CN116286261A - Single-cell pretreatment device, application and manufacturing method of single-cell capturing membrane - Google Patents

Abstract

The invention discloses a single-cell pretreatment device, application and a manufacturing method of a single-cell capture membrane, wherein the single-cell pretreatment device comprises a shell; the fluid channel is arranged in the shell and comprises a first channel and a second channel, one end of the first channel is an input port, the other end of the first channel is a first output port, one end of the second channel is communicated with the first channel, the other end of the second channel is a second output port, and the second channel is positioned at the lower layer of the first channel; the single-cell capturing membrane is arranged at the communication part of the first channel and the second channel; the pressure detection assembly detects the second output port; the single-cell capturing membrane comprises a first chamber, a second chamber and a third chamber which are sequentially arranged and longitudinally communicated, wherein the first chamber is used for capturing magnetic beads, the second chamber is used for capturing cells, and the third chamber is used for flowing liquid. Through setting gradually and vertical intercommunication first cavity, second cavity and third cavity, use the micro-fluidic to control single cell and correspond the falling hole and the capture of magnetic bead to can greatly improve target cell's capture and mark efficiency.

Description

Single-cell pretreatment device, application and manufacturing method of single-cell capturing membrane

Technical Field

The invention relates to the technical field of biological cell detection, in particular to a single-cell pretreatment device, application and a manufacturing method of a single-cell capturing membrane.

Background

Single cell sequencing technology is a new technology for high throughput sequencing analysis of genome, transcriptome and apparent group at single cell level. The gene expression system can reveal the gene structure and the gene expression state of single cells, reflect the heterogeneity among cells, play an important role in the fields of tumor, developmental biology, microbiology, neuroscience and the like, and are becoming the focus of life science research. In tumor tissues, the genetic information of tumor center cells, tumor surrounding cells, primary foci and metastasis cells, genome and transcriptome are different, which leads to the phenotype of different tumor cells such as immunity, growth speed, invasion capacity, and the like, and finally leads to the sensitivity to different antitumor drugs or the sensitivity to radiotherapy. The core idea of single cell sequencing is to sequence a large number of single cells individually, and this is accomplished based on a single cell identification technology of a tag (barcode), and the core idea is that: when reverse transcription is performed before mRNA sequencing of each cell, unique tag sequences are added to the mRNA sequences, so that when a large number of cells are mixed and sequenced, RNA fragments carrying the same tag sequences (barcode) can be regarded as being from the same cell, and by means of the strategy, information of tens of thousands of single cells can be measured through one-time library establishment.

Currently the mainstream commodity comprises: 10xGenomics based on droplet technology and BDRhapsody based on microwell plates. However, these techniques follow poisson distribution, i.e. the encapsulation of cells is random, and in general, the cell encapsulation rate is about 10%, which results in loss of a large number of cells that are introduced, and the single cell sequencing information obtained is only about 10%. This technique is only suitable for capture analysis of high proportion of cell types, and rare cells are difficult to obtain for efficient capture. Aiming at the problems that the single cell mark in the prior art has lower capture efficiency, is not suitable for capturing cell types with lower occupation ratio, and the like, and has a large initial sample amount, waste reagents and the like, the development of a novel single cell capture device is needed.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a single-cell pretreatment device which can solve the problems of low efficiency, low activity, unsatisfactory flux, large pressure difference and the like of the existing single-cell capture and labeling as far as possible.

The invention also provides a manufacturing method of the single-cell capturing membrane.

The invention also provides application of the single-cell pretreatment device.

The invention also provides an application of the single-cell pretreatment device in cell analysis.

According to an embodiment of the first aspect of the present invention, a single cell pretreatment device includes:

a housing;

the fluid channel is arranged in the shell, one end of the first channel is used as an input port, the other end of the first channel is used as a first output port, one end of the second channel is communicated with the first channel, the other end of the second channel is used as a second output port, and the second channel is positioned at the lower layer of the first channel;

a single cell capturing membrane disposed at a communication point between the first channel and the second channel;

the pressure detection component is used for detecting the second output port to judge the single cell capturing condition;

the single-cell capturing membrane comprises a first chamber, a second chamber and a third chamber which are sequentially arranged and longitudinally communicated, wherein the first chamber is used for capturing magnetic beads, the second chamber is used for capturing cells, and the third chamber is used for flowing liquid.

