JP5499840B2 - Sample analysis chip 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 biochemical reaction detection and analysis methods.

  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 sample solution. . 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, an analysis chip having an inlet and an outlet at both ends of a main channel and a reaction container for storing a solution in the main channel is disclosed. Is put into a sealed container, and the solution is sent from the main channel to the reaction container by reducing the pressure with a vacuum pump. Therefore, equipment and an apparatus for reducing pressure are required like a vacuum pump, and automation is difficult. Further, when the main flow path connecting the reaction vessel and another reaction vessel is curved, it is not possible to distribute the liquid only by the pressure difference, and it is necessary to rotate the chip, and a complicated mechanism and space are required.

JP 2009-178146 A

  In light of the above-described problems of the prior art, the present invention is to provide a sample analysis chip capable of performing detection and analysis with simple equipment and a small amount of equipment, in a sample analysis chip that supplies liquid to a well. And It is another object of the present invention to provide a sample analysis chip that does not contaminate each sample when analyzing a plurality of samples simultaneously. The term “contamination” as used herein means that reagents that should not be mixed are mixed. There is no limitation on the amount to be mixed, and contamination occurs even when the amount of one reagent to be mixed is very small relative to the amount of the other reagent. Contamination increases the possibility of an unreliable reagent reaction, so it is necessary to prevent it from occurring as much as possible.

The invention according to claim 1 of the present invention, which has been made to solve the above-described problems, includes a main channel and an injection port for injecting a first solution that opens to one of the end portions of the main channel. An inlet for injecting a second solution that opens to the other end of the main flow path, and a plurality of straight portions of the main flow path, which can accommodate the first and second solutions Do, has a plurality of reaction field is depression asymmetric, each of the plurality of reaction fields were referenced to the main channel of the inlet side of the first solution, the first solution angle a of the end face of the inlet side to the main channel, relative to the said main channel of the inlet side of the second solution, forming of the second solution the main channel and the inlet side end face of the The sample analysis chip is characterized by being larger than the angle B.
The invention according to claim 2 of the present invention is the sample analysis chip according to claim 1, wherein the angle A falls within a range of 100 ° to 140 °.
The invention according to claim 3 of the present invention is the sample analysis chip according to claim 1 or 2, wherein the angle B is within 20 ° to 100 °.
The invention according to claim 4 of the present invention is the sample analysis chip according to any one of claims 1 to 3, wherein at least the reaction field is subjected to a hydrophilic treatment.
In the invention according to claim 5 of the present invention, the sample analysis chip includes a first base material on which the reaction field and the flow path are formed, and a second base material bonded to the base material. A sample analysis chip according to any one of claims 1 to 4.
Further, in the invention according to claim 6 of the present invention, any one of the base materials is formed of a light-transmitting material, and the sample analysis according to any one of claims 1 to 5 Chip.
In the invention according to claim 7 of the present invention, the first base material is a light-transmitting resin material, and the second base material is a metal material. The sample analysis chip according to any one of the above.
Moreover, in the invention according to claim 8 of the present invention, the first solution flowing in the main channel of the sample analysis chip according to any one of claims 1 to 7 is hydrophilic, and the second solution The main channel is formed from a different inlet from the one into which the first solution is injected after the step of distributing the solution to the main channel, wherein the solution is a substance having a low affinity with the first solution. A sample analysis method comprising the step of injecting the second solution into the well and the reaction / detection step in each well.
The invention according to claim 9 of the present invention is a gene analysis method characterized by using the sample analysis method according to claim 8.

According to the sample analysis chip of the present invention, a simple, functional and inexpensive reaction chip can be realized. Furthermore, the reaction can be observed with respect to one type of specimen using a plurality of reagents.

  Further, according to the sample analysis chip of the present invention, when the liquid is sent from the main channel to the reaction field, the surplus can be discharged from an inlet different from the inlet into which the solution is injected. Therefore, by supplying more liquid than desired in all wells, a uniform amount of liquid can be distributed to all reaction fields, and variations can be eliminated.

  In addition, when the different solutions are hydrophobic, the hydrophobic solution filled in the main channel seals the reaction field, so that the solution distributed in the reaction field is different from the solutions in other reaction fields. Contact is eliminated and contamination between reagents can be prevented.

