JP2015121493A - Sample analysis chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample analysis chip capable of performing a plurality of processes and also preventing effects of an external environment on reagents.SOLUTION: A sample analysis chip is for injecting sample liquid and causing a biochemical reaction. The sample analysis chip includes: an initial reaction part 21 capable of storing sample liquid; and a flow channel part 22 disposed in a periphery of the initial reaction part 21 and connected to the initial reaction part 21 in a planar view, and having a plurality of wells 24.

Description

  The present invention relates to a sample analysis chip, and more particularly to a sample analysis chip that can be used for detection and analysis of biochemical reactions and the like and can perform a plurality of processes.

  Conventionally, in the field of biochemical reactions such as DNA reaction and protein reaction, as a reaction apparatus for processing a small amount of sample solution, a technique called μ-TAS (Total Analysis System) or Lab-on-Chip is known. . This is one chip or cartridge provided with a plurality of reaction chambers (hereinafter referred to as wells) and flow paths, 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 merit is, for example, that the amount of strong acid and strong alkali chemicals that have been used in the past is reduced to a very small amount, and the influence on the human body and the environment is significantly reduced, and it is also used for biochemical reactions, etc. The amount of expensive 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, reagent reagents that react with different chemicals, specimens, and enzymes are placed in multiple wells and react with these chemicals, specimens, and enzymes. Must flow from one or several main conduits into the well to produce different reactions.
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. Is possible.

  However, there are many expensive or valuable samples to be poured at this time. In many cases, these samples are first subjected to several biochemical reaction treatments. Therefore, there are many usage methods in which a reaction is performed in a well using a sample that has been processed once in advance.

  In other words, when using this type of technique, the technology that processes the entire sample first, the technology that sends an equal amount of sample to multiple reaction fields, and the contents of each well after feeding are not mixed. Three technologies to make are important. The following is mentioned as a prior art about the chip | tip which performs liquid feeding to such a well.

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

  In patent document 2, the dispersion | variation in the amount of liquid feeding to each well is solved by interweaving rotation and revolution in centrifugation. However, this method requires a complicated mechanism and space for the chip to rotate and revolve, and when trying to add multiple treatments to the sample solution, if multiple treatments are performed after distribution, The dispersion for each reaction well overlaps and stable treatment cannot be performed.

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

  In Patent Document 4, there is a method of performing fluid control in a microchannel by moving a hot-melt material previously filled in a microchannel by air pressure from an inlet while controlling the viscosity with a heater. Have been described. However, this method requires an air pressure control mechanism, and in a solution processing chip that performs many reactions at once, the shape and mechanism for controlling the movement of the hot-melt material is complicated.

  In general, when an enzyme reaction is performed, if the enzyme and the substrate come into contact before reaching the reaction start temperature, an undesirable side reaction or reaction inhibition may occur, resulting in a problem that the reaction efficiency is lowered. In the enzyme reaction on the chip, for example, when each reaction chamber is filled with the enzyme and the substrate is fed as a sample solution, the enzyme and the substrate come into contact with each other when the liquid feeding is completed, and side reactions and reaction inhibition occur. May happen.

  Furthermore, when a biochemical reaction is performed on the chip, the reaction chamber is often heated after the sample solution is distributed, and an evaporation prevention material such as mineral oil is further injected to prevent the sample solution from evaporating. In some cases, the chip must be physically sealed, which increases the number of work steps.

JP-T-2004-502164 Japanese Patent No. 3699721 JP 2008-83017 A US Pat. No. 7,195,036

  In view of the above circumstances, an object of the present invention is to provide a sample analysis chip capable of performing a plurality of processes while preventing the influence of an external environment on reagents.

  The present invention is a sample analysis chip for injecting a sample liquid and performing a biochemical reaction, the initial reaction part capable of storing the sample liquid, and the initial reaction part around the initial reaction part in plan view And a flow path section having a plurality of wells.

  The sample analysis chip of the present invention may further include a plurality of weighing units having a predetermined volume, which are provided in communication with the plurality of wells.

The sample analysis chip of the present invention further includes a diffusion control unit that is provided between the initial reaction unit and the flow channel unit and controls the behavior of the sample liquid that moves from the initial reaction unit to the flow channel unit. May be.
At this time, a wall portion provided between the initial reaction portion and the diffusion control portion may be further provided, or a grind portion provided between the diffusion control portion and the flow path portion may be further provided. May be.

