Micro-fluidic chip for tissue cell nucleus space transcriptome sequencing

Abstract

The invention discloses a micro-fluidic chip for tissue cell nucleus space transcriptome sequencing, and belongs to the technical field of particle control in micro-fluidic chips. Comprising a free transcriptome capture layer, a micro-well layer, a coding A channel layer, a coding B channel layer and a nuclear transcriptome capture layer. A to-be-detected tissue slice is placed on the micro-well layer, and single cells dissociated from the tissue slice are captured through a micro-well. The lower part of the micro-well layer is attached to a free transcriptome capture layer, and an mRNA capture sequence is fixed on the free transcriptome capture layer exposed at the bottom of the micro-well for capture. And the coding A runner layer and the coding B runner layer are used for fixing a cross-type DNA microarray on the surface of the nuclear transcriptome capture layer. The coding A runner layer and the coding B runner layer are simultaneously used for fixing the cross type DNA microarray on the free transcriptome capture layer, so that common sequencing of a single cell space free transcriptome and a space nuclear transcriptome is realized; the blank of single-cell nuclear space transcriptome sequencing and common sequencing of a space free transcriptome and a space nuclear transcriptome is filled.

Description

Technical Field

[0001] The invention belongs to the technical field of particle control in a microfluidic chip, and in particular relates to a microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei. Background Art

[0002] The transcriptome is the sum of all RNAs transcribed from a specific tissue or cell at a certain developmental stage or functional state, reflecting the types of genes expressed in the tissue or cell, as well as the level and pattern of expression. Through the analysis of the transcriptome, it is possible to study gene function and gene structure at the overall level, which plays an important role in understanding the development of the body. In 2009, Helicos BioSciences first proposed transcriptome sequencing analysis (RNA sequencing, RNA-Seq) based on second-generation sequencing technology. Since then, with the continuous improvement of high-throughput sequencing platforms, transcriptome sequencing technology has been widely used in biological research, medical research, clinical research, and drug research as a powerful tool for comprehensively obtaining transcriptome information and analyzing transcript structure and expression levels.

[0003] Although high-throughput single cell RNA sequencing (scRNA-seq) technology can obtain the gene expression status of a single cell, for many frozen tissue samples, the ice crystals formed in the cells will pierce the cell membrane when the tissue is quickly frozen, making it difficult or impossible to prepare high-quality single cell suspensions, and thus it is impossible to use scRNA-seq to solve biological problems. In contrast, the nuclear membrane is more solid, and after the cell membrane of frozen tissue is ruptured, the nucleus can remain intact. In order to gain a deeper understanding of the functions and regulatory mechanisms of some rare frozen samples, Rashel V. Grindberg et al. proposed RNA-sequencing from single nuclei in 2013. Single nuclei RNA sequencing (snRNA-seq) technology not only makes up for the shortcomings of frozen tissues, the difficulty in preparing high-quality single cell suspension samples, and the inability to perform single cell transcriptome sequencing, but also reduces the stress response of cells. At the same time, it has unique application value for studying non-coding RNA in the nucleus and exploring cell differentiation and development, and has been widely used in research in recent years. For example, in 2017, Naomi Habib et al. used snRNA-seq to study the dynamic process of adult neurogenesis; in 2020, SelewaA et al. explored cellular composition and cell characteristics by performing snRNA-seq on mammalian heart; in 2021, TS Andrews et al. revealed the existence of rare subtypes of hepatic parenchymal cells through snRNA-seq.

[0004] Since genes are expressed differently in different cell types, locations, and time points, spatial transcriptomics technology has emerged to gain a deeper understanding of the distribution of genes in organisms and the specificity of their expression. The emergence of spatial transcriptomics enables researchers to more accurately determine the spatial expression distribution of genes in organisms, thereby better understanding the regulation and function of biological processes. One of the earliest spatial transcriptomics technologies was in situ hybridization. With further development, a new generation of spatial transcriptomics technologies has emerged, such as RNA in situ hybridization (RNA-ISH), sequential fluorescence in situ hybridization (seqFISH), and so on. However, these methods are based on a limited set of probes that hybridize to known messenger RNA (mRNA) sequences, which limits the potential of spatial transcriptomics technology in discovering new sequences and variants. They also require complex experimental processes and professional equipment, including expensive instruments and consumables. In addition, the emergence of spatial transcriptomics has made genome-scale gene expression maps possible, but research areas such as single-cell nuclear spatial transcriptome sequencing and co-sequencing of spatial free transcriptome and spatial nuclear transcriptome are still blank. Summary of the invention

[0005] In view of the shortcomings and deficiencies of the above technologies, the purpose of the present invention is to provide a microfluidic chip for spatial bar coding of the transcriptome of tissue cell nuclei. The chip adopts an innovative three-dimensional structure, and obtains the gene information and spatial position information of the transcriptome in the tissue cell nucleus by spatial bar coding of the transcriptome in the tissue cell nucleus, so as to more accurately study the localization of genes in the cell nucleus; the chip can also realize the spatial coding of the nuclear and cytoplasmic transcriptome of tissue cells at the same time, which is used to explore the interaction between the nucleus and the cytoplasm, and then more comprehensively understand the regulatory mechanism of gene expression. Compared with the prior art, the design of the microwell in the present invention improves the transcriptome sequencing throughput; the process of spatial coding using microfluidic technology greatly reduces the consumption of samples and reagents, and reduces costs; the device is small and easy to operate, and does not require professional equipment to support complex experimental processes, which improves its application potential in the development of portable systems.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A microfluidic chip for spatial bar coding of nuclear transcriptomes of tissue cells, characterized in that the microfluidic chip comprises a free transcriptome capture layer (1), a microwell layer (2), a coding A flow channel layer (3), a coding B flow channel layer (4), and a nuclear transcriptome capture layer (5) which are combined from bottom to top.

