A microfluidic chip for spatial transcriptome sequencing of tissue nuclei

Abstract

The application discloses a microfluidic chip for tissue cell nucleus spatial transcriptome sequencing, and belongs to the technical field of particle control in microfluidic chips; the microfluidic chip comprises a free transcriptome capturing layer, a micro-well layer, a code A flow channel layer, a code B flow channel layer and a nucleus transcriptome capturing layer. The micro-well layer is used for placing a tissue slice to be measured, and single cells after dissociation of the tissue slice are captured through the micro-well. The micro-well layer is attached to the free transcriptome capturing layer below, and the free transcriptome capturing layer exposed at the bottom of the micro-well is fixed with mRNA capturing sequences. The code A flow channel layer and the code B flow channel layer are used for fixing cross-type DNA microarrays on the surface of the nucleus transcriptome capturing layer. The code A flow channel layer and the code B flow channel layer are simultaneously used for fixing cross-type DNA microarrays on the free transcriptome capturing layer, so that the common sequencing of single cell spatial free transcriptome and spatial nucleus transcriptome is realized, and the blank of single cell nucleus spatial transcriptome sequencing and the common sequencing of spatial free transcriptome and spatial nucleus transcriptome is filled.

Description

Technical Field

[0001] This invention belongs to the field of microparticle control technology in microfluidic chips, specifically relating to a microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei. Background Technology

[0002] The transcriptome is the sum of all RNA transcribed from a specific tissue or cell at a particular developmental stage or functional state. It reflects the types of genes expressed in that tissue or cell, as well as their expression levels and patterns. Analysis of the transcriptome allows for the study of gene function and structure at a holistic level, playing a crucial role in understanding organismal development. In 2009, Helicos BioSciences first proposed RNA sequencing (RNA-Seq) based on next-generation sequencing technology. Since then, with continuous improvements in high-throughput sequencing platforms, transcriptome sequencing technology has become a powerful tool for comprehensively acquiring transcriptome information and analyzing transcript structure and expression levels, and is now widely used in biological research, medical research, clinical research, and drug research.

[0003] Although high-throughput single-cell RNA sequencing (scRNA-seq) technology can obtain the gene expression status of individual cells, for many frozen tissue samples, the ice crystals formed inside the cells during rapid freezing can puncture the cell membranes, making it difficult or impossible to prepare high-quality single-cell suspensions. This hinders the application of scRNA-seq to solve biological problems. In contrast, the nuclear membrane is more robust; after the cell membrane of frozen tissue cells ruptures, the nucleus remains intact. To gain a deeper understanding of the function and regulatory mechanisms of certain rare frozen samples, in 2013, Rashel V. Grindberg et al. proposed RNA-sequencing from single nuclei. Single-nuclei RNA sequencing (snRNA-seq) technology not only overcomes the limitations of frozen tissue and the difficulty in preparing high-quality single-cell suspensions for single-cell transcriptome sequencing, reducing stress on cells, but also has unique application value for studying non-coding RNA in the cell nucleus and exploring cell differentiation and development. It 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 neuronal development; in 2020, Selewa A et al. explored cellular components and characteristics by performing snRNA-seq on mammalian hearts; and in 2021, TS Andrews et al. revealed the existence of rare subtypes of hepatocytes using snRNA-seq.

[0004] Because gene expression varies across different cell types, locations, and time points, spatial transcriptomics has emerged to provide a deeper understanding of gene distribution and expression specificity within organisms. Spatial transcriptomics allows researchers to more precisely determine the spatial distribution of gene expression within organisms, thereby better understanding the regulation and function of biological processes. One of the earliest spatial transcriptomics techniques was in situ hybridization. With further development, new generations of spatial transcriptomics techniques have emerged, such as RNA in situ hybridization (RNA-ISH) and sequential fluorescence in situ hybridization (seqFISH). However, these methods are all based on a limited probe set that hybridizes with known messenger RNA (mRNA) sequences, which limits the potential of spatial transcriptomics in discovering new sequences and variants. They also require complex experimental procedures and specialized equipment, including expensive instruments and consumables. In addition, while the emergence of spatial transcriptomics has made it possible to map gene expression at the genome scale, research areas such as single-cell nuclear spatial transcriptome sequencing and co-sequencing of spatial free transcriptome and spatial nuclear transcriptome are still in the blank stage. Summary of the Invention

