JP2015206715A - sample analysis chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample analysis chip capable of preventing affection of a reagent to an external environment, and performing plural kinds of treatment.SOLUTION: A sample analysis chip 1 comprises: a main flow channel 24 which is formed into a spiral state on a base material, disposed around an initial reaction part, and whose one end part is communicated with the initial reaction part; plural sawtooth-state storage parts 25 disposed in an inner face 14a of the base material in a radial direction outside on the main flow channel; and plural main storage parts 26 disposed on an opposite side of the main flow channel, on the sawtooth state storage part. The respective sawtooth-state storage part is a space regulated between a first inner face 17 and a second inner face 18 whose one ends 17a, 18a are communicated with the inner face of the main flow channel, and the first inner face is disposed on an upstream side D1 of the second inner face. When a reference line L2 coupling one ends of the first inner face and the second inner face is regulated, a downstream side angle α2 formed of the reference line and second inner face on the sawtooth-state storage part side, is larger than an upstream side angle α1 formed of the reference line and first inner face on the sawtooth-state storage part side.

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. . In this technique, one sample analysis chip (hereinafter also abbreviated as “chip”) or cartridge is provided with a plurality of wells (main housing portions) and flow paths as reaction chambers. By using the sample analysis chip, analysis of a plurality of specimens (samples) or a plurality of reactions can be performed. These technologies have been considered to have various merits by reducing the size of chips and cartridges, thereby reducing the amount of chemicals to be handled.

  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. Need to flow from one to several main conduits (main flow paths) 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, many samples (specimens) poured at this time are expensive or valuable. In many cases, these samples are previously subjected to several biochemical reaction treatments. Therefore, there are many usage methods in which a reaction is performed in a well using a sample subjected to biochemical reaction treatment 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.
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.
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.

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.
Patent Document 5 discloses a sample analysis chip including a flat circular base material, a channel having no opening, and first and second sample wells.

JP-T-2004-502164 Japanese Patent No. 3699721 JP 2008-083017 A US Pat. No. 7,195,036 JP-T-2007-527517

However, the method described in Patent Document 1 requires a mechanism for crushing the flow path, and is difficult to automate. 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.
The method described in Patent Document 2 requires a complicated mechanism and space for the tip to rotate and revolve. In addition, when a plurality of processes are to be added to the sample solution, if a plurality of processes are performed after distribution, variations in each reaction well overlap and stable processing cannot be performed.

In Patent Document 3, the liquid distribution property of the liquid is not considered, and conversely, the fluid is controlled by pressing 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 the method described in Patent Document 4, an air pressure control mechanism is required, 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 are complicated. End up.
In the sample analysis chip described in Patent Document 5, since the user introduces the sample, reagent, or liquid into the sample analysis chip using a pipette or a syringe, the burden on the user's work is large.

  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.

  Further, when biochemical reaction is performed on the chip, the reaction is often performed by heating the inside of the reaction chamber after distributing the sample solution. In this case, in order to prevent evaporation of the sample solution, it may be necessary to inject an evaporation preventing material such as mineral oil or to physically seal the chip, which increases the number of work steps. There's a problem.

  The present invention has been made in view of such problems, and provides a sample analysis chip capable of performing a plurality of processes while preventing reagents from affecting the external environment. The purpose is to provide.

In order to solve the above problems, the present invention proposes the following means.
The sample analysis chip of the present invention is a disk-shaped base material, an initial reaction portion that is disposed in the center of the base material and communicates with an injection port and can store a liquid sample, and the thickness direction of the base material The main flow path is formed around the initial reaction section and is disposed around the initial reaction section, and one end thereof is recessed from the radially inner surface of the base material in the main flow path. A plurality of serrated accommodation portions provided in the manner described above, and a sample analysis chip in which a plurality of main accommodation portions provided on the opposite side of the main flow path in each of the serrated accommodation portions is formed, The sawtooth-shaped accommodation portion is a space defined between a first inner surface and a second inner surface, one end of which is continuous with the inner surface of the main flow path and the other end extending outward in the radial direction. The first inner surface is more along the main flow path than The reference line and the first inner surface are arranged on the upstream side which is the one end portion side of the main flow path and define a reference line connecting the one end of the first inner surface and the one end of the second inner surface. Is characterized in that the downstream angle between the reference line and the second inner surface on the sawtooth accommodation portion side is larger than the upstream angle formed on the sawtooth accommodation portion side.

