JP2011214943A - Chip, method and device for analyzing sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample analysis chip and a method of analyzing a sample for solving fluorescence intensity detection obstruction by bubbles without obstructing reaction of biomolecules and measurement fluorescence.SOLUTION: In the sample analysis chip including a plurality of wells, channels connected to the respective wells, and an inlet for injecting solution to the channels in a base material, and distributes the solution to the wells by rotating the base material, the channels include main channels connected to the inlet and provided at the side of a rotation center as compared with the well, and the upper surface of the well is in a recessed shape projecting toward the center of the upper part of the well.

Description

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

    Currently, techniques using fluorescent dyes are known as methods for elucidating biomolecules, such as real-time PCR (polymerase chain reaction) and double-stranded DNA quantification methods. These are called μ-TAS (Total Analysis System), and are provided with a plurality of reaction chambers (hereinafter referred to as wells) and flow paths on one chip or cartridge, and can be used for analysis of a plurality of samples or a plurality of samples. The reaction can be carried out. 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 consumption of fluorescent reagents can be reduced to reduce the cost for analysis and reaction.

  In order to perform biochemical reactions efficiently using chips and cartridges, different types of chemicals, specimens, and enzymes are dispensed into multiple wells, and one or more reagents that react with these chemicals, specimens, and enzymes are used. It is necessary to flow from several main conduits into a well and generate multiple 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, greatly reducing the time and effort required in the past. Is possible.

    However, not a little air is mixed in the sample liquid containing the specimen. When the sample liquid mixed with air in the well is reacted with the fluorescent dye, bubbles are generated in the mixed solution.

    Bubbles generally float on the surface of the solution. When trying to measure the intensity of the fluorescent dye in the well in this state (hereinafter photometry), the fluorescence wavelength emitted from the molecule changes when passing through the bubble, and the detection probe that recognizes the fluorescence intensity at a specific wavelength can recognize it. Disappear. Thereby, the detected fluorescence intensity is significantly reduced.

  In Patent Document 1, in order to suppress bubbles generated in the well, heat is applied to the flow path. However, it is impossible to completely suppress generation of bubbles at a temperature of 20 to 30 degrees. .

  In Patent Document 2, hydrophilizing the well is a technique for suppressing the occurrence. However, hydrophilizing the well surface suggests the possibility of generating non-specific binding with a biomolecule.

JP2008-151772A JP2007-333440

  In view of the prior art as described above, it is an object of the present invention to provide a sample analysis chip and a sample analysis method capable of solving the fluorescence intensity detection inhibition due to bubbles without inhibiting biomolecule reaction and fluorescence measurement. And

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 on the rotation center side of the well. A sample analysis chip having a path, wherein the upper surface of the well has a concave shape protruding toward the upper center of the well.
The invention according to claim 2 is the sample analysis chip according to claim 1, characterized in that the main flow path leading from the injection port to each well is formed to be inclined with respect to the rotation center direction. .
The invention according to claim 3 is the sample analysis chip according to claim 1 or 2, wherein the substrate is formed of a light-transmitting material.
According to a fourth aspect of the present invention, there is provided a sample analysis method characterized in that a sample is distributed in the well by centrifugation on the sample analysis chip according to any one of the first to third aspects.
The invention according to claim 5 is the sample analysis method according to claim 4, characterized in that a sample to be measured for fluorescence is accommodated in the well and optical information of the sample is measured.
According to a sixth aspect of the present invention, there is provided a sample having means for installing and rotating the sample analysis chip according to any one of the first to fifth aspects, and detection and measurement means for detecting a reaction in the well. It is an analysis device.
The invention according to claim 7 is the sample analyzer according to claim 6, characterized in that detection measurement is performed at the center of the well.

  Furthermore, according to the present invention, since the upper shape in the well is not a flat type, bubbles can be unevenly distributed at the end just by centrifuging the chip, thereby inhibiting the reaction of biomolecules and fluorescence measurement. Sample analysis is possible. In addition, according to the sample analysis chip of the present invention, a simple, functional, safe and inexpensive sample analysis chip can be realized.