[0005] To address the shortcomings and deficiencies of the above-mentioned technologies, the present invention aims to provide a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells. This chip employs an innovative three-dimensional structure to spatially barcode the transcriptome within the tissue cell nucleus, obtaining both gene information and spatial location information, thereby enabling more accurate study of gene localization within the cell nucleus. Furthermore, the chip can simultaneously spatially encode the nucleoplasmic transcriptome of tissue cells, allowing for the exploration of interactions between the nucleus and cytoplasm, and ultimately providing a more comprehensive understanding of the regulatory mechanisms of gene expression. Compared to existing technologies, the microwell design in this invention increases transcriptome sequencing throughput; the spatial encoding process using microfluidic technology significantly reduces sample and reagent consumption, lowering costs; the device is compact and easy to operate, requiring no specialized equipment to support complex experimental procedures, thus enhancing its application potential in portable system development.

[0006] The objective of this invention is achieved through the following technical solution:

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

[0008] like Figure 1As shown, the microwell layer (2) is a rectangular thin layer with a neatly arranged array of microwells (8) in its central region. The array of microwells (8) penetrates the microwell layer (2) in depth. The depth and cross-sectional area of ​​each microwell are required to accommodate only one cell, so that when the tissue slice is decomposed into single cells, the cells can vertically sink into the microwell array (8) under the action of gravity. The microwell array (8) has i rows and j columns, where each row and each column corresponds one-to-one with the i-th row of the parallel channel in the middle of the A channel layer (3) and the j-th column of the parallel channel in the middle of the B channel layer (4). The lower surface of the microwell layer (2) is attached and fixed to the upper surface of the free transcriptome capture layer (1). The area of ​​the free transcriptome capture layer (1) exposed to the microwell layer (2) is fixed with mRNA capture sequences for capturing free transcriptome in tissue cells, thereby achieving the capture of free transcriptome in tissue cells and avoiding its interference with nuclear transcriptome capture.

[0009] like Figure 1 As shown, the nuclear transcriptome capture layer (5) consists of a rectangular thin substrate (6) and a cross-shaped DNA microarray (7) with unique positional information fixed in the central region of the lower surface of the substrate. The cross-shaped DNA microarray (7) has i rows and j columns, and the cross-sites in the i-th row and j-th column of the cross-shaped DNA microarray (7) correspond one-to-one with the microwells in the i-th row and j-th column of the microwell array (8).

[0010] like Figure 3 As shown, each crossover site in the crossover DNA microarray (7) is fixed with tens of thousands of specific DNA sequences. Each specific DNA sequence contains a specific primer for identifying the gene fragment to be amplified during PCR amplification, a spatial barcode A, a spatial barcode B, a ligation linker for connecting the two sets of DNA barcodes A and B, a specific molecular tag (UMI), and a transcriptome capture region. Feature points in different rows of the crossover DNA microarray (7) have different spatial barcodes Ai (i = 1, 2, 3...i), and feature points in different columns have different spatial barcodes Bj (j = 1, 2, 3...i). The combination of the two sets of spatial barcodes Ai and Bj ensures that each feature point in the crossover DNA microarray (7) has a different spatial barcode.

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

[0012] During the process of immobilizing mRNA capture sequences for capturing free transcriptome within tissues on the surface of the free transcriptome capture layer (1), such as Figure 4 As shown, the lower surface of the microwell layer (2) is closely attached to and fixed to the upper surface of the free transcriptome capture layer (1), and both are immersed 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 microwell layer (2).

[0013] During the operation of the cross-shaped DNA microarray (7) with unique positional information immobilized on the surface of the cuboid substrate (6) in the nuclear transcriptome capture layer (5), such as Figure 5 As shown, the lower surface of the A-encoding channel layer (3) is attached to the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5), forming i closed parallel channels, thereby allowing the nucleotide sequences A1, A2, A3... to Ai flowing through the parallel channels (9) to be fixed on the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5). When the B-encoding channel layer (4) is in use, its lower surface is attached to the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5), forming j closed parallel channels, so that the nucleotide sequences B1, B2, B3... to Bj flowing through the parallel channels (10) are fixed on the nucleotide sequences (A1, A2, A3... to Ai) formed by the A-encoding channel layer (3), forming intersecting nucleotide sequences with two-dimensional positional information. When the A-channel layer (3) and the B-channel layer (4) are attached to the lower surface of the nuclear transcriptome capture layer (5), their elongated grooves are perpendicular to each other. The intersection of their projections in the vertical direction onto the nuclear transcriptome capture layer (5) is the cross-type DNA microarray (7) with unique two-dimensional positional information.

