JP2017053650A - Sample analysis chip, sample analysis method, and sample analyzer - Google Patents

Abstract

Provided are a simple sample analysis chip, a sample analysis method, and a sample analysis apparatus capable of measuring a biochemical reaction while performing temperature control necessary for the biochemical reaction with high efficiency.
A sample having a base material on which a plurality of reaction fields and a channel into which a solution to be supplied to the plurality of reaction fields is introduced, and inducing a biochemical reaction by controlling the temperature of the solution in the reaction field. In the analysis chip, the reaction field includes a main reaction unit 104 capable of controlling temperature control from at least two directions, and a sub reaction unit 105 communicated with the main reaction unit 104. And at least a measurement surface for measuring a biochemical reaction by the measurement mechanism. By performing measurement on the measurement surface of the secondary reaction unit 105 while controlling the temperature of the main reaction unit 104 from two directions, sample analysis is performed while performing highly efficient temperature control.
[Selection] Figure 2

Description

  The present invention relates to a sample analysis chip, a sample analysis method, and a sample analysis apparatus.

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 and generate a plurality of reactions. These technologies are said 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 for 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 a plurality of wells, and a single reagent or reagent that reacts with these chemicals, specimens, and enzymes is used. It is necessary to pour into several wells from several main conduits to produce different reactions.
By using this method, multiple types of specimens can be processed simultaneously with the same reagent, and conversely, multiple types of specimens can be processed simultaneously. This saves time, effort, and cost. It can be greatly reduced.
When using this type of technique, it is important to use a technique that delivers equal amounts of sample to multiple reaction fields and a technique that prevents the contents of each well from being mixed together. The measurement method is important.

In Patent Document 1, a technique is provided in which liquid is fed to a circular reaction portion arranged on the circumference, and a biochemical reaction in the reaction portion is optically detected.
Patent Document 2 provides a technique for controlling the temperature of a reaction field on a circular substrate in a non-contact manner and performing various detections in the reaction field.

Japanese Patent No. 555041 Japanese Patent No. 4773035

However, the chip described in Patent Document 1 has a problem in that the temperature of one side of the reaction field cannot be controlled at least when measuring the reaction field, and efficiency is deteriorated only by controlling the temperature from one side.
In addition, the non-contact temperature control described in Patent Document 2 controls the temperature of the entire space including the chip, and thus is affected by the room temperature and the temperature condition of the apparatus, and measures the reaction result simultaneously with the temperature control. For this purpose, there is a problem that the reaction result must be detected from the rotating chip.

  The present invention has been made in view of the above-described circumstances, and the purpose thereof is a reaction that can suitably perform a chemical reaction by heating and cooling or heating, a biochemical reaction such as an enzyme reaction, and the like, and has few restrictions. An object of the present invention is to provide a low-cost sample analysis chip, a sample analysis method, and a sample analysis apparatus for measuring results.

A sample analysis chip according to an embodiment of the present invention includes a substrate on which a plurality of reaction fields and flow paths for supplying solutions to the plurality of reaction fields are formed, and controls the temperature of the solution in the reaction field. A sample analysis chip for inducing a biochemical reaction, wherein the reaction field includes a main reaction part capable of controlling temperature control from at least two directions, and a side reaction part communicating with the main reaction part, The side reaction part has at least a measurement surface for measuring a biochemical reaction by the measurement mechanism.
In addition, the sample analysis method according to another embodiment of the present invention includes at least the main reaction part of the sample analysis chip in the embodiment described above sandwiched by the temperature control mechanism from at least two directions, and the temperature control mechanism controls the temperature of the main reaction part. The chemical reaction is measured by the measurement mechanism on the measurement surface of the side reaction part.
Furthermore, a sample analyzer according to another embodiment of the present invention is a temperature controller that performs temperature control by sandwiching the sample analysis chip in the above embodiment and the sample analysis chip from at least two directions at least at the position of the main reaction unit. A mechanism and a measurement mechanism for measuring a biochemical reaction on the measurement surface of the side reaction part.

  According to one embodiment of the present invention, the temperature adjustment necessary for the biochemical reaction can be applied to the reaction field while sandwiching the sample analysis chip, and at the same time, the result of the biochemical reaction can be measured. Therefore, high-speed biochemical reaction can be performed, and at the same time, biochemical reaction can be measured and analyzed.

