CN217265655U

CN217265655U - Microporous plate

Info

Publication number: CN217265655U
Application number: CN202221084456.7U
Authority: CN
Inventors: 陈岱
Original assignee: Jiangxi Liebing Biotechnology Co ltd
Current assignee: Jiangxi Liebing Biotechnology Co ltd
Priority date: 2022-05-08
Filing date: 2022-05-08
Publication date: 2022-08-23
Anticipated expiration: 2032-05-08

Abstract

The utility model discloses a microporous plate, which comprises an L1 laminate, wherein an L2 laminate is arranged on the L1 laminate, and a chip is arranged on the L2 laminate; l1 plywood includes panel, panel is transparent rectangle structure, and the lower left corner contains the chamfer structure, the top of panel is equipped with toper shrinkage pool and square pit, square pit's internal surface is equipped with the U-shaped boss, the below of panel is equipped with trapezoidal boss, the panel bottom is equipped with semi-circular boss along the profile. The device can be used for rapid and nondestructive cell separation; the time spent is short; the ability to isolate lower concentrations of cells; separating cells with high efficiency and softness; cell numbers can be analyzed in real time using fluorescence imaging.

Description

Microporous plate

Technical Field

The utility model relates to a single cell separation technology field, concretely relates to micropore board.

Background

In the past decade, high throughput sequencing technology has been widely used in various fields of biology and medicine, greatly facilitating related research and applications. Among them, transcriptional sequencing is widely used to determine and characterize the expression of genes or transcripts in various species. However, conventional transcriptome sequencing techniques are based on a population of cells, each sample containing thousands of cells, and so ultimately reflect the average expression level of genes in the population of cells, thereby masking expression heterogeneity between different cells. In recent years, sequencing of single-cell transcriptome (the technology is vigorously developed, so that the expression conditions of all genes in a whole genome range can be revealed at a single-cell level, and the study on expression heterogeneity among cells is very beneficial). at present, the sequencing technology of the single-cell transcriptome is widely applied to different types of tissues and cell lines of various species, including normal cells, diseased cells and the like.

Before single cell sequencing is carried out, single cells need to be separated firstly, different types of single cell transcriptome sequencing technologies are adopted, and the used cell separation technologies may be different. In general, there are currently mainly micropipetting techniques, laser capture microdissection techniques, flow cytometry techniques, microdroplet techniques, and microfluidic techniques. The micro-suction technology and the laser capture micro-cutting technology are mainly applied to samples with small cell amount or more precious, any tissue can be subjected to cell separation by adopting the technology, and cell selection is carried out based on morphological or fluorescent marker visualization. It takes a long time. The flow cytometry technology can separate a large number of cells of any tissue, and cell selection is carried out according to the size of the cells or fluorescent markers, so that the separation speed is high. The microdroplet technique can separate a large number of cells with high separation speed, but can not select the cells. Microfluidic technology is capable of isolating larger numbers of cells, does not allow for selection of cells, but allows visualization of cells, which can be used in conjunction with flow cytometry isolation techniques to order cells. Sorting cells using microfluidic technology is much more gentle and less costly for cells, with the disadvantage of requiring presumption of cell loss and deviation in cell size.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a micropore board to use this micropore board in order to solve the problem that mentions in the background art. In order to achieve the above object, the utility model provides a following technical scheme: a microplate comprising an L1 lamina, the L1 lamina having an L2 lamina mounted thereon, the L2 lamina having a chip mounted thereon;

the L1 laminate comprises a plate, the plate is of a transparent rectangular structure, the lower left corner of the plate is provided with a chamfer structure, the top of the plate is provided with a conical concave hole and a square depression, the inner surface of the square depression is provided with a U-shaped boss, a trapezoidal boss is arranged below the plate, and the bottom of the plate is provided with a semicircular boss along the outline;

