WO2020067212A1

WO2020067212A1 - Sheet gasket

Info

Publication number: WO2020067212A1
Application number: PCT/JP2019/037701
Authority: WO
Inventors: 鈴木　誠一郎
Original assignee: 株式会社エンプラス
Priority date: 2018-09-28
Filing date: 2019-09-25
Publication date: 2020-04-02
Also published as: JP2020051574A

Abstract

The present invention pertains to a sheet gasket that is capable of satisfactorily sealing prescribed wells. A gasket 200 according to the present invention is a gasket 200 for sealing a plurality of droplet wells 103, which are arranged in a straight line on a microfluidic chip 100, when the gasket is sandwiched between a cantilevered depressor 310 and the plurality of droplet wells 103, the gasket comprising: a lower surface 206 for contacting the plurality of droplet wells 103; and an upper surface 205 for contacting the depressor 310. The thickness of the gasket 200 gradually decreases toward the fulcrum of the cantilevered depressor 310.

Description

Sheet gasket

<< The present invention relates to a sheet gasket for sealing a well of a microchip.

2. Description of the Related Art Conventionally, a specific region of DNA is amplified by a polymerase chain reaction (hereinafter, also referred to as “PCR”) for various tests and studies. In the PCR, usually, a step of denaturing the DNA into a single strand, a step of annealing a primer to a desired region of the DNA, and a step of extending the DNA with a polymerase are performed. When these steps performed 1 cycle, the number of a particular region of the DNA is doubled, the 2 ⁿ times in the reaction of n cycles theoretically.

In recent years, a technique called digital PCR has been proposed as a technique for specifying the amount of DNA fragments or RNA fragments contained in cells. In digital PCR, a specimen is sufficiently diluted, and the diluted liquid is distributed to a large number of droplets (or a large number of wells). At this time, a droplet (or well) containing only one DNA fragment (or cDNA fragment) and a droplet (or well) containing no DNA fragment are generated. When PCR is performed on these droplets, DNA is amplified only in droplets (or wells) containing the desired DNA fragment or RNA fragment. Therefore, the amount of the DNA fragment or the RNA fragment contained in the specimen can be specified by confirming the presence or absence of the amplification of the DNA in the droplet (or well) by the detection unit.

As an apparatus for such digital PCR, Patent Literature 1 discloses droplet generation using a microchannel chip in which wells for oil, a sample, and a droplet are arranged in parallel. A system is disclosed. In such a droplet generation system, by applying a positive pressure or a negative pressure to a predetermined well, oil and a sample flow into a microchannel connecting each well, and a droplet including the sample is obtained. The generated droplet is stored in the droplet well.

JP 2018-86008 A

According to the technique disclosed in Patent Document 1, a positive pressure or a negative pressure is applied to a predetermined well by a flow path formed in a manifold engaged with a gasket that seals the predetermined well. At this time, if the predetermined well is not properly sealed with the gasket, the positive or negative pressure applied to the well may be insufficient, and a desired droplet may not be obtained.

Therefore, an object of the present invention is to provide a sheet-like gasket capable of suitably sealing a predetermined well.

In order to solve the above-mentioned problem, the sheet-like gasket according to the present invention includes a plurality of liquid storage portions arranged in a straight line and a plurality of liquid storage portions when sandwiched by a cantilevered pressing portion. A sheet-like gasket for sealing the plurality of liquid storage sections, the first gasket having a first plane for contacting the plurality of liquid storage sections, and a second plane for contacting the pressing section. The thickness gradually decreases as approaching the fulcrum of the pressing portion.

According to the present invention, a predetermined well can be suitably sealed.