It is a perspective exploded view showing a sample analysis chip of a first embodiment of the present invention. It is a top view of the 1st base material of the sample analysis chip | tip of FIG. It is a top view which shows the other example of a 1st base material. It is a top view which shows the other example of a 1st base material. It is a top view which shows the other example of a 1st base material. It is a perspective exploded view showing a sample analysis chip of a second embodiment of the present invention. It is a top view of the 1st base material of the sample analysis chip | tip of FIG. It is a top view which shows the other example of a 1st base material. It is a top view which shows the other example of a 1st base material. It is a lineblock diagram showing an example of a sample analysis device using a sample analysis chip of a first embodiment. It is a block diagram which shows the other example of the sample analyzer using the sample analysis chip of 1st embodiment. It is a block diagram which shows an example of the sample analyzer using the sample analysis chip of 2nd embodiment.

Below, the sample analysis chip concerning one embodiment of the present invention is explained based on a drawing.
In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent, however, that one or more embodiments may be practiced without such specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.
The sample analysis chip according to the present embodiment sends a solution containing a sample containing a substance to be detected and mixed with a detection reagent mixed in advance or contained in the sample analysis chip. In the sample analysis chip in which a biochemical reaction occurs under a predetermined temperature condition, the reaction result is measured while the temperature of the sample analysis chip is controlled from at least two directions. Here, the case where the detection reagent is included in the sample analysis chip in advance will be described. However, the solution containing the sample is included in the sample analysis chip in advance, and the solution including the detection reagent is sent. You may make it liquid.

FIG. 1 is an exploded perspective view showing an embodiment of a sample analysis chip according to the first embodiment of the present invention.
As shown in FIG. 1, the sample analysis chip 1 can be formed by bonding a first base material 101 and a second base material 102 having a circular plan view. In this case, the first base material 101 or the second base material 102 is provided with a flow path structure, and another base material is bonded to the base material provided with the flow path structure, so that the first base material 101 The formed channel structure is sealed. The first base material 101 and the second base material 102 do not have to be a single base material, and can be prepared by further laminating the respective base materials in layers.

FIG. 2 is a plan view of the first substrate 101 of the sample analysis chip of FIG.
A flow path 103 and a reaction field 110 are formed in the first substrate 101, and the reaction field 110 includes a main reaction unit 104 and a sub reaction unit 105.
A flow path 103 having a concentric concave portion of the first base material 101 is formed in the center of the first base material 101 in plan view. Further, a groove-like side passage 107 extending radially from the center of the first base material 101 is formed. The main reaction part 104 is formed in a concave shape, and communicates with the flow path 103 via the corresponding side passage 107. Each of the main reaction portions 104 is formed with a groove-like sub reaction portion 105 that is located on the extension of the corresponding side passage 107 and extends from the main reaction portion 104 in the edge direction of the first base material 101. .

Further, a sample liquid inlet 106 is formed at the center of the flow path 103.
In FIG. 2, the sample solution inlet 106 is provided on the first base material 101 side, but may be provided on the second base material 102 side as long as the sample can be introduced into the flow path 103.
The sample liquid can be introduced into the sample liquid inlet 106 by a pipette tip generally used in biochemistry. The introduction of the sample liquid may be performed manually, or may be performed by an automated instrument or device.
The flow path 103 and the main reaction part 104 communicate with each other through a side path 107, and the sample liquid introduced into the flow path 103 is rotated about the center of the circular base material by using a centrifugal force to circulate the side path 107. Then, the liquid can be sent to the main reaction unit 104 and the sub reaction unit 105.

  In the case of a rectangular substrate or a polygonal substrate such as the chip of the second embodiment to be described later, the sample solution is introduced from the flow path 103 into the main reaction unit 104 and the sub reaction unit 105 using the pipette chip described above. May be. When the solution cannot be sent to all the reaction fields 110 with a pipette tip, the solution can be sent under pressure with air or the like. In the case of a rectangular base material or a polygonal base material, as in the case of the circular base material in FIG. 2, the reaction fields 110 are arranged radially around the sample solution inlet 106 provided at the center of gravity. For example, the sample liquid inlet 106, the main reaction unit 104, and the side reaction unit 105 may be sent by centrifugal rotation about the center of gravity. In the case where the liquid is supplied to the unit 105 in a positional relationship where the liquid can be supplied, the liquid may be sent by centrifugal rotation.