[0008] like Figure 1As shown, the micro-well layer (2) is a rectangular thin layer, and the central area of ​​the micro-well array (8) is neatly arranged. The micro-well array (8) penetrates the micro-well layer (2) in depth. The depth and cross-sectional area of ​​each micro-well are required to be able to accommodate and only accommodate one cell, so as to ensure that when the tissue slice is decomposed into a single cell, the cell can vertically settle into the micro-well array (8) under the action of gravity. The micro-well array (8) has i rows and j columns, wherein each row and each column respectively corresponds to the i-th row of the middle parallel flow channel of the coding A flow channel layer (3) and the j-th column of the middle parallel flow channel of the coding B flow channel layer (4). The lower surface of the micro-well layer (2) is fixedly attached to the upper surface of the free transcriptome capture layer (1), and the area of ​​the free transcriptome capture layer (1) exposed to the micro-well layer (2) is fixed with an mRNA capture sequence for capturing the free transcriptome in the tissue cell, thereby achieving the capture of the free transcriptome in the tissue cell to avoid its interference with the capture of the nuclear transcriptome.

[0009] like Figure 1 As shown, the nuclear transcriptome capture layer (5) is composed of a rectangular thin layer substrate (6) and a cross-type DNA microarray (7) with unique position information fixed in the central area of ​​the lower surface of the substrate, wherein the cross-type DNA microarray (7) has i rows and j columns, and the cross-sites of the i-th row and j-th column in the cross-type DNA microarray (7) correspond one-to-one to the micro-wells of the i-th row and j-th column in the micro-well array (8).

[0010] like Figure 3 As shown, tens of thousands of specific DNA sequences are fixed at each intersection site in the cross-type DNA microarray (7), and each specific DNA sequence includes specific primers for identifying gene fragments to be amplified during PCR amplification technology, spatial barcode A (Spatial Barcode A), spatial barcode B (Spatial Barcode B), a connection handle (Ligation Linker) for connecting the two groups of DNA barcodes A and B, a specific molecular label (UMI), and a transcriptome capture handle (mRNA capture region). The feature points of different rows in the cross-type DNA microarray (7) have different spatial barcodes Ai (Spatial Barcode Ai, i=1, 2, 3...i), and the feature points of different columns have different spatial barcodes Bj (Spatial Barcode Bj, j=1, 2, 3...i). Through the combination of the two groups of spatial barcodes Ai and Bj, it is ensured that each feature point in the cross-type DNA microarray (7) has a different spatial barcode.

[0011] like Figure 1-2As shown, the coding A flow channel layer (3) and the coding B flow channel layer (4) are composed of four parts: intermediate parallel microchannels (9, 10), an inlet (11), an outlet (12), and a vacuum chamber (13) for pumping liquid. The lower surface of the coding A flow channel layer (3) has i long strip grooves (14) with the same number of rows as the cross-type DNA microarray (7) and the microwell array (8), and each groove (14) is respectively connected to the i inlets (11) and the i outlets (12) that are connected. The lower surface of the coding B flow channel layer (4) has j long strip grooves (14) with the same number of columns as the cross-type DNA microarray (7) and the microwell array (8), and each groove is respectively connected to the j inlets (11) and the j outlets (12) that are connected.

[0012] In the process of fixing the mRNA capture sequence for capturing the free transcriptome in the tissue on the surface of the free transcriptome capture layer (1), as Figure 4 As shown, the lower surface of the micro-well layer (2) is tightly attached to and fixed to the upper surface of the free transcriptome capture layer (1), and they are immersed in a solution rich in mRNA capture sequences together, ensuring that the mRNA capture sequences are fixed to the area of ​​the free transcriptome capture layer (1) exposed to the micro-well layer (2).

[0013] In the process of fixing the cross-type DNA microarray (7) with unique position information on the surface of the rectangular substrate (6) in the nuclear transcriptome capture layer (5), as Figure 5 As shown, the lower surface of the coding A flow channel layer (3) is attached to the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) to form i closed parallel flow channels, so that the nucleotide sequences A1, A2, A3... to Ai flowing through the parallel flow channels (9) can be fixed on the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5). When the coding B flow channel layer (4) is in use, its lower surface is attached to the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) to form j closed parallel flow channels, so that the nucleotide sequences B1, B2, B3... to Bj flowing through the parallel flow channels (10) are fixed on the nucleotide sequences (A1, A2, A3... to Ai) formed by the coding A flow channel layer (3) to form a cross nucleotide sequence with two-dimensional position information. When the coding A flow channel layer (3) and the coding B flow channel layer (4) are respectively attached to the lower surface of the nuclear transcriptome capture layer (5), their long strip grooves are perpendicular to each other, and the intersection of the two projections on the nuclear transcriptome capture layer (5) in the vertical direction is a cross-type DNA microarray (7) with unique two-dimensional position information.