Sectional drawing of the one aspect | mode of the sample analysis chip | tip of this invention Enlarged view of reaction field in sample analysis chip of the present invention

A sample analysis chip of the present invention will be described with reference to the drawings.
FIG. 1 is a sectional view showing an embodiment of the sample analysis chip of the present invention. The chip of the present invention has a plurality of reaction fields 102 on a first substrate, a channel for sending a solution, for example, a liquid sample, to the reaction field, and a solution, for example, a liquid sample, is injected into the channel. It has an inlet for. The flow path has a main flow path 101 that communicates with each reaction field in order to send a solution to each reaction field. The flow path has an injection port 103 that communicates with one end of the main flow path for injecting the solution, and an injection port 104 that communicates with the other end of the main flow path.

  The sample analysis chip of the present invention distributes a hydrophilic solution to the reaction field 102 by injecting a hydrophilic solution (first solution) from the injection port 103 into the main flow channel 101, and the injection port at the other end. A hydrophobic solution (second solution) is injected from 104 into the main channel 101. Since the hydrophilic solution accumulated in the reaction field is sealed with the hydrophobic solution, it is desirable that the hydrophobic solution does not flow into the reaction field. An angle A201 formed between the main flow path on the injection port 103 side for injecting the hydrophilic solution and the reaction field 102 is larger than an angle B202 formed between the main flow path on the injection port 104 side for injecting the hydrophobic solution and the reaction field 102. Thus, the hydrophobic solution can be flowed through the main channel without involving the hydrophilic solution accumulated in the reaction field. The hydrophilic solution is an aqueous solution containing at least water as a solvent and a reagent for analysis, and detection or analysis is performed for each hydrophilic solution distributed in each reaction field.

  According to the present invention, the hydrophobic solution filled in the main channel seals the reaction field, so that the solution distributed in the reaction field does not come into contact with the solution in the other reaction field, Contamination between reagents can be prevented. In addition, by supplying more hydrophilic solution to all wells than desired, a uniform amount of hydrophilic solution can be distributed to all reaction fields, and variations can be eliminated. .

  The angle A201 of 0 ° to 180 ° formed by the main flow channel 101 on the inlet side for injecting the hydrophilic solution and the reaction field 102 is preferably 100 ° to 140 °. If it is 0 ° to 99 ° or 141 ° to 180 °, it is difficult to fill the reaction field with a hydrophilic solution. As shown in FIG. 2, when the main channel 101 and the reaction field 102 are connected by a curved surface, an angle (0) formed by a tangent line A203 extending from the main channel 101 side and a tangent line B204 from the linear part of the reaction field. (° to 180 °).

  The angle B202 between 0 ° and 180 ° formed by the reaction channel 102 and the main channel 101 on the inlet side for injecting the hydrophobic solution is preferably 20 ° to 100 °. If it is 0 ° to 19 ° or 101 ° to 180 °, a hydrophobic solution may flow into the reaction field, which is not desirable.

  The volume of the reaction field 102 is desirably 5 to 20 μL.

  In addition, the hydrophilicity of the reaction field 102 or the entire inner surface of the flow path including the reaction field increases the affinity between the hydrophilic solution and the reaction field 102 compared to the hydrophobic solution. Variations can be reduced. Examples of the hydrophilic treatment include plasma treatment, corona discharge treatment, chemical treatment, and fine processing.

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

  The sample analysis chip of the present invention can be prepared by bonding a second substrate to a first substrate having a reaction field, a main channel, and an inlet. At this time, the second base material needs to have an area that can cover the reaction field, the main flow path, and the injection port of the first base material in order to prevent the distributed solution from leaking.

  The substrate is not particularly limited as long as it does not affect the reactivity of the reagent and the solution to be distributed, but a substrate that can ensure good visible light transmittance, for example, polypropylene and acrylic are preferable. The light transmittance in the present invention is 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, it is easy to grasp the sample state in the chip, but this is not restrictive.

  Further, as a material other than the resin, a metal material, for example, aluminum, copper, silver, nickel or the like is desirable. When using a metal material, it is excellent in thermal conductivity and bonding performance.

  The reagent is fixed to the reaction field 102 before the substrates are bonded. Since each reaction field is isolated, it is possible to fix different reagents in each reaction field. By fixing different reagents for each reaction field, a plurality of reactions can be examined at one time for one specimen.