FIG. 3 is a plan view of the first substrate 101 showing another example of the sample analysis chip according to the present embodiment.
In the sample analysis chip according to the present embodiment, the side reaction unit 105 is not limited to being arranged on the extension of the side passage 107 as shown in FIG. 2, but the side reaction unit 105 as shown in FIG. However, it may be arranged in a direction slightly inclined from the extension of the side path 107. In this case, the side reaction part 105 may be arranged at a position where the sample liquid is sufficiently introduced into the side reaction part 105 via the side passage 107 and the main reaction part 104.

FIG. 4 is a plan view of the first substrate 101 showing another example of the sample analysis chip according to the present embodiment.
As shown in FIG. 4, the flow path 103 has an annular groove shape arranged on the circumference of a concentric circle of the first base material 101, one end of the ring being the sample solution inlet 106 and the other end. The sample solution may be introduced into the flow path 103 using the sample solution outlet. In this case, the side passages 107 are not arranged radially with respect to the center of the first base material 101, but the edge of the first base material 101 with respect to the traveling direction of the sample liquid introduced from the inlet 106. The side channel 107 and the side reaction unit 105 may be arranged so that the sample liquid is sufficiently introduced into the side reaction unit 105 through the side channel 107 and the main reaction unit 104.

FIG. 5 is a plan view of the first substrate 101 showing another example of the sample analysis chip according to the present embodiment.
As shown in FIG. 5, the flow path 103 is arranged radially with respect to the center of the first base material 101, and one end of the flow path 103 on the edge side of the first base material 101 is connected to the main reaction unit 104, By providing the introduction port 106 at the other end of the channel 103, the sample solution is introduced from the introduction port 106 for each channel 103, and the sample solution is introduced into the side reaction unit 105 through the channel 103 and the main reaction unit 104. You may make it do. Moreover, the side reaction part 105 may be arrange | positioned so that it may connect with the main reaction part 104 on the extension of the flow path 103 arrange | positioned radially as shown in FIG. 2, and the flow path 103 is shown in FIG. It may be arranged in a direction slightly tilted from the extension.

FIG. 6 is an exploded perspective view showing a uniform state of the sample analysis chip 1 according to the second embodiment of the present invention.
In the sample analysis chip in the second embodiment, as shown in FIG. 6, the first base material 101 and the second base material 102 have, for example, a rectangular shape in plan view. Here, the first base material 101 and the second base material 102 will be described in the case where the plan view is rectangular. However, the first base material 101 and the second base material 102 are not limited to the rectangular shape, and the plan view has a shape such as an ellipse, a trapezoid, and a triangle. And having a longitudinal direction and a lateral direction. Of course, it can also be applied to a base material having a shape such as a square or a regular pentagon, but by using a base material having a longitudinal direction and a short direction, a useless area within the substrate area is reduced. can do.
In order to send the sample solution in these forms, the substrate surface is hydrophilized to promote introduction of the solution, or the solution is centrifuged so that the sample solution inlet is at the most central side when it is centrifuged. However, the method is not limited as long as the main reaction part and the side reaction part are filled with the sample.

FIG. 7 is a plan view of the first substrate 101 of the sample analysis chip 1 according to the second embodiment of FIG.
As shown in FIG. 7, the main reaction portion 104 has a concave shape that is circular in plan view, and a plurality of main reaction portions 104 are arranged along the longitudinal direction of the first base material 101. A groove-like flow path 103 is formed between the main reaction portions 104. A groove-like sub-reaction portion 105 is formed so as to be orthogonal to the direction in which the main reaction portions 104 are arranged in the main reaction portion 104.
Further, the end portion of the flow path 103 formed near one end in the longitudinal direction of the first base material 101 is opposed to the sample liquid inlet 106 provided in the second base material 102 as shown in FIG. Arranged.
Then, the first base material 101 and the second base material 102 are bonded together, and the sample liquid is introduced from the inlet 106 so that the first base material passes through the flow path 103 and the main reaction portion 104 alternately. 101, the sample liquid is introduced from one end side to the main reaction section 104 on the other end side, and the sample liquid introduced into each main reaction section 104 is introduced into the sub reaction section 105 provided in each main reaction section 104. Is done.