Further, in the sample analysis chip, in the pair of serrated accommodation units adjacent to each other along the inner surface of the main flow path, the one end of the second inner surface of the upstream serrated accommodation unit and the mainstream It is more preferable that the one end of the first inner surface of the serrated accommodation portion on the other end side of the main flow path along the path is in contact with the first flow passage.
In the sample analysis chip, it is more preferable that a surplus liquid container is formed at the other end of the main channel.
In the sample analysis chip, it is more preferable that the upstream angle is smaller than 80 ° and the downstream angle is larger than 80 °.

In the sample analysis chip described above, in the pair of serrated accommodation portions adjacent to each other along the inner surface of the main flow path, the upstream angle of the upstream serrated accommodation section is along the main flow path. More preferably, the angle is equal to or smaller than the upstream angle of the serrated housing portion on the other end side of the main flow path.
In the sample analysis chip, it is more preferable that the initial reaction part is formed in a truncated cone shape having an axis parallel to the central axis of the substrate.

In the sample analysis chip, the main flow path may be configured such that the axis of the main flow path and the main flow path with respect to a rotation angle θ around the central axis of the base material when a and b are constants. It is more preferable that the distance r to the axis is formed so as to satisfy the equation (1).
r = a + b × θ (1)
In the sample analysis chip, the main flow path may be configured such that the axis of the main flow path has a center with respect to a rotation angle θ around the central axis of the substrate when a and b are constants and e is the Napier number. More preferably, the distance r between the axis and the axis of the main flow path is expressed by the equation (2).
r = a × e ^(bθ) (2)

  According to the sample analysis chip of the present invention, it is possible to prevent a reagent from affecting the external environment while enabling a plurality of processes to be performed.

It is a figure which shows the top view, side view, and bottom view of the sample analysis chip of 1st Embodiment of this invention collectively. It is a perspective view of the sample analysis chip. It is the top view which permeate | transmitted the cover part of the sample analysis chip | tip. It is a principal part enlarged view in FIG. It is the top view which permeate | transmitted the cover part explaining operation | movement of the sample analysis chip | tip. It is the top view which permeate | transmitted the cover part explaining operation | movement of the sample analysis chip | tip. It is the top view which permeate | transmitted the cover part explaining operation | movement of the sample analysis chip | tip. It is the top view which permeate | transmitted the cover part explaining operation | movement of the sample analysis chip | tip. It is the top view which permeate | transmitted the cover part explaining operation | movement of the sample analysis chip | tip. It is a principal part enlarged view of the sample analysis chip in the modification of 1st Embodiment of this invention. It is the top view which permeate | transmitted the cover part of the sample analysis chip of 2nd Embodiment of this invention.

(First embodiment)
Hereinafter, a first embodiment of a sample analysis chip 1 according to the present invention will be described with reference to FIGS. 1 to 10.
As shown in FIGS. 1 to 3, the sample analysis chip 1 according to the present embodiment includes an initial reaction unit 23, a spiral channel (main channel) 24, a sawtooth storage unit 25, and a well (main storage unit) described later. 26, a main body 10 in which concave portions 13, 14, 15, and 16 corresponding to 26 are formed, and a lid portion 30 that is joined to the main body 10 and serves as a lid for the concave portions 13, 14, 15, and 16.
In FIGS. 3 to 11, the cover 30 is shown in a transparent manner.
The main body 10 and the lid part 30 constitute a base material 40.