the L2 laminate comprises a sheet material I, wherein a boss is arranged at the top of the sheet material I, a liquid channel groove is formed in the boss, a U-shaped channel liquid inlet hole and a liquid outlet are formed in two ends of the liquid channel groove respectively, the liquid outlet is of a conical boss structure, symmetrical U-shaped channels are arranged in the center of the sheet material I, five equal-large circular holes are distributed around the U-shaped channel, a liquid injection port is formed in the top of the sheet material I and communicated with the U-shaped channel liquid inlet hole and the liquid injection port, an annular bulge is arranged along the circumference of the liquid injection port, an airtight rubber plug clamping groove is formed inside the annular bulge, a rubber airtight pad is mounted inside the airtight rubber plug clamping groove, the position of the liquid injection port corresponds to that of a conical concave hole, a chip is mounted on the back of the sheet material I through a quadrangular frustum bulge, and is located below the U-shaped channel liquid inlet hole, the circular hole, the U-shaped channel liquid inlet hole and the liquid injection port, the chip is in sealing contact with the back face of the plate I, and the square recess corresponds to the boss structure in position.

Preferably, linear protrusions are arranged on the front side and the back side of the U-shaped channel and on the periphery of the liquid channel groove.

Preferably, trapezoidal reinforcing ribs are arranged in the inner contour of the bottom of the plate.

Preferably, the chip is of a transparent cuboid structure and is provided with a fool-proof chamfer, the working face of the chip is stamped with regularly arranged hexagonal pot holes by using a precision casting technology, cross-shaped marks corresponding to the positions of the round holes are stamped on the chip, the non-working face of the chip is a smooth surface, the side length of each hexagonal pot hole is 25 micrometers, the center distance is 56.3 micrometers, and the depth is 50 micrometers.

Preferably, the L1 laminate and the L2 laminate are made of ABS material, and the chip is made of PMMA (polymethyl methacrylate) material.

Preferably, the surface area of the U-shaped shaft is 344.5 square millimeters, the thickness of the U-shaped shaft is 2mm, the inner wall of the U-shaped shaft is in a slope shape, and after the L1 laminate and the L2 laminate are assembled, the height of a cavity corresponding to the U-shaped shaft is 0.8mm to 1 mm.

The utility model discloses a technological effect and advantage: the micro-porous plate disclosed by the utility model can be used for quickly and nondestructively separating cells; the time spent is short; the ability to isolate lower concentrations of cells; separating cells with high efficiency and softness; cell numbers can be analyzed in real time using fluorescence imaging.

Drawings

FIG. 1 is a front side view of a schematic diagram of a microplate structure of the present invention;

FIG. 2 is a rear side view of the schematic diagram of the microporous plate structure of the present invention;

FIG. 3 is a front side view of the L1 laminate of the microplate of the present invention;

FIG. 4 is a rear side view of the L1 laminate of the microplate of the present invention;

FIG. 5 is a front side view of the L2 laminate of the microplate of the present invention;

fig. 6 is a rear side view of the L2 laminate of the microplate of the present invention.

In the figure: 1. a chip; 2. a plate material; 3. a chamfering structure; 4. a conical concave hole; 5. a square recess; 6. a U-shaped boss; 7. trapezoidal reinforcing ribs; 8. a plate I; 9. a boss; 10. a liquid channel groove; 11. a U-shaped channel liquid inlet hole; 12. a liquid outlet; 13. a U-shaped channel; 14. a circular hole; 15. a liquid injection port; 16. an annular projection; 17. an airtight rubber pad; 18. the quadrangular frustum is convex; 19. a linear protrusion; 20. a trapezoidal boss; 21. a semicircular boss; 22. and (5) preventing the fool and chamfering.

Detailed Description

In order to make the technical means, the creative features, the objectives and the functions of the present invention easily understood and appreciated, the present invention will be further described with reference to the specific drawings, and in the description of the present invention, unless otherwise specified or limited, the terms "mounted," connected "and" connected "should be understood broadly, and for example, the terms" fixed connection, "detachable connection," integral connection, mechanical connection, and electrical connection may be used; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements.