[0014] In the process of capturing tissue cell-free transcriptome, such as Figure 7As shown in step I and step II, the tissue slices are fixed on the surface of the microwell layer (2), and the tissue is processed to be digested into single cells, and then vertically falls into the microwell array (8) below under the action of gravity. A relatively mild cell membrane lysis solution is added thereto, and the cells are incubated for 10 minutes to lyse the tissue cells in the microwell array (8) and achieve nuclear-cytoplasmic separation. The transcriptome in the tissue cytoplasm is free in the microwell array (8), and is complementary to the base pairing of the mRNA capture sequence on the bottom surface, thereby achieving the capture of the free transcriptome in the tissue cells.

[0015] In the process of capturing the transcriptome in the nucleus of tissue cells, a nuclear membrane lysis solution is added to the microwell array (8) to closely fit and fix the nuclear transcriptome capture layer (5) and the microwell layer (2). Figure 7 As shown in step III and step IV, the projection of each cross site in the upper cross-type DNA microarray (7) in the depth direction is completely contained in the lower microwell array (8), and it is ensured that each cross site of the cross-type DNA microarray (7) with specific position information corresponds to each microwell in the microwell array (8). The shaking device fully dissolves the nuclear membrane, and the transcriptome in the cell nucleus is free in the microwell array (8), and is complementary to the DNA sequence base with specific position information on the top surface, thereby realizing the spatial bar coding of the transcriptome in the tissue cell nucleus.

[0016] The rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) is preferably made of glass material, and has a thickness of 1-2 mm.

[0017] The coding A flow channel layer (3) and the coding B flow channel layer (4) are preferably made of polydimethylsiloxane (PDMS) material, the width of the intermediate parallel flow channels (9, 10) is preferably 20-70 μm, and the diameters of the flow channel inlet (11) and outlet (12) are preferably 1 mm. The vacuum chamber (13) for pumping liquid above the outlet is made of transparent materials, including but not limited to polymethyl methacrylate, glass, polydimethylsiloxane, etc., preferably polydimethylsiloxane (PDMS). The shape of the internal bottomless cavity (16) includes but is not limited to a cuboid, a sphere, etc. The height of the bottomless cavity (16) is preferably 7-10 mm, and the area covered should include all outlets (12). The thickness of the cavity side walls and the top layer is preferably 5-8 mm.

[0018] The shape of the microwell array (8) in the middle area of ​​the microwell layer (2) includes but is not limited to rectangle, ellipse, dumbbell, etc. The material is preferably SU-8, and the size of the microwell array (8) is preferably 1.2-1.5 times the cell diameter. The material used for the free transcriptome capture layer (1) includes but is not limited to polydimethylsiloxane (PDMS), glass, etc., preferably glass.

[0019] The reagents used to decompose the tissue slices placed on the micro-well layer (2) into single cells include but are not limited to trypsin, pronase, collagenase, etc., preferably trypsin.

[0020] After the free transcriptome in the cytoplasm is captured by the mRNA capture sequence fixed on the surface of the free transcriptome capture layer (1), the method for passivating the excess mRNA capture sequence on the surface of the free transcriptome capture layer (1) includes but is not limited to adding a small amount of a short nucleotide sequence with a polyA tail, a nuclease, etc. to the microwell, preferably a short nucleotide sequence with a polyA tail.

[0021] The coding A flow channel layer (3) and the coding B flow channel layer (4) are realized by a one-step molding reverse molding process; the free transcriptome capture layer (1) and the micro-well layer (2) are realized simultaneously by a single-layer photolithography process, and the process is as follows:

[0022] (1) Processing of the free transcriptome capture layer (1) and the micro-well layer (2): using a coating machine to spin-coat a layer of photoresist as the micro-well layer (2) on the surface of the free transcriptome capture layer (1), exposing with a UV photolithography machine, developing with a developer, and heating and curing to form a capture groove array (8) in the middle area of ​​the micro-well layer (2) for capturing single cells obtained by decomposing tissue slices under trypsin digestion. The material of the free transcriptome capture layer (1) is preferably glass, and the material of the micro-well layer (2) is preferably SU-8;

[0023] (2) Processing of molds for the coding A flow channel layer (3) and the coding B flow channel layer (4): The molds required for the coding A flow channel layer (3) and the coding B flow channel layer (4) are prepared by single-layer SU-8 photolithography; once the molds are processed, they can be reused;

[0024] (3) Processing of the mold of the vacuum chamber (13) for pumping liquid above the outlet of the coding A flow channel layer (3) and the coding B flow channel layer (4): a rectangular parallelepiped mold is made by laser cutting technology, and the material is preferably polymethyl methacrylate; once the mold is processed, it can be reused;

[0025] (4) The coded A flow channel layer (3) and the coded B flow channel layer (4) are formed by inverting the mold of the coded A flow channel layer (3) and the coded B flow channel layer (4), and the material is preferably PDMS, and the mold is removed after heating and curing;

[0026] (5) The vacuum chamber (13) for pumping liquid above the outlet of the coding A flow channel layer (3) and the coding B flow channel layer (4) is formed by inverting the mold of the vacuum chamber (13) for pumping liquid, and the material is preferably PDMS, and the thickness is preferably 5-8 mm, and demolding is performed after heating and curing;

[0027] (6) Plasma bonding the coded A flow channel layer (3) and the coded B flow channel layer (4) obtained in step 4 to the vacuum chamber (13) for pumping liquid formed in the previous step, which is PDMS-PDMS bonding.