The sample analysis chip of the present invention may further include an accelerating unit that is provided in the initial reaction unit and accelerates the sample liquid that moves from the initial reaction unit toward the channel unit.
The acceleration unit may be formed so as to protrude from the bottom of the initial reaction unit, or is provided on an inner surface of the initial reaction unit, and is inclined so as to form an angle with respect to the axis of the initial reaction unit. A formed fin may be included.

  According to the sample analysis chip of the present invention, the influence of the external environment on the reagents can be prevented while enabling a plurality of processes to be performed.

It is a figure showing a sample analysis chip concerning a first embodiment of the present invention. It is a disassembled perspective view of the sample analysis chip. It is a top view of the main body of the sample analysis chip. It is a figure which shows the main body of the sample analysis chip which concerns on 2nd embodiment of this invention. It is a figure which shows the state by which the sample solution and the lid | cover solution were inject | poured into the sample analysis chip | tip. It is a figure which shows the behavior of a sample liquid and a lid | cover solution when the sample analysis chip | tip is rotated. It is a figure which shows the behavior of a sample liquid and a lid | cover solution when the sample analysis chip | tip is rotated. It is a figure which shows the behavior of a sample liquid and a lid | cover solution when the sample analysis chip | tip is rotated. It is a perspective view which shows the main body of the modification of the sample analysis chip | tip. It is a perspective view which shows the main body of the modification of the sample analysis chip | tip. It is sectional drawing which shows the main body of the modification of the sample analysis chip | tip. It is sectional drawing which shows the main body of the modification of the sample analysis chip | tip. It is a top view which shows the main body of the modification of the sample analysis chip. It is sectional drawing which shows the main body of the sample analysis chip concerning 3rd embodiment of this invention. It is sectional drawing which shows the other example of an acceleration part. (A)-(e) is a top view which shows the cover part in the modification of the sample analysis chip | tip of this invention. It is a disassembled perspective view which shows the modification of the sample analysis chip | tip of this invention. (A) And (b) is a front view which shows the main body in the modification of the sample analysis chip | tip of this invention. It is a perspective view which shows the main body in the modification of the sample analysis chip | tip of this invention. It is a top view which shows the main body. It is a front view which shows the main body. It is sectional drawing of the main body.

A first embodiment of the present invention will be described with reference to FIGS. 1 to 3.
FIG. 1 is a view showing a sample analysis chip 1 of the present embodiment, and is a plan view, a front view, and a bottom view, respectively, from above. FIG. 2 is an exploded perspective view of the sample analysis chip 1. As shown in FIGS. 1 and 2, the sample analysis chip 1 includes a main body 2 in which a space for storing a sample liquid is formed, and a lid portion 3 that is joined to the main body 2 and serves as a lid for the storage space. Yes.

The material of the main body 2 and the lid portion 3 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 is obtained. And an optical detection method can be used during initial reaction or sample analysis in a well. 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, it becomes excellent in thermal conductivity and sealing performance.
In the present invention, “transparent” and “light transmission” mean that the average transmittance in the wavelength region of detection light is 70% or more. If a material having optical transparency in the visible light region (wavelength 350 to 780 nm) is used, it is easy to visually recognize the sample state in the chip. However, materials that can be used in the present invention are not limited thereto.

  The lid 3 is a substantially disk-shaped member, and includes an inlet 31 for injecting the sample liquid into the main body 2 and a deaeration port 32 for smoothly injecting the sample liquid.

FIG. 3 is a plan view of the main body 2. As shown in FIGS. 2 and 3, the main body 2 has an initial reaction part 21 provided in the center part in a plan view and a flow path part 22 positioned around the initial reaction part 21 in a plan view. Yes.
The initial reaction part 21 is formed in a substantially truncated cone shape having a large upper diameter, and the sample liquid is stored in the internal space to perform the first-stage biochemical reaction. In the initial reaction unit 21, chemicals used for the first stage biochemical reaction may be arranged.

  The flow path part 22 is connected to the upper end part of the initial reaction part 21 and is formed so as to spread around the initial reaction part 21 in plan view. The flow path part 22 has a plurality of measuring parts 23 formed closer to the initial reaction part 21 and a plurality of wells 24 formed in communication with the measuring part 23. The measuring unit 23 has a predetermined volume corresponding to the volume of the well 24, and one well 24 communicates with one measuring unit 23.