  In addition, if the volume of the main channel from the channel crest to the adjacent channel crest is arbitrarily designed, a sample with the same capacity can be sent to the well connected from the channel trough sandwiched between the previous channel crests. Since it can be liquidated, the amount of the solution sample to be used can be arbitrarily set for each well.

Plan view of an embodiment of the sample analysis chip of the present invention Sectional drawing of the uniform state of the well in the sample analysis chip of this invention The perspective view of the sample analysis chip of this invention Cross-sectional view of well after sample distribution before bubbles are excluded Sample fluorescence measurement result before bubble removal Cross-sectional view of well after sample distribution after bubble removal Sample fluorescence measurement results after excluding bubbles

A sample analysis chip of the present invention will be described with reference to the drawings.
FIG. 1 is a plan view showing an embodiment of the sample analysis chip of the present invention. 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. The flow path has at least one main flow path 103 that communicates with each well and supplies a side path 105 that connects the main flow path and the well in order to send liquid to each well. The flow path has an inlet for injecting the solution. In the embodiment of FIG. 1, an inlet (INLET) is provided at the end of the main channel, and an outlet (OUTLET) for excess solution that also serves as an air outlet for the other end.

  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. It is important to apply a centrifugal force evenly to distribute wells to the wells, but the tip is designed so as to have rotational symmetry about the center point except for the area of INLET / OUTLET107. It can be easily realized. That is, assuming that there are N wells, centrifugal force can be applied evenly if N wells are provided. Since the wells are arranged concentrically, all wells can be analyzed in one inspection region by rotating the substrate.

  The main channel 103 is formed closer to the center point than the well. Furthermore, the sample analysis chip of the present invention is characterized in that the main channel is formed so as to have one peak in the direction of the center point between adjacent wells. Here, the wells adjacent to each other mean wells in which the liquid supply flow path from the main flow path to the well is moving back and forth. Further, having a mountain in the direction of the center point means having a maximum point (main channel peak portion 103a) in the direction of the center point. In this way, the liquid injected into the main channel is naturally interrupted at the peak of the main channel when the chip rotates by forming a single peak in the center point direction between adjacent wells. Variation in the amount of liquid distribution to the liquid can be reduced.

  The connection point between the well 102 and the main channel 103, that is, the connection point between the main channel 103 and the side channel 105 is preferably a valley 130b between the peaks of the main channel. A trough is a point farthest from the center point between the mountains of the main channel. By connecting the well and the main flow path at this point, it is possible to reduce the remaining of the solution in the main flow path at the time of liquid distribution.

  Further, as described in the processing method using the sample analysis chip described later, the connection port between the main channel 103 and the well 102 has a width and a disconnection enough to prevent the solution from entering the well before the chip is rotated. It needs to be an area.

  Further, in order not to leave air in the well 102, it is preferable to connect to the main channel at a point closest to the center point of the well. That is, when the side path 105 is formed, it is preferable to form the side path 105 so as to connect the point closest to the center point on the well side and the valley on the main flow path side.

  In the embodiment of FIG. 1, the main channel width is narrow at the main channel peak 103a and wide at the main channel valley 103b. The smaller the solution present in the region corresponding to the main channel peak 103a, the smaller the distribution variation. Therefore, the cross-sectional area of the main channel in the peak is preferably smaller than the cross-sectional area of other portions. Therefore, it is preferable to narrow the flow path width of the peak and / or to reduce the depth. For the same reason, it is preferable to make the cross-sectional area of the main flow path smaller as it approaches the peak.

  Furthermore, the amount of liquid distribution to each well 102 can be controlled by widening the width of the main channel valley 103b. Therefore, as in the sample analysis chip of FIG. 1, if the channel between the peaks is made chamber-like and the volume of the main channel from the main channel peak to the adjacent main channel peak is arbitrarily designed, it is equivalent. Since a sample of the volume can be sent to a well communicating from a valley portion sandwiched between both mountain portions, an arbitrary amount of solution can be set in each well.

  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 liquid feeding to the well is difficult to perform, and 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. Such a phenomenon may occur.

