WO2020067187A1 - Sheet-like gasket - Google Patents

Abstract

The present invention relates to a sheet-like gasket that can suitably seal a predetermined well. A gasket 200, which is a sheet-like gasket for sealing a droplet well 103 when an opening edge of the gasket is sandwiched between the circular droplet well 103 and a pressing portion 310, comprises: a sheet-like base portion 210; and a protrusion portion 212 provided on a lower surface 206 of the base portion 210. The protrusion portion 212 has a curved surface of which a distance from the base portion 210 increases as the curved surface approaches a central axis of the protrusion portion 212. The cross-section perpendicular to the central axis of the protrusion portion 212 is circular. The maximum value of the diameter of the cross-section is larger than the diameter of the opening edge of the droplet well 103.

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 problems, a sheet-like gasket according to the present invention has a sheet-like gasket for sealing the liquid storage section when an opening edge is sandwiched between a circular liquid storage section and a pressing section. A gasket, comprising: a sheet-like base; and a protrusion provided on a first surface of the base, wherein the protrusion has a greater distance from the base as approaching a central axis of the protrusion. A cross section orthogonal to the central axis of the protrusion is circular, and a maximum value of a diameter of the cross section is larger than a diameter of the opening edge of the liquid storage section.

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 cross-sectional view for explaining a microchannel chip, a gasket, and a pressing portion when a gasket according to a modification of the present 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 and have an opening with a circular edge at the upper end. 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. These flow paths are formed, for example, on the back surface of the substrate 104 and covered with the film 105, as shown in FIG. 2B. 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 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 has a sheet-like base 210 formed substantially in a rectangular shape in plan view similarly to the microchannel chip 100. The base 210 has long sides 201 and 202 and short sides 203 and 204 in plan view. 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 substantially 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.

The base 210 has an upper surface 205 and a lower surface 206. The upper surface 205 and the lower surface 206 are formed to be substantially parallel, and the gasket 200 has a substantially uniform thickness.

取 付 At the base 210 of the gasket 200, mounting holes 211 for mounting are provided near four corners. As shown in FIG. 3B, a plurality of protrusions 212 protruding from the lower surface 206 are provided on a side closer to the long side 201 than the long side 202 of the base 210. FIG. 3B is a diagram of the gasket 200 viewed from the lower surface 206 side. Further, a through hole 213 is provided for each protruding portion 212 so as to penetrate from the vicinity of the tip to the upper surface 205. These protrusions 212 and through holes 213 are provided at positions corresponding to the droplet wells 103 when the gasket 200 is fixed to the microchannel chip 100.

The projecting portion 212 has a round shape as shown in FIG. 3B and FIG. 5 described later. In the present specification, the round shape is a shape in which a cross section including the central axis of the protrusion 212 is an arc (for example, a semi-arc), an elliptical arc (for example, a semi-elliptical arc), or a shape obtained by combining these with a straight line or a curve. , And a shape that increases as the distance from the base 210 approaches the central axis.

(4) The cross-sectional shape orthogonal to the central axis of the protrusion 212 is circular, and the diameter is formed so as to gradually decrease as approaching the tip of the protrusion 212. The maximum value of the diameter of the protrusion 212 is formed to be larger than the diameter of an edge of an opening (hereinafter, referred to as an opening edge) provided at an upper end of the droplet well 103.

複数 A plurality of openings 214 are provided on the side closer to the long side 202 than the protrusion 212 of the base 210. These openings 214 are provided so as to be located 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 213 and the opening 214 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 213 and the opening 214 May be different or the shapes may be different.

[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.

As described above, in the gasket 200, the protruding portion 212 protrudes from the lower surface 206 of the base 210 (see FIGS. 3A and 3B). Therefore, when the gasket 200 is put on the microchannel chip 100, each of the plurality of protrusions 212 comes into contact with the upper end of the plurality of droplet wells 103 of the microchannel chip 100. Thereby, the plurality of droplet wells 103 are sealed by the protruding portions 212. In this state, the microchannel chip 100 and the gasket 200 are fixed to the base 300 by hooking the mounting holes 211 provided in the base 210 to projections (not shown) provided on the base 300. The digital PCR experiment apparatus is desirably installed such that the base 300 is substantially horizontal.

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.