Examples

The microplate shown in FIGS. 1-6 comprises an L1 plate, wherein the L1 plate is connected with an L2 plate, and a chip 1 is mounted on the L2 plate;

the L1 laminate is made of ABS material acrylonitrile-butadiene-styrene copolymer) and comprises a plate 2, the plate 2 is of a transparent rectangular structure, the lower left corner of the plate 2 contains a chamfer structure 3, a conical concave hole 4 and a square recess 5 are formed in the top of the plate 2, the bottom of the square recess 5 is in contact with an L2 laminate, a U-shaped boss 6 is arranged on the inner surface of the square recess 5, a trapezoidal boss 20 is arranged below the plate 2, and the trapezoidal boss 20 can be matched with a buckle grabbing boss arranged on an experimental instrument and fixed in position; the bottom of the plate 1 is provided with a semicircular boss 21 along the outline, and when the die is demolded, the demolding thimble demolds the product by extruding the position of the semicircular boss 21; a trapezoidal reinforcing rib 7 is arranged in the inner outline at the bottom of the plate 2, and the trapezoidal reinforcing rib 7 can enhance the deformation resistance of the plate;

the L2 laminate is made of ABS material and has a black quadrangular frustum pyramid structure, and comprises a plate I8, the top of the plate I8 is provided with a boss 9, the boss 9 is provided with a liquid channel groove 10, two ends of the liquid channel groove 10 are respectively provided with a U-shaped channel liquid inlet hole 11 and a U-shaped channel liquid outlet 12, the liquid outlet 12 is of a conical boss structure, symmetrical U-shaped channels 13 are arranged in the center of the plate I8, the surface area of the U-shaped channel 13 is 344.5 square millimeters, the thickness of the U-shaped channel 13 is 2mm, the inner wall of the U-shaped channel 13 is in a slope shape, the height of the U-shaped boss 6 can be controlled by adjusting the mould, the U-shaped boss 6 is embedded into the U-shaped channel 13 for positioning, meanwhile, the height of the cavity formed by the U-shaped channel 13 can be adjusted by utilizing the height of the U-shaped boss 6, so as to achieve the purpose of controlling the volume of the channel, after the L1 laminate and the L2 laminate are assembled, the height of a corresponding cavity formed by the U-shaped channel 13 is 0.8mm to 1 mm; five circular holes 14 with equal size are distributed around the U-shaped channel 13, and a liquid injection port 15 is formed in the top of the plate I8; the U-shaped channel 13 is communicated with the U-shaped channel liquid inlet hole 11 and the liquid injection port 15, the liquid channel groove 10 can prevent liquid in the U-shaped channel 13 from flowing out and air from entering the U-shaped channel 13, and the liquid in the channel can flow out only when the liquid injection amount exceeds the volume of the channel and is lifted to a high position; when air enters from the liquid outlet 12, the air stays in the liquid channel groove 10 on the boss 9; an annular bulge 16 is arranged at the liquid injection port 15 along the circumference, an airtight rubber plug clamping groove is formed in the annular bulge 16, a rubber airtight pad 17 is installed in the airtight rubber plug clamping groove, the airtight rubber pad 17 is in contact with the bottom of the conical concave hole 4, the back of the plate I8 is fixedly limited by a chip 1 through a quadrangular frustum bulge 18, and the chip 1 is located below the U-shaped channel 13, the round hole 14, the U-shaped channel liquid inlet hole 11 and the liquid injection port 15; linear protrusions 19 are arranged on the front side and the back side of the periphery of the U-shaped channel 13 and the periphery of the liquid channel groove 10, the linear protrusions 19 are melted by using an ultrasonic welding technology and are tightly welded, and the airtight effect of an L1 laminate, an L2 laminate, an L2 laminate and the chip 1 can be realized; the chip 1 is in sealing contact with the back surface of the plate I8, and the square recess 5 corresponds to the boss 9 in structure position;

the chip 1 is made of PMMA (polymethyl methacrylate) material, is in a high-transparent cuboid structure, and is provided with a fool-proof chamfer 22, and the fool-proof chamfer 22 can help to identify the front side, the back side and the mounting direction of the chip 1; the working surface of the chip 1 is stamped with regularly arranged hexagonal pits by using a precision casting technology, the chip 1 is stamped with cross-shaped marks corresponding to the positions of the round holes, the non-working surface of the chip 1 is a smooth surface, the side length of each hexagonal pit is 25 micrometers, the center distance is 56.3 micrometers, and the depth is 50 micrometers.