The single-cell pretreatment device according to the embodiment of the first aspect of the present invention has at least the following advantageous effects: through setting gradually and vertical intercommunication first cavity, second cavity and third cavity, use the micro-fluidic to control single cell and correspond the falling hole and the capture of magnetic bead to can greatly improve target cell's capture and mark efficiency.

According to an embodiment of the first aspect of the present invention, the single-cell pretreatment device comprises a first layer, a second layer and a third layer, wherein the first layer, the second layer and the third layer are sequentially arranged, a plurality of first micropores are arranged on the first layer at intervals and are respectively used as the first chambers, a plurality of second micropores are arranged on the second layer at intervals and are respectively used as the second chambers, a plurality of groups of third micropores are arranged on the third layer at intervals and are respectively used as the third chambers, and each first micropore, each second micropore and each group of third micropore are respectively communicated.

According to the single-cell pretreatment device of the embodiment of the first aspect of the present invention, the sizes of the first micro-holes are the same, the sizes of the second micro-holes are the same, the sizes of the third micro-holes are the same, and the sizes of the first micro-holes, the second micro-holes and the third micro-holes are sequentially set from large to small.

According to an embodiment of the first aspect of the present invention, the number of the first microwells and the number of the second microwells are in one-to-one correspondence, and the third microwells of each group include one or more third microwells.

According to an embodiment of the first aspect of the present invention, the first layer, the second layer and the third layer are integrally formed;

and/or the height and diameter of the first micro-holes is 50-100um, the height and diameter of the second micro-holes is 10-40um, and the height and diameter of the third micro-holes is 0.5-5um;

and/or the first layer, the second layer and the third layer are made of a photosensitive transparent polymer or a thermosetting transparent polymer.

According to the single-cell pretreatment device disclosed by the embodiment of the first aspect of the invention, the shell comprises a first plate, a second plate and a third plate which are sequentially stacked from top to bottom, and the second plate is provided with a first pair of interfaces and a second pair of interfaces;

the first channel, the input port, the first output port and the second output port are formed in the first plate, the second channel is formed in the third plate, one end of the second channel is communicated with the first channel through the first pair of interfaces, and the other end of the second channel is communicated with the second output port through the second pair of interfaces.

According to an embodiment of the first aspect of the present invention, the first pair of ports are arranged to extend in a longitudinal direction, and the first channel and the second channel are arranged in parallel to each other.

According to a second aspect of the present invention, the method for manufacturing a single-cell capturing membrane, which is the single-cell capturing membrane of the single-cell pretreatment device according to the first aspect of the present invention, includes the steps of:

providing a silicon wafer substrate, coating photoresist on the surface of the silicon wafer substrate, and obtaining a third microporous layer structure through photoetching and developing;

coating photoresist on the third microporous layer structure, and obtaining a second microporous layer structure through photoetching and developing;

coating photoresist on the second microporous layer structure, and obtaining a first microporous layer structure through photoetching and developing;

duplicating the obtained structure to obtain a mould of the single-cell capturing membrane;

providing a glass substrate, reversely attaching the mold surface downwards on the glass substrate, and forming a single-cell capturing film by utilizing a vacuum adsorption photo-curing technology;

and stripping the single-cell capturing film from the glass substrate to obtain the single-cell capturing film.

According to an embodiment of the third aspect of the present invention, the use of a single cell pretreatment device comprises the steps of:

inputting cells into the first channel and flowing into the second chamber of the single-cell capturing membrane until the value detected by the pressure detection assembly exceeds a preset value, and removing the remaining cells which are not captured;

the magnetic beads are input into the first channel and flow into the first chamber of the single-cell capturing membrane, and naturally settle for a certain time, so that single cells and single magnetic beads form a one-to-one correspondence and react, and the residual non-captured magnetic beads are removed.

According to a fourth aspect of the present invention, the use of a single cell pretreatment device in cell analysis.

It will be appreciated that the method for manufacturing a single-cell pretreatment device according to the second aspect of the present invention, the application of the single-cell pretreatment device according to the third aspect of the present invention, and the application of the single-cell pretreatment device according to the fourth aspect of the present invention have the technical effects of the single-cell pretreatment device according to the first aspect of the present invention, and thus will not be described in detail.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The invention is further described below with reference to the drawings and examples;

FIG. 1 is a schematic diagram of an embodiment of the present invention;

FIG. 2 is a schematic illustration of a single cell capture membrane according to an embodiment of the invention;

FIG. 3 is a schematic illustration of single cell and magnetic bead capture according to an embodiment of the present invention;

FIG. 4 is a flow chart of the fabrication of a single cell capture membrane according to an embodiment of the present invention.