The material forming the main body 10 and the lid 30 is not particularly limited as long as it does not affect the sample accommodated in the sample analysis chip 1. If a resin material containing polypropylene, polycarbonate, acrylic, cyclic polyolefin, or the like is used as this material, good visible light transmission can be secured, and an optical detection method can be used during initial reaction or sample analysis in the well 26. Can be used.
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 the chip | tip 1 are also securable.

Examples of materials other than the resin material forming the main body 10 and the lid 30 include metal materials such as aluminum, copper, silver, nickel, brass, and gold. If a metal material is used, the main body 10 and the cover part 30 will be excellent in thermal conductivity and sealing performance.
In the present invention, “light transmittance” means 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 (nanometer)) is used, it is easy to visually recognize the state of the sample in the chip 1, but the material that can be sampled in the present invention is not limited to this. I can't.

First, the lid 30 will be described.
The lid 30 is formed in a disc shape. The lid 30 includes an inlet 31 for injecting a sample liquid (not shown), which is a liquid sample, into the main body 10, and a deaeration port 32 for smoothly injecting the sample liquid. It is formed to penetrate.

The main body 10 is formed in a disk shape.
On one surface 10a of the main body 10, a protruding portion 11 protruding in a truncated cone shape is formed. The protrusion 11 is formed on the central axis C <b> 1 of the base material 40. By forming the protrusions 11, the temperature of the sample liquid stored in the initial reaction part 23 formed in the protrusions 11 can be reduced as compared to the case where the protrusions 11 are not formed on the main body 10 as will be described later. It becomes easy to raise.

On the other surface 10 b of the main body 10, an initial reaction portion recess 13 disposed in the central portion of the main body 10, formed in a spiral shape when viewed in the thickness direction X of the base material 40, is an initial reaction portion recess. A plurality of sawtooth shapes provided so as to be recessed radially outward from the radially outer inner surface 14a of the base material 40 in the spiral flow path recess 14 disposed around 13; A plurality of well recesses 16 provided on the side opposite to the spiral channel recesses 14 in each of the serrated recess recesses 15 are formed.
As used herein, the term “spiral” refers to an increase in the rotation angle (center angle) with respect to the target line around a predetermined axis (for example, the central axis C1 of the base material 40). It means the shape to be.

  By joining the lid portion 30 to the other surface 10 b of the main body 10, the initial reaction portion 23 is configured by the initial reaction portion recess 13 and the one surface 30 a of the lid portion 30 (see FIG. 2). Similarly, a spiral channel 24 is constituted by the spiral channel recess 14 and the one surface 30a of the lid portion 30, and the serrated housing recess portion 15 and the one surface 30a of the lid portion 30 are serrated. The accommodating part 25 is comprised, and the well 26 is comprised by the recessed part 16 for wells and the one surface 30a of the cover part 30. FIG.

The initial reaction part 23 is formed in a truncated cone shape having an axis parallel to the central axis C1, and is arranged coaxially with the central axis C1. The outer diameter of the initial reaction part 23 increases as it approaches the other surface 10 b of the main body 10.
As shown in the plan view in FIG. 1, the inlet 31 and the deaeration port 32 of the lid part 30 are formed in the edge part on the other surface 10 b side of the initial reaction part 23. Thereby, the inlet 31 and the deaeration port 32 and the initial reaction part 23 are connected.
The initial reaction unit 23 can store the sample solution injected through the injection port 31, and the first stage biochemical reaction can be performed in the initial reaction unit 23. The initial reaction unit 23 is a storage unit that stores a sample solution.
In the initial reaction unit 23, chemicals and the like used for the first stage biochemical reaction may be arranged.