  In the present embodiment, the measuring unit 23 and the well 24 communicate with each other via the narrow channel 25, but the connection mode between the measuring unit 23 and the well 24 is not limited to this, and both are directly connected to communicate with each other. May be. However, the communication through the narrow channel has the advantage that the formation position of the well can be freely set by changing the width and length of the narrow channel, the extending direction, and the like. In addition, there is an advantage that the measurement error generated in the measurement unit can be received as a buffer by the narrow channel.

Next, an example of the manufacturing procedure of the sample analysis chip 1 will be described.
The sample analysis chip 1 can be manufactured by attaching the lid 3 to the main body 2 in which the flow path portion 22 is formed.

  As a processing method for forming the flow path portion 22 in the main body 2, 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, and pressing or drawing a thin metal sheet.

  When fixing a chemical such as a reagent for reaction in the internal space of the main body 2, it is performed before the main body and the lid are bonded together. For example, a reaction reagent for common processing may be arranged in the initial reaction unit 21, and a different reagent may be arranged in the well 24 for each well. By fixing different reagents in each well 24, a plurality of treatments can be performed on one specimen (sample).

As a reagent fixing method, for example, a liquid reagent is dropped into the main body 2 with a pipette or the like, and centrifuged at 2000 to 3000 revolutions / minute (rpm) for about 5 minutes with a centrifuge, so that an appropriate amount of the liquid reagent flattens the liquid surface. It remains in the state. This can be fixed by drying.
Moreover, a hot-melt material can be used as a reagent fixing method. By dripping the melted hot melt substance on the reagent, the hot melt substance is arranged so as to cover the reagent, and the reagent is fixed. As the heat-meltable substance, if it satisfies the conditions such as solidification-liquefaction at the temperature to be used, no adverse effect on the reaction and liquid feeding, the heat-meltable substance can be replaced with the sample liquid, Those that meet the usage requirements can be selected as appropriate. For example, in the case of performing a biochemical reaction, a commercially available dedicated wax can be used. In other application forms, paraffin wax can be used. A plurality of waxes having different melting points can be mixed and used so as to melt at a desired temperature.

As a method of bonding the main body and the lid portion, there is a method in which a resin coating layer is provided on one side as an adhesive layer, and this is melted to bond them together. 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.
When the resin coating layer is formed on the main body or the lid, fusion using a laser is possible by forming an anchor layer as a base of the resin coating layer. When carbon black (light-absorbing material) that absorbs laser wavelength light is kneaded into the anchor layer, the resin coating layer can be melted and bonded by irradiating the 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. Unlike laser welding, laser welding does not require heating of the chip, so that the bonding can be performed with almost no effect on the chip and the reagent fixed to the chip.

The operation at the time of using the sample analysis chip 1 of the present embodiment configured as described above will be described.
The user takes the sample solution into a pipette or the like, and inserts the tip portion of the pipette or the like into the injection port 31 to inject the sample solution into the sample analysis chip 1. The sample solution is stored in the initial reaction unit 21.
Next, the user supplies necessary chemicals and the like to the initial reaction unit 21 and performs a first-stage biochemical reaction on the sample solution. As described above, the chemical or the like may be disposed in the initial reaction unit 21 in advance, or may be supplied from the inlet 31 before or after the sample solution. Alternatively, the first stage biochemical reaction may be performed with stirring by placing the sample analysis chip 1 in a centrifuge and rotating it lightly.
When the first-stage biochemical reaction is not started only by mixing, for example, a PCR reaction, heat treatment or the like may be performed on the initial reaction unit 21 as necessary. Examples of the mechanism for performing the heat treatment include a cartridge heater, a heating wire, and a Peltier element.

  When the first-stage biochemical reaction is completed, the user installs the sample analysis chip 1 on a device such as a centrifuge and rotates the sample analysis chip 1 around the axis of the initial reaction unit 21. The sample liquid moves toward the radially outer side of the substantially frustoconical initial reaction part 21 while connecting the inner surface of the initial reaction part 21 due to the centrifugal force generated with the rotation. It moves so that the part 23 may be filled. As a result, the sample solution is weighed by each measuring unit 23 by the volume of the measuring unit 23. When the centrifugal force is further applied to the sample solution, the sample solution in each measuring unit 23 moves into the well 24 through the narrow channel 25. As a result, a predetermined amount of sample liquid is distributed to each of the plurality of wells 24. If different reaction reagents and the like are arranged inside the well 24, it is possible to perform a second stage process different for each well on the sample solution.