The shape of the side reaction part 105 is arbitrary. As shown in FIG. 8, in the side reaction part 105, a measurement part 105a that performs measurement and a measurement part 105a that communicates the measurement part 105a and the main reaction part 104. It is also possible to provide a communication portion 105b having a narrow width in a plan view and reduce the volume of the communication portion 105b.
The measurement unit 105a that actually performs the measurement can increase the amount of light and enhance the sensitivity and accuracy by making it an effective size to some extent according to the size of the detection unit of the measurement mechanism described later. In 105b, since the temperature is not adjusted and the photometry is not performed, it is preferable that the amount of the sample solution is small.

FIG. 9 is a plan view showing another example of the first base material 101 of the sample analysis chip 1 according to the second embodiment.
In the first base material 101 shown in FIG. 9, a plurality of main reaction portions 104 are arranged along the flow path 103 arranged along the longitudinal direction of the first base material 101, and in a direction substantially orthogonal to the flow path 103. Each main reaction part 104 and the flow path 103 communicate with each other by the branched side path 107. Further, on the extension of the side passage 107, a groove-like sub reaction portion 105 extending from the main reaction portion 104 is provided. As shown in FIG. 7, the side reaction part 105 may have a constant groove width in a plan view, and may have a shape including a measurement part 105a and a communication part 105b as in FIG.
In the sample analysis chip 1 in the first and second embodiments, there is no problem even if the depth is set freely in each of the flow path 103, the main reaction section 104, the sub reaction section 105, and the side path 107. In order to reduce the volume of the reaction unit 105 and improve the reaction efficiency, the side reaction unit 105 is preferably made shallow.

FIG. 10 is a configuration diagram showing an example of a sample analyzer using the circular sample analysis chip 1 in the first embodiment.
The sample analysis chip 1 is disposed so as to be sandwiched between the main temperature adjustment mechanism 201 and the sub temperature adjustment mechanism 202, and temperature adjustment is performed from both surfaces of the sample analysis chip 1. Specifically, the first base material 101 side of the sample analysis chip 1 and the main temperature control mechanism 201 are disposed so as to contact each other, and the second base material 102 and the sub temperature control mechanism 202 are disposed so as to contact each other.
At this time, the main temperature adjustment mechanism 201 is configured to be able to adjust the temperature covering at least the entire area of the main reaction unit 104. For example, as shown in FIG. Alternatively, it may be formed, or a donut-shaped region covering all the main reaction parts 104 in a plan view may be formed to be temperature-controllable.

The sub-temperature control mechanism 202 is disposed so as to cover the entire area of the second base material 102 that overlaps the main reaction part 104 in a plan view and does not cover at least a part of the area that overlaps the sub-reaction part 105. A region of the second base material 102 that is not covered with the sub temperature control mechanism 202 is a measurement surface for measuring a biochemical reaction.
With the main temperature adjustment mechanism 201 and the auxiliary temperature adjustment mechanism 202 arranged in this manner, the measurement mechanism 203 is moved along the outer periphery of the auxiliary temperature adjustment mechanism 202, and the annular region on the upper surface of the auxiliary reaction unit 105 is moved. By measuring, most of the reaction solution can be measured while strongly controlling the temperature from two directions.
The measurement mechanism 203 is formed by combining, for example, a photomultiplier tube and an optical fiber. By adjusting the temperature from both sides of the sample analysis chip 1, a fluorescence detection reaction is caused to the sample liquid in the sample analysis chip 1, and the fluorescence wavelength generated by the fluorescence detection reaction is measured by the measurement mechanism 203. The sample solution is analyzed by measuring.

  Further, as another example of the sample analyzer using the sample analysis chip 1 in the first embodiment, as shown in FIG. 11, the main temperature adjustment mechanism 201 is dug into a size larger than that of the sample analysis chip 1. Thus, the first base material 101 side including the side surface of the sample analysis chip 1 is completely covered by the main temperature control mechanism 201, and the second base material 102 side is sub-temperature controlled except for a part of the side reaction part 105. By covering with the mechanism 202, the strength of temperature control and the temperature change rate can be improved.