As shown in FIG. 3, the spiral channel recess 14 has an axis C <b> 2 of the spiral channel recess 14 as the rotation angle θ around the center axis C <b> 1 of the substrate 40 increases with respect to the target line L <b> 1. Is formed so as to be separated from the central axis C1.
The depth (length in the thickness direction X) of the spiral channel recess 14 is shallower (shorter) than the depth of the initial reaction recess 13, and the depth of the serrated housing recess 15 and The depth is substantially equal to the depth of the well recess 16.
One end portion 14 b of the spiral channel recess 14 is continuous with the inner peripheral surface of the initial reaction recess 13. That is, the one end 24 b of the spiral channel 24 communicates with the initial reaction unit 23. In the present embodiment, the initial reaction part 23 and the one end part 24 b of the spiral channel 24 are in communication with each other by the direct contact between the initial reaction part 23 and the one end part 24 b of the spiral channel 24.
When viewed in the thickness direction X shown in FIG. 3, the spiral channel 24 is formed in a spiral shape and is disposed around the initial reaction unit 23. In other words, the spiral flow path 24 is disposed so as to surround the initial reaction portion 23.
The distance between the other end 14c of the spiral flow path recess 14 and the central axis C1 is longer than the distance between the one end 14b of the spiral flow path recess 14 and the central axis C1.
In this example, the central angle of the spiral channel 24 is about 360 °.

As will be described later, the sample liquid stored in the initial reaction section 23 is directed from the one end 24 b side of the spiral flow path 24 toward the other end 24 c side of the spiral flow path 24 along the spiral flow path 24. Flowing. For this reason, hereinafter, the one end 24b side along the spiral channel 24 is referred to as upstream D1, and the other end 24c side along the spiral channel 24 is referred to as downstream D2.
In the other end 24c of the spiral flow path 24, an excess liquid storage part 27 for storing excess sample liquid is formed. The surplus liquid container 27 is formed in a shape in which the width of the flow path is narrowed toward the downstream side D2.

As shown in FIG. 4, the serrated housing recess 15 has a first inner surface extending at one end 17 a to the inner surface 14 a of the spiral channel recess 14 and extending at the other end 17 b toward the radially outer side of the substrate 40. 17, and one end 18 a is connected to the inner surface 14 a of the spiral channel recess 14, and the other end 18 b has a second inner surface 18 that extends outward in the radial direction of the substrate 40. The first inner surface 17 is disposed on the upstream side D <b> 1 rather than the second inner surface 18 of the serrated housing recess 15.
The serrated housing portion 25 is formed between the first inner surface 17 and the second inner surface 18 of the serrated housing portion recess 15, and between the bottom surface 15 a of the serrated housing portion recess 15 and one surface 30 a of the lid portion 30. It is a space defined between.

Here, a reference line L2 connecting the one end 17a of the first inner surface 17 and the one end 18a of the second inner surface 18 is defined. The concave portion 15 for the serrated accommodation portion is such that the reference line L2 and the second inner surface 18 are closer to the serrated accommodation portion 25 side than the upstream angle α1 formed by the reference line L2 and the first inner surface 17 on the serrated accommodation portion 25 side. Is formed so that the downstream angle α2 is larger.
The upstream angle α1 is larger than 0 ° and smaller than 90 °. The downstream angle α2 is larger than 0 ° and smaller than 180 °.
The concave portion 15 for the serrated accommodation portion becomes narrower as it approaches the well 26, and a constricted portion 15 b is formed between the concave portion 15 and the well 26.

Here, of the pair of serrated accommodation portions 25 adjacent to each other along the inner surface 24a of the spiral channel 24, the sawtooth accommodation portion 25 on the upstream side D1 is the sawtooth accommodation portion 25A and the sawtooth accommodation portion on the downstream side D2. 25 will be referred to as a sawtooth accommodating portion 25B. The upstream angle α1 of the serrated housing portion 25A is referred to as an upstream angle α11, and the upstream angle α1 of the sawtooth housing portion 25B is referred to as an upstream angle α12.
One end 18a of the second inner surface 18 of the serrated housing portion 25A is in contact with one end 17a of the first inner surface 17 of the serrated housing portion 25B. For this reason, the distance between the serrated accommodation portion 25A and the serrated accommodation portion 25B is shortened.
The upstream angle α11 of the serrated housing portion 25A is equal to or smaller than the upstream angle α12 of the sawtooth housing portion 25B.