[0014] In the process of capturing the free transcriptome of tissue cells, such as Figure 7As shown in steps I and II, tissue slices are fixed on the surface of the microwell layer (2), processed to digest into single cells, and then vertically dropped into the microwell array (8) below under gravity. A relatively mild cell membrane lysis buffer is added, and the cells are incubated for 10 minutes to lyse the tissue cells in the microwell array (8), achieving nucleocytoplasmic separation. The transcriptome in the tissue cytoplasm is free in the microwell array (8) and pairs complementaryly with the bases of the mRNA capture sequence on the bottom surface, achieving the capture of the free transcriptome in the tissue cells.

[0015] During the process of capturing the nuclear transcriptome of tissue cells, nuclear membrane lysis buffer is added to the microwell array (8) to tightly adhere and fix the nuclear transcriptome capture layer (5) to the microwell layer (2). Figure 7 As shown in steps III and IV, the projection of each crossover site in the upper crossover DNA microarray (7) along the depth direction is completely contained within the lower microwell array (8), ensuring that each crossover site in the crossover DNA microarray (7) with specific location information corresponds one-to-one with each microwell in the microwell array (8). The shaking device fully dissolves the nuclear membrane, allowing the transcriptome within the cell nucleus to be freed in the microwell array (8), where it pairs complementaryly with the DNA sequence bases with specific location information on the top surface, thus achieving spatial barcoding of the transcriptome within the tissue cell nucleus.

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

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

[0018] The microwell array (8) in the middle region of the microwell layer (2) has a shape including but not limited to rectangle, ellipse, dumbbell, etc., and the material is preferably SU-8. The size of the microwell array (8) is preferably 1.2-1.5 times the cell diameter. The materials used for the free transcriptome capture layer (1) include but are not limited to polydimethylsiloxane (PDMS), glass, etc., with glass being preferred.

[0019] The reagents used to decompose the tissue slices placed on the microwell layer (2) into single cells include, but are not limited to, trypsin, streptomycin, collagenase, etc., with trypsin being preferred.

[0020] The method for passivating the excess mRNA capture sequences on the surface of the free transcriptome capture layer (1) after capturing the free transcriptome in the cytoplasm by using the mRNA capture sequences fixed on the surface of the free transcriptome capture layer (1) includes, but is not limited to, adding a small amount of short nucleotide sequences with polyA tails, endonucleases, etc., to the microwell, preferably short nucleotide sequences with polyA tails.

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

[0022] (1) Processing of free transcriptome capture layer (1) and microwell layer (2): A layer of photoresist is spin-coated on the surface of free transcriptome capture layer (1) as microwell layer (2) using a spin coater. After exposure by ultraviolet lithography, development by developing solution, and curing by heating, a capture microwell array (8) is formed in the middle region of microwell layer (2) to capture single cells obtained by digestion of tissue sections by trypsin. The material of free transcriptome capture layer (1) is preferably glass, and the material of microwell layer (2) is preferably SU-8.

[0023] (2) Processing of molds for coding A flow channel layer (3) and coding B flow channel layer (4): The molds required for coding A flow channel layer (3) and 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) The vacuum chamber (13) above the outlet of the coded A flow channel layer (3) and the coded B flow channel layer (4) for pumping liquid is processed as follows: A cuboid mold is made by laser cutting technology, and the preferred material is polymethyl methacrylate; once the mold is processed, it can be reused.

[0025] (4) The code A flow channel layer (3) and the code B flow channel layer (4) are formed by molding the mold of the code A flow channel layer (3) and the code B flow channel layer (4). The preferred material is PDMS. After heating and curing, the material is demolded.

[0026] (5) The vacuum chamber (13) for pumping liquid above the outlet in the code A flow channel layer (3) and code B flow channel layer (4) is formed by molding the mold of the vacuum chamber (13) for pumping liquid. The preferred material is PDMS, and the preferred thickness is 5-8mm. After heating and curing, it is demolded.