FIG. 12 is a configuration diagram showing an example of a sample analyzer using the rectangular sample analysis chip 1 in the second embodiment of the present invention.
The sample analysis chip 1 is arranged such that the first base material 101 side is in contact with the main temperature control mechanism 201 and the second base material 102 side is in contact with the sub temperature control mechanism 202, whereby the main temperature control mechanism 201 and the sub temperature control mechanism are arranged. The temperature is adjusted so as to be sandwiched by 202.
At this time, the main temperature adjustment mechanism 201 is formed so that the temperature of the region including all the main reaction units 104 can be adjusted, and as shown in FIG. 12, the temperature of the entire surface of the sample analysis chip 1 on the first substrate 1 side is adjusted. Alternatively, the region including only the main reaction unit 104 in a plan view of the surface on the first base material 1 side may be configured to be temperature-controllable.

The sub-temperature control mechanism 202 is disposed so as to cover the entire surface of all the main reaction units 104 and at the same time not cover at least a part of the sub-reaction units 105.
As described above, the measurement mechanism 203 moves along the side surface of the sub temperature control mechanism 202 in a state where at least a part of the sub reaction unit 105 is not covered by the sub temperature control mechanism 202 or the main temperature control mechanism 201. By measuring the upper surface of 105 linearly, most of the reaction solution can be measured while strongly controlling the temperature from two directions.
The main reaction unit 104 and the sub reaction unit 105 can improve the reaction efficiency according to the volume ratio, and the volume ratio of the sub reaction unit 105 and the main reaction unit 104 is 1: 1 or less. It is desirable that the volume of the secondary reaction unit 105 is equal to or less than the volume of the main reaction unit 104. For example, if the volume of the main reaction unit 104 is 10 μl with respect to the volume of 0.1 μl of the secondary reaction unit 105, the reaction solution is almost from two directions. Therefore, it is possible to obtain a highly efficient temperature control capability, and sufficient efficiency can be obtained even with about 3 μl of the main reaction part with respect to 0.5 μl of the secondary reaction part. I can do things.

If there is only one reaction part and photometry is performed while applying temperature control to the reaction part, a surface that contacts the temperature control mechanism and a surface to be measured are required. Therefore, even if heat is applied from one side, the heat escapes from the side that is not in contact with the temperature control mechanism. The same applies to cooling.
Therefore, it is possible to realize a highly efficient temperature control-measurement function by sandwiching most of the liquid whose temperature is to be adjusted so that the temperature can be changed suddenly, and by measuring and measuring the result without inserting a part of the liquid. .
The sample analysis chip 1 is particularly effective in a reaction in which a temperature control program such as a PCR (polymerase chain reaction) method is complicated, or in a temperature reaction in which heat is more than 90 degrees, which easily escapes heat.

Next, the manufacturing method of the sample analysis chip of the present invention will be described.
First, the first substrate 101 is formed by molding using a mold.
The 1st base material 101 can be selected in the thickness range of about 100 micrometers or more and 10 mm or less.
When the thickness of the 2nd base material 102 exists in the range of 10 micrometers or more and 300 micrometers or less, both the heat conductivity of the 1st base material 101 and lid | cover performance can be satisfied suitably. When the thickness of the second base material 102 is larger than 300 μm, the heat capacity becomes large and the thermal responsiveness may be lowered.

  As a material of the first base material 101, a resin having optical transparency can be used. In addition, when optical analysis (fluorescence measurement, colorimetric measurement, or the like) is performed on the solution in the side reaction unit 105, it is preferable that the first substrate 101 has high transparency. For example, the material of the first base material 101 is not particularly limited as long as it does not affect the sample. However, if a resin material containing any of polypropylene, polycarbonate, and acrylic is used, good visible light transmittance can be obtained. Can be secured. 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. In the case where a metal material is used, in addition, it has excellent thermal conductivity and sealing performance, but overlaps with the side reaction part 105 in top view on at least one side of the first base material 101 side or the second base material 102 side. Need to be partially changed to a light transmissive material.