One well 26 communicates with each serrated accommodation portion 25. The well 26 is provided on the opposite side (radially outer side) from the spiral flow path 24 in the serrated accommodation portion 25.
In this example, the volumes of the plurality of wells 26 are equal to each other. Each serrated accommodation portion 25 has a predetermined volume corresponding to the volume of the well 26.
For joining the main body 10 and the lid 30, welding or a known adhesive is used.

Next, an example of a method for manufacturing the sample analysis chip 1 configured as described above will be described.
The sample analysis chip 1 can be manufactured by bonding and bonding the lid 30 to the main body 10 in which the recesses 13, 14, 15, and 16 are formed.

  As a processing method for forming the recesses 13, 14, 15, and 16 in the main body 10, when the main body 10 is formed of a resin material, various resin molding methods such as injection molding and vacuum molding, and machine cutting Etc. can be used. When the main body 10 is formed 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 chemicals such as a reagent for reaction in the recesses 13, 14, 15, and 16 of the main body 10, it is performed before the main body 10 and the lid 30 are bonded together. For example, a reaction reagent for common processing may be disposed in the initial reaction unit 23, and a different reagent may be disposed in the well 26 for each well 26. By fixing different reagents in each well 26, a plurality of processes can be performed on one specimen (sample).

As a reagent fixing method, for example, a liquid reagent is dropped into the recesses 13, 14, 15, 16 of the main body 10 with a pipette or the like, and the sample analysis chip 1 is rotated around the central axis C1 by 2000 to 3000 rotations / min ( rpm) After centrifuging for about 5 minutes, an appropriate amount of the liquid reagent remains with the liquid surface flattened. The reagent can be fixed by drying it.
In addition, 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 10 and the lid part 30, there is a method in which a resin coating layer is provided as an adhesive layer on one side, and this is melted to bond them together. The resin coating layer is preferably provided on a base material made of a metal material having a 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 10 or the lid 30, 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 by irradiating light of an infrared photodiode laser having a wavelength of about 900 nm. Laser welding, unlike thermal welding, does not require heating of the chip, so that bonding can be performed with almost no influence on the chip and the reagent fixed to the chip.

Next, 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, inserts the tip of the pipette or the like into the injection port 31, and injects the sample solution into the initial reaction portion 23 of the sample analysis chip 1. As shown in FIG. 5, the sample liquid M is stored in the initial reaction unit 23. The sample liquid M can be easily injected into the initial reaction part 23 by the air in the initial reaction part 23 being removed from the deaeration port 32.
Next, the user may supply necessary chemicals to the initial reaction unit 23 and perform the first-stage biochemical reaction on the sample liquid M. As described above, the medicine or the like may be disposed in the initial reaction unit 23 in advance, or may be supplied from the inlet 31 before or after the sample liquid M.
Alternatively, the first stage biochemical reaction may be performed with stirring by installing the sample analysis chip 1 in a centrifuge and rotating it lightly around the central axis C1.
When the first stage biochemical reaction is not started only by mixing, such as a PCR (Polymerase Chain Reaction) reaction, heat treatment or the like may be performed on the initial reaction unit 23 as necessary. Examples of the mechanism for performing the heat treatment include a cartridge heater, a heating wire, and a Peltier element.

The user installs the sample analysis chip 1 in the centrifuge and rotates the sample analysis chip 1 around the central axis C1 at a relatively low rotational speed in order to distribute the sample liquid M. The relatively slow rotation speed is, for example, about several hundred rotations / minute to 2000 rotations / minute.
The sample liquid M moves toward the outside in the radial direction of the initial reaction part 23 as shown in FIG. 24 enters one end 24b. As shown in FIG. 7, the sample liquid M moves to the downstream side D2 along the inner surface 14a of the spiral flow path 24, and moves so as to fill the saw-tooth containing portion 25 in order from the upstream side D1 to the downstream side D2.