FIG. 1 is a plan view of the microchannel chip. FIG. 2A is an enlarged view for explaining details of one set of droplet forming units of the microchannel chip. FIG. 2B is a sectional view taken along line AA of FIG. 2A. FIG. 3A is a plan view of the gasket. FIG. 3B is a perspective view for explaining the gasket. FIG. 4 is a diagram illustrating a state in which a gasket is placed from above the microchannel chip in a state where the microchannel chip is placed on the base. FIG. 5 is a cross-sectional view for explaining a state in which the microchannel chip and the gasket are fixed to the base. FIG. 6 is a view showing a state in which a gasket having a uniform thickness in a short side direction is pressed against a microchannel chip by a cantilevered pressing portion as a comparative example of the present invention. FIG. 7 is a cross-sectional view illustrating a microchannel chip, a gasket, and a pressing portion when a gasket according to a modified example of the invention is applied.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[Microchannel chip 100]
First, the microchannel chip 100 will be described. The microchannel chip 100 is a chip that generates droplets for digital PCR.

FIG. 1 is a plan view of the microchannel chip 100. As shown in FIG. 1, the microchannel chip 100 is formed in a substantially rectangular shape in plan view. The microchannel chip 100 has an oil well 101, a sample well 102, and a droplet well 103. These wells are, for example, chimney-type wells. The droplet well 103 is an example of the liquid storage unit of the present invention.

Further, as shown in FIG. 1, in the microchannel chip 100, a plurality of oil wells 101, a plurality of sample wells 102, and a plurality of droplet wells 103 are respectively arranged and arranged to form a row. ing. As shown in FIG. 1, the oil well row 101R, the sample well row 102R, and the droplet well row 103R are arranged in parallel with each other. The plurality of oil wells 101 constituting the oil well row 101R are formed so that the height from the substrate 104 to the upper end is the same. The same applies to the sample well 102 and the droplet well 103. The substrate 104 of the microchannel chip 100 is a portion of the microchannel chip 100 other than each well. The height of the oil well 101, the height of the sample well, and the height of the droplet well 103 are not necessarily the same.

{Circle around (1)} In the microchannel chip 100, one set of the oil well 101, the sample well 102, and the droplet well 103 constitute one set of the droplet forming unit 110. In the example shown in FIG. 1, eight sets of droplet forming units 110 are arranged in the microchannel chip 100.

FIG. 2A is an enlarged view for explaining details of one set of droplet forming units 110 of the microchannel chip 100. Two oil flow paths 111 are connected to the oil well 101, and a sample flow path 112 is connected to the sample well 102. Two oil channels 111 are connected to both sides of one sample channel 112. A liquid drop channel 113 connected to the liquid drop well 103 is provided at the intersection of the oil flow path 111 and the sample flow path 112. The oil channel 111, the sample channel 112, and the droplet channel 113 are, for example, microchannels having a width of several tens μm to several hundred μm. As shown in FIG. 2B, these flow paths are formed on the back surface of the substrate 104 and are covered with the film 105. FIG. 2B is a sectional view taken along line AA of FIG. 2A. The method of forming the flow path is not limited to this, and may be formed inside the substrate 104, for example.

[Gasket 200]
Next, the gasket 200 according to the present invention, which is used by being fixed to the microchannel chip 100, will be described. FIG. 3A is a plan view of the gasket 200. FIG. FIG. 3B is a perspective view for explaining the gasket 200.

The gasket 200 is a sheet-like member formed of a material such as an elastomer (for example, silicone rubber) having flexibility and / or elasticity. As shown in FIG. 3A, the gasket 200 is formed in a substantially rectangular shape in plan view, like the microchannel chip 100. That is, the gasket 200 has the

long sides

201 and 202 and the

short sides

203 and 204, and the length of the long side 201 and the long side 202 and the length of the short side 203 and the short side 204 are almost the same. The long side 201 is an example of a first long side of the present invention, and the long side 202 is an example of a second long side of the present invention.

Gasket 200 has upper surface 205 and lower surface 206. As shown in FIG. 3B, the upper surface 205 and the lower surface 206 are not parallel, and are formed so that the thickness gradually decreases (gradually decreases) toward the long side 201 along the short side direction. Thus, when the thickness T1 of the gasket 200 on the long side 201 side and the thickness T2 of the gasket 200 on the long side 202 side are compared, T1 <T2. In addition, the thickness of the gasket 200 along the long side direction is formed uniformly. The upper surface 205 is an example of a second plane of the present invention, and the lower surface 206 is an example of a first plane of the present invention.