[0028] Compared with the existing tissue cell spatial transcriptome sequencing technology, the beneficial effects of the present invention are as follows:

[0029] The present invention proposes a microfluidic chip for sequencing the spatial transcriptome of tissue cell nuclei. The chip uses an upper and lower double-layer structure, the upper layer is a cross-type DNA microarray with position information, and the lower layer is a microwell array with an mRNA capture sequence. The tissue slice fixed on the surface of the microwell array is dissociated into a single cell under the action of a trypsin solution, and falls vertically into a capture groove due to gravity. The captured single cell realizes nuclear-cytoplasmic separation under the action of a cell membrane lysis solution, and the transcriptome free in the cytoplasm is combined with the mRNA capture sequence on the bottom of the array through base complementary pairing, completing the capture of the free transcriptome in the tissue slice to avoid its interference with subsequent and transcriptome capture; afterwards, by one-to-one correspondence between the cross-type DNA microarray with position information and the microwell array, under the action of a nuclear membrane lysis solution, the free cell nuclei in the microwell array are lysed to release the nuclear transcriptome, and the partial transcriptome is connected to the DNA sequence on the top surface of the microwell array according to base complementary pairing, realizing the capture of the nuclear transcriptome in the cell nucleus of the tissue slice at this position, so that the capture of the tissue cell nuclear transcriptome is completed and its position information is obtained.

[0030] Compared with the prior art, the chip designed by the present invention changes the traditional method of using microbeads to capture single-cell transcriptomes, and at the same time realizes the separation of nuclear and cytoplasmic transcriptomes in tissue cells, and uses the mechanism of spatial bar coding to complete the simultaneous acquisition of gene information and position information of nuclear transcriptomes in tissue cells, thereby realizing the spatial transcriptome sequencing in the nucleus of tissue cells. The design of the capture array and the cross-type DNA microarray allows the sequencing of the transcriptome of tissue sections to be almost accurate to the single-cell level, which improves the detection throughput; in the present invention, the transcriptome in the nucleus of tissue cells is captured by complementary pairing with the DNA sequence bases with specific primers on the top surface of the microwell array, further shortening the capture time; the present invention only needs to further perform spatial coding on the microwell array to realize the simultaneous sequencing of the spatial transcriptome inside and outside the nucleus of tissue sections; the operation process of the present invention does not require steps such as flipping the chip, nor does it require the support of equipment such as valve control, and the chip structure is simple, easy to operate, and has low processing cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an overall structural diagram of a microfluidic chip for spatial bar coding of tissue cell nuclear transcriptomes according to the present invention.

[0032] Figure 2 This is an overall structural diagram of the coded A flow channel layer and the coded B flow channel layer described in the present invention.

[0033] Figure 3 The overall structure diagram of the vacuum chamber for pumping liquid according to the present invention

[0034] Figure 4 This is a design diagram of a DNA sequence for spatial bar coding of tissue cell nuclear transcriptome according to the present invention.

[0035] Figure 5 This is a schematic diagram of forming a DNA sequence on the surface of the free transcriptome capture layer for capturing the free transcriptome in the tissue according to the present invention.

[0036] Figure 6 This is the working mode of forming a cross-type DNA microarray on the surface of the nuclear transcriptome capture layer described in the present invention.

[0037] Figure 7 The present invention is a processing flow chart of a microfluidic chip for spatial bar coding of tissue cell nuclear transcriptome.

[0038] Figure 8 This is a workflow diagram for spatial bar coding of tissue cell nuclear transcriptomes according to the present invention.

[0039] Fig. 9 This is the working mode of forming a cross-type DNA microarray on the surface of the free transcriptome capture layer described in the present invention.

[0040] The accompanying drawings are marked as follows: 1. Free transcriptome capture layer, 2. Microwell layer, 3. Coding A flow channel layer, 4. Coding B flow channel layer, 5. Nuclear transcriptome capture layer, 6. Rectangular thin layer substrate, 7. Cross-type DNA microarray, 8. Microwell array, 9. Middle parallel microchannel (horizontal direction), 10. Middle parallel microchannel (vertical direction), 11. Inlet, 12. Outlet, 13. Vacuum chamber for pumping liquid, 14. Long strip groove, 15. Vacuum hole, 16. Bottomless cavity DETAILED DESCRIPTION

[0041] In order to further describe the present invention in detail, the following is a description with reference to embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Example 1

[0043] The present invention proposes a microfluidic chip for spatial bar coding of tissue cell nuclear transcriptome, the complete structure of which is as follows: Figure 1As shown, it includes a free transcriptome capture layer 1, a microwell layer 2, a coding A flow channel layer 3, a coding B flow channel layer 4, and a nuclear transcriptome capture layer 5. The nuclear transcriptome capture layer 5 includes a rectangular thin layer substrate 6 and a cross-type DNA microarray 7 fixed in the central area of ​​the lower surface of the substrate. The rectangular thin layer substrate 6 is 2 mm high, and the cross-type DNA microarray 7 contains 70×70 feature points. The top view of the coding A flow channel layer 3 and the coding B flow channel layer 4 is shown in FIG. Figure 2 As shown, there are 70 parallel flow channels 9 and 10 with a width of 50 μm in the middle, the flow channel inlet 11 and outlet 12 have a diameter of 1 mm, the vacuum chamber height for pumping liquid is 15 mm, and the thickness of the cavity side wall and top layer is 5 mm. The thickness of the microwell layer 2 and the free transcriptome capture layer 1 are 1 mm and 2 mm respectively. A microwell array 8 is neatly arranged in the middle of the microwell layer 2. The microwell array 8 includes 70×70 feature points, each of which has a diameter of 26 μm and a depth of 1 mm. The distance between the centers of two adjacent feature points is 100 μm.