According to the sample analysis chip 1 of the present embodiment, since the initial reaction unit 21 is provided, a common process that must be performed on the sample liquid, such as a first-stage biochemical reaction, is hygienic in the initial reaction unit 21. Moreover, it can carry out efficiently and can also suppress the influence of the external environment on a reagent.
In addition, since the initial reaction part 21 is arranged in the center part in the plan view and the flow path part 22 is provided around the initial reaction part 21 in the plan view, the initial reaction part 21 is rotated around the central axis in the plan view. The sample liquid can be suitably fed into the flow path section 22 from the reaction section 21 and further into the well 24 from the measurement section 23 using centrifugal force.

  A second embodiment of the present invention will be described with reference to FIGS. The difference between the sample analysis chip 51 of the present embodiment and the sample analysis chip 1 of the first embodiment is that a diffusion control unit is further provided. In the following description, the same components as those already described are denoted by the same reference numerals and redundant description is omitted.

  FIG. 4 is a view showing the main body 60 of the sample analysis chip 51, where the upper side is a plan view and the lower side is a front view. In the main body 60, the structure of the flow path part 22 is the same as that of the first embodiment, but a diffusion control part 62 is provided above the initial reaction part 61, and the flow path part 22, the initial reaction part 61, Are connected via the diffusion control unit 62.

  The diffusion control unit 62 is formed in a ring-like slope shape in a plan view extending from the upper end of the initial reaction unit 61 to the outside in the radial direction and upward of the initial reaction unit 61. The inclination angle θ1 that is an angle formed by the diffusion control unit 62 with the plane orthogonal to the axis X of the initial reaction unit 61 is set to be larger than the inclination angle θ2 of the inner surface of the initial reaction unit 61.

An operation at the time of using the sample analysis chip 51 of the present embodiment configured as described above will be described.
The procedure until the first-stage biochemical reaction is completed is the same as in the first embodiment. When the first-stage biochemical reaction is completed, the user supplies a lid solution for preventing evaporation or contamination of the sample liquid from the inlet 31 into the sample analysis chip 51. Although there is no restriction | limiting in particular in a lid | cover solution, For example, the solution which is a stable substance like mineral oil and silicone oil and whose specific gravity is smaller than a sample solution is preferable. If the lid solution does not inhibit the first-stage biochemical reaction, it may be supplied into the sample analysis chip 51 at an earlier stage.

When a lid solution having a specific gravity smaller than that of the sample solution is used, the lid solution 101 is located in the upper layer and the sample solution 102 is located in the lower layer in the initial reaction unit 61 as shown in FIG.
Next, when the sample analysis chip 51 is rotated around the axis of the initial reaction unit 21, the sample solution 102 and the lid solution 101 move upward along the inner surface of the initial reaction unit 61 by centrifugal force, and the diffusion control unit 62 is reached. Since the inclination angle of the diffusion control unit 62 is smaller than the inclination angle of the initial reaction unit 61 and the sample solution 102 has a higher specific gravity than the lid solution 101, the centrifugal force acting on the sample solution 102 is the centrifugal force acting on the lid solution 101. Bigger than. As a result, as shown in FIG. 6, in the diffusion control unit 62, the behavior of the sample solution is such that the sample solution 102 moves radially outward from the lid solution 101 and reaches the flow path unit 22 first. As shown in FIG. 7, the measuring unit 23 is filled with the sample liquid 102.

  Subsequently, when the sample analysis chip 51 is rotated at a higher rotation speed, the sample solution 102 fills the well 24 and the lid solution 101 moves to the measuring unit 23 as shown in FIG. When the rotation is stopped, a part of the lid solution 101 flows from the metering unit 23 to the diffusion control unit 62, but the lid solution 101 remaining in the metering unit 23 blocks the communication portion to the narrow channel 25, and evaporates, contaminates, etc. Is suitably suppressed.

In the sample analysis chip 51 of the present embodiment as well, as in the first embodiment, common processing that must be performed on the sample liquid can be performed hygienically and efficiently in the initial reaction unit, and the external environment to the reagent The influence of can also be suppressed.
Furthermore, since the diffusion control unit 62 is provided between the initial reaction unit 61 and the flow channel unit 22, even when the lid solution is used, the sample solution is first preferably moved to the flow channel unit, and then the lid is used. The well can be suitably sealed with the solution.