[0027] (6) The coded A channel layer (3) and coded B channel layer (4) obtained in step 4 are plasma bonded together with the vacuum cavity (13) of the pumping liquid formed in the previous step to form PDMS-PDMS bonding.

[0028] Compared with existing tissue cell spatial transcriptome sequencing technologies, the advantages of this invention are as follows:

[0029] This invention proposes a microfluidic chip for spatial transcriptome sequencing of tissue cell nuclei. The chip uses a two-layer structure: the upper layer is a cross-shaped DNA microarray with location information, and the lower layer is a microwell array with mRNA capture sequences. Tissue slices immobilized on the surface of the microwell array are dissociated into single cells by trypsin solution. Due to gravity, they fall vertically into the capture grooves. The captured single cells achieve nucleocytoplasmic separation under the action of cell membrane lysis buffer. The transcriptome in the cytoplasm binds to the mRNA capture sequence on the bottom surface of the array through base complementarity pairing, completing the capture of free transcriptome within the tissue slice and avoiding its interference with subsequent transcriptome capture. Subsequently, by corresponding the cross-shaped DNA microarray with the microwell array one-to-one, the free cell nuclei in the microwell array are lysed under the action of nuclear membrane lysis buffer, releasing the nuclear transcriptome. This portion of the transcriptome is linked to the DNA sequence on the top surface of the microwell array according to base complementarity pairing, realizing the capture of the nuclear transcriptome of the tissue slice at that location. Thus, the capture of the nuclear transcriptome of the tissue cell is completed, and its location information is obtained.

[0030] Compared with existing technologies, the chip designed in this invention changes the traditional method of capturing single-cell transcriptomes using microbeads, while simultaneously achieving the separation of the nuclear cytoplasmic transcriptome from tissue cells. It employs a spatial barcoding mechanism to simultaneously acquire gene and location information of the nuclear transcriptome, thereby enabling spatial transcriptome sequencing within the tissue cell nucleus. The design of the capture array and the cross-shaped DNA microarray allows for near-single-cell level sequencing of the tissue slice transcriptome, improving throughput. In this invention, the transcriptome within the tissue cell nucleus is captured by complementary base pairing with DNA sequences containing specific primers on the top surface of the microwell array, further shortening the capture time. This invention only requires further spatial coding of the microwell array to achieve simultaneous spatial transcriptome sequencing inside and outside the tissue slice nucleus. The operation of this invention does not require chip flipping or valve control equipment; the chip structure is simple, easy to operate, and has low processing costs. Attached Figure Description

[0031] Figure 1 This is an overall structural diagram of a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, as described in this invention.

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

[0033] Figure 3 This is an overall structural diagram of the vacuum chamber for pumping liquids according to the present invention.

[0034] Figure 4 This is a design diagram of a DNA sequence for spatial barcoding of the nuclear transcriptome of tissue cells, as described in this invention.

[0035] Figure 5 This is a schematic diagram illustrating the formation of DNA sequences on the surface of a free transcriptome capture layer, as described in this invention, for capturing free transcriptome within tissues.

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

[0037] Figure 7 This is a flowchart illustrating the fabrication process of a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, as described in this invention.

[0038] Figure 8 This is a flowchart illustrating the spatial barcoding process for the nuclear transcriptome of tissue cells as described in this invention.

[0039] Figure 9 This is the working mode of forming a cross-shaped DNA microarray on the surface of the free transcriptome capture layer as described in this invention.

[0040] The attached diagram is labeled as follows: 1. Free transcriptome capture layer; 2. Microwell layer; 3. A-coder layer; 4. B-coder layer; 5. Nuclear transcriptome capture layer; 6. Rectangular thin substrate; 7. Cross-shaped DNA microarray; 8. Microwell array; 9. Parallel microchannel in the middle (horizontal direction); 10. Parallel microchannel in the middle (vertical direction); 11. Inlet; 12. Outlet; 13. Vacuum chamber for pumping liquid; 14. Long strip groove; 15. Vacuum vent; 16. Bottomless cavity. Detailed Implementation

[0041] To further describe the present invention in detail, the following description is provided in conjunction with embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0042] Example 1