In the embodiment of the present invention, “transparent” and “light transmissive” mean that the total average transmittance in the visible light region (wavelength of 350 nm or more and 780 nm or less) when formed is 70% or more. When the measurement (photometry) target is in a wavelength range other than visible light, it is preferable that the measurement target has a light transmittance that is measurable in the wavelength range of the measurement target.
As a method of bonding the first base material 101 and the second base material 102, there is a method in which a resin coating layer is provided as an adhesive layer on one base material, and this is melted to bond both base materials. 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 polyethylene terephthalate (PET), polyacetal, polyester, or polypropylene can be used.

In the bonding process of the first base material 101 and the second base material 102, a finely transmissive and light-transmitting resin material suitable for fluorescence measurement is used for the first base material 101. On the other hand, it is preferable to use a material that can be easily bonded by fusion bonding for the second substrate 102.
As the material of the second base material 102, a metal material having a high thermal conductivity and having a resin coating layer for fusion bonding can be suitably used. By forming the resin coating layer on the surface of the metal material, it is not necessary to consider the chemical resistance of the metal material itself when selecting the material.

Further, when a resin coating layer is formed on the surface of the metal material in the second base material 102, if an anchor layer is formed as a base of the resin coating layer, fusion using a laser is possible. The anchor layer suitable for fusing using a laser is preferably incorporated with carbon black (light absorbing material) that absorbs laser wavelength light. In this case, by irradiating the laser beam, the anchor layer generates heat and the resin coating layer can be melt bonded.
Further, 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 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 entire sample analysis chip 1, so that the first base material hardly affects the sample analysis chip 1 or the reagent fixed to the sample analysis chip 1. 101 and the 2nd base material 102 can be bonded together.

Next, details of the sample analysis method will be described using a specific example of the sample analysis method of the present embodiment.
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.

Germline mutation detection Germline mutations can be detected by identifying SNPs. As one of the methods for identifying SNPs, for example, a PC-PHFA (PCR-Preferred Modulation Formation Assay) method using fluorescence is used. According to the method, mutation is detected by the difference in luminescence of the fluorescent reagent, but by using the sample analysis chip 1 of the present invention, since there is little liquid distribution variation in each well, accurate analysis is possible. Suitable for detecting SNPs. In addition, a sample analysis chip can be similarly used for the Invader (registered trademark) method and the like as a method for detecting SNPs other than the above.

In the case of fluorescence measurement, there is one that measures fluorescence generated by irradiating excitation light. In that case, the measurement mechanism is a photometry mechanism, and preferably includes an optical fiber, a filter for selecting a specific wavelength range, a light source for excitation light, a photomultiplier tube, and the like.
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 injection into the sample analysis chip 1 of the present embodiment. For example, VK4, VK1, CYP2C9 * 3, CYP2C9 * 2, and positive controls (such as MTHFR) are used to amplify gene fragments in the sample by multiplex PCR. Transfer sample and perform PCR.

In the above detection method, two main reaction units 104 and sub reaction units 105 for detection are required to determine one SNP, and therefore, 10 or more main reaction units 104 and sub reaction units 105 are required for each specimen sample. It is preferable to use a sample analysis chip on which SNP is formed, and a reagent for SNP detection is fixed to each reaction part.
The sample whose nucleic acid is amplified by the PCR is distributed to each main reaction unit 104. Each main reaction unit 104 is temperature-controlled, the measurement of the sub-reaction unit 105 is performed, and a mutation is detected based on the difference in luminescence of the fluorescent reagent mixed in the SNP detection reagent. If only one of the two side reaction parts 105 is positive with respect to one SNP, it can be determined to be homo, and if two are positive, it can be determined to be hetero.

-Detection of K-ras gene mutation In this example, the sample analysis chip 1 different from the sample analysis chip 1 used for the detection of the germ cell mutation is used.
A reagent containing a probe nucleic acid is fixed to the main reaction part 104 and the side reaction part 105 for detecting the gene mutation. The reagent containing the probe nucleic acid contains a fluorescent reagent. Since there are 13 types of mutations in the detection of the K-ras gene, a sample analysis chip in which at least 14 main reaction parts 104 and side reaction parts 105 are formed is used, and corresponding to each of the main reaction parts 104 It is preferable that the reagent is immobilized.
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 1 of the present invention. The sample is fed, PCR is performed, and detection is performed in the same manner.

