CN115161198B - High-capture-rate single-cell marking device based on microporous microfluidic chip and application - Google Patents
High-capture-rate single-cell marking device based on microporous microfluidic chip and application Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and in particular relates to a high-capture-rate single-cell marking device based on a microporous microfluidic chip and application thereof. The single-cell marking device prepared by the invention has the advantages of high flux, easy integration, easy automation, small volume, less reagent consumption, cost saving, high single-cell capturing efficiency up to 95.3%, high chip utilization rate, high single-cell and encoding microbead pairing efficiency up to 91.8%, suitability for analysis of rare cells such as circulating tumor cells, stem cells and the like, and single-cell utilization rate improvement.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a high-capture-rate single-cell marking device based on a microporous microfluidic chip and application thereof.
Background
Single cell detection technology refers to a technology of performing high-throughput analysis of genetic information such as genome, transcriptome, epigenetic group, proteome, metabolome and the like and functional products thereof at the single cell level. However, single cell analysis does not perform sequencing studies on only a single or a few cells, and the number of cells analyzed at single cell resolution can reach thousands of cells, depending on sample type and application requirements. Traditional analytical methods are performed at the tissue level or multicellular level, and the resulting data is in fact an average value obtained from the tissue or multicellular, with loss of information about cellular heterogeneity. And single cell analysis can obtain genetic information at a finer single cell level, so that the genetic information of each cell is revealed and the state of each cell is read, the heterogeneous information which cannot be obtained by sequencing a mixed sample and the genetic information of rare cells are reflected, and the research visual angle has finer resolution.
In recent years, single-cell sequencing technology has been rapidly developed, accurate gene expression patterns of thousands of cells have been rapidly determined, and genetic heterogeneity of each cell has been analyzed, thereby playing an important role in various fields of neurobiology, organ growth, cancer biology, clinical diagnosis, immunology, microbiology, embryology, prenatal gene diagnosis, etc., and being one of the focus of life science research. For single cell sequencing, the first challenge is the requirement for single cell isolation techniques, how to isolate the captured single cells quickly and efficiently. The microfluidic technology integrates or basically integrates basic operation units such as sample preparation, reaction, separation, detection, cell culture, sorting, cracking and the like in the related fields of biology and chemistry on a chip with a size of a few square centimeters (even smaller), and a network is formed by micro channels, so that controllable fluid penetrates through the whole system to replace various functions of a conventional chemistry or biology laboratory. Microfluidic technology has unique advantages in single cell analytical research. At present, the high-throughput single-cell detection mainly comprises two implementation modes, namely a water-in-oil-based droplet technology and a microwell plate-based beads marking technology. Water-in-oil based droplet technology is represented by the 10xGenomics, drop-Seq and inDrop platforms. The technology wraps the microbeads marked by the barcode and single cells in single oil drops through microfluidics to achieve single-cell capturing. The beads marking technology based on the micro-pore plates is represented by BD Rhapody, seqwell and Microwell-seq, and the technology ensures the single-cell porosity in the micro-pore array with the cell number of more than ten times by naturally settling the cells, and the micro-pore utilization rate is about 10%. The droplet technique and the microplate technique are only suitable for capturing and analyzing high-proportion cell types, and rare cells are difficult to obtain effective capturing.