[0044] The microfluidic chip for spatial bar coding of tissue cell nuclear transcriptome according to the present invention is as follows: Figure 1 As shown, the microwell array 8 penetrates the microwell layer 2 in depth, the free transcriptome capture layer 1 is tightly attached to the microwell layer 2, and the surface of the free transcriptome capture layer 1 is exposed to the outside through the microwell array 8; the nuclear transcriptome capture layer 5 is located above the free transcriptome capture layer 1 and aligned up and down, and each crossover site of the cross-type DNA microarray 7 in the nuclear transcriptome capture layer 5 corresponds one-to-one to each microwell in the microwell array 8.

[0045] A schematic diagram of a microfluidic chip for spatial barcoding of tissue cell nuclear transcriptomes according to the present invention, wherein an mRNA capture sequence is fixed on the surface of a free transcriptome capture layer 1 is shown in FIG. Figure 4 As shown, the micro-well layer 2 and the free transcriptome capture layer 1 are tightly fitted and fixed, and are immersed together in a solution rich in mRNA capture sequences to ensure that the mRNA capture sequences are fixed in the area of ​​the free transcriptome capture layer 1 exposed to the micro-well layer 1.

[0046] The microfluidic chip for spatial bar coding of nuclear transcriptome of tissue cells of the present invention forms a cross-type DNA microarray 7 on the surface of a rectangular thin layer substrate 6 in a nuclear transcriptome capture layer 5 as shown in the flowchart. Figure 5As shown, first, the coding A channel layer 3 is attached and fixed to the rectangular substrate 6, and the A group DNA primer solution (A1, A2, A3, A4...A70) is injected into the horizontal channel through the inlet 11, and the outlet 12 is evacuated, and the fluid flow is driven by atmospheric pressure. The A group DNA primers are fixed on the surface of the substrate 6, so as to form a DNA barcode in the X direction on the rectangular substrate; then, the coding B channel layer 4 is attached and fixed to the rectangular substrate 6 with the X direction DNA barcode fixed, and the middle parallel channel 10 of the coding B channel layer 4 is aligned with the DNA barcode in the X direction on the surface of the rectangular substrate 5 in position and intersects with it in direction. The B group DNA primer solution (B1, B2, B3, B4...B70) is injected into the vertical channel through the inlet 11, and the A and B groups of DNA primers are connected. After the coding B channel layer 4 is removed, a cross-type DNA microarray 7 with specific position information can be formed on the surface of the rectangular substrate 6.

[0047] The processing flow chart of the microfluidic chip for spatial bar coding of tissue cell nuclear transcriptome, the microwell layer 2 and the free transcriptome capture layer 1 of the present invention is as follows: Figure 7 As shown in (a), the processing process only requires a single-layer photolithography process. The processing flow of the coding A channel layer 3 and the coding B channel layer 4 is as follows Figure 7 As shown in (b), the processing process only needs one mold casting, and there is no need for micro-machining of alignment photolithography process during the mold casting process. The specific steps include:

[0048] (1) Design and production of photolithography masks: Draw the design of the microfluidic device and use high-precision printing to output multiple masks. Each mask has a light-transmitting and opaque pattern to form a pattern in the exposure step.

[0049] First, the processing flow of the microwell layer 2 and the free transcriptome capture layer 1:

[0050] (2) The micro-well layer 2 and the free transcriptome capture layer 1 are simultaneously fabricated by a single-layer photolithography process. The main steps are to sequentially undergo SU8 spin coating, soft bake, exposure, post-expose bake, development, and hard bake on a glass sheet to obtain the designed SU8 pattern. Figure 7 (a) As shown in steps Ⅰ-Ⅱ.

[0051] The second is the processing flow of code A flow channel layer 3 and code B flow channel layer 4:

[0052] (3) Mold processing: that is, a single-layer SU8 mold for the coding flow channel layer 3 and 4 is processed by photolithography. The mold processing is completed and can be used repeatedly. The molds of the coding flow channel layer 3 and the coding flow channel layer are obtained by sequentially performing SU8 spin coating, soft baking, exposure, post-expose baking, development, and hard baking on the silicon wafer, such as Figure 7 (b) As shown in steps Ⅰ-Ⅱ.

[0053] (4) Fabrication of the code A and B flow channel layers 3 and 4: a layer of PDMS is solidified on the surface of the mold, and after solidification, the structure is demolded from the mold, and holes are punched in the demolded structure to form an inlet and an outlet, and a vacuum cavity 13 for pumping liquid is bonded to the demolded structure, which is PDMS-PDMS plasma bonding, such as Figure 7 (b) As shown in steps III-VI.

[0054] This example uses a microfluidic chip for spatial barcoding of tissue cell nucleus transcriptome obtained in the above-mentioned embodiment to test the spatial transcriptome sequencing effect of tissue cell nucleus. Figure 7 As shown, the specific steps are as follows:

[0055] (1) Tissue slice decomposition: The tissue slices are placed on the surface of the micro-well layer 2 and treated with 0.1% trypsin solution to decompose the tissue slices into single cells, which then naturally settle into the micro-well array 8 in the lower layer under the action of gravity.