  Further, the side path 105 is formed inclined from the direction of the center point. By forming the side passages in this way, the well air moves along the inner side of the side passage in the direction of the main flow path when a centrifugal force is applied, while the solution flows along the outer side of the side passage. Since it moves in the 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 side path is preferably between 10 degrees and 80 degrees. If it is less than 10 degrees, the exhaust from the well and the penetration of the solution into the well may interfere to prevent the entry of the solution. If it exceeds 80 degrees, the centrifugal force in the lateral direction is weak, and the solution is in the well May not move to.

  FIG. 2 shows a cross-sectional view of each well form in the sample analysis chip. The first substrate 401 includes a solution injection port 203 penetrating the chip, a groove 103 serving as a main flow path for the injection solution to flow into the chip, and a side path communicating with each well extending to the outer periphery of the chip. A groove 105 to be formed and a recess 102 to be a well on the outer peripheral portion of the chip are formed. The cross-sectional view of FIG. 2 schematically shows the path from the inlet (INLET / OUTLET) 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 is fixed to the well, the amount of the liquid sample to be put into the reaction well is reduced accordingly, so that the volume of the flow channel flowing in may be reduced accordingly.

  In the present invention, when performing detection measurement on the first substrate side for fluorescence reaction or measurement, the bubbles are placed at the end by centrifugation so that the bubbles contained in the sample liquid do not interfere with the detection measurement in the recesses at the top of the well. It is molded into a structure that brings it together. That is, the upper surface 501 of the well 102 formed on the light-transmitting first base material has a concave shape protruding inward. When air bubbles are mixed in the reagent and the sample, there is a possibility that the optical measurement is greatly affected. However, by forming a concave shape with the upper part of the well protruding inward as in each of the wells 102 described above, the air bubbles are reduced. It is possible to remove from the center of the well that is the measurement region. When bubbles are mixed, it is considered that the bubbles are dispersed in the upper part of the well. This is because the bubbles move from the concave portion to the outside of the center of the centrifugal portion due to the specific gravity with the reagent and the centrifugal force during rotation. In FIG. 2, the entire upper surface 501 of the well 102 has an arcuate shape on the inside, but only the measurement region on the upper surface of the well may have a concave shape protruding inward.

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

FIG. 3 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 manufactured by bonding the second base material 402 to the first base material 401 in which the well and the flow path (including the main flow path and the side 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 coincides with the end of the main channel 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 substrate is preferably one that does not affect the sample and that has visible light permeability. If a resin material containing any of polypropylene, polycarbonate, and acrylic is used, good visible light transmittance can be obtained. 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 transmittance” mean that the 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 second base material is in the range of 50 μm to 3 mm, good light transmittance, heat resistance, and strength can be ensured, and the recess can be reliably processed.

  Moreover, when the thickness of the 2nd base material exists in the range of 10 micrometers-300 micrometers, both the heat conductivity and sealing performance of a 1st base material can be satisfied. When the thickness of the first base material is larger than 300 μm, the heat capacity becomes large and the thermal responsiveness may be lowered.

  A reaction reagent is fixed to the well 102 before the substrates are bonded together. 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 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 detection and analysis of biochemical substances in samples such as DNA and proteins. 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. Or a sample is fixed to each well, and a liquid reagent is distributed to each well. In this case, a different sample can be used for each well.

  Next, with respect to the sample analysis chip of the present invention in which the first substrate 401 and the second substrate 402 are bonded together, first, a solution such as a reagent is injected into the main channel 103 from the injection port 107. At this stage, only the main channel is filled with the solution and does not enter the side channel 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.

  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 form of the chip, it is preferably about 1000 rpm or more. If the rotation speed of the chip is less than about 1000 rpm, the solution does not flow into the well, and the liquid volume may not be constant.

  After dispensing the liquid 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 wax 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.

  When air bubbles are mixed in the reagent and the sample, there is a possibility that the optical measurement is greatly affected. However, as described above, by forming the well upper surface 501 of each well 102 into a concave shape protruding inward, the air bubbles Can be removed from the center of the well, which is the measurement region. When bubbles are mixed, it is considered that they are dispersed in the upper part of the well. This is because the bubbles move from the concave surface to the outside of the center of the centrifugal portion due to the specific gravity with the reagent and the centrifugal force during rotation.