At present, a single-cell marking system based on a microfluidic technology is low in general capture efficiency. Laboratory platform Drop-seq (Macosko E Z, basu a, satija R, et al high Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets [ J ]. Cell,2015,161 (5): 1202-1214) based on droplet microfluidic technology uses encoded magnetic beads with an efficiency of only about 5%, commercial platform 10xGenomics Chromium controller in scientific research service field and its companion chip use encoded gel beads with a single Cell capture efficiency of about 65%, hua Dazhi DNBelab C4 portable single Cell system single Cell capture efficiency of about 3%. Whereas the laboratory platform Cyto-Seq (Fan H C, fu G K, fodor S P A. Expression profiling.combineral labeling of single cells for gene expression cytometry. [ J ]. Science,2015,347 (6222): 1258367), microwell-Seq (Han X, wang R, zhou Y, et al mapping the Mouse Cell Atlas by Microwell-Seq [ J ]. Cell,2018,172 (5): 1091-1107.e17), the commercial platform BD Rhapsody, the New grid Matrix system were well-dropped by gravity sedimentation due to both encoded microbeads and single cells, the Cell marker system was open, resulting in a final Cell capture rate of about 30% and chip use efficiency of no more than 10%. The capturing efficiency is low, the requirement of initial sample quantity can be improved, the method is not suitable for clinical samples such as puncture biopsy, and meanwhile, a large amount of single cells, reagents and microporous materials are wasted in the capturing and library building process, and the quality of finally produced single cell data can be affected. The existing single-cell marking system based on the microfluidic chip cannot capture a small amount of rare particles, for example, drop-seq and other droplet microfluidic technologies are based on a poisson distribution principle, most droplets generated by droplet generation do not wrap cells, only about 1% of droplets contain single cells, and the encoded microspheres are also based on poisson distribution, so that capture analysis of relatively high cell types in a sample can only be realized, and therefore capture analysis of relatively low rare cell types in the sample, such as circulating tumor cells, in general, the circulating tumor cells in 10mL of blood of a patient only contain dozens of circulating tumor cells, and single-cell analysis cannot be performed by a conventional single-cell sequencing method; chinese patent CN111774110a discloses a biological analysis chip capable of capturing and fixing cells, the chip comprises a biological analysis substrate and a microporous array chip, the microporous array chip comprises a substrate and a microporous array formed by a plurality of micropores on the substrate, the inner diameter of the micropore opening of the micropores is smaller than that of the micropore cavity, and the single cell capturing can be realized by slightly larger than the cell diameter through the micropore opening diameter, but the chip is mainly used for improving the retention rate of the captured single cells in the experimental process, and the micropore opening and the micropore cavity are in an up-down structure, so that the capturing efficiency of the chip structure for the single cells is lower; chinese patent CN110628567a discloses a real-time fluorescent quantitative analysis chip for ultra-high throughput single cell nucleic acid molecules, which comprises a micro-pore array chip with micro-pores of the order of hundred thousand and millions and a microfluidic packaging structure, and by performing hydrophilic and hydrophobic treatment on the micro-pore array chip, capillary force of the micro-pores can be increased, single cell inhalation of the micro-pores is facilitated, single cell capture is realized, and uniform distribution of samples is realized by designing a flow channel of a multi-branched tree-shaped bifurcation structure, but the capturing process has low efficiency and slow flow velocity, and the effects of capturing single cells and performing cell analysis are difficult to realize rapidly and efficiently.
Aiming at the problems that the single-cell labeling in the current microfluidic technology application has lower capturing efficiency, is not suitable for capturing cell types with lower occupation ratio, and the like, and the problems of large initial sample quantity, reagent waste and the like, the development of a single-cell labeling microfluidic chip and a device with high capturing rate are needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a microporous microfluidic chip-based single-cell marking device with high capture rate and application thereof.
In a first aspect, the invention provides a high capture rate single-cell marking device based on a microporous microfluidic chip, the device comprises an upper control layer, a lower control layer and an intermediate capture layer, the upper control layer comprises a sample inlet, an upper liquid flow channel and a first peristaltic pump interface, the lower control layer comprises a lower liquid flow channel and a second peristaltic pump interface, the intermediate capture layer is the microporous microfluidic chip, the microporous microfluidic chip is designed as a double-layer microporous membrane, and the intermediate capture layer is arranged between the upper liquid flow channel and the lower liquid flow channel.
Further, the double-layer microporous membrane comprises an upper microporous structure layer and a lower microporous structure layer, wherein the upper microporous structure layer comprises a plurality of groups of micropores, each group of micropores consists of a first micropore and a second micropore which are connected with each other, the first micropore and the second micropore are consistent in depth and different in diameter, the interconnecting part between the first micropore and the second micropore is smaller than the diameter of a captured cell, the lower microporous structure layer comprises a plurality of third micropores with uniform size, and the number of the third micropores of the lower microporous structure layer is not less than the sum of the number of the first micropore and the second micropore of the upper microporous structure layer
Further, the lower microporous structure layer is provided with a third micropore corresponding to each first micropore and each second micropore of the upper microporous structure layer.