[0056] (2) Cell membrane lysis: 0.01% Tween-20 solution was added dropwise to the microwell array and allowed to stand at room temperature for 15 min to allow the cells to lyse.

[0057] (3) Capture of free transcriptome: After the cell membrane of the cells in the microwell array 8 is lysed, the transcriptome in the cytoplasm will be free in the microwell array 8 and bind to the mRNA capture sequence fixed on the surface of the free transcriptome capture layer 1.

[0058] (4) Passivation of excess mRNA capture sequences. A small amount of a short nucleotide sequence solution rich in polyA at the tail is added to the microwell array 8, and the short sequence is used to passivate the excess mRNA capture sequences on the surface of the free transcriptome capture layer 1 to avoid interference with subsequent tissue cell nuclear spatial transcriptome sequencing.

[0059] (5) Nuclear membrane lysis. TRIzol solution was added dropwise to the microwell array 8, and the microwell array was incubated at 15-30° C. for 5 minutes. The device was shaken to fully dissolve the nuclear membrane and obtain the transcriptional substances in the nuclei of tissue cells.

[0060] (6) Capture and spatial encoding of nuclear transcriptome. The nuclear transcriptome capture layer 5 is aligned with the microwell layer 2, and is tightly fitted and fixed, so that the projection of each cross site in the upper cross-type DNA microarray 7 in the depth direction is completely contained in the lower microwell array 8, and it is ensured that each cross site of the cross-type DNA microarray 7 with specific position information corresponds to each microwell in the microwell array 8. The device is gently shaken so that the free nuclear transcriptome in the microwell array 8 is captured by the upper cross-type DNA microarray 7, thereby realizing the spatial encoding of the nuclear transcriptome of tissue cells.

[0061] (7) Collection and sequencing of nuclear transcriptome. Reverse transcription is performed on the nuclear transcriptome capture layer 5, and the reverse transcribed sequence is collected from the substrate 6 into a test tube for subsequent amplification, sequencing and other operations to complete spatial transcriptome sequencing of tissue cell nuclei.

[0062] Example 2

[0063] The present invention provides a microfluidic chip for spatial bar coding of tissue cell nuclear transcriptome, the complete structure of which is as follows: Figure 1 Except for the spatial bar coding of the free transcriptome capture layer, the chip structure and processing are the same as those in Example 1.

[0064] This example uses a microfluidic chip for spatial barcoding of tissue cell nucleus transcriptome obtained in the above embodiment to test the spatial transcriptome sequencing effect inside and outside the tissue cell nucleus. The specific steps are as follows:

[0065] (1) Spatial encoding of microwell arrays. Figure 8 As shown, the middle parallel channels of the coding A channel layer and the coding B channel layer are respectively matched one by one with each row and each column of the microwell array, and the coding A and B channel layers are successively attached and fixed to the microwell array, and two groups of A and B DNA primer solutions with spatial position information are introduced in turn, and finally a cross-type DNA microarray is formed on the bottom surface of each characteristic point in the microwell array, and different DNA microarrays are fixed on the bottom surfaces of different characteristic points.

[0066] (2) Tissue slice decomposition: The tissue slices are placed on the surface of the micro-well layer 2 and treated with 0.1% trypsin solution to decompose the tissue slices into single cells, which then naturally settle into the micro-well array 8 in the lower layer under the action of gravity.

[0067] (3) Cell membrane lysis: 0.01% Tween-20 solution was added dropwise to the microwell array and allowed to stand at room temperature for 15 min to allow the cells to lyse.

[0068] (4) Capture and spatial encoding of free transcriptomes. After the cell membrane of the cells in the microwell array 8 is lysed, the transcriptomes in their cytoplasm will be free in the microwell array 8 and bind to the cross-type DNA microarray fixed on the bottom surface of the microwell array 8, thereby achieving spatial encoding of the free transcriptome of tissue cells.

[0069] (5) Passivation of excess DNA sequences on the surface of the free transcriptome capture layer 1. A small amount of a short nucleotide sequence solution rich in polyA at the tail is added to the microwell array 8, and the short sequence is used to passivate the excess DNA sequences on the surface of the free transcriptome capture layer 1 to avoid interference with subsequent tissue cell nuclear spatial transcriptome sequencing.

[0070] (6) Nuclear membrane lysis. TRIzol solution was added to the microwell array and incubated at 15-30°C for 5 minutes. The device was shaken to fully dissolve the nuclear membrane and obtain the transcriptional substances in the nuclei of tissue cells.

[0071] (7) Capture and spatial encoding of nuclear transcriptome. Align the nuclear transcriptome capture layer and the microwell layer up and down, fit tightly and fix them, so that the projection of each cross site in the upper cross-type DNA microarray 7 in the depth direction is completely contained in the lower microwell array 8, and ensure that each cross site of the cross-type DNA microarray 7 with specific position information corresponds to each microwell in the microwell array 8. Gently shake the device so that the free nuclear transcriptome in the microwell array 8 is captured by the upper cross-type DNA microarray 7, thereby realizing the spatial encoding of the nuclear transcriptome of tissue cells.

[0072] (8) Collection and sequencing of nuclear and cytoplasmic transcriptomes. Reverse transcription is performed on the nuclear transcriptome capture layer 5 and the free transcriptome capture layer 1, respectively. The reverse transcribed sequences are collected from the substrate 6 and the surface of the free transcriptome capture layer 1 into two test tubes, respectively, and subsequent amplification, sequencing and other operations are performed to complete the simultaneous spatial transcriptome sequencing of the nucleus and cytoplasm of tissue cells.