  In the present embodiment, the inclination angle θ1 of the diffusion control unit can be appropriately set in consideration of the inclination angle θ2 of the initial reaction unit, the specific gravity of the sample solution and the lid solution, and the like. It is preferable that a difference of, for example, about 100 rpm occurs between the rotational speed for moving to the measuring unit 23 and the rotational speed for moving the sample liquid to the well 24. In this way, the two processes can be clearly distinguished, and the distribution process can be suitably performed.

  In addition, as in the modification shown in FIG. 9, an annular wall portion 65 in plan view may be provided between the initial reaction portion 61 and the diffusion control portion 62. In this way, by adjusting the height of the wall portion 65, the amount of liquid that moves from the initial reaction portion 61 to the diffusion control portion 62 at a predetermined rotational speed can be adjusted, and the above setting is easy. Become.

  About a wall part, besides adjusting height, as shown in FIG. 10, the adjustment by providing the notch 66 is also possible, and it is also possible to combine both. In the part provided with the notch 66, the movement of the liquid to the diffusion control unit 62 becomes easier than the part without the notch. Therefore, in addition to adjusting the amount of liquid that moves from the initial reaction unit 61 to the diffusion control unit 62 by providing, for example, notches 66 at equal intervals, the moving liquid has a specific phase in the circumferential direction of the sample analysis chip. It is possible to prevent the bias and to move the liquid evenly around the axis of the initial reaction unit 61. As a result, the variation in the amount of liquid in each well can be suppressed. In addition, the notch does not need to extend over the height of the wall portion, and may end at an intermediate portion in the height direction.

  In the modification shown in FIG. 11, a circular cut portion 67 having a ring shape in plan view is provided between the diffusion control unit 62 and the flow path unit 22. By providing the scraping portion 67, the liquid from the diffusion control portion 62 toward the measuring portion 23 of the flow path portion 22 hits the scraping portion 67 and diffuses in the circumferential direction. As a result, a part of the liquid is easily circulated to other measuring units and is more easily distributed to each measuring unit. Further, there is an advantage that the liquid that has moved to the measuring unit 23 is less likely to return to the diffusion control unit 62 and is preferably held by the measuring unit 23 even after the rotation is stopped.

  In this modification, the cross-sectional shape of the ground portion 67 is substantially hemispherical, but the cross-sectional shape may be a rectangle or other shapes such as a triangle, as with the wall portion 65 described above. Similar changes can be made to the cross-sectional shape of the wall portion 65.

Further, various changes can be made to the inner surface shape of the diffusion control section.
In the modification shown in FIG. 12, a plurality of annular grooves 62 a are formed concentrically in the diffusion control unit 62. If it does in this way, the liquid which flows on the spreading | diffusion control part 62 will accelerate when entering the groove | channel 62a, and will decelerate when exiting the groove | channel 62a. By repeating this, the liquid is suitably dispersed in the circumferential direction, and is uniformly supplied to each measuring unit 23.
The shape exhibiting such a function is not limited to the annular groove, and may be a spiral groove, or a shape such as the above-mentioned wall portion or filed portion. Further, these shapes may be provided intermittently at intervals without extending in the circumferential direction. Furthermore, a plurality of protrusions 62b and a plurality of recesses as in the modification shown in FIG. 13 may be provided. Although FIG. 13 shows an example in which a plurality of protrusions 62b are arranged in an annular and concentric manner, the protrusions and recesses may be formed randomly.

  A third embodiment of the present invention will be described with reference to FIGS. 14 and 15. The difference between the sample analysis chip 71 of the present embodiment and each of the above-described embodiments is that an acceleration unit is provided in the initial reaction unit. The acceleration unit is a structure for controlling the position and flow direction of the solution so that the centrifugal force is easily applied when the sample solution in the initial reaction unit is fed by centrifugal force.

  FIG. 14 is a cross-sectional view of the main body 80 of the sample analysis chip 71. From the bottom surface of the initial reaction part 61, a substantially truncated cone-like acceleration part 81 projects. The axis of the accelerating unit 81 is coincident (including substantially coincident) with the axis X of the initial reaction unit 61.