Further, the sample inlet and the first peristaltic pump interface are respectively positioned at two ends of the upper control layer, the sample inlet is connected with one end of the upper liquid flow passage, the first peristaltic pump interface is connected with the other end of the upper liquid flow passage, one end of the lower liquid flow passage in the lower control layer is connected with the second peristaltic pump interface, and the first peristaltic pump interface and the second peristaltic pump interface are respectively connected with the first peristaltic pump and the second peristaltic pump.
Further, the upper flow channel and the lower flow channel completely cover the double-layer microporous membrane, the bottom of the upper flow channel is the upper surface of the upper microporous structure layer of the double-layer microporous membrane, the top of the lower flow channel is the lower surface of the lower microporous structure layer of the double-layer microporous membrane, the top of the front section of the lower flow channel is aligned with the bottom of the upper flow channel and communicated with each other through the double-layer microporous membrane, and the double-layer microporous membrane is intercepted in a capturing channel of the aligned part of the upper flow channel and the lower flow channel.
Further, the upper microporous structure layer and the lower microporous structure layer are integrally formed, the thickness of the upper microporous structure layer is 20-100 mu m, and the thickness of the lower microporous structure layer is 1-20 mu m.
Further, in the upper layer of microporous structure layer, the first micropore is used for capturing the encoded microbead, the diameter of the first micropore is larger than that of the captured encoded microbead and is 1.1-1.5 times of that of the encoded microbead, the second micropore is used for capturing single cells, the diameter of the second micropore is larger than that of the captured single cells and is 1.1-1.5 times of that of the captured single cells, and the depth of the first micropore and the depth of the second micropore are consistent with the thickness of the upper layer of microporous structure layer.
Further, in the lower layer micropore structure layer, the diameter of the third micropore is smaller than the diameter of a single cell and is 0.1-0.5 times of the diameter of the captured single cell, and the depth of the third micropore is consistent with the thickness of the lower layer micropore structure layer.
Further, the bilayer microporous membrane captures the larger size encoded microbeads first and then the smaller size single cells.
In a second aspect, the invention provides a method for preparing a microporous microfluidic chip double-layer microporous membrane for capturing single cells and single coding microbeads, which comprises the following steps:
(1) Coating a seed layer on the surface of the substrate;
(2) Coating a first photoresist layer on the seed layer, obtaining a lower microporous structure layer through photoetching and developing, and processing to obtain a plurality of third micropores with uniform size of the lower microporous structure layer;
(3) Coating a second photoresist layer on the first photoresist layer, manufacturing an upper photoresist microporous layer on the second photoresist layer to obtain an upper microporous structure layer, processing to obtain first micropores and second micropores of the upper microporous structure layer, and aligning the first micropores and the second micropores of the upper microporous structure layer with third micropores of the lower microporous structure layer according to a set condition;
(4) The seed layer on the substrate is stripped using a reagent to obtain a bilayer microporous membrane with bilayer pairing trapped single cells and single encoded microbeads.
Further, the seed layer in step 1) includes, but is not limited to, chromium metal or LOR stripper rubber or a combination of the two;
further, the reagent in the step 4) includes, but is not limited to, a chromium etching solution or an az 300mif developing solution.
In a third aspect, the present invention provides an application of the microporous microfluidic chip-based single cell labeling device with high capture rate, specifically:
when the buffer solution, the coding microbeads or the single cells are introduced from the sample inlet, the coding microbeads and the single cells respectively enter the corresponding first micropores and the corresponding second micropores in sequence, the buffer solution flows out from the first peristaltic pump interface through the upper liquid flow passage or flows out from the second peristaltic pump interface through the double-layer microporous membrane through the lower liquid flow passage.
Further, a preferred method of application is provided as follows:
(1) Firstly, introducing buffer solution from a sample inlet to dip the double-layer microporous membrane, and flowing out of a second peristaltic pump interface through a lower liquid flow channel;
(2) The large-size coding microbeads are introduced from the sample inlet, the coding microbeads are controlled by liquid flow to enter first micropores of a microporous structure layer on the upper layer of the double-layer microporous membrane, and the liquid flow flows out from a second peristaltic pump interface through a lower liquid flow passage;
(3) Introducing buffer solution from a sample inlet, cleaning redundant coded microbeads which are not perforated on the upper surface of a microporous structure layer of the upper layer of the double-layer microporous membrane, and enabling the buffer solution to flow out of the first peristaltic pump interface through an upper liquid flow channel;
(4) Introducing single cells with smaller size from a sample inlet, controlling the single cells to enter second micropores of a microporous structure layer on the upper layer of the double-layer microporous membrane by liquid flow, and enabling the liquid flow to flow out of the second peristaltic pump interface through a lower liquid flow channel;
(5) And (3) introducing buffer solution from the sample inlet, cleaning redundant single cells without holes on the upper surface of the microporous structure layer of the upper layer of the double-layer microporous membrane, and enabling liquid flow to flow out of the first peristaltic pump interface through the upper liquid flow channel.