As described above, the sample analysis chip 1 according to the present embodiment includes the first base material 101 including the flow path for feeding the sample, the main reaction unit 104, the sub reaction unit 105, and the flow path 103. It has at least a second base material 102 for covering, and a reaction field for performing a biochemical reaction by adjusting temperature, a main reaction unit 104 for performing a temperature control reaction, and a sub-field for measuring a biochemical reaction. The reaction part 105 was divided.
In order to adjust the temperature, the sample analysis chip 1 is sandwiched by a temperature control mechanism from at least two directions to adjust the temperature of the main reaction unit 104. At this time, the side reaction unit 105 is at least in one direction so that measurement is possible. It is released from the temperature control mechanism. Therefore, this biochemical reaction can be measured by the side reaction unit 105 while the temperature of the main reaction unit 104 is adjusted to cause a biochemical reaction. That is, the temperature is efficiently adjusted by adjusting the temperature in at least two directions by the temperature adjustment mechanism in the main reaction unit 104. On the other hand, because the temperature of the secondary reaction unit 105 is controlled only by the temperature control mechanism from one direction, the temperature control efficiency is lower than that of the main reaction unit 104, but the main reaction unit 104 communicates with the secondary reaction unit 105. Therefore, the solution causing the biochemical reaction caused by the temperature change in the main reaction unit 104 and the solution in the sub reaction unit 105 come and go to each other and are mixed. In particular, in biochemical reactions that repeat temperature changes such as PCR and biochemical reactions that use high temperatures, the solution flow is intense, and as a result, the reactions of the main reaction unit 104 and the sub reaction unit 105 occur at almost the same timing. Become.

At this time, since the volume of the side reaction part 105 is set to be equal to or less than the volume of the main reaction part 104, the generation rate of the biochemical reaction in the main reaction part 104 is dominant, and the side reaction rapidly follows the situation. A biochemical reaction can be caused in the sample in the unit 105, and the biochemical reaction in the main reaction unit 104 can be measured with high accuracy in the sub-reaction unit 105.
In addition, since the main reaction unit 104 is arranged on the circumference and the sub reaction unit 105 is arranged on a concentric circle outside the main reaction unit 104, the circular temperature control mechanism is not covered by the sub reaction unit 105 and the main reaction unit 105 is not covered. By disposing the reaction unit 104 so as to cover it, the measurement mechanism can be easily positioned at a position facing the sub-reaction unit 105 by moving the measurement mechanism along the outer periphery of the temperature control mechanism.

Similarly, the main reaction unit 104 is arranged on a straight line, and the sub reaction unit 105 is arranged along the main reaction unit 104 on one side of the row of the main reaction units 104. By disposing the reaction unit 105 so as to cover the main reaction unit 104 without covering it, the measurement mechanism can be easily moved to a position facing the sub reaction unit 105 by moving the measurement mechanism along the edge of the temperature control mechanism. Can be positioned.
In order to efficiently perform measurement by the measurement mechanism, it is preferable that the main reaction units 104 are provided in a circular shape and linearly. However, efficient measurement can be performed by combining the temperature control mechanism and the measurement mechanism. This is not the case if possible.

Further, in order to efficiently perform the measurement by the measurement mechanism, it is preferable that the side reaction parts 105 are provided in a circular shape and linearly, but the temperature adjustment mechanism, the measurement mechanism, and the main reaction part 104 are arranged in a line. This is not necessarily the case as long as efficient measurement is possible by combination.
Further, by making the first base material 101 and the second base material 102 into a circular shape, it is possible to make it easy to use centrifugal force for feeding the sample.
In the sample analysis chip 1 in which the first base material 101 and the second base material 102 are circular, the main reaction unit 104 is arranged on the circumference of the concentric circle on the first base material 101 or the second base material 102. In addition, the side reaction part 105 can be measured on the side reaction part 105 while efficiently adjusting the temperature by sandwiching the main reaction part 104 by arranging the side reaction part 105 on the outer circumference of the concentric circle.