Reference numerals:

a first plate 100, a first channel 110, an input port 111, a first output port 112;

a second board 200, a first pair of interfaces 210, a second pair of interfaces 220;

a third plate 300, a second channel 310, a second output 311;

single cell capture membrane 400, first chamber 410, second chamber 420, third chamber 430;

a pressure detection assembly 500;

magnetic beads 610, cells 620.

Detailed Description

Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application.

In the description of the present application, it should be understood that references to orientation descriptions, such as directions of up, down, front, back, left, right, etc., are based on the orientation or positional relationship shown in the drawings, are merely for convenience of describing the present application and simplifying the description, and do not indicate or imply that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present application.

In the description of the present application, the meaning of several is one or more, the meaning of a plurality is at least two, greater than, less than, exceeding, etc. is understood to not include the present number, and above, below, within, etc. is understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

In the description of the present application, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present application can be reasonably determined by a person skilled in the art after combining the specific contents of the technical solutions.

Referring to fig. 1 to 4, the single-cell pretreatment device according to the first aspect of the present application solves the problems of low efficiency, low activity, non-ideal flux, large pressure difference, etc. of the existing single-cell capture and labeling, and can greatly improve the capture efficiency and labeling of target cells by using microfluidic control of single-cells and corresponding dropping holes and capture of encoded magnetic beads, and the single-cell pretreatment device includes a housing, a fluid channel, a pressure detection assembly 500, and a single-cell capture membrane 400.

Wherein the fluid channel is disposed in the housing, the fluid channel includes a first channel 110 and a second channel 310, one end of the first channel 110 is used as an input port 111, the other end of the first channel 110 is used as a first output port 112, one end of the second channel 310 is communicated with the first channel 110, the other end of the second channel 310 is used as a second output port 311, and the second channel 310 is located at the lower layer of the first channel 110; the single cell capturing membrane 400 is disposed at the communication of the first channel 110 and the second channel 310; the pressure detection assembly 500 is used for detecting the second output port 311 to determine the capturing condition of the single cell 620; wherein the single-cell capturing membrane 400 comprises a first chamber 410, a second chamber 420 and a third chamber 430 which are sequentially arranged and longitudinally communicated, the first chamber 410 is used for capturing the magnetic beads 610, the second chamber 420 is used for capturing the cells 620, and the third chamber 430 is used for liquid flow. By arranging the first chamber 410, the second chamber 420 and the third chamber 430 in sequence and communicating them longitudinally, the microfluidic control of single cells 620 and the falling holes and capturing of the corresponding magnetic beads 610 can be used, so that the capturing and labeling efficiency of the target cells 620 can be greatly improved.

It should be noted that the housing may be introduced into the cells 620, the magnetic beads 610, the buffer, and the like as a sample container in a specific operation, and thus the housing is preferably provided as a sealed container. The input port 111 is used for inputting the cells 620, the magnetic beads 610, and buffering agents, etc. and flows in the first channel 110, since the second channel 310 is located at the lower layer and is in communication with the first channel 110, the cells 620 and the magnetic beads 610 have a tendency to flow to the second channel 310 based on gravity, and flow through the single-cell capturing membrane 400, wherein the first chamber 410, the second chamber 420, and the third chamber 430 are sequentially arranged from top to bottom, the third chamber 430 ensures that the liquid can smoothly flow from the first channel 110 to the second channel 310, but cannot pass the cells 620, the second chamber 420 can accommodate one biological sample or the cells 620, but cannot pass the magnetic beads 610, and when in use, the cells 620 are introduced, and the third chamber 430 has a tendency to flow in the longitudinal direction, so that the cells 620 are more easily accommodated by the second chamber 420, after the second chamber 420 is accommodated, the liquid tends to flow to the next third chamber 430 more easily, and the liquid is similarly driven to enter each of the second chambers 420, so as to increase the capturing rate of the single cells 620. The flow rate of the third chamber 430 is reduced to zero, which not only reduces the fluid pressure experienced by the single cell 620, but also further avoids subsequent influx of cells 620, thereby creating a single biological sample or capture of cells 620. After the cells 620 are captured to a certain extent, since the third chamber 430 is blocked or opened to be reduced, the internal pressure difference can be monitored by the pressure detecting assembly 500 to determine the capturing condition of the single cells 620. Finally, enough magnetic beads 610 are introduced, and because the first chamber 410 and the second chamber 420 are longitudinally arranged and the first chamber 410 is positioned at the uppermost part, adjacent interference does not occur, thereby greatly improving the capturing efficiency and the labeling effect of the target cells 620.