In a fourth aspect, the present invention provides an application of the microporous microfluidic chip-based high capture rate single cell labeling device in rare cell analysis, wherein the rare cells include but are not limited to rare cells such as Circulating Tumor Cells (CTCs) and stem cells.
Compared with the prior art, the invention has the following beneficial effects:
1. the high-capture-rate single-cell marking device based on the microporous microfluidic chip has high single-cell capturing efficiency, can realize single-cell capturing of up to 95.3% especially when the number of single cells is small, has little cell loss, and is particularly suitable for analyzing rare samples, such as rare cells of Circulating Tumor Cells (CTC), stem cells and the like.
2. The single-cell marking device has high chip use rate, can enable most of capturing micropores to capture single cells under the condition of limited single cells and the number of coded microbeads, can control the capturing number of the single cells according to the number of the capturing micropores of the chip, and improves the chip use efficiency.
3. The single cell and coded microbead pairing efficiency of the single cell marking device prepared by the invention is high and can reach 91.8%, so that analysis of most captured single cells can be realized, and the single cell utilization rate is improved.
4. The single-cell marking device designed by the invention has the advantages of high flux, easy integration and easy automation, small volume, less reagent consumption and cost saving.
Drawings
Fig. 1 is a schematic structural diagram of a high capture rate single cell labeling device based on a microporous microfluidic chip according to the present invention.
FIG. 2 shows a structure of a microporous membrane for paired capturing of single cells and microbeads, wherein A is a schematic diagram of paired capturing of single cells and microbeads, and B is a schematic diagram of paired capturing of single cells and microbeads.
FIG. 3 is a process flow for making a double layer microporous membrane according to the present invention.
FIG. 4 is a flow chart of the single cell and microbead paired capture process of the present invention.
FIG. 5 is a graph showing the results of single cell capture efficiency and quality control according to the present invention.
Reference numerals: 1. a sample inlet; 2. an upper flow path; 3. a first peristaltic pump interface; 4. an intermediate trapping layer; 5. a lower flow path; 6. a second peristaltic pump port; 40. an upper microporous structure layer; 42. a lower microporous structure layer; 400. a first microwell; 402. a second microwell; 404. an interconnecting portion between the first microwell and the second microwell; 406. the spacing between two groups of micropores in the upper microporous structure layer; 420. and a third microwell.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
All other embodiments obtained by persons of ordinary skill in the art without making any creative effort based on the embodiments of the present invention are within the protection scope of the present invention, and the following embodiments are further described for the present invention, but are not meant to limit the protection scope of the present invention.
Example 1 microporous microfluidic chip-based high Capture Rate Single cell labelling device
The high capture rate single-cell marking device based on the microporous microfluidic chip comprises an upper control layer, a lower control layer and an intermediate capture layer, wherein the upper control layer comprises a sample inlet 1, an upper liquid flow channel 2 and a first peristaltic pump interface 3, the lower control layer comprises a lower liquid flow channel 5 and a second peristaltic pump interface 6, the intermediate capture layer 4 is a microporous microfluidic chip, the microporous microfluidic chip is designed into a double-layer microporous membrane and is used for capturing single cells and coded microbeads in a pairing manner, and the intermediate capture layer 4 is arranged between the upper liquid flow channel 2 and the lower liquid flow channel 5.
The double-layer microporous membrane comprises an upper microporous structure layer 40 and a lower microporous structure layer 42, as shown in fig. 2, the upper microporous structure layer 40 comprises a plurality of microporous groups, each microporous group is composed of two first micropores 400 and second micropores 402 which are connected with each other, the first micropores 400 and the second micropores 402 are identical in depth and different in diameter, the width of an interconnecting portion 404 between the first micropores 400 and the second micropores 402 is smaller than the diameter of captured cells, the lower microporous structure layer 42 comprises a plurality of third micropores 420 with identical sizes, the number of the third micropores of the lower microporous structure layer is not smaller than the sum of the number of the first micropores and the second micropores of the upper microporous structure layer, and the lower microporous structure layer is provided with one third micropore 420 corresponding to each of the first micropores 400 and the second micropores 402 of the upper microporous structure layer.