  When a liquid such as a sample solution or a lid solution is supplied to the initial reaction unit 61 of the sample analysis chip 71, no liquid can exist in a portion where the acceleration unit 81 exists, and the liquid is arranged around or above the acceleration unit 81. . As a result, the amount of liquid located in the vicinity of the axis X of the initial reaction unit 61 is reduced. As a result, when the sample analysis chip 71 is rotated about the axis X, a larger centrifugal force acts on the liquid, and the liquid can be suitably moved to the flow path section 22.

  The shape of the accelerating portion 81 is not limited to the truncated cone shape, and may be a truncated pyramid shape, a column shape such as a cylinder or a prism, or a shape such as a cone or a pyramid. Further, the acceleration portion may have a hollow shape as shown in FIG. 14 or a solid shape. However, if it is hollow, a heater, a sensor, or the like can be inserted into the accelerating portion from the outside of the sample analysis chip 71 to perform heating, heating, various measurements, or the like of the sample liquid.

FIG. 15 is an example in which the acceleration unit is provided at a position other than the bottom surface of the initial reaction unit. In this modification, a plurality of fins 82 that form an angle with respect to the axis of the initial reaction part are provided on the inner side surface 61b of the initial reaction part 61 as an acceleration part.
When the sample analysis chip is rotated, the liquid that moves up the inner surface 61b by centrifugal force hits the surface of the fin 82 that faces the bottom surface 61a of the initial reaction unit 61 and is accelerated. Thereby, the liquid can be suitably moved to the flow path part 22.

Further, the fin 82 exhibits a function of prompting stirring of the liquid in the initial reaction unit 61 by rotating the sample analysis chip at a low speed during the first stage biochemical reaction. When chemicals or the like are placed in the initial reaction unit 61 in advance, they are stored in a state where they are fixed to either the bottom or the inner surface by some method, such as drying, solidification, freeze drying, wax coating, gel coating, etc. Sometimes. In such a case, even if the sample solution is injected, it may not mix well with chemicals, etc., and a reaction failure may occur or the reaction may take longer than usual. Occurrence of various problems can be suppressed.
In addition, the shape of the acceleration part formed in the inner surface 61b is not restricted to a fin, For example, a helical groove | channel etc. may be sufficient. Moreover, although it does not correspond to the acceleration part of this embodiment, you may provide a processus | protrusion, a recessed part, etc. instead of a fin on the inner surface 61b in order to exhibit only the function of stirring assistance.

  As mentioned above, although each embodiment and modification of this invention were demonstrated, the technical scope of this invention is not limited to the said embodiment and modification, In the range which does not deviate from the meaning of this invention, it is the combination of a component. Can be changed, various changes can be made to each component, and it can be deleted.

  For example, in the sample analysis chip of the present invention, the lid portion does not necessarily need to be provided with a deaeration port. That is, if the shape of the inlet is a non-circular shape such as the inlets 31a to 31e as illustrated in FIGS. 16 (a) to 16 (e), a gap is created when a general pipette or the like is inserted, The gap functions as a deaeration port. As a result, the liquid can be smoothly injected into the sample analysis chip without providing a degassing port.

  Further, as in the modification shown in FIG. 17, in the lid portion 110, the member that forms the injection port 111 may be a separate member to increase the rigidity. Thereby, a lid | cover part can be stabilized at the time of liquid injection | pouring, and injection | pouring can be performed simply. Here, when the support portion 113 extending downward is provided on the member 112 in which the injection port 111 is formed, the lid portion 110 is prevented from being bent due to a pressing force or the like generated when a pipette or the like is inserted into the injection port 111. The posture of the lid 110 can be further stabilized. Of course, the shape of the inlet formed in the member 112 may be the non-circular shape described above.

Furthermore, the shape of the initial reaction part is not limited to that described above, and can be variously changed. For example, a hemispherical shape such as the initial reaction portion 115 shown in FIG. 18A may be used, or a substantially truncated cone shape having a hemispherical bottom portion such as the initial reaction portion 116 shown in FIG. But you can.
In the main body 121 in the sample analysis chip of the modification shown in FIG. 19, the region surrounded by the wall portion 122 having a plurality of cuts 122a that does not reach the lower end portion is the initial reaction portion 123, and the flow passage portion 124 and the initial portion are formed. The reaction part 123 is located at substantially the same height. With such a shape, as shown in FIG. 21, the lower surface of the main body 121 becomes nearly flat, so that handling becomes easy.