As shown in FIG. 5, with the plurality of protrusions 212 protruding from the lower surface 206 of the base 210 being in contact with and fixed to the upper ends of the plurality of droplet wells 103 of the microchannel chip 100, the pressing portion 310 is The gasket 200 is pressed from the upper surface 205 side of the gasket 200 to the lower side, that is, 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 and fixed 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. 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).

下 When the downward pressing force is applied by the obliquely pressing portion 310, the lower surface 206 of the base 210 of the gasket 200 may be lifted from the microchannel chip 100. FIG. 5 shows a state in which the lower surface 206 of the gasket 200 rises from the upper end of each well of the microchannel chip 100. When the lower surface 206 of the base 210 rises from the upper end of the droplet well 103 and the droplet well 103 is no longer sealed, even if an attempt is made to apply a negative pressure to the droplet well 103 through the conduit 312, the liquid It is difficult to make the inside of the drop well 103 negative pressure. In a state where no negative pressure is applied to the droplet well 103, the oil and the sample do not flow out of the oil well 101 and the sample well 102 properly, so that the droplet is not suitably generated. It should be noted that no pressure is applied to the oil well 101 and the sample well 102 during the generation of droplets, so that there is no problem even if the gasket 200 is lifted.

According to the present invention, the protrusion 212 has a round shape and is formed such that the maximum value of the diameter of the cross section orthogonal to the central axis is larger than the diameter of the opening edge of the droplet well 103. For this reason, even if the gasket 200 is inclined, somewhere of the round-shaped curved surface of the projecting portion 212 is flat as shown in FIG. 1 and the like as long as the upper end of the droplet well 103 is formed flat. It always contacts the opening edge of the droplet well 103 formed in a circular shape when viewed. Therefore, even when the gasket 200 is inclined, the projecting portion 212 seals the droplet well 103 properly.

貫通 As described above, the protrusion 212 is provided with the through-hole 213 that penetrates from the vicinity of the tip to the upper surface 205 (see FIGS. 3A and 3B). Further, the pressing portion 310 is provided with a conduit 312 for transmitting pressure. One end of the conduit 312 is connected to the upper surface 205 side of the through hole 213, 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 the present embodiment, the case where the gasket 200 is not inclined obliquely, that is, the case where the lower surface 206 of the gasket 200 is in contact with the lower surface 206 of the gasket 200 at the upper ends of the oil well 101 and the sample well 102. 3 and the like, the oil well 101 and the sample well 102 are open to the atmosphere. Alternatively, even when the gasket 200 is fixed without being inclined obliquely, it may be formed in advance 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 is provided with the protrusion 212 projecting from the lower surface 206 of the sheet-like base 210. Such a gasket 200 is fixed while being covered with the microchannel chip 100 mounted on 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 to the base portion 300 is pressed against the upper surface 205 of the base 210. Then, a part of the base 210 of the gasket 200 may be lifted from the microchannel chip 100.

However, since the protrusion 212 has a round shape and the maximum value of the diameter of the cross section perpendicular to the central axis is larger than the diameter of the opening edge of the droplet well 103, no matter how the base 210 is inclined, Somewhere on the round curved surface of the protrusion 212 comes into contact with the opening edge of the droplet well 103 having a circular shape in plan view. Therefore, the projections 212 can reliably seal the droplet well 103. By applying a negative pressure to the droplet well 103 in this state, droplets are suitably generated.

<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.

[Modification 1]
In the embodiment described above, the protrusion 212 has a round shape. However, the present invention is not limited to this, and the protrusion 212 may have a curved surface whose distance from the base 210 increases as approaching the central axis.

具体 Specific examples of the protruding portion 212 include a tapered shape, a truncated cone shape, and a shape in which a tapered shape is connected to a cylindrical shape. The taper shape in this specification includes a linear taper shape, an exponential taper shape, a parabolic taper shape, and the like.

Thus, even when the protrusion 212 has a shape other than the round shape, the protrusion 212 has a circular cross section orthogonal to the central axis, and the maximum value of the diameter of the cross section is the opening edge of the droplet well 103. What is necessary is just to be formed so that it may become larger than the diameter of.