The sample inlet 1 and the first peristaltic pump interface 3 are respectively positioned at two ends of the upper control layer, the sample inlet 1 is connected with the inlet end of the upper liquid flow channel 2, the first peristaltic pump interface 3 is connected with the outlet end of the upper liquid flow channel 2, the outlet end of the lower liquid flow channel 5 in the lower control layer is connected with the second peristaltic pump interface 6, and the first peristaltic pump interface and the second peristaltic pump interface are respectively connected with the first peristaltic pump and the second peristaltic pump.
The upper liquid flow passage 2 completely covers the double-layer microporous membrane, the bottom of the upper liquid flow passage 2 is the upper surface of the upper microporous structure layer of the double-layer microporous membrane, the lower liquid flow passage 5 completely covers the double-layer microporous membrane, the top of the lower liquid flow passage 5 is the lower surface of the lower microporous structure layer of the double-layer microporous membrane, the top of the front section of the lower liquid flow passage 5 is aligned with the bottom of the upper liquid flow passage 2 and communicated with each other through the double-layer microporous membrane, and the double-layer microporous membrane is intercepted in a capturing channel of the aligned part of the two.
The upper microporous structure layer 40 and the lower microporous structure layer 42 in the double-layer microporous membrane are integrally formed, the thickness of the upper microporous structure layer is 20-100 μm, the thickness of the lower microporous structure layer is 1-20 μm, the first micropores in the upper microporous structure layer of the double-layer microporous membrane are used for capturing coded microbeads, the second micropores are used for capturing single cells, and the third micropores in the lower microporous structure layer are used for bearing coded microbeads or single cells in the micropores of the upper microporous structure layer.
The diameter of the large-size micropores in the first microporous structure layer is slightly larger than the diameter of the captured encoded microbeads, and is 1.1-1.5 times of the diameter of the captured encoded microbeads, the diameter of the second micropores is slightly larger than the diameter of the captured single cells, and is 1.1-1.5 times of the diameter of the captured single cells, and the diameter of the micropores of the third microporous structure layer is smaller than the diameter of the single cells and is 0.1-0.5 times of the diameter of the captured single cells.
In the micropores of the upper microporous structure layer: capture single cell diameter < first microwell diameter < encoded microbead diameter < first microwell diameter.
The double-layer microporous membrane for capturing the single cells and the coded microbeads in a pairing way captures the coded microbeads with larger sizes first and then captures the single cells with smaller sizes.
Example 2 preparation of microporous microfluidic chip double-layer microporous Membrane capturing encoded microbeads and Single cells
The specific preparation method of the double-layer microporous membrane in the embodiment 1 comprises the following steps:
the double-layer microporous membrane material is selected from SU8-3005 and SU8-3050 photoresist of Microchem company, the thickness of the upper layer microporous structure layer of the double-layer microporous membrane is 50 μm, the first micropores and the second micropores are all round, the microporous array formed by the first micropores and the second micropores in the embodiment is 50 multiplied by 50, and the total number of the microporous array is 2500, wherein the first micropores and the second micropores are 2500, and the microporous array can be designed to be more or less according to the requirement. The first microwell diameter is 50 μm for capturing encoded microbeads, the second microwell diameter is 30 μm for capturing single cells, the width of the interconnecting portion 404 between the first microwell and the second microwell is 5 μm, the depth of both the first microwell and the second microwell is 50 μm, the width of the space 406 between the two groups of microwells in the upper microwell structure layer is 10 μm, the thickness of the lower microwell structure layer of the double-layer microwell film is 5 μm, the inner diameter of the third microwell in the lower microwell structure layer is 5 μm, the depth is 5 μm, and the third microwells are respectively arranged below the centers of the first microwells and the second microwells in the upper microwell structure layer.