  At this time, the downstream angle α2 is larger than the upstream angle α1 in the sawtooth accommodation portion 25. As a result, the sample liquid M can easily enter the serrated container 25. When the sample liquid M flows so as to hit the second inner surface 18, the sample liquid M that has once entered the sawtooth storage unit 25 is dragged by the flow of the sample liquid M toward the downstream side D <b> 2, and the inside of the sawtooth storage unit 25. It is suppressed that it comes out of.

When the rotation speed around the central axis C1 of the sample analysis chip 1 is constant, the sample liquid M on the downstream side D2 that is farther from the central axis C1 than the sample liquid M on the upstream side D1 that is closer to the distance from the central axis C1. The centrifugal force received by the person increases. Therefore, even if the upstream angle α12 of the sawtooth accommodation portion 25B on the downstream side D2 is designed to be larger than the upstream angle α11 of the sawtooth accommodation portion 25A on the upstream side D1, the sample liquid M will be the same in the sawtooth accommodation portion 25. It is possible to enter. It is possible to increase the upstream angle α1 of the sawtooth storage portion 25 toward the downstream side D2, that is, it is possible to provide a sawtooth storage portion 25 that stands up with respect to the spiral flow path 24. 25 and the well 26 can be densely integrated in the main body 10.
Further, in the sample analysis chip 1 of the present embodiment, since the sample liquid M can be fed without requiring a large centrifugal force, the same is true even if the upstream angle α1 is the same angle in all of the sawtooth accommodating portions 25. Is possible. In this case, all the serrated accommodation portions 25 can be formed in the same shape, and the sample analysis chip 1 can be designed very easily.

In this manner, as shown in FIG. 8, the sample liquid M is weighed by the volume of the sawtooth accommodation portion 25 in each sawtooth accommodation portion 25.
The sample liquid M that does not fully enter the plurality of sawtooth-shaped storage units 25 is stored in the surplus liquid storage unit 27.

The rotation speed of the sample analysis chip 1 is increased, and the sample analysis chip 1 is rotated around the central axis C1 at a relatively high rotation speed. The relatively fast rotation speed is, for example, about 1000 rotations / minute to 10000 rotations / minute.
As the centrifugal force is increased, the sample liquid M moves from the serrated container 25 to the well 26 through the constricted portion 15b as shown in FIG.
As a result, a predetermined amount of sample liquid M is distributed to each of the plurality of wells 26. If different reaction reagents and the like are arranged inside the well 26, the sample solution M can be subjected to different treatments for each well 26.

As described above, according to the sample analysis chip 1 of the present embodiment, since the initial reaction unit 23 is provided, a common process that is always performed on all the sample liquids M, such as a first-stage biochemical reaction. Can be performed hygienically and efficiently in the initial reaction section 23, and the influence of the external environment on the reagent can also be suppressed.
In addition, an initial reaction unit 23 is disposed in the center of the base material 40, and a spiral channel 24 is provided around the initial reaction unit 23. For this reason, by rotating the sample analysis chip 1 around the central axis C1, it is preferable to use the centrifugal force from the initial reaction part 23 to the well 26 via the spiral flow path 24 and the serrated accommodation part 25. The sample liquid M can be fed.
Since the downstream angle α2 is larger than the upstream angle α1 of the serrated container 25, the sample liquid M easily enters the sawtooth container 25, and the sample liquid once enters the sawtooth container 25. It is possible to suppress M from exiting from the serrated housing portion 25.

Since the constricted portion 15b is formed in the serrated container 25, the sample analysis chip 1 is rotated at a rotational speed lower than the rotational speed that is a threshold value between the relatively slow rotational speed and the relatively fast rotational speed. Then, the sample liquid M stays in the serrated container 25, and when the sample analysis chip 1 is rotated at a rotational speed that exceeds the threshold speed, the sample liquid M enters the well 26 from the serrated container 25. Moving. For this reason, a relatively slow rotation speed or a relatively fast rotation speed can be set with a certain width, and the rotation speed of the sample analysis chip 1 can be easily controlled.
The user only has to inject the sample liquid M into the initial reaction unit 23, and the user himself / herself does not need to inject the sample liquid M into the spiral flow path 24, thereby reducing the work load on the user. be able to.