In some embodiments of the present application, the single-cell capturing membrane 400 includes a first layer, a second layer, and a third layer that are sequentially disposed, wherein a plurality of first micropores are disposed on the first layer at intervals and serve as the first chambers 410, a plurality of second micropores are disposed on the second layer at intervals and serve as the second chambers 420, a plurality of groups of third micropores are disposed on the third layer at intervals and serve as the third chambers 430, and each first micropore, each second micropore, and each group of third micropores are respectively communicated.

It will be appreciated that any one of the first microwells has a shape and size that can accommodate only one particle or magnetic bead 610, i.e., the pore size is larger than the particle or magnetic bead 610 size, any one of the second microwells has a shape and size that can accommodate only one biological sample or cell 620, i.e., the particle or magnetic bead 610 size is larger than the second microwell size and smaller than the first microwell size, and any one of the third microwells has a size that is smaller than the particle or magnetic bead 610, biological sample or cell 620, facilitating liquid flow.

Preferably, the size of each first micro-hole is the same, preferably 1.1-1.5 times the diameter of the magnetic bead 610; the size of each second micro-pore is the same, preferably 1.1-1.5 times the diameter of the cell 620; the size of each third microwell is the same, preferably 0.1-0.5 times the diameter of the captured cells 620, and the sizes of the first microwell, the second microwell and the third microwell are sequentially set from large to small. The first micropores and the second micropores are in one-to-one correspondence, and each group of third micropores comprises one or more third micropores, and the number of the third micropores can be an integral multiple of the number of the second micropores if the area allows. Preferably, the first layer, the second layer and the third layer are integrally formed, and the first micropores, the second micropores and the third micropores are round, triangular or any polygonal. The first micro-holes have a height and diameter of 50-100um, the second micro-holes have a height and diameter of 10-40um, and the third micro-holes have a height and diameter of 0.5-5um. The first layer, the second layer and the third layer are made of photosensitive transparent polymer or thermosetting transparent polymer.

In some embodiments of the present application, the housing includes a first plate 100, a second plate 200, and a third plate 300 that are sequentially stacked from top to bottom, and the second plate 200 is provided with a first pair of interfaces 210 and a second pair of interfaces 220; wherein, the first channel 110, the input port 111, the first output port 112 and the second output port 311 are formed in the first plate 100, the second channel 310 is formed in the third plate 300, one end of the second channel 310 communicates with the first channel 110 through the first pair of interfaces 210, and the other end of the second channel 310 communicates with the second output port 311 through the second pair of interfaces 220. Preferably, the first pair of ports 210 are disposed to extend longitudinally, and the first channel 110 and the second channel 310 are disposed parallel to each other.

It should be noted that the processing of the first plate 100, the second plate 200 and the third plate 300 are matched and corresponding to each other so as to splice the second channel 310 through the features processed on the first plate 100, the second plate 200 and the third plate 300, wherein the second plate 200 is also used for placing the single cell capturing membrane 400, and thus a groove structure for limiting the single cell capturing membrane 400 can be processed inside. After the first plate 100, the second plate 200 and the third plate 300 are assembled and matched, sealing treatment is needed at the joint of the first plate 100, the second plate 200 and the third plate 300 so as to ensure the measurement accuracy of pressure and the normal flow of liquid. While the provision of the first pair of interfaces 210 along the longitudinally extending preset length enables the cells 620 and the magnetic beads 610 to move downward so as to be more easily captured. The parallel arrangement of the first and second channels 110, 310 further facilitates the flow and discharge of liquid. Further, the thickness of the second plate 200 is less than 2mm by integrally injection molding the channel and the first and second pairs of interfaces 210, 220, so as to facilitate the capturing and observation of the single cell 620.

Referring to fig. 1 to 4, a method for manufacturing a single-cell trapping membrane according to an embodiment of the second aspect of the present application is a single-cell trapping membrane 400 of a single-cell pretreatment device according to an embodiment of the first aspect of the present invention, the method for manufacturing the single-cell trapping membrane 400 comprising the steps of:

providing a silicon wafer substrate, coating photoresist on the surface of the silicon wafer substrate, and obtaining a third microporous layer structure through photoetching and developing;

coating photoresist on the third microporous layer structure, and obtaining a second microporous layer structure through photoetching and developing; wherein the micropores of the second microporous structure layer are aligned with the micropores of the third microporous structure layer according to a set condition;

coating photoresist on the second microporous layer structure, and obtaining a first microporous layer structure through photoetching and developing; wherein the micropores of the first microporous structure layer are aligned with the micropores of the second microporous structure layer according to a set condition;

duplicating the above obtained structure to obtain a mold of the single-cell trapping membrane 400;

providing a glass substrate, reversely attaching the die face down on the glass substrate, and forming the single-cell capturing film 400 by utilizing a vacuum adsorption photo-curing technology;

the single-cell trapping film 400 was peeled off from the glass substrate to obtain a single-cell trapping film 400.