Claims

1. A microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei, characterized in that: It includes a free transcriptome capture layer (1), a microwell layer (2), a coding A flow channel layer (3), a coding B flow channel layer (4) and a nuclear transcriptome capture layer (5) which are sequentially assembled from bottom to top, and the five-layer structure is aligned vertically; A microwell array (8) is neatly arranged in the central area of ​​the microwell layer (2); the microwell array (8) has i rows and j columns and penetrates the microwell layer (2) in depth, and each microwell of the microwell array (8) accommodates only one cell; The lower surface of the micro-well layer (2) is in contact with the upper surface of the free transcriptome capture layer (1), and the area of ​​the free transcriptome capture layer (1) exposed at the bottom of the micro-well layer (2) is fixed with an mRNA capture sequence for capturing the free transcriptome in tissue cells; The nuclear transcriptome capture layer (5) is composed of a top rectangular thin layer substrate (6) and a cross-type DNA microarray (7) having unique cell nucleus position information fixed in the middle area of ​​the lower surface of the substrate. The cross-type DNA microarray (7) is fixed by sequentially combining the coding A flow channel layer (3) and the coding B flow channel layer (4) with the thin layer substrate (6) in the nuclear transcriptome capture layer (5). The cross-type DNA microarray has i rows and j columns. When capturing the nuclear transcriptome, the original sample, i.e., the tissue slice, is first fixed on the upper surface of the micro-well layer (2), and the single cells in the tissue slice that have been processed and decomposed are allowed to settle into the micro-well array (8) under the action of gravity, and the free transcriptome in the cell is captured by the mRNA capture sequence fixed on the surface of the free transcriptome capture layer (1) through cell lysis treatment, thereby avoiding interference with subsequent nuclear transcriptome sequencing; Afterwards, a nuclear membrane lysis treatment is performed, and the lower surface of the nuclear transcriptome capture layer (5) is bonded to the upper surface of the microwell layer (2), and each crossover site in the cross-type DNA microarray (7) fixed on the lower surface of the nuclear transcriptome capture layer (5) corresponds one-to-one with each microwell in the microwell array (8), thereby ensuring that the DNA sequence at each crossover site can capture the nuclear transcriptome in its corresponding microwell.

2. A microfluidic chip for tissue cell nuclear spatial transcriptome sequencing according to claim 1, characterized in that: The mRNA capture sequence fixed on the surface of the free transcriptome capture layer (5) for capturing the free transcriptome in the cell is a DNA sequence with a polythymine nucleotide poly T at the tail, wherein the poly T is used to connect with the polyadenylic acid sequence polyA at the end of the free transcriptome in the tissue cell, thereby achieving the capture of the free transcriptome in the tissue cell; 3. A microfluidic chip for tissue cell nuclear spatial transcriptome sequencing according to claim 1, characterized in that: The coding A flow channel layer (3) and the coding B flow channel layer (4) are composed of four parts: intermediate parallel flow channels (9, 10), an inlet (11), an outlet (12), and a vacuum chamber (13) for pumping liquid; wherein the lower surface of the coding A flow channel layer (3) has i long strip grooves (14) with the same number of rows as the cross-type DNA microarray (7) and the microwell array (8), and each groove (14) is respectively connected to the i continuous inlets (11) and the i continuous outlets (12); wherein the lower surface of the coding B flow channel layer (4) has j long strip grooves (14) with the same number of columns as the cross-type DNA microarray (7) and the microwell array (8), and each groove is respectively connected to the j continuous inlets (11) and the j continuous outlets (12); when the coding A flow channel layer (3) is in use, its lower surface is attached to the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) to form i closed parallel flow channels, from The nucleotide sequences A1, A2, A3, ... to Ai flowing through the parallel flow channels (9) can be fixed on the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5); when the coding B flow channel layer (4) is in use, its lower surface is bonded to the lower surface of the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) to form j closed parallel flow channels, so that the nucleotide sequences B1, B2, B3, ... to Bj flowing through the parallel flow channels (10) are fixed on the nucleotide sequence Ai formed by the coding A flow channel layer (3) to form a crossed nucleotide sequence with two-dimensional position information AiBj; when the coding A flow channel layer (3) and the coding B flow channel layer (4) are respectively bonded to the lower surface of the nuclear transcriptome capture layer (5), their long strip grooves are perpendicular to each other, and the intersection of the two projections on the nuclear transcriptome capture layer (5) in the vertical direction is the cross-type DNA microarray (7) with unique two-dimensional position information.

4. A microfluidic chip for tissue cell nuclear spatial transcriptome sequencing according to claim 3, characterized in that: The vacuum chamber (13) for pumping liquid in the coding A flow channel layer (3) and the coding B flow channel layer (4) is a rectangular parallelepiped structure having a bottomless cavity (16), and the bottom of the bottomless cavity (16) is connected to the air extraction hole (15) and extends to the upper surface of the vacuum chamber (13); when in use, the lower surface of the vacuum chamber (13) for pumping liquid is completely in contact with the upper surface of the coding A flow channel layer (3) or the coding B flow channel layer (4), and the bottom opening of the bottomless cavity (16) completely covers the coding A flow channel layer (3) or the upper surface of the coding B flow channel layer (4). The outlet (12) of the flow channel layer (3) or the outlet (12) of the coding B flow channel layer (4); when the vacuum chamber (13) for pumping liquid is used for the coding A flow channel layer (3) or the coding B flow channel layer (4), a negative pressure is formed through the air pumping hole (15), so that the nucleotide sequence Ai loaded on the i inlets (11) flows into the parallel flow channel (9) and flows out from the outlet (12), or the nucleotide sequence Bj loaded on the j inlets (11) flows into the parallel flow channel (10) and flows out from the outlet (12).