  As a method for fixing the reagent, for example, the reagent can be fixed to the reaction field by dropping a liquid reagent onto the reaction field 102 of the first substrate with a pipette or the like and drying it.

  Alternatively, wax may be dropped after fixing the reagent on the reaction field 102. For example, wax dissolved on a block bath is dropped onto a reagent fixed on the reaction field with a pipette or the like. At this time, it is possible to prevent the reagent from flowing out by dripping the wax so as to cover the fixed reagent. If the first substrate is below the melting temperature of the wax, the dropped wax will solidify immediately.

  As a bonding method of a base material, the method of adhere | attaching a 1st base material and a 2nd base material by providing resin coating as an adhesive layer in one base material, and making it melt | dissolve by applying heat is mentioned. For example, when the second substrate is made of metal, the resin coating layer is desirably provided on the metal material side having high thermal conductivity and melt-bonded.

  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 protein. A reagent is fixed to each reaction field 102, and wax is dropped on the fixed reagent. In this case, a different reagent can be used for each reaction field.
Thereby, it can suppress that a reagent melt | dissolves in a solution and flows out during liquid distribution.

Next, a liquid sample is distributed to each well.

  Next, a solution such as a reagent is first injected into the main channel 101 from the injection port 103 with respect to the sample analysis chip of the present invention in which the first substrate and the second substrate are bonded together. Thereby, the main channel 101 and the reaction field 102 are filled with the solution.

  Next, a hydrophobic solution such as mineral oil that does not inhibit the reaction of the sample and the reagent is injected into the main channel 101 from the injection port 104 to the sample analysis chip into which the solution such as the reagent has been injected. As a result, the evaporation of the liquid during the reaction can be prevented, the outflow of the solution such as the reagent accumulated in the reaction field can be prevented, and contamination can be prevented. By using a mineral oil having a specific gravity lighter than the previously distributed solution, it plays the role of a plug for each reaction field on the main channel side. The type of mineral oil is not particularly limited as long as it does not inhibit the reaction of the sample or reagent.

  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.

  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 the mutation of SNP can be detected, it can be used for detection of mutated cells and genes. In addition, since a liquid reagent can be simultaneously mixed with a plurality of dried reagents, application as a reaction container is possible.

101: Main channel 102: Reaction field 103: Inlet A
104: Inlet B
201: Angle A formed by tangent line A and tangent line B
202: Angle B formed by the main flow path on the inlet 104 side and the reaction field 102
203: Tangent line A on the main channel side of the wall surface on the side where the main channel and the reaction field are connected
204: Tangent line B on the reaction field side of the wall surface on the side where the main channel and the reaction field are connected

Claims (9)

A main channel, an inlet for injecting a first solution that opens to one of the ends of the main channel, and an inlet for injecting a second solution that opens to the other end of the main channel And a plurality of reaction fields that are asymmetric depressions that are provided at a plurality of locations of the linear portion of the main flow path and can contain the first and second solutions ,
Each of the plurality of reaction fields, the first relative to the said main channel of the inlet side of the solution, the angle A of the main channel and the end face of the inlet side of the first solution, the second second the inlet side of the solution relative to the main channel, the second solution sample analysis chip being larger than the angle B of the primary flow passage and the inlet side end face of the. The sample analysis chip according to claim 1, wherein the angle A is within a range of 100 ° to 140 °. The sample analysis chip according to claim 1, wherein the angle B is within a range of 20 ° to 100 °. The sample analysis chip according to any one of claims 1 to 3, wherein at least the reaction field is subjected to a hydrophilic treatment. The said sample analysis chip | tip has the 1st base material in which the said reaction field and the said flow path were formed, and the 2nd base material bonded together with this base material. Sample analysis chip. 6. The sample analysis chip according to claim 1, wherein any one of the substrates is made of a light transmissive material. The sample analysis chip according to any one of claims 1 to 6, wherein the first base material is a light-transmitting resin material, and the second base material is a metal material.   The first solution that flows through the main channel of the sample analysis chip according to any one of claims 1 to 7 is hydrophilic, and the second solution has a low affinity with the first solution. A step of injecting the second solution into the main channel from an inlet different from the direction of injecting the first solution after the step of distributing the solution into the main channel, each well A sample analysis method characterized by comprising a reaction / detection step. A gene analysis method using the sample analysis method according to claim 8.

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