Method and device for acquiring single cell data

Abstract

The embodiment of the invention provides a single cell data acquisition method and device, which are applied to the technical field of single cell omics, and the method comprises the following steps: separating a plurality of single cells in a cell suspension through a micro-well array of a micro-well layer, each micro-well of the micro-well array containing one single cell; the fence layer is attached to the micro-well layer, so that single cells in the multiple micro-wells of the micro-well array are subjected to a molecular reaction of target omics through a reaction reagent; attaching the coding layer to the micro-well layer so as to chain a nucleotide sequence carrying spatial coding data into nucleic acid molecules of single cells of a plurality of micro-wells; cracking the single cells in the plurality of micro-wells through chemical cracking or electric cracking to release nucleic acid molecules with nucleotide sequences; collecting nucleic acid molecules with nucleotide sequences, and performing sequencing analysis of target omics; and determining single cell space coding data of each nucleic acid molecule based on the nucleotide sequence, and obtaining target omics data of each single cell.

Description

Technical Field

[0001] The embodiments in this specification relate to the field of single-cell omics technology, specifically to a method and apparatus for acquiring single-cell data. Background Technology

[0002] In recent years, with the development of single-cell sequencing technology, researchers have been able to reveal cellular heterogeneity and transcriptional dynamics within tissues at single-cell resolution. However, as research continues, the scientific community has gradually realized that transcriptional information alone cannot fully reveal cellular state and biological processes. Cellular biological functions are also regulated at multiple levels, including translation, protein modification, and chromatin spatial structure. Therefore, relying solely on transcriptome data is still insufficient to fully characterize cellular state and its regulatory mechanisms.

[0003] 96-well and 384-well plates are now widely used in single-cell omics research. These microplate platforms allow researchers to process hundreds of samples simultaneously within a small volume, offering a simple and cost-effective workflow. However, due to the size limitation, even the 384-well plate, which currently handles the largest number of analyses, can only process 384 samples at a time, making it difficult to analyze tens of thousands of samples, resulting in very low throughput. Furthermore, existing methods for acquiring single-cell omics data essentially provide overall measurements of a large mixture of cells—a statistically integrated summary of signals from all cells in the population. This method completely masks the heterogeneity between cells, failing to distinguish or acquire the independent, specific omics information of each individual cell. Consequently, it is difficult to reveal key biological characteristics such as the existence of rare cell subpopulations, the continuous evolution of cell states, and significant differences in expression among cells. Summary of the Invention

[0004] The purpose of the embodiments in this specification is to provide a method and apparatus for acquiring single-cell data, so as to overcome the problems of low throughput and inability to distinguish or acquire independent and specific omics information of single cells in existing methods.

[0005] To solve the above-mentioned technical problems, the specific technical solutions of the embodiments in this specification are as follows:

[0006] On the one hand, embodiments of this specification provide a method for acquiring single-cell data, including:

[0007] Multiple single cells in a cell suspension are separated by a microwell array of microwell layers, wherein each microwell in the microwell array contains one single cell.

[0008] By attaching the fence layer to the microwell layer, molecular reactions of targeted omics can be performed on single cells in multiple microwells of the microwell array using reactive reagents.

[0009] The coding layer is attached to the microwell layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of single cells in multiple microwells;

[0010] Nucleic acid molecules with nucleotide sequences are released by cleaving single cells in multiple microwells through chemical or electrolytic cleavage.

[0011] Collect nucleic acid molecules with nucleotide sequences and perform sequencing analysis for the target omics;

[0012] Based on nucleotide sequences, the single-cell spatial coding data of each nucleic acid molecule is determined, and the target omics data of each single cell are obtained.

[0013] On another front, embodiments of this specification provide a device for acquiring single-cell omics data, comprising:

[0014] A separation module is used to separate multiple single cells from a cell suspension through a microwell array of microwell layers, wherein each microwell of the microwell array contains one single cell.

[0015] The reaction module is used to attach the fence layer to the microwell layer so that single cells in multiple microwells of the microwell array can undergo molecular reactions for targeted omics through reaction reagents.

[0016] The coding module is used to attach the coding layer to the microwell layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of single cells in multiple microwells;

[0017] The lysis module is used to lyse single cells in multiple microwells via chemical or electrolysis to release nucleic acid molecules with nucleotide sequences.

[0018] The analysis module is used to collect nucleic acid molecules with nucleotide sequences and perform sequencing analysis for target omics.

[0019] The acquisition module is used to determine the single-cell spatial coding data of each nucleic acid molecule based on the nucleotide sequence and to acquire the target omics data of each single cell.