[0043] This invention proposes a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, the complete structure of which is shown below. Figure 1As shown, the structure includes a free transcriptome capture layer 1, a microwell layer 2, an A-coding channel layer 3, a B-coding channel layer 4, and a nuclear transcriptome capture layer 5. The nuclear transcriptome capture layer 5 comprises a rectangular thin substrate 6 and a cross-shaped DNA microarray 7 fixed in the central region of the substrate's lower surface. The rectangular thin substrate 6 is 2 mm high, and the cross-shaped DNA microarray 7 contains 70 × 70 feature points. Top views of the A-coding channel layer 3 and the B-coding channel layer 4 are shown below. Figure 2 As shown, the middle section contains 70 parallel channels 9 and 10, each 50 μm wide. The inlet 11 and outlet 12 of the channels have a diameter of 1 mm. The vacuum chamber used for pumping liquid has a height of 15 mm, and the thickness of the chamber sidewalls and top layer is 5 mm. The microwell layer 2 and the free transcriptome capture layer 1 have thicknesses of 1 mm and 2 mm, respectively. Microwell layer 2 has a neatly arranged microwell array 8 in the middle. The microwell array 8 includes 70×70 feature points, each with 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 present invention describes a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, such as... Figure 1 As shown, the microwell array 8 penetrates the microwell layer 2 in depth, the free transcriptome capture layer 1 is closely 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 is aligned vertically, and each crossover site of the cross-shaped DNA microarray 7 in the nuclear transcriptome capture layer 5 corresponds one-to-one with each microwell in the microwell array 8.

[0045] The present invention describes a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, with a schematic diagram of the mRNA capture sequence immobilized on the surface of the free transcriptome capture layer 1 as shown in the figure. Figure 4 As shown, the microwell layer 2 and the free transcriptome capture layer 1 are tightly attached and fixed, and then 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 microwell layer 2.

[0046] The flowchart of the microfluidic chip for spatial bar coding of the nuclear transcriptome of tissue cells described in this invention, in which a cross-shaped DNA microarray 7 is formed on the surface of a cuboid thin substrate 6 in the nuclear transcriptome capture layer 5, is shown below. Figure 5As shown, firstly, the A-coded flow channel layer 3 is bonded and fixed to the cuboid substrate 6. DNA primer solution of group A (A1, A2, A3, A4…A70) is injected into the horizontal flow channel through inlet 11. A vacuum is drawn at outlet 12, and atmospheric pressure drives fluid flow. The mixture is allowed to stand, allowing the DNA primers of group A to adhere to the surface of substrate 6, thus forming a DNA barcode in the X direction on the cuboid substrate. Subsequently, the B-coded flow channel layer 4 is rebonded and fixed to the cuboid substrate 6 with the DNA barcode fixed in the X direction. The middle parallel flow channel 10 of the B-coded flow channel layer 4 is aligned with the DNA barcode in the X direction on the surface of the cuboid substrate 6 and intersects it in the X direction. DNA primer solution of group B (B1, B2, B3, B4…B70) is injected into the vertical flow channel through inlet 11. The mixture is allowed to stand, allowing the A and B DNA primers to connect. After removing the B-coded flow channel layer 4, a cross-shaped DNA microarray 7 with specific positional information is formed on the surface of the cuboid substrate 6.

[0047] The microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells described in this invention, with a processing flowchart of microwell layer 2 and free transcriptome capture layer 1 as shown below. Figure 7 As shown in (a), the fabrication process requires only a single-layer photolithography process. The fabrication flow for encoding A flow channel layer 3 and encoding B flow channel layer 4 is as follows: Figure 7 As shown in (b), the processing only requires a single molding step, and the molding process does not require the micromachining steps of alignment photolithography. Specifically, it includes the following steps:

[0048] (1) Design and fabrication of photomasks: Draw the design diagram of the microfluidic device and use high-precision printing to output multiple photomasks. Each photomask has light-transmitting and opaque patterns to form patterns in the exposure step.

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

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

[0051] The second is the processing flow for encoding flow channel layer 3 (A) and encoding flow channel layer 4 (B):

[0052] (3) Mold processing: This involves photolithography to fabricate single-layer SU8 molds for encoding flow channel layers 3 and 4. The molds are reusable after processing. The molds for encoding flow channel layer A and layer B are obtained by sequentially processing the silicon wafer through SU8 spin coating, soft bake, exposure, post-expose bake, development, and hard bake. Figure 7 (b) As shown in steps I-II.