One end 18a of the second inner surface 18 of the serrated housing portion 25A is in contact with one end 17a of the first inner surface 17 of the serrated housing portion 25B. Therefore, the plurality of sawtooth accommodating portions 25 and wells 26 can be formed in a narrower area of the base material 40.
Since the surplus liquid container 27 is formed at the other end 24c of the spiral flow path 24, the surplus sample liquid M can be reliably stored.

In the present embodiment, as shown in FIG. 10, the one end 18a of the second inner surface 18 of the serrated housing portion 25A and the one end 17a of the first inner surface 17 of the serrated housing portion 25B are not in contact with each other. The inner surface 14a of the spiral flow path 24 may be provided between the one end 18a of the second inner surface 18 of the cylindrical housing portion 25A and the one end 17a of the first inner surface 17 of the serrated housing portion 25B.
The pitches of the sawtooth-shaped accommodation portions 25 and the wells 26 in the direction along the spiral flow path 24 can be appropriately set according to the specifications required for the sample analysis chip 1.

  The upstream angle α1 of the serrated housing portion 25 may be smaller than 80 °. By doing in this way, sample liquid M comes into the sawtooth accommodation part 25 more easily. Further, the downstream angle α2 may be larger than 80 °. By doing so, the sample liquid M once entering the serrated container 25 becomes more difficult to get out of the serrated container 25, and the amount of the sample liquid M weighed in the serrated container 25 is further stabilized. Can be made.

In addition, the spiral channel can have an axis shape described below.
For example, when each of a and b is a constant, the distance r between the center axis C1 and the axis of the spiral channel with respect to the rotation angle θ around the center axis C1 of the substrate 40 is expressed by the following equation (3): A spiral channel may be formed.
r = a + b × θ (3)
Further, when a and b are constants and e is a Napier number (Eulerian number: 2.718...), The central axis C1 and the spiral shape with respect to the rotation angle θ around the central axis C1 of the substrate 40 A spiral channel may be formed so that the distance r from the channel axis is expressed by the equation (4). The line according to equation (4) is also called a logarithmic spiral.
r = a × e ^(bθ) (4)
By forming the spiral channel using the equations (3) and (4), the design of the spiral channel is facilitated, and the position of each part of the spiral channel in the sample analysis chip 1 ( It can be easily obtained from the equations (3) and (4).

(Second Embodiment)
Next, a second embodiment of the present invention will be described with reference to FIG. 11, but the same parts as those of the above-described embodiment will be denoted by the same reference numerals, the description thereof will be omitted, and only different points will be described.
As shown in FIG. 11, the sample analysis chip 2 of the present embodiment includes a spiral flow path 51, a solution instead of the spiral flow path 24 and the excess liquid storage portion 27 of the sample analysis chip 1 of the first embodiment. The discharge part 52 and the excess liquid storage part 53 are provided.

The central angle of the spiral channel 51 is about 720 °.
In the present embodiment, the initial reaction part 23 and the one end part 51 b of the spiral channel 51 are communicated via the solution discharge part 52 without being in direct contact. The solution discharge part 52 is a flow path extending in the radial direction of the substrate 40.
By forming the solution discharge part 52 on the sample analysis chip 2, the sample liquid M supplied from the initial reaction part 23 can be concentrated in a narrow range of the one end part 51 b of the spiral channel 51.
In the present embodiment, the sawtooth accommodating portions 25 and the wells 26 are arranged with a higher arrangement density per unit area than in the first embodiment.
The surplus liquid storage portion 53 of the present embodiment is formed in an elliptical shape that is wider than the spiral flow path 51.

  According to the sample analysis chip 2 of the present embodiment configured as described above, it is possible to perform a plurality of processes and prevent the reagents from affecting the external environment.