The gasket 200 has mounting holes 211 for mounting in the vicinity of four corners. A plurality of through holes 212 are provided on a side closer to the long side 201 than the long side 202 of the gasket 200. These through holes 212 are provided at positions corresponding to the droplet wells 103 when the gasket 200 is fixed to the microchannel chip 100.

複数 Further, on the side closer to the long side 202 than the through hole 212, a plurality of openings 213 are provided. These openings 213 are provided at positions corresponding to the oil well 101 and the sample well 102 when the gasket 200 is fixed to the microchannel chip 100. 3A and 3B, the through hole 212 and the opening 213 are shown to have substantially the same size and shape (circular), but the present invention is not limited to this, and the size of the through hole 212 and the opening 213 is not limited to this. May be different, or the shapes may be different.

{Circle around (2)} On the upper surface 205 of the gasket 200, a direction indicator 214 indicating the direction in which the gasket 200 is fixed to the microchannel chip 100 is provided. Details of the fixing direction will be described later. The direction indicator 214 indicates a direction such as an arrow, and is provided on the upper surface 205 of the gasket 200 by printing, for example. Note that the direction indicator 214 is not limited to a printed mark, and may be an engraved mark on the upper surface 205, or a portion of the upper surface 205 protruding from the upper surface 205 to form the direction indicator 214. Good. Further, the shape of the direction indicator 214 need not be an arrow, but may be any as long as the direction is suggested.

[Method of Using Micro Channel Chip 100 and Gasket 200]
Hereinafter, a method of generating droplets using the microchannel chip 100 and the gasket 200 will be described.

{Circle around (1)} First, oil for generating droplets is introduced into the oil well 101 of the microchannel chip 100 and stored (see FIGS. 1 and 2A). The introduction of oil into the oil well 101 is performed by, for example, an experimenter performing digital PCR using a pipette or the like. The oil introduced into the oil well is not particularly limited as long as it is a component that is hardly compatible with the components in the sample described later. Specific examples include oils that are liquid at room temperature, such as mineral oil and silicone oil. Further, a surfactant may be added to the oil.

{Circle around (4)} A desired nucleic acid (for example, DNA or RNA) and a reagent for amplifying a specific region of the nucleic acid are introduced and stored in the sample well 102 of the microchannel chip 100. The introduction of the sample into the sample well 102 is performed, for example, by an experimenter, similarly to the oil. Specific examples of the reagent include, for example, primers, polymerases or mutant polymerases, salts, buffers for adjusting pH, nucleotides, fluorescent dyes that emit fluorescence by binding to nucleic acids, and diluents. In this specification, a nucleic acid and a reagent are collectively referred to as a sample.

In a state where oil is stored in the oil well 101 and the sample is stored in the sample well 102, the microchannel chip 100 is mounted on a base 300 of a digital PCR experiment device (not shown) (see FIGS. 4 and 5 described later). ). Note that oil and a sample may be introduced after the microchannel chip 100 is placed on the base 300. In a state where the microchannel chip 100 is placed on the base 300, the gasket 200 is covered from above the microchannel chip 100. FIG. 4 is a diagram showing a state in which the gasket 200 is put on the microchannel chip 100 from above in a state where the microchannel chip 100 is placed on the base 300. The arrow in FIG. 4 indicates the direction in which the gasket 200 is placed.

The mounting hole 211 of the gasket 200 is provided in the base 300 while the lower surface 206 of the gasket 200 (see FIGS. 3A and 3B) is in contact with the upper ends of the plurality of droplet wells 103 of the microchannel chip 100. The microchannel chip 100 and the gasket 200 are fixed to the base 300 by being hooked on a projection or the like that does not. It is desirable that the digital PCR experiment apparatus be installed such that the base 300 is substantially horizontal. The wells other than the droplet well 103 of the microchannel chip 100, that is, the upper ends of the oil well 101 and the sample well 102 may or may not be in contact with the gasket 200.

FIG. 5 is a cross-sectional view for explaining a state in which the microchannel chip 100 and the gasket 200 are fixed to the base 300. FIG. 5 shows a cross section corresponding to one of the droplet forming units 110 shown in FIG. 2A. In other words, FIG. 5 shows a cross section of the microchannel chip 100 and the gasket 200 along the short side direction.

