquid reservoir and the second liquid reservoir are partitioned by independent island portions . The second liquid reservoir is in communication with an adjacent second liquid reservoir, and the communication passage is in communication with the adjacent second liquid reservoir. The sample analysis chip according to claim 1. 3. The sample analysis chip according to claim 2, wherein each of the communication channels communicates with two adjacent second liquid reservoirs. The sample analysis chip according to any one of claims 1 to 3, wherein the first liquid reservoir, the second liquid reservoir, the well, the branch channel, and the communication passage communicate with each other on the same plane.   5. The sample analysis chip according to claim 1, wherein a channel cross-sectional area of the communication channel is larger than a channel cross-sectional area of the branch channel.   The sample analysis chip according to claim 1, wherein the branch channel is formed to be inclined with respect to a rotation center direction.   The sample analysis chip according to claim 1, wherein the well and the second liquid reservoir are arranged circumferentially with respect to the center of rotation.   The sample analysis chip according to any one of claims 1 to 7, wherein the sample analysis chip includes a first base material on which the well and the flow path are formed, and a second base material bonded to the base material. .   9. The sample analysis chip according to claim 8, wherein any one of the substrates is formed of a light transmissive material.   The sample analysis chip according to claim 9, wherein the first base material is a light-transmitting resin material, and the second base material is a metal material. Means for installing and rotating the sample analysis chip according to any one of claims 1 to 10;
A sample analyzer having detection and measurement means for detecting a reaction in the well. A step of injecting a solution into the first liquid reservoir of the sample analysis chip according to claim 1;
Rotating the sample chip at a first rotation speed to fill the second liquid reservoir with a solution;
Rotating the sample chip at a second rotation speed greater than the first rotation speed to distribute the solution to the wells;
A sample analysis method comprising:   A gene analysis method using the sample analysis method according to claim 12.

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