5. A microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei according to claim 1, characterized in that: The free transcriptome capture layer (1) is encoded by using the encoding A flow channel layer (3) and the encoding B flow channel layer (4), and the free space transcriptome inside and outside the tissue cell nucleus is sequenced at the same time, that is, the spatial sequencing of the transcriptome inside and outside the cell nucleus is simultaneously achieved.

6. A microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei according to claim 5, characterized in that: The free transcriptome capture layer (1) is bonded to the microwell layer (2), and the lower surface of the coding A flow channel layer (3) is bonded to the upper surface of the microwell layer (2) to form i closed parallel flow channels, so that the nucleotide sequences A1, A2, A3, ... to Ai flowing through the parallel flow channels (9) can be fixed to the area of ​​the free transcriptome capture layer (5) exposed at the bottom of the microwell layer (2); the lower surface of the coding B flow channel layer (4) is bonded to the upper surface of the microwell layer (2) to form j closed parallel flow channels, so that the nucleotide sequences B1, B2, ... to Ai flowing through the parallel flow channels (10) can be fixed to the area of ​​the free transcriptome capture layer (5) exposed at the bottom of the microwell layer (2). B3... to Bj are fixed on the nucleotide sequence formed by the coding A flow channel layer (3) to form a cross-shaped nucleotide sequence with two-dimensional position information; when the coding A flow channel layer (3) and the coding B flow channel layer (4) are respectively attached to the upper surface of the micro-well layer (2), their long strip grooves are perpendicular to each other, and the intersection of the two projections on the micro-well layer (2) in the vertical direction is the position of each micro-well in the micro-well array (8), thereby ensuring that a cross-shaped DNA microarray with unique position information is fixed at the bottom of each micro-well, thereby realizing the spatial sequencing of the free transcriptome in the cell.

7. A microfluidic chip for tissue cell nuclear spatial transcriptome sequencing according to claim 1, characterized in that: The reagents used to decompose the tissue slices placed on the surface of the micro-well layer (2) into single cells include trypsin, pronase and collagenase.

8. The microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei according to claim 1, characterized in that: After the free transcriptome in the cytoplasm is captured by the mRNA capture sequence fixed on the surface of the free transcriptome capture layer (1), the method for passivating the excess mRNA capture sequence on the surface of the free transcriptome capture layer (1) includes but is not limited to adding a small amount of a short nucleotide sequence with a polyA tail, a nuclease, etc. to the microwell, preferably a short nucleotide sequence with a polyA tail.

9. The microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei according to claim 1, characterized in that: After the nuclear transcriptome capture layer (5) completes the spatial encoding of the nuclear transcriptome of the tissue cell, reverse transcription is first performed on the rectangular thin layer substrate (6) in the nuclear transcriptome capture layer (5) to generate a long cDNA sequence with both nuclear transcriptome gene information and position information. Afterwards, the sequence is collected and amplified, sequenced, and data analyzed to finally complete the spatial transcriptome sequencing of the tissue cell nucleus. The free transcriptome capture layer (1), the encoding A flow channel layer (3), the encoding B flow channel layer (4), and the nuclear transcriptome capture layer (4) are all made of transparent materials.

10. The microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei according to claim 1, characterized in that: The coding A flow channel layer (3) and the coding B flow channel layer (4) are realized by a one-step molding reverse molding process; the free transcriptome capture layer (1) and the micro-well layer (2) are realized simultaneously by a single-layer photolithography process, and the process is as follows: Step 1, processing of the free transcriptome capture layer (1) and the micro-well layer (2): using a coating machine to spin-coat a layer of photoresist on the surface of the free transcriptome capture layer (1) as the micro-well layer (2), exposing with an ultraviolet photolithography machine, developing with a developer, and heating and curing to form a capture groove array (8) in the middle area of ​​the micro-well layer (2) for capturing single cells obtained by decomposing tissue slices under trypsin digestion; Step 2, processing the molds of the coding A flow channel layer (3) and the coding B flow channel layer (4): preparing the molds required for the coding A flow channel layer (3) and the coding B flow channel layer (4) by photolithography processing of a single-layer SU-8 structure; Step 3, processing the mold of the vacuum chamber (13) for pumping liquid above the outlet of the coding A flow channel layer (3) and the coding B flow channel layer (4): making a rectangular parallelepiped mold by laser cutting technology; Step 4, the coded A flow channel layer (3) and the coded B flow channel layer (4) are formed by inverting the molds of the coded A flow channel layer (3) and the coded B flow channel layer (4); Step 5, the vacuum chamber (13) for pumping liquid above the outlets in the coding A flow channel layer (3) and the coding B flow channel layer (4) is formed by inverting the mold of the vacuum chamber (13) for pumping liquid; Step 6, plasma bonding the coded A flow channel layer (3) and the coded B flow channel layer (4) obtained in step 4 to the vacuum chamber (13) for pumping liquid formed in the previous step, which is PDMS-PDMS bonding.

Images