厚み As described above, the thickness of the gasket 200 gradually decreases toward the long side 201 along the short side direction. As shown in FIG. 5, the long side 202 having a relatively large thickness is closer to the oil well 101 such that the longer side 201 of the gasket 200 is relatively thinner is closer to the droplet well 103 of the microchannel chip 100. The gasket 200 is fixed such that As a result, the upper surface 205 of the gasket 200 in a fixed state has a longer side 201 side (side covering the droplet well 103), a longer side 202 side (side covering the oil well 101), and a higher side. The state is such that the part 300 is inclined obliquely. On the other hand, the lower surface 206 of the fixed gasket 200 is parallel to the base 300.

(4) When the experimenter fixes the microchannel chip 100 and the gasket 200 to the base 300, it is necessary to make sure that the gasket 200 is correctly oriented. For this reason, as described above, the direction indicator 214 is provided on the upper surface 205 of the gasket 200 so that the experimenter does not mistake the mounting direction of the gasket.

With the lower surface 206 of the gasket 200 fixed in contact with the upper ends of the plurality of droplet wells 103 of the microchannel chip 100 as described above, the pressing portion 310 moves the gasket 200 downward from the upper surface 205 side of the gasket 200. That is, it is pressed against the microchannel chip 100 side. More specifically, the pressing portion 310 contacts the long side 201 side (the side that covers the droplet well 103) in the short side direction of the gasket 200, and the other portions (the oil well 101 and the sample well 102) (Covering side). In other words, the gasket 200 is sandwiched between the upper ends of the plurality of droplet wells 103 of the microchannel chip 100 and the pressing portion 310. Thereby, the degree of adhesion between the gasket 200 and the upper end of the droplet well 103 is increased.

The pressing unit 310 is a part of the digital PCR experiment apparatus, and is, for example, a plate facing the base unit 300. The pressing portion 310 is installed so as to be able to move in the vertical direction in FIG. 5 by a motor or the like (not shown). Further, the pressing portion 310 is supported in a cantilever state by the support portion 311. Therefore, the support portion 311 is a fixed end of the pressing portion 310 in a cantilever state, and serves as a fulcrum when the pressing portion 310 moves in the vertical direction. The length of the pressing portion 310 in the depth direction of the paper surface in FIG. 5 is formed so as to cover all of the plurality of droplet wells 103 of the microchannel chip 100.

Since the pressing portion 310 is supported so as to be able to move in the up and down direction in the cantilever state, when the pressing portion 310 contacts the upper surface 205 of the gasket 200 and presses the gasket 200, the pressing portion 310 is inclined obliquely. Can happen. More specifically, the side closer to the droplet well 103 of the microchannel chip 100 (the long side 201 side of the gasket 200) is closer to the oil well 101 of the microchannel chip 100. It can be inclined to be lower than the side (the long side 202 side of the gasket 200).

However, in the present invention, the gasket 200 is fixed with the upper surface 205 of the gasket 200 inclined as described above. In other words, the thickness of the gasket 200 gradually decreases as it approaches the support 311 serving as a fulcrum of the pressing portion 310 in the cantilever state. For this reason, the lower surface of the pressing portion 310 and the upper surface 205 of the gasket 200, which are inclined at an angle, are preferably in contact with each other, and the downward pressing force of the pressing portion 310 is suitably transmitted to the gasket 200. Thereby, the droplet well 103 of the microchannel chip 100 is suitably sealed.

から From the viewpoint of suitably contacting the lower surface of the obliquely inclined pressing portion 310, the degree of gradually decreasing the thickness of the gasket 200 in the short side direction may be determined based on the degree of inclination of the pressing portion 310.

(3) A through hole 212 is provided in a portion of the gasket 200 covering the droplet well 103, as shown in FIGS. 3A, 3B, and 5. Further, as shown in FIG. 5, a conduit 312 for transmitting pressure is provided in the pressing portion 310. One end of the conduit 312 is connected to the through hole 212, and the other end is connected to a pressure source (for example, a pump) not shown. With such a configuration, a negative pressure is applied to the droplet well 103 through the conduit 312 by operating the pump.