The preparation method of the double-layer microporous membrane comprises the following steps:
(1) Uniformly spreading LOR stripping adhesive on the surface of the silicon wafer subjected to surface activation treatment;
(2) Uniformly coating photoresist SU8-3005 on LOR stripping adhesive at 2000rpm, exposing and developing the photoresist layer by photoetching to obtain a lower microporous structure layer, and processing micropores in the lower microporous structure layer of the double-layer microporous membrane;
(3) Uniformly spreading photoresist SU8-3050 on the lower microporous structure layer at 1800rpm, exposing and developing the photoresist layer by photoetching to obtain an upper microporous structure layer, and processing into first micropores and second micropores in the upper microporous structure layer of the double-layer microporous membrane;
(4) And (3) stripping the silicon wafer and the LOR stripping adhesive by using an az 300mif developing solution to obtain the double-layer microporous membrane of the microporous microfluidic chip for capturing the coded microbeads and the single cells in a double-layer pairing manner, as shown in the figure 3.
Example 3 Single cell Capture test
The bilayer microporous membrane used in this example was prepared as in example 2, the encoded microbeads used were 40 μm diameter encoded microbeads with molecular barcodes for single cell capture, and the captured cells were 293T cell lines;
the flow of single-cell capturing experiment based on the double-layer microporous membrane prepared in example 2 and the high-capturing-rate single-cell marking device of the microporous microfluidic chip prepared in example 1 is shown in fig. 4, and the specific experimental steps are as follows:
(1) Adding PBS solution from a sample inlet 1, starting a first peristaltic pump and a second peristaltic pump at the same time, controlling the flow rate at 500uL/min, driving the solution to infiltrate the integral flow path and the double-layer microporous membrane through an upper flow path 2 and a lower flow path 5, and enabling the solution to flow out from a second peristaltic pump interface 6 through the lower flow path 5;
(2) 2500 coded gel microbeads are added from a sample inlet 1, only a second peristaltic pump is started, the flow speed is controlled at 100uL/min, the coded microbeads are driven to enter first micropores in an upper microporous structure layer of the double-layer microporous membrane, and the solution flows out from a second peristaltic pump interface 6 through a lower liquid flow channel 5;
(3) Adding PBS solution from a sample inlet 1, starting a first peristaltic pump only, controlling the flow rate at 300uL/min, driving the solution to pass through an upper flow channel 2, cleaning redundant microbeads without holes on the upper surface of the double-layer microporous membrane, and enabling the solution to flow out from a first peristaltic pump interface 3 through the upper flow channel 2;
(4) 2500 293T cells are added from a sample inlet 1, only a second peristaltic pump is started, the flow speed is controlled at 100uL/min, the 293T cells are driven to enter second micropores in a microporous structure layer on an upper layer of the double-layer microporous membrane, and the solution flows out from a second peristaltic pump interface 6 through a lower liquid flow channel 5;
(5) Adding PBS solution from a sample inlet 1, starting a first peristaltic pump only, controlling the flow rate at 100uL/min, driving the solution to pass through an upper liquid flow channel 2, cleaning superfluous cells without holes on the upper surface of the double-layer microporous membrane, and flowing out of a first peristaltic pump interface 3 through the upper liquid flow channel 2;
quality inspection of the number and efficiency of coded microbeads, the number and efficiency of single-cell capture, and the chip use efficiency on a double-layered microporous membrane was performed using a microscope, in this example, the chip use efficiency (%) = (coded microbead-cell pairing)/2500×100%, and the obtained quality inspection results are shown in table 1 and fig. 5;
table 1: quality inspection results
| Quality inspection project | Number of captures | Percent capture |
| Coded microbead capture | 2335 pieces | 93.4％ |
| Single cell capture | 2384 | 95.3％ |
| Encoding microbead-cell pairing | 2295 pair | 91.8％ |
According to quality inspection results, the high-capture-rate chip device designed by the invention can greatly improve single-cell capture efficiency, and under the condition of small cell number, the single-cell capture rate can reach more than 95.3 percent, in addition, the calculation shows that the whole chip use efficiency of the high-capture-rate single-cell microporous microfluidic chip device designed by the invention is 91.8 percent, which is far superior to that of a conventional microporous plate single-cell marking technology (the chip use efficiency is about 10 percent), thereby being beneficial to high-efficiency capture of rare cell types and reducing consumption of reagents and chip consumables for single-cell capture.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being within the scope of the invention, obvious variations or modifications may be made thereto.