[0053] (4) Fabrication of A and B flow channel layers 3 and 4: A layer of PDMS is cured on the mold surface. After curing, this structure is demolded from the mold. Holes are punched in the demolded structure to form inlets and outlets. The vacuum chamber 13 for pumping liquid is bonded to the demolded structure, which is a PDMS-PDMS plasma bond. Figure 7 (b) As shown in steps III-VI.

[0054] This embodiment uses a microfluidic chip obtained from the above-described embodiments for spatial barcoding of tissue cell nuclear transcriptomes to test the spatial transcriptome sequencing performance of tissue cell nuclei. Figure 7 As shown, the specific steps are as follows:

[0055] (1) Tissue section decomposition. The tissue section was placed on the surface of the microwell layer 2 and treated with 0.1% trypsin solution to decompose it into single cells. Under the action of gravity, the tissue section naturally settled into the lower microwell array 8.

[0056] (2) Cell membrane lysis. Add 0.01% Tween-20 solution to the microwell array and let it stand at room temperature for 15 min to lyse the cells.

[0057] (3) Capture of free transcriptome. After cell membrane lysis, the transcriptome in the cytoplasm of cells in microwell array 8 is released into microwell array 8 and binds to the mRNA capture sequence fixed on the surface of free transcriptome capture layer 1.

[0058] (4) Passivation of excess mRNA capture sequences. A small amount of short nucleotide sequence solution rich in polyA tails was added to the microwell array 8 to passivate the excess mRNA capture sequences on the surface of the free transcriptome capture layer 1, thus avoiding their interference with subsequent tissue cell nuclear spatial transcriptome sequencing.

[0059] (5) Nuclear membrane lysis. TRIzol solution was added to the microwell array 8 and incubated at 15-30℃ for 5 minutes. The device was shaken to fully dissolve the nuclear membrane and obtain the transcription material from the cell nucleus.

[0060] (6) Capture and spatial encoding of the nuclear transcriptome. The nuclear transcriptome capture layer 5 is aligned vertically and tightly fitted and fixed to the microwell layer 2, so that the projection of each crossover site in the upper crossover DNA microarray 7 in the depth direction is completely contained in the lower microwell array 8, and each crossover site of the crossover DNA microarray 7 with specific location information corresponds one-to-one with each microwell in the microwell array 8. The device is gently shaken to capture the free nuclear transcriptome in the microwell array 8 by the upper crossover 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 was performed on nuclear transcriptome capture layer 5. The reverse transcribed sequence was collected from substrate 6 into a test tube for subsequent amplification, sequencing and other operations to complete the spatial transcriptome sequencing of tissue cell nuclei.

[0062] Example 2

[0063] This invention proposes a microfluidic chip for spatial barcoding of the nuclear transcriptome of tissue cells, the complete structure of which is shown below. Figure 1 As shown. Except for spatial bar coding of the free transcriptome capture layer, the chip structure and fabrication process are the same as in Example 1.

[0064] This embodiment uses a microfluidic chip obtained from the above-described embodiments for spatial barcoding of the nuclear transcriptome of tissue cells to test the spatial transcriptome sequencing performance inside and outside the tissue cell nucleus. The specific steps are as follows:

[0065] (1) Spatial coding of microwell arrays. For example... Figure 8 As shown, the parallel channels in the middle of the A-channel layer and the B-channel layer are respectively matched one-to-one with each row and column of the microwell array. The A-channel layer and the B-channel layer are successively attached and fixed to the microwell array, and the two sets of DNA primer solutions with spatial position information, A and B, are sequentially introduced. Finally, a cross-shaped DNA microarray is formed on the bottom surface of each feature point in the microwell array, and different DNA microarrays are fixed on the bottom surface of different feature points.

[0066] (2) Tissue section decomposition. The tissue section was placed on the surface of microwell layer 2 and treated with 0.1% trypsin solution to decompose it into single cells. Under the action of gravity, the tissue section naturally settled into the lower microwell array 8.

[0067] (3) Cell membrane lysis. Add 0.01% Tween-20 solution to the microwell array and let it stand at room temperature for 15 min to lyse the cells.

[0068] (4) Capture and spatial encoding of free transcriptome. After cell membrane lysis, the transcriptome in the cytoplasm of cells in microwell array 8 is released into microwell array 8 and binds to the cross-shaped DNA microarray fixed on the bottom surface of microwell array 8, thereby realizing the spatial encoding of free transcriptome of tissue cells.