The photoresist may be SU83005 photoresist. The specific way to obtain the mold of the single cell trapping membrane 400 is: mixing the PDMSA glue and the B glue according to the ratio of 10:1, vacuumizing to remove bubbles, pouring the mixture on the photoetching mould, and copying the structure. The specific way of forming the single-cell capturing film 400 by vacuum adsorption photo-curing technology is as follows: pouring the photo-curing adhesive into a mold under the action of vacuum adsorption technology, curing the photo-curing adhesive in the mold under the action of 365nm ultraviolet light, and stripping the photo-curing adhesive from glass after the mold is removed to obtain the final three-layer microporous film.

Referring to fig. 1 to 4, the application of the single-cell pretreatment device according to the embodiment of the third aspect of the present application may be the method for using the single-cell pretreatment device according to the embodiment of the first aspect of the present application, the application of the single-cell pretreatment device including the steps of:

the cells 620 are input into the first channel 110 and flow into the second chamber 420 of the single cell capture membrane 400 until the value detected by the pressure detection assembly 500 exceeds a preset value and the remaining non-captured cells 620 are removed;

the magnetic beads 610 are input into the first channel 110 and flow into the first chamber 410 of the single-cell trapping membrane 400, and naturally settle for a certain time, so that the single cells 620 and the single magnetic beads 610 form a one-to-one correspondence and react, and the remaining non-trapped magnetic beads 610 are removed.

It should be noted that, the input of the cells 620 and the magnetic beads 610 may be performed by the aspirator, and when the value of the pressure detecting component 500 exceeds a certain preset value, the aspirator automatically stops moving, so as to avoid the accumulation of a large number of cells 620 blocking the single-cell capturing membrane 400 and exceeding the tolerance pressure of the cells 620. Further, the pressure detection assembly 500 may also be used to monitor the number and efficiency of captured cells 620 by the single cell capture membrane 400, which is coupled to the second output port 311 to provide a sensor to terminate or increase or decrease the fluid driving force to the aspirator.

Referring to fig. 1 to 4, a method for using a single cell pretreatment device according to a third aspect of the present application includes:

1) Buffer solution is introduced from the inlet 111 to wash the single-cell capture membrane 400, and flows out through the first outlet 112 and the second outlet 311.

2) Passing single cells 620 of smaller size from input port 111, single cells 620 being controlled by the fluid flow into the second micropores of single cell capture membrane 400, and the fluid flow out through first output port 112;

3) Buffer solution is introduced from the inlet 111 to wash the excess single cells 620 on the upper surface of the single cell trapping membrane 400 without the holes, and the liquid flows out through the first outlet 112.

4) Passing larger size encoded microbeads from input port 111, the encoded microbeads being flow-controlled into the first microwells of single-cell capture membrane 400, the flow exiting through first output port 112;

5) Introducing buffer solution from the input port 111, cleaning redundant coded microbeads without holes on the upper surface of the single-cell capturing membrane 400, and enabling liquid flow to flow out through the first output port 112;

the quality inspection is carried out on the coded microspheres and the capturing condition of the cells 620 on the double-layer microporous membrane by using a microscope, and the quality inspection result shows that the high-capturing-rate chip device designed by the invention can greatly improve the capturing efficiency of the single cells 620, and the capturing rate of the single cells 620 can reach more than 90% under the condition of smaller number of the cells 620.

Referring to fig. 1 to 4, the application of the single cell pretreatment device according to the fourth aspect of the present application may be an application in cell analysis such as biological experiments, such as second generation sequencing (NGS), etc.

It can be appreciated that the present application not only adopts the mode of stacking the single-cell capturing membrane 400, but also has more efficient matching efficiency and more sufficient reaction compared to the horizontal communication of the prior art. And the first plate 100, the second plate 200 and the third plate 300 which form the fluid channels are designed in the same way, compared with the design mode of the flow channels which are communicated from side to side, the capturing efficiency can be improved by about 5-10%. Meanwhile, compared with the method of directly stripping after manufacturing the grinding tool by utilizing the photoetching in the prior art, the method has higher molding efficiency and higher success rate.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The embodiments of the present application have been described in detail above with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application.