When a negative pressure is applied to the droplet well 103, the oil stored in the oil well 101 flows out to the oil channel 111, and the sample stored in the sample well 102 flows to the sample channel 112. (See FIGS. 2A and 5). Here, in order for the oil and the sample to flow out of the oil well 101 and the sample well 102, respectively, the oil well 101 and the sample well 102 need to be open to the atmosphere. In this embodiment, when the upper ends of oil well 101 and sample well 102 are in contact with lower surface 206 of gasket 200, oil well 101 and sample well 102 are formed by opening 213 shown in FIG. Open to the atmosphere. Alternatively, the gasket 200 may be fixed so that the upper ends of the oil well 101 and the sample well 102 do not contact the lower surface 206 of the gasket 200. In this case, for example, the height of the oil well 101 and the height of the sample well 102 may be lower than the height of the droplet well 103.

{Circle around (4)} The oil flowing through the oil flow path 111 and the sample flowing through the sample flow path 112 generate droplets at the intersections of the oil flow path 111 and the sample flow path 112. The generated droplet flows into the droplet well 103 through the droplet channel 113 and is stored.

(5) Thereafter, in the digital PCR experiment apparatus, a process of amplifying the nucleic acid by accelerating the PCR reaction, a process of detecting the amount of the nucleic acid, and the like are performed. A known method may be applied to these processes, and a description thereof will be omitted in this specification.

<Action and effect>
As described above, the gasket 200 according to the present invention has the lower surface 206 that contacts the upper end of the droplet well 103 and the upper surface 205 that contacts the pressing portion 310 supported in a cantilever state. And the lower surface 206 are formed so as not to be parallel to each other. In addition, the thickness of the gasket 200 according to the present invention gradually decreases as approaching the fulcrum of the pressing portion 310 in the cantilever state. In other words, the thickness of the gasket 200 having a substantially rectangular shape in a plan view gradually decreases toward the long side 201 along the short side direction. By fixing such a gasket 200 so as to cover the microchannel chip 100 mounted on the base 300, the upper surface 205 of the gasket 200 is inclined with respect to the base 300.

In this state, by being supported so as to be able to move up and down in a cantilevered state, the pressing portion 310 inclined obliquely with respect to the base portion 300 is pressed against the upper surface 205 of the gasket 200. As a result, the lower surface of the pressing portion 310 that is obliquely inclined and the upper surface 205 of the gasket 200 are preferably in contact with each other, and the downward pressing force of the pressing portion 310 is suitably transmitted to the gasket 200. Therefore, the droplet well 103 of the microchannel chip 100 is suitably sealed. By applying a negative pressure to the droplet well 103 in this state, droplets are suitably generated.

Here, as a comparative example of the present invention, a case where a gasket 200C having a uniform thickness in the short side direction is used will be described. FIG. 6 is a diagram illustrating a state in which the gasket 200C having a uniform thickness in the short side direction is pressed against the microchannel chip 100 by the cantilever pressing portion 310. As shown in FIG. 6, when a downward pressing force is applied by the obliquely pressing portion 310, the gasket 200 </ b> C may float up from the microchannel chip 100. With respect to the oil well 101 and the sample well 102, there is no problem if the gasket 200C rises, but if the gasket 200C rises from the upper end of the droplet well 103, the droplet well 103 is sealed. Will be gone. In this state, even if an attempt is made to apply a negative pressure to the droplet well 103 through the conduit 312, it is difficult to make the inside of the droplet well 103 negative. Therefore, in this state, the oil and the sample do not appropriately flow out of the oil well 101 and the sample well 102, so that droplets are not generated appropriately.

According to the present invention, it is possible to prevent the gasket 200 from floating as described above, and it is possible to preferably generate droplets.

<Modification>
Although various embodiments have been described with reference to the drawings, the present disclosure is not limited to such examples. It will be apparent to those skilled in the art that various changes or modifications can be made within the scope of the claims, and these naturally belong to the technical scope of the present disclosure. I understand. Further, each component in the above embodiment may be arbitrarily combined without departing from the spirit of the disclosure.