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

[0070] (6) Nuclear membrane lysis. TRIzol solution was added dropwise to the microwell array and incubated statically for 5 minutes at 15-30°C. The device was shaken to fully dissolve the nuclear membrane and obtain the transcription material from the cell nucleus.

[0071] (7) Capture and spatial encoding of the nuclear transcriptome. The nuclear transcriptome capture layer is aligned vertically and tightly fitted and fixed to the microwell layer, ensuring that the projection of each crossover site in the upper crossover DNA microarray 7 in the depth direction is completely contained in the lower microwell array 8, and that each crossover site of the crossover DNA microarray 7 with specific location information corresponds one-to-one with each microwell in the microwell array 8. The device is gently shaken to capture the free nuclear transcriptome in the microwell array 8 by the upper crossover DNA microarray 7, thereby achieving spatial encoding of the tissue cell nuclear transcriptome.

[0072] (8) Collection and sequencing of the nucleoplasmic transcriptome. Reverse transcription was performed on the nuclear transcriptome capture layer 5 and the free transcriptome capture layer 1, respectively. The reverse transcribed sequences were collected from the substrate 6 and the surface of the free transcriptome capture layer 1 into two test tubes, respectively, for subsequent amplification, sequencing and other operations, to complete the spatial transcriptome sequencing of the nucleoplasm of tissue cells.

Claims

1. A microfluidic chip for spatial transcriptome sequencing of tissue cells, characterized in that, The structure consists of five layers arranged vertically from bottom to top: a free transcriptome capture layer (1), a microwell layer (2), an A-channel encoding layer (3), a B-channel encoding layer (4), and a nuclear transcriptome capture layer (5). The microwell layer (2) has a neat array of microwells (8) arranged in the central region; the microwell array (8) has i rows and j columns and penetrates the microwell layer (2) in depth, and each microwell in the microwell array (8) contains only one cell; The lower surface of the microwell layer (2) is attached to 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 microwell layer (2) is fixed with mRNA capture sequences for capturing free transcriptome in tissue cells; The nuclear transcriptome capture layer (5) consists of a top rectangular thin substrate (6) and a cross-shaped DNA microarray (7) with unique nuclear position information fixed in the middle region of the lower surface of the substrate. The cross-shaped DNA microarray (7) is fixed by combining the A-channel layer (3) and the B-channel layer (4) in sequence with the thin substrate (6) in the nuclear transcriptome capture layer (5). The cross-shaped 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 microwell layer (2). The single cells of the tissue slice after processing and decomposition sink into the microwell array (8) under the action of gravity. 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, so as to avoid interference with subsequent nuclear transcriptome sequencing. Afterwards, the nuclear membrane was lysed and the lower surface of the nuclear transcriptome capture layer (5) was attached to the upper surface of the microwell layer (2). Each crossover site in the cross-shaped 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 on each crossover site can capture the nuclear transcriptome in its corresponding microwell. The coding A flow channel layer (3) and coding B flow channel layer (4) are both composed of four parts: a central parallel flow channel (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 elongated grooves (14) with the same number of rows as the cross-shaped DNA microarray (7) and microwell array (8), each groove (14) being connected to i through inlets (11) and i outlets (12). The lower surface of the coding B flow channel layer (4) has j elongated grooves (14) with the same number of columns as the cross-shaped DNA microarray (7) and microwell array (8), each groove being connected to j through inlets (11) and j outlets (12). When in use, the lower surface of the coding A flow channel layer (3) is attached to the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5), forming i closed parallel flow channels, from which liquid flows... This allows the nucleotide sequences A1, A2, A3... to Ai flowing through the parallel channels (9) to be fixed on the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5); when the B-encoding channel layer (4) is in use, its lower surface is attached to the lower surface of the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5) to form j closed parallel channels, so that the nucleotide sequences B1, B2, B3... to Bj flowing through the parallel channels (10) are fixed on the nucleotide sequence Ai formed by the A-encoding channel layer (3) to form a cross-shaped nucleotide sequence with two-dimensional position information AiBj; when the A-encoding channel layer (3) and the B-encoding channel layer (4) are attached to the lower surface of the nuclear transcriptome capture layer (5) respectively, their long strip grooves are perpendicular to each other, and the intersection of the two in the vertical direction projected on the nuclear transcriptome capture layer (5) is the cross-shaped DNA microarray (7) with unique two-dimensional position information.