A single-cell separation process based on a microporous plate specifically comprises the following steps:

the microporous plate is arranged on a workbench, and a waste liquid recovery cylinder is arranged below the liquid outlet 12; the experimental instrument grabs the trapezoidal boss 20 below the plate 1 of the L1 laminate through the buckle grabbing boss, and fixes the position;

pumping the buffer into a flushing U-shaped channel 13 and a liquid channel groove 10 from a liquid injection port by using a liquid transfer gun to soak the surface of the chip 1; during an experiment, a pipette head of the pipette is pricked into a central circular hole of the rubber airtight pad 17, a chamfer angle near a clamping groove of the rubber pad can provide an expansion space, the rubber airtight pad 17 is extruded by manual pressure to be tightly attached to the pipette head, and meanwhile, the diameter of the clamping groove of the rubber pad can prevent the pipette head from deeply contacting the surface of the chip 1, so that a liquid-tight effect is realized, a reagent can be guaranteed to be squeezed into the U-shaped channel 13, and liquid cannot be injected because the pipette head is propped against the chip 1;

pumping the cell tissue into the U-shaped channel 13 from the liquid injection port by using a liquid transfer gun, and waiting for the cells to fall into the hole; the inner wall of the U-shaped channel 13 is slope-shaped, the height of a cavity is about 0.8mm to 1mm after the L1 laminate and the L2 laminate are assembled, and cells and magnetic beads slowly descend and fall into hexagonal pits on the surface of the chip 1; after the liquid is injected, the U-shaped channel 13 utilizes the recoil force generated when the liquid washes the bent channel to more uniformly lay the cell tissues in the U-shaped channel 13 through the U-shaped structure, so that the double-cell rate is reduced;

the method comprises the following steps that a microporous plate is placed in a scanner, a rectangular outline and the lower left corner of a plate 1 of an L1 laminate contain a chamfer structure 3 and are matched with the shape of a clamping groove of an experimental instrument, when the instrument runs, a hook claw of a bullet cabin applies thrust to the position of the chamfer structure 3, the microporous plate is pushed to the upper right of the clamping groove, and orientation correction and locking of the microporous plate are carried out; during scanning, an instrument observes the cross-shaped marks on the chip 1 through the positions of five equal-size circular holes 14 distributed around the U-shaped channel 13, observes the cross-shaped marks inside the circular holes through an optical system, records the Z coordinate of the chip 1 by utilizing the Z-axis height of a microscope objective during focusing, establishes a focusing map and ensures the focusing definition during high-speed scanning photographing;

carrying out high-speed photographing and fluorescent irradiation photographing on the position of the U-shaped channel 13, and calculating the cell activity rate of the cells falling into the bottom of the hexagonal pot hole of the chip 1 by observing the number of green fluorescent points; at the moment, because the focal plane is the bottom of the hexagonal pot hole, only the cells falling into the hexagonal pot hole can be clearly observed and counted, only the active cells can reflect fluorescence when the fluorescence is irradiated, and the cell survival rate is calculated by observing the number of green fluorescence points;

pumping the magnetic beads into the U-shaped channel 13 from the liquid injection port 15 by using a pipette, standing for 3 minutes, and waiting for the magnetic beads to fall into the holes; the side length of a hexagonal cavity on the surface of the chip 1 is 25 micrometers, the center distance is 56.3 micrometers, and the depth is 50 micrometers; the function is to capture cells, capture magnetic beads, and serve as a lysis reaction vessel to contain RNA tissue attached to the magnetic beads. The depth of the hexagonal pot hole can just accommodate one magnetic bead to fall into the hexagonal pot hole, when a second magnetic bead falls into the hexagonal pot hole, the second magnetic bead is higher than the edge of the hexagonal pot hole, when the U-shaped channel 13 is washed, the second magnetic bead cannot be blocked by the wall of the hexagonal pot hole and is smoothly washed away, and one pot and one bead are realized;