In the flow path portion 124 formed in the main body 121, a larger number of wells 124b can be arranged as compared with the flow path portion 22 by reducing the width dimension that is a dimension in the circumferential direction of the measuring portion 124a. is made of. As described above, various changes can be made to each configuration of the flow path section.
For reference, FIG. 20 shows a plan view of the main body 121 and FIG. 22 shows a cross-sectional view.

  In the present invention, it is not important which of the main body and the lid portion is provided with the flow path portion. Therefore, the cover part may be formed thick and the flow path part may be formed in the cover part, or the flow path part may be formed by forming a space to be the flow path part on both sides and bonding them together.

Finally, a sample analysis method using the sample analysis chip of the present invention will be described.
The sample analysis chip of the present invention can be used, for example, for detection and analysis of biochemical substances in a sample such as DNA or protein.

  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)
In the human genome, there are differences in individual base sequences in about 0.1%. This 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. However, when the sample analysis chip of the present invention is used, the sample solution can be evenly distributed to each well, and accurate SNPs can be detected. I can do it. 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.

  Below, the analysis example using PCR-PHFA method is demonstrated about the SNP which concerns on the side effect with respect to warfarin (Anti-coagulant. Used as a medicine for heart disease and hypertension) using this invention.

  A sample nucleic acid obtained from blood or the like is purified to obtain a solution sample. After injecting into the sample analysis chip of the present invention, the sample nucleic acid is amplified in the initial reaction part before liquid distribution. 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. What is necessary is just to fix the reagent for SNP detection for performing competitive strand displacement reaction.

  The sample solution in which the nucleic acid is amplified by the PCR is filled in each well. Each well is temperature-controlled, and a mutation is detected by a difference in luminescence of a fluorescent reagent mixed in a reagent arranged in the well. 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)
Most of the mutations characteristic of cancer cells and mutations that are resistant to molecularly targeted drugs are somatic mutations. In the case of germ cell mutations (SNP, etc.), a common mutation is seen in all cells, whereas in somatic mutations, cells are mutated only in the mutated cells, and cells that are not mutated (usually normal cells) ) Shows no mutation.

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

  The K-ras gene is a gene whose molecular target drug has been shown to be ineffective in most patient groups when mutations are present in cancer cells. This gene can be detected simply, quickly, inexpensively and with high accuracy. It is being hoped for. Below, the example of analysis by PCR-PHFA method of a K-ras gene is demonstrated.

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

  Collect cancer cells such as colorectal cancer and purify the sample nucleic acid to obtain a sample solution. After injection into the sample analysis chip of the present invention, the sample nucleic acid is first amplified in the initial reaction part.

  The sample solution in which the nucleic acid is amplified by the PCR is filled in each well. The temperature of the well is controlled, and the mutation can be detected by the difference in luminescence of the fluorescent reagent mixed in the reagent arranged in the well.

1, 51, 71, 121 Sample analysis chip 21, 61, 115, 116, 123 Initial reaction part 22, 124 Flow path part 23, 124a Weighing part 24, 124b Well side 61b Diffusion control part 65 Wall part 67 Grinding part 81 Accelerator 82 Fin (Accelerator)
102 Sample liquid X (Initial reaction part) axis

Claims (8)

A sample analysis chip for injecting a sample liquid and performing a biochemical reaction,
An initial reaction part capable of storing the sample liquid;
A flow path portion provided in connection with the initial reaction portion around the initial reaction portion in plan view, and having a plurality of wells;
A sample analysis chip comprising:   The sample analysis chip according to claim 1, further comprising a plurality of measuring units having a predetermined volume, which are provided in communication with each of the plurality of wells.   The diffusion control part which is provided between the initial reaction part and the flow path part, and controls the behavior of the sample liquid which moves from the initial reaction part to the flow path part is provided. Sample analysis chip.   The sample analysis chip according to claim 3, further comprising a wall portion provided between the initial reaction unit and the diffusion control unit.   The sample analysis chip according to any one of claims 3 and 4, further comprising a grind part provided between the diffusion control unit and the flow path unit.   6. The sample analysis chip according to claim 1, further comprising an accelerating unit provided in the initial reaction unit and accelerating the sample liquid moving from the initial reaction unit toward the flow channel unit. .   The sample analysis chip according to claim 6, wherein the acceleration part is formed so as to protrude from a bottom part of the initial reaction part. The sample analysis chip according to claim 6 or 7, wherein the acceleration unit includes a fin provided on an inner surface of the initial reaction unit and formed obliquely so as to form an angle with respect to an axis of the initial reaction unit. .

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