2. The microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, The mRNA capture sequence fixed on the surface of the free transcriptome capture layer (5) for capturing intracellular free transcriptome is a DNA sequence with polythymidine nucleotide poly T at the end, wherein poly T is used to connect with the polyadenylated polyA sequence at the end of the free transcriptome in tissue cells, thereby achieving the capture of free transcriptome in tissue cells.

3. A microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, The vacuum chamber (13) for pumping liquid in the coded A flow channel layer (3) and the coded B flow channel layer (4) is a cuboid structure with a bottomless cavity (16). The bottomless cavity (16) is connected to a suction port (15) and extends to the upper surface of the vacuum chamber (13). The lower surface of the vacuum chamber (13) for pumping liquid is completely in contact with the upper surface of the coded A flow channel layer (3) or the coded B flow channel layer (4) during use, and the bottom opening of its bottomless cavity (16) completely covers the coded A flow channel layer. The outlet (12) of the flow channel layer (3) or the outlet (12) of the B-encoding flow channel layer (4); when the vacuum chamber (13) for pumping liquid is used to encode the A-encoding flow channel layer (3) or the B-encoding flow channel layer (4), a negative pressure is formed through the air extraction through hole (15) so that the nucleotide sequence Ai loaded in i inlets (11) flows into the parallel flow channel (9) and flows out from the outlet (12), or so that the nucleotide sequence Bj loaded in j inlets (11) flows into the parallel flow channel (10) and flows out from the outlet (12).

4. A microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, The reagents used to break down tissue sections placed on the surface of the microwell layer (2) into single cells include trypsin, streptomycin, and collagenase.

5. A microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, After capturing the free transcriptome in the cytoplasm using the mRNA capture sequence fixed on the surface of the free transcriptome capture layer (1), the method of passivating the excess mRNA capture sequence on the surface of the free transcriptome capture layer (1) includes adding a small amount of short nucleotide sequences with polyA tails and endonucleases to the microwell.

6. A microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, After completing the spatial encoding of the nuclear transcriptome of tissue cells, the nuclear transcriptome capture layer (5) first performs reverse transcription on the cuboid thin substrate (6) in the nuclear transcriptome capture layer (5) to generate a long cDNA sequence that carries both nuclear transcriptome gene information and location information. Then, the sequence is collected and amplified, sequenced, and analyzed to finally complete the spatial transcriptome sequencing of the tissue cell nucleus. The free transcriptome capture layer (1), the A-channel encoding layer (3), the B-channel encoding layer (4), and the nuclear transcriptome capture layer (4) are all made of transparent materials.

7. A microfluidic chip for spatial transcriptome sequencing of tissue cells according to claim 1, characterized in that, The coding A channel layer (3) and coding B channel layer (4) are realized by a one-step molding process; the free transcriptome capture layer (1) and microwell layer (2) are realized simultaneously by a single-layer photolithography process, as follows: Step 1, processing of free transcriptome capture layer (1) and microwell layer (2): A layer of photoresist is spin-coated on the surface of free transcriptome capture layer (1) as microwell layer (2) using a spin coater. After exposure by ultraviolet lithography, development by developing solution, and curing by heating, a capture groove array (8) is formed in the middle region of microwell layer (2) to capture single cells obtained by digestion of tissue sections by trypsin. Step 2, processing of the molds for encoding A flow channel layer (3) and encoding B flow channel layer (4): The molds required for encoding A flow channel layer (3) and encoding B flow channel layer (4) are prepared by photolithography processing of a single-layer SU-8 structure; Step 3, processing of the mold for the vacuum chamber (13) above the outlet of the coded A flow channel layer (3) and the coded B flow channel layer (4): a cuboid mold is made by laser cutting technology; Step 4, the code A flow channel layer (3) and the code B flow channel layer (4) are formed by casting the mold of the code A flow channel layer (3) and the code B flow channel layer (4); Step 5, the vacuum chamber (13) for pumping liquid above the outlet in the code A flow channel layer (3) and the code B flow channel layer (4) is formed by casting the mold of the vacuum chamber (13) for pumping liquid; Step 6: The coded A flow channel layer (3) and coded B flow channel layer (4) obtained in step 4 are plasma bonded together with the vacuum cavity (13) of the pumping liquid formed in the previous step, which is a PDMS-PDMS bond.

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