placing a magnet below the microporous plate, attracting magnetic beads to the bottom of the hexagonal pot hole, driving a buffer into the U-shaped channel 13 from the liquid injection port 15 by using a liquid transfer gun, and flushing away the magnetic beads which are not settled in the hexagonal pot hole and second magnetic beads which fall into the hexagonal pot hole to fall into a waste liquid cylinder; placing the microporous plate in a scanning machine, scanning, and observing and recording the hole falling condition of the magnetic beads;

injecting the lysate into the U-shaped channel 13 from the liquid injection port 15 by using a pipette, standing for 2 minutes, and waiting for cell lysis in the hexagonal pit; after lysis, cellular RNA was attached to the magnetic beads; placing the microporous plate in a scanning machine, scanning, and observing and recording cell lysis conditions;

placing a magnet above the microporous plate, wherein the magnetic beads are influenced by magnetic force and are moved out of the hexagonal pits, placing a collecting cylinder below the liquid outlet 12, driving a buffer into the collecting cylinder from the liquid injection port 15 by using a liquid-moving gun, flushing the inside of the U-shaped channel 13, and collecting the magnetic beads in the collecting cylinder; and (3) placing the plate in a sheet sweeping machine, sweeping the sheet, and observing and recording the condition of the U-shaped channel 13.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions on some technical features, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A micropore plate comprises an L1 layer plate, and is characterized in that: the L2 laminated plate is arranged on the L1 laminated plate, and a chip is arranged on the L2 laminated plate;

the L2 plywood comprises a plate I, wherein a boss is arranged at the top of the plate I, a liquid channel groove is formed in the boss, a U-shaped channel liquid inlet hole and a liquid outlet are formed in the two ends of the liquid channel groove respectively, the liquid outlet is of a conical boss structure, a symmetrical U-shaped channel is arranged in the center of the plate I, five equal-size round holes are distributed around the U-shaped channel, a liquid injection port is formed in the top of the plate I and communicated with the U-shaped channel liquid inlet hole and the liquid injection port, an annular bulge is arranged along the circumference of the liquid injection port, an airtight rubber plug clamping groove is formed in the annular bulge, a rubber airtight pad is mounted in the airtight rubber plug clamping groove, the position of the liquid injection port corresponds to that of a conical concave hole, a chip is mounted on the back of the plate I through a four-pyramid-shaped boss bulge, and is located below the U-shaped channel liquid inlet hole and the liquid injection port, the chip is in sealing contact with the back face of the plate I, and the square recess corresponds to the boss structure in position.

2. The microplate according to claim 1, wherein: linear bulges are arranged on the front surface and the back surface of the periphery of the U-shaped channel and the periphery of the liquid channel groove.

3. The microplate according to claim 1, wherein: trapezoidal reinforcing ribs are arranged in the inner outline at the bottom of the plate.

4. The microplate according to claim 1, wherein: the chip is transparent cuboid structure, is equipped with prevents slow-witted chamfer, the working face of chip utilizes the hexagon pothole of precision casting technique impression regular arrangement, the cross shape mark that has impressed with the round hole position on the chip corresponds, the non-working face of chip is smooth surface, hexagon pothole length of side is 25 microns, and the centre-to-centre spacing is 56.3 microns, and the degree of depth is 50 microns.

5. The microplate according to claim 4, wherein: the L1 plywood and the L2 plywood are made of ABS materials, and the chip is made of PMMA (polymethyl methacrylate) materials.

6. The microplate according to claim 2, wherein: the surface area of the U-shaped channel is 344.5 square millimeters, the thickness of the U-shaped channel is 2mm, the inner wall of the U-shaped channel is in a slope shape, and after the L1 laminate and the L2 laminate are assembled, the height of a cavity corresponding to the U-shaped channel is 0.8mm to 1 mm.