As mentioned above, although 1st Embodiment and 2nd Embodiment of this invention were explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The structure of the range which does not deviate from the summary of this invention Changes, combinations, deletions, etc. are also included. Furthermore, it goes without saying that the configurations shown in the embodiments can be used in appropriate combinations.
For example, in the first embodiment and the second embodiment, the surplus liquid container may not be formed in the spiral flow path. This is because the amount of the sample liquid M to be injected into the initial reaction unit 23 may be set to be equal to or less than the volume of the entire serrated container 25.
The upstream angle α11 of the serrated housing portion 25A may be larger than the upstream angle α12 of the sawtooth housing portion 25B.
The protrusion 11 may not be formed on the main body 10. The deaeration port 32 may not be formed in the lid part 30.

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 making use of this in medical practice and examining the “genotype” of each patient, tailor-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. it can. 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, 2 Sample analysis chip 14a Inner surface 17 First inner surface 17a, 18a One end 17b, 18b Other end 18 Second inner surface 23 Initial reaction part 24, 51 Spiral channel (main channel)
24b, 51b One end portion 25 Sawtooth-shaped accommodation portion 26 Well (main accommodation portion)
27, 53 Excess liquid storage part 31 Inlet 40 Base material C1 Central axis C2 Axis L2 Reference line M Sample liquid (sample)
X Thickness direction α1, α11, α12 Upstream angle α2 Downstream angle

Claims (8)

On a disk-shaped substrate,
An initial reaction part arranged in the central part of the base material and communicating with the inlet and accommodating a liquid sample;
A main channel formed in a spiral shape when viewed in the thickness direction of the base material and disposed around the initial reaction part and having one end communicating with the initial reaction part,
A plurality of serrated accommodation portions provided so as to be recessed from the radially inner inner surface of the base material in the main channel;
And a sample analysis chip in which a plurality of main housing portions provided on the opposite side to the main flow path in each of the sawtooth housing portions are formed,
The serrated container is a space defined between a first inner surface and a second inner surface, one end of which is continuous with the inner surface of the main channel and the other end of which extends toward the radially outer side,
The first inner surface is arranged on the upstream side, which is the one end side of the main channel along the main channel, than the second inner surface,
When defining a reference line connecting the one end of the first inner surface and the one end of the second inner surface,
The downstream angle formed by the reference line and the second inner surface on the serrated housing portion side is larger than the upstream angle formed by the reference line and the first inner surface on the sawtooth housing portion side. A sample analysis chip characterized by In the pair of sawtooth accommodating portions adjacent along the inner surface of the main flow path,
The one end of the second inner surface of the serrated housing portion on the upstream side contacts the one end of the first inner surface of the serrated housing portion on the other end side of the main channel along the main channel. The sample analysis chip according to claim 1, wherein the sample analysis chip is provided.   The sample analysis chip according to claim 1, wherein a surplus liquid container is formed at the other end of the main channel. The upstream angle is less than 80 °,
The sample analysis chip according to any one of claims 1 to 3, wherein the downstream side angle is larger than 80 °. In the pair of sawtooth accommodating portions adjacent along the inner surface of the main flow path,
The upstream angle of the upstream serrated accommodation portion is less than or equal to the upstream angle of the serrated accommodation portion on the other end side of the main flow path along the main flow path. Item 5. The sample analysis chip according to any one of Items 1 to 4.   The sample analysis chip according to any one of claims 1 to 5, wherein the initial reaction part is formed in a truncated cone shape having an axis parallel to a central axis of the base material. The main channel has a distance r between the center axis and the axis of the main channel with respect to the rotation angle θ around the center axis of the base material when the axis of the main channel is a and b are constants (1 The sample analysis chip according to claim 1, wherein the sample analysis chip is formed so that
r = a + b × θ (1) The main flow path has an axis between the central axis and the axis of the main flow path with respect to a rotation angle θ around the central axis of the substrate, where a and b are constants and e is the Napier number. The sample analysis chip according to any one of claims 1 to 6, wherein the distance r is formed to satisfy formula (2).
r = a × e ^(bθ) (2)

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