In the above-described embodiment, the liquid well 103 of the microchannel chip 100 is sealed and a negative pressure is applied, so that the oil and the sample flow out of the oil well 101 and the sample well 102, respectively. The case where droplets are generated has been described. However, the present invention is not limited to this. In the present invention, the oil well 101 and the sample well 102 of the microchannel chip 100 are sealed and a positive pressure is applied, so that the oil and the sample respectively flow out of the oil well 101 and the sample well 102, Droplets may be generated.

FIG. 7 is a cross-sectional view for describing the microchannel chip 100, the gasket 200M, and the pressing portion 310 when the gasket 200M according to the modification of the present invention is applied. In FIG. 7, the pressing portion 310 is supported in a cantilever state by a support portion 311 provided on the right side of the drawing to seal the oil well 101 and the sample well 102. As a result, the pressing portion 310 is closer to the oil well 101 of the microchannel chip 100 (to the longer side 201M side of the gasket 200M) than the side closer to the droplet well 103 of the microchannel chip 100 (to the oil well 101). The gasket 200M is inclined so that the long side 202M side of the gasket 200M is lower. In this modification, the oil well 101 and the sample well 102 are examples of the liquid storage unit of the present invention.

ガス The gasket 200M of this modification is formed such that the thickness on the long side 202M side is smaller than the thickness on the long side 201M side. Accordingly, the lower surface of the pressing portion 310 that is obliquely inclined preferably contacts the upper surface 205M of the gasket 200M, and the downward pressing force of the pressing portion 310 is suitably transmitted to the gasket 200M. Therefore, the oil well 101 and the sample well 102 of the microchannel chip 100 are suitably sealed. In this state, a positive pressure is applied to the oil well 101 and the sample well 102 through the through hole 212M of the gasket 200M, so that the oil and the sample respectively flow out of the oil well 101 and the sample well 102, and the droplets are discharged. It is preferably generated.

In the above-described embodiment and modified examples, the gasket 200 (200M) according to the present invention was used to seal the microchannel chip 100 used for generating droplets for digital PCR. However, the present invention is not limited to this, and for example, a microchannel chip used for droplet generation other than PCR can also be applied.

This application claims the priority of Japanese Patent Application No. 2018-183723 filed on Sep. 28, 2018. The entire contents described in the specification and drawings of the application are incorporated herein by reference.

According to the present invention, there is provided a gasket used for a digital PCR or the like and applied to a microchannel chip that generates droplets.

REFERENCE SIGNS LIST 100 Micro flow path chip 101 Oil well 101R Oil well row 102 Sample well 102R Sample well row 103 Drop well 103R Drop well row 104 Substrate 105 Film 110 Drop forming unit 111 Oil flow path 112 sample Flow path 113

Droplet flow path

200, 200C,

200M Gasket

201, 201M, 202, 202M

Long side

203, 204

Short side

205, 205M Upper surface 206 Lower surface 211

Mounting hole

212, 212M Through hole 213 Opening 214 Direction indicating unit 300 Base part 310 Press part 311 Support part 312 Conduit

Claims

A plurality of liquid storage units aligned in a straight line, when sandwiched by a cantilevered pressing unit, a sheet-like gasket for sealing the plurality of liquid storage units,
A first plane for contacting the plurality of liquid storage units, and a second plane for contacting the pressing unit,
The thickness gradually decreases as it approaches the fulcrum of the cantilevered pressing portion,
Sheet gasket.
Rectangular in plan view,
The sheet gasket according to claim 1.
The thickness gradually decreases toward the first long side of the rectangle,
A portion closer to the first long side than a second long side of the rectangle is configured to be sandwiched between the plurality of liquid storage units and the pressing unit.
The sheet gasket according to claim 2.
A plurality of through holes are arranged in a line closer to the first long side than the second long side of the rectangle.
The sheet gasket according to claim 3.
A portion closer to the second long side than the through hole covers another plurality of liquid storage portions different from the plurality of liquid storage portions,
The sheet gasket according to claim 4.