Claims (10)

1. A single-cell pretreatment apparatus, comprising:

a housing;

the fluid channel is arranged in the shell, one end of the first channel is used as an input port, the other end of the first channel is used as a first output port, one end of the second channel is communicated with the first channel, the other end of the second channel is used as a second output port, and the second channel is positioned at the lower layer of the first channel;

a single cell capturing membrane disposed at a communication point between the first channel and the second channel;

the pressure detection component is used for detecting the second output port to judge the single cell capturing condition;

the single-cell capturing membrane comprises a first chamber, a second chamber and a third chamber which are sequentially arranged and longitudinally communicated, wherein the first chamber is used for capturing magnetic beads, the second chamber is used for capturing cells, and the third chamber is used for flowing liquid.

2. The single-cell pretreatment device according to claim 1, wherein: the single-cell capturing membrane comprises a first layer, a second layer and a third layer which are sequentially arranged, wherein a plurality of first micropores are formed in the first layer at intervals and serve as the first chamber respectively, a plurality of second micropores are formed in the second layer at intervals and serve as the second chamber respectively, a plurality of groups of third micropores are formed in the third layer at intervals and serve as the third chamber respectively, and the first micropores, the second micropores and the third micropores are communicated respectively.

3. The single-cell pretreatment device according to claim 2, wherein: the sizes of the first micropores are the same, the sizes of the second micropores are the same, the sizes of the third micropores are the same, and the sizes of the first micropores, the second micropores and the third micropores are sequentially arranged from large to small.

4. The single-cell pretreatment device according to claim 2, wherein: the first micropores and the second micropores are in one-to-one correspondence, and one or more third micropores are contained in the third micropores of each group.

5. The single-cell pretreatment device according to claim 2, wherein: the first layer, the second layer and the third layer are integrally formed;

and/or the height and diameter of the first micro-holes is 50-100um, the height and diameter of the second micro-holes is 10-40um, and the height and diameter of the third micro-holes is 0.5-5um;

and/or the first layer, the second layer and the third layer are made of a photosensitive transparent polymer or a thermosetting transparent polymer.

6. The single-cell pretreatment device according to claim 1, wherein: the shell comprises a first plate, a second plate and a third plate which are sequentially stacked from top to bottom, and a first pair of interfaces and a second pair of interfaces are arranged on the second plate;

the first channel, the input port, the first output port and the second output port are formed in the first plate, the second channel is formed in the third plate, one end of the second channel is communicated with the first channel through the first pair of interfaces, and the other end of the second channel is communicated with the second output port through the second pair of interfaces.

7. The single-cell pretreatment device according to claim 6, wherein: the first pair of connectors are arranged along the longitudinal extension, and the first channel and the second channel are arranged in parallel.

8. A method for producing a single-cell trapping membrane, which is the single-cell trapping membrane of the single-cell pretreatment device according to any one of claims 1 to 7, comprising the steps of:

providing a silicon wafer substrate, coating photoresist on the surface of the silicon wafer substrate, and obtaining a third microporous layer structure through photoetching and developing;

coating photoresist on the third microporous layer structure, and obtaining a second microporous layer structure through photoetching and developing;

coating photoresist on the second microporous layer structure, and obtaining a first microporous layer structure through photoetching and developing;

duplicating the obtained structure to obtain a mould of the single-cell capturing membrane;

providing a glass substrate, reversely attaching the mold surface downwards on the glass substrate, and forming a single-cell capturing film by utilizing a vacuum adsorption photo-curing technology;

and stripping the single-cell capturing film from the glass substrate to obtain the single-cell capturing film.

9. Use of a single cell pretreatment device according to any of claims 1 to 7, comprising the steps of:

inputting cells into the first channel and flowing into the second chamber of the single-cell capturing membrane until the value detected by the pressure detection assembly exceeds a preset value, and removing the remaining cells which are not captured;

the magnetic beads are input into the first channel and flow into the first chamber of the single-cell capturing membrane, and naturally settle for a certain time, so that single cells and single magnetic beads form a one-to-one correspondence and react, and the residual non-captured magnetic beads are removed.

10. Use of a single cell pretreatment device according to any of claims 1 to 7 in cell analysis.

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