In addition, if the first base material 101 and the second base material 102 are long in one direction and the main reaction unit 104 and the sub reaction unit 105 are arranged on a straight line, the measurement mechanism is sequentially moved in one direction. Therefore, the measurement efficiency in the side reaction unit 105 can be increased.
In the above embodiment, the case where the main reaction part and the sub reaction part are arranged on the circumference of the concentric circle on the circular base material has been described. However, the present invention is not limited to this, for example, a polygonal shape. You may make it arrange | position a main reaction part and a sub reaction part on the base material on the circumference of a concentric circle.
Although the present invention has been described above with reference to specific embodiments, the present invention is not limited to these descriptions. From the description of the invention, other embodiments of the invention will be apparent to persons skilled in the art, along with various variations of the disclosed embodiments. Therefore, it is to be understood that the claims encompass these modifications and embodiments that fall within the scope and spirit of the present invention.

  The sample analysis chip of the present invention can be used for detection and analysis of biochemical substances in a sample such as a nucleic acid. In particular, since the mutation of SNP can be detected, it can be used for a technique 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 First base material 102 Second base material 103 Channel 104 Main reaction part 105 Sub reaction part 106 Inlet 107 Side path

Claims (13)

A substrate having a plurality of reaction fields and a flow path for supplying a solution to the plurality of reaction fields;
A sample analysis chip for inducing a biochemical reaction by controlling the temperature of the solution in the reaction field,
The reaction field is a main reaction part capable of controlling the temperature from at least two directions,
A secondary reaction part communicated with the main reaction part,
The side reaction part has at least a measurement surface for measuring the biochemical reaction by a measurement mechanism. The sample analysis chip according to claim 1,
The substrate has a circular shape or a polygonal shape,
The reaction field is
The main reaction part is located on the circumference;
The sample analysis chip, wherein the side reaction part is arranged so as to be positioned on a circumference of a concentric circle outside the main reaction part. The sample analysis chip according to claim 1,
The substrate has a longitudinal direction and a transverse direction;
The reaction field is
The main reaction part is located on a straight line along the longitudinal direction;
The sample analysis chip, wherein the side reaction part is arranged on one side of the main reaction part so as to be positioned along a line of the main reaction part. The sample analysis chip according to claim 1 or 2,
A sample analysis chip, wherein each main reaction part has a side path whose one end communicates with the main reaction part, and the other end of the side path communicates with the flow path. The sample analysis chip according to any one of claims 1 to 4,
A sample analysis chip, wherein an introduction port for introducing the solution into the channel is provided. The sample analysis chip according to any one of claims 1 to 5,
The volume of the said main reaction part is larger than the volume of the said secondary reaction part, The sample analysis chip characterized by the above-mentioned. The sample analysis chip according to any one of claims 1 to 6,
The side reaction unit includes a communication unit communicating with the main reaction unit and a measurement unit for measuring the biochemical reaction. The sample analysis chip according to any one of claims 1 to 7,
A sample analysis chip comprising: a first base on which the reaction field and the flow path are formed; and a second base that is bonded to the first base and seals the first base. . The sample analysis chip according to claim 8, wherein
A sample analysis chip, wherein at least one of the first base material and the second base material is formed of a light transmissive material. The sample analysis chip according to claim 8, wherein
A sample analysis chip, wherein at least one of the first substrate and the second substrate is made of a metal material. At least the main reaction part of the sample analysis chip according to any one of claims 1 to 10, is sandwiched by a temperature control mechanism from at least two directions,
A sample analysis method, wherein the biochemical reaction is measured by the measurement mechanism on the measurement surface of the sub-reaction part while the temperature of the main reaction part is adjusted by the temperature adjustment mechanism. The sample analysis chip according to any one of claims 1 to 10,
A temperature control mechanism for adjusting the temperature by sandwiching the sample analysis chip at least at the position of the main reaction part from at least two directions;
And a measurement mechanism for measuring the biochemical reaction on the measurement surface of the side reaction unit. The temperature control mechanism includes a main temperature control mechanism that covers the entire main reaction part and the sub reaction part, and
A sub-temperature control mechanism that is disposed on the opposite side of the main temperature control mechanism across the sample analysis chip and covers the entire main reaction unit and a region excluding at least the measurement surface of the sub-reaction unit. The sample analyzer according to claim 12.

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