With such a configuration, even when the base 210 of the gasket 200 is obliquely inclined by the pressing portion 310, any part of the curved surface of the protrusion 212 comes into contact with the opening edge of the droplet well 103. 103 can be suitably sealed and droplets are suitably generated.

[Modification 2]
In the above-described embodiment and the first modification, the droplet well 103 of the microchannel chip 100 is sealed and a negative pressure is applied, so that the oil and the sample are respectively discharged from the oil well 101 and the sample well 102. The case where the liquid flows out and 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 flow out of the oil well 101 and the sample well 102, respectively. Droplets may be generated.

FIG. 6 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. 6, the pressing portion 310 is supported in a cantilevered 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 201 of the gasket 200) than the side closer to the droplet well 103 of the microchannel chip 100 (the side closer to the oil well 101). The long side 202 side of the gasket 200) is inclined to be lower. In the second modification, the oil well 101 and the sample well 102 are examples of the liquid storage unit of the present invention.

In the above-described embodiment, when the gasket 200 is placed on the microchannel chip 100, the protrusion 212 is formed at a position corresponding to the droplet well 103. In the second modification, the protrusions 212M1 and 212M2 are formed at portions corresponding to the oil well 101 and the sample well 102. Thereby, the oil well 101 and the sample well 102 of the microchannel chip 100 are suitably sealed. The size of the protrusions 212M1 and 212M2 (such as the diameter of a cross section orthogonal to the central axis) may be appropriately designed according to the size of the oil well 101 and the sample well 102. In this state, when a positive pressure is applied to the oil well 101 and the sample well 102, the oil and the sample flow out of the oil well 101 and the sample well 102, respectively, and droplets are suitably generated.

[Other Modifications]
In the above-described embodiment and modified examples, the gasket 200 according to the present invention is used for sealing the microchannel chip 100 used for generating droplets for digital PCR. However, the present invention is not limited to this, and can be applied to, for example, a microchannel chip used for droplet generation other than PCR.

In the above-described embodiment and the modified example, the gasket 200 according to the present invention has the plurality of protrusions 212 (212M1, 212M2), and is configured to be in contact with the opening edges of the plurality of droplet wells 103. It had been. However, the present invention is not limited to this, and the number of protrusions 212 may be one.

In addition, in the above-described embodiment and the modified example, the gasket 200 according to the present invention is provided with the plurality of protrusions 212 (212M1, 212M2) arranged in a straight line. This was to seal the droplet wells 103 provided in a straight line. However, the present invention is not limited to this. For example, when the plurality of droplet wells 103 are not provided in a linear array, the protrusions 212 ( 212M1 and 212M2) may be provided.

This application claims priority based on Japanese Patent Application No. 2018-183728 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, 200M Gasket 201, 202 Long side 203, 204 Short side 205 Upper surface 206 Lower surface 210 Base 211 Mounting hole 212, 212M1, 212M2 Projecting portion 213 Through hole 214 Opening 300 Base 310 Pressing portion 311 Support part 312 Conduit

Claims (7)

When the opening edge is sandwiched between the circular liquid storage portion and the pressing portion, a sheet gasket for sealing the liquid storage portion,
A sheet-like base, and a protrusion provided on a first surface of the base,
The projecting portion has a curved surface whose distance from the base increases as approaching the central axis of the projecting portion,
The cross section of the protrusion orthogonal to the central axis is circular, and the maximum value of the diameter of the cross section is larger than the diameter of the opening edge of the liquid storage section.
Sheet gasket. The curved surface of the protrusion is configured to contact the opening edge of the liquid storage unit,
The sheet gasket according to claim 1. A plurality of the protrusions are provided in line with each other in a straight line, and each of the curved surfaces is configured to contact each of the opening edges of the plurality of liquid storage units.
The sheet gasket according to claim 2. A plurality of the liquid storage section, and the pressing section in a cantilever state, is configured to be sandwiched,
The sheet gasket according to claim 3. The protrusion has a round shape in a cross section including the central axis,
The sheet gasket according to any one of claims 1 to 4. The base is rectangular in plan view,
The protrusion is disposed closer to a second long side than to a first long side of the rectangle.
The sheet gasket according to any one of claims 1 to 5. The protrusion has a through-hole that opens in the protrusion and the second surface of the base.
The sheet gasket according to any one of claims 1 to 6.

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