Claims (7)
1. The device is characterized by comprising an upper control layer, a lower control layer and an intermediate capturing layer, wherein the upper control layer comprises a sample inlet, an upper liquid flow channel and a first peristaltic pump interface, the lower control layer comprises a lower liquid flow channel and a second peristaltic pump interface, the intermediate capturing layer is a microporous microfluidic chip, the microporous microfluidic chip is designed into a double-layer microporous membrane, and the intermediate capturing layer is arranged between the upper liquid flow channel and the lower liquid flow channel;
the double-layer microporous membrane comprises an upper microporous structure layer and a lower microporous structure layer, wherein the upper microporous structure layer comprises a plurality of groups of micropores, each group of micropores consists of two first micropores and second micropores which are connected with each other, the first micropores and the second micropores are consistent in depth and different in diameter, the width of the interconnection part between the first micropores and the second micropores is smaller than the diameter of captured cells, the lower microporous structure layer comprises a plurality of third micropores with uniform size, and the number of the third micropores of the lower microporous structure layer is not less than the number of the first micropores and the second micropores of the upper microporous structure layer;
the lower microporous structure layer is provided with a third micropore corresponding to each first micropore and each second micropore of the upper microporous structure layer;
in the upper layer of micropore structure layer, a first micropore is used for capturing the encoded microbeads, the diameter of the first micropore is 1.1-1.5 times of the diameter of the encoded microbeads, a second micropore is used for capturing single cells, the diameter of the second micropore is 1.1-1.5 times of the diameter of the captured single cells, and in the lower layer of micropore structure layer, the diameter of the third micropore is 0.1-0.5 times of the diameter of the captured single cells.
2. The single cell marker device according to claim 1, wherein said sample inlet and said first peristaltic pump interface are positioned at two ends of said upper control layer, respectively, said sample inlet is connected to one end of said upper flow path, said peristaltic pump interface is connected to the other end of said upper flow path, and one end of said lower flow path in said lower control layer is connected to a second peristaltic pump interface.
3. The single cell marker device according to claim 1, wherein said upper flow channel and said lower flow channel are each entirely covered with a double-layered microporous membrane, said upper flow channel bottom being an upper surface of an upper microporous structure layer, said lower flow channel top being a lower surface of a lower microporous structure layer, and a front section top of said lower flow channel being aligned with a bottom of said upper flow channel and communicating with each other through said double-layered microporous membrane.
4. The single cell marker of claim 1, wherein said upper microporous structure layer is integrally formed with said lower microporous structure layer, said upper microporous structure layer having a thickness of 20-100 μm and said lower microporous structure layer having a thickness of 1-20 μm.
5. A method of preparing a bilayer microporous membrane, the bilayer microporous membrane being a microporous microfluidic chip-based high capture rate single cell labeling device according to claim 1, comprising the steps of:
(1) Coating a seed layer on the surface of the substrate;
(2) Coating a first photoresist layer on the seed layer, and obtaining a lower microporous structure layer through photoetching and developing, wherein the lower microporous structure layer comprises a plurality of third micropores with uniform size;
(3) Coating a second layer of photoresist on the first layer of photoresist, and manufacturing an upper layer of photoresist microporous layer on the second layer of photoresist to obtain an upper layer of microporous structure layer, wherein the upper layer of microporous structure layer comprises a plurality of groups of first micropores and second micropores, and the first micropores and the second micropores of the upper layer of microporous structure layer are aligned with the third micropores of the lower layer of microporous structure layer according to a set condition;
(4) The seed layer on the substrate is stripped using a reagent to obtain a bilayer microporous membrane with bilayer pairing trapped single cells and single encoded microbeads.
6. The use of a high capture rate single cell labelling device according to any of claims 1 to 4, wherein when the encoded microbeads, single cells or buffer solution is introduced from the sample inlet, the encoded microbeads and single cells enter the corresponding first and second microwells respectively, the buffer solution flows out of the first peristaltic pump interface through the upper flow channel or through the double layer microporous membrane, and flows out of the second peristaltic pump interface through the lower flow channel.
7. The use of the high capture rate single cell labelling device based on microporous microfluidic chip according to any of claims 1-4 in rare cell analysis.
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