  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 K-ras gene mutation and detection of germline mutation. The identification of K-ras gene mutation is desired to establish a specific method for cancer treatment, and the identification of germ cell mutation is considered to be usable for estimation of drug efficacy.

-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 heterogeneous.

-Detection of K-ras gene mutation A reagent containing a probe nucleic acid is fixed to the well for detection of the gene 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. Transfer sample and perform PCR.

  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.

  Examples of the present invention are shown below, but the present invention is not limited thereto.

<Example 1> SNPs analysis chip Using a polypropylene resin as a SNPs chip base material, it has a disk-like outer shape as shown in FIG. 1, and is connected to a corrugated main flow path 103 and a main flow path trough 103b on a concentric circle. A side passage 105 having a mouth and a chip having a well 102 at the end of the side passage were formed by injection molding. In this base material (polypropylene base material), 23 wells and side paths are respectively formed. As shown in FIG. 4, the chip of this example was designed such that the well shape was a concave shape with the well upper surface 501 protruding inward. As a comparative example, a chip having a flat upper surface 502 (non-concave shape) was manufactured as shown in FIG. In addition, the main flow paths of the examples and the comparative examples were designed so that the area was periodically changed, and the volume of the main flow path between the adjacent main flow path peak portions 103a was 12 μL.

  As the second substrate to be bonded to the polypropylene substrate, an aluminum sheet substrate 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.

  Further, an anchor layer in which carbon is kneaded is provided between the aluminum layer and the resin coating layer, and the resin coating layer is melted even by heat generated by laser light irradiation.

  To the wells on the polypropylene substrate, an invader reaction probe reagent, an enzyme such as DNA polymerase and chestnut were dropped with a pipette and dried and fixed.

  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 to melt the resin coating layer and weld the polypropylene base material and the aluminum base material.

  A buffer solution in which purified genome was added to the chip produced in the above process was pipetted as a solution sample and filled into the main channel 103. At this stage, no sample had entered the wells and the sideways.

  When the tip was rotated around the tip center at 5000 rpm after feeding, 11 μL of the sample was fed to each well. 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.

  As for the direction of rotation of the tip during centrifugation, even if it is rotated in any direction with respect to the direction of inclination of the side passage, the liquid behavior in the tip is affected while the number of rotations is increasing. It was confirmed that it does not affect the variation of

  Subsequently, when a mineral oil that does not inhibit the reaction was fed in the same manner, the sample filled the well, the remaining solution filled about half of the side passage, and the oil filled the remaining half of the side passage and the channel valley. 80% was satisfied.

  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.

  FIG. 5 shows the result of one well that was reacted using a chip having a concave well upper surface 501 according to this example, and it was confirmed that no inhibition of fluorescence intensity detection by the bubbles 701 occurred.

  FIG. 7 is a graph of analysis results of SNPs by fluorescence reaction detected using a chip having a non-concave-shaped well upper surface 502 as a comparative 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. As indicated by the arrows, the fluorescence intensity detection inhibition by the bubbles 701 was confirmed.

  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 103 ... Main channel 103a ... Main channel peak 103b ... Main channel valley 105 ... Side 107 ... INLET / OUTLET
401 ... first base material 402 ... second base material 405 ... supporting portion 501 ... well upper surface (concave shape)
502 ... Well upper surface (non-concave shape)
701 ... Bubble

Claims (7)

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 injection port, has a main flow path provided closer to the center of rotation than the well, and the upper surface of the well has a concave shape protruding toward the upper center of the well. A featured sample analysis chip.   2. The sample analysis chip according to claim 1, wherein a main flow path leading from the injection port to each well is formed to be inclined with respect to a rotation center direction.   The sample analysis chip according to claim 1 or 2, wherein the substrate is made of a light-transmitting material.   A sample analysis method, comprising: dispensing a sample into the well by centrifugation on the sample analysis chip according to any one of claims 1 to 3.   The sample analysis method according to claim 4, wherein a sample to be measured for fluorescence is accommodated in the well and optical information of the sample is measured. Means for installing and rotating the sample analysis chip according to any one of claims 1 to 5;
A sample analyzer having detection and measurement means for detecting a reaction in the well.   7. The sample analyzer according to claim 6, wherein detection measurement is performed at the center of the well.

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