[0020] As can be seen from the technical solutions provided in the embodiments of this specification above, the embodiments of this specification can separate multiple single cells from a cell suspension using a micro-well array of micro-well layers, where each micro-well in the micro-well array contains one single cell; a fence layer is attached to the micro-well layer to allow the single cells in the multiple micro-wells of the micro-well array to undergo molecular reactions for target omics using reaction reagents; a coding layer is attached to the micro-well layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of the single cells in the multiple micro-wells; the single cells in the multiple micro-wells are lysed by chemical cleavage or electrolysis to release nucleic acid molecules carrying nucleotide sequences; the nucleic acid molecules carrying nucleotide sequences are collected and subjected to sequencing analysis for target omics; the single-cell spatial coding data of each nucleic acid molecule is determined based on the nucleotide sequences, and the target omics data of each single cell is obtained. Compared with existing methods, the embodiments of this specification, through the micro-well array structure, utilize gravity sedimentation and hydrodynamics to achieve parallel capture and separation of single cells, with each micro-well confining one cell, allowing thousands to tens of thousands of cells to be processed simultaneously in a single experiment. By adding customizable reaction reagents in situ within microwells, various omics analysis needs can be supported, and different omics pretreatment types can be achieved simply by changing the reagents. Introducing spatially encoded nucleotide sequences ensures a unique spatial coding combination on the nucleic acid molecular markers of each single cell. This coding allows for accurate tracing of the cell origin of each sequencing read in subsequent mixed sequencing, preventing cross-contamination of molecular information between cells and ensuring the purity and reliability of single-cell omics data. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments or prior art of this specification, the accompanying drawings used in the description of the embodiments or prior art will be briefly introduced below.

[0022] Figure 1 This is a flowchart illustrating a method for acquiring single-cell omics data provided in the embodiments of this specification;

[0023] Figure 2 This is a cross-sectional view of a microfluidic chip used in a cell capture state, as provided in the embodiments of this specification.

[0024] Figure 3 This is a schematic diagram of a microfluidic chip used for multi-step continuous molecular reactions, provided in the embodiments of this specification.

[0025] Figure 4 This is a schematic diagram of the process of using a microfluidic chip for nucleotide sequence pairing and ligation, as provided in the embodiments of this specification.

[0026] Figure 5 This is a schematic diagram of a microfluidic chip used for cell lysis provided in the embodiments of this specification;

[0027] Figure 6This is an overall structural diagram of a microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0028] Figure 7 This is a schematic diagram of the process of using a microfluidic chip for nucleotide sequence pairing and ligation, as provided in the embodiments of this specification.

[0029] Figure 8 This is a schematic diagram illustrating the bonding method of the vacuum cover layer, the coded A microchannel layer, and the microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0030] Figure 9 This is a schematic diagram illustrating the bonding method of the vacuum cover layer, the coded B microchannel layer, and the microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0031] Figure 10 This is an overall structural diagram of the coded A microchannel layer and the coded B microchannel layer of a microfluidic chip provided in the embodiments of this specification;

[0032] Figure 11 This is an overall structural diagram of the vacuum cover layer of a microfluidic chip provided in the embodiments of this specification;

[0033] Figure 12 This is an overall structural diagram of the conductive microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0034] Figure 13 This is a schematic diagram of a microfluidic chip used for cell electrolysis reaction provided in the embodiments of this specification;

[0035] Figure 14 This is an overall structural diagram of the silver-PDMS cover layer of a microfluidic chip provided in the embodiments of this specification;

[0036] Figure 15 This is an overall structural diagram of the conductive cover layer of a microfluidic chip provided in the embodiments of this specification;

[0037] Figure 16 This is an overall structural diagram of a non-conductive microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0038] Figure 17 This is a schematic diagram of the bonding method between the interdigitated electrode capping layer and the microwell layer of a microfluidic chip provided in the embodiments of this specification;

[0039] Figure 18 This is an overall structural diagram of the interdigitated electrode capping layer of a microfluidic chip provided in the embodiments of this specification;

[0040] Figure 19 This is a schematic diagram of the structural composition of a single-cell omics data acquisition device provided in the embodiments of this specification.

[0041] The reference numerals in the above figures are as follows:

[0042] 1. Micro-well layer;

[0043] 2. Encode the A microchannel layer;

[0044] 3. Encoding B microchannel layer;

[0045] 4. Vacuum cover layer;

[0046] 5. Fence layer;

[0047] 6. Microwell array;

[0048] 7. First glass substrate;

[0049] 8. Second through hole;

[0050] 9. Entrance;

[0051] 10. First parallel microchannel;

[0052] 11. Second parallel microchannel;

[0053] 12. Export;

[0054] 13. Groove;

[0055] 14. Vent hole;

[0056] 15. First through hole;

[0057] 16. Conductive substrate layer;

[0058] 17. Conductive cover layer;

[0059] 18. Silver-PDMS block;

[0060] 19. Conductive material layer;

[0061] 20. Second glass substrate;

[0062] 21. PDMS material;

[0063] 22. Interdigitated electrode cover layer;

[0064] 23. PDMS material;

[0065] 24. Interdigitated structure. Detailed Implementation

[0066] The technical solutions in the embodiments of this specification will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0067] It should be noted that the terms "first," "second," etc., used in this specification, claims, and the foregoing drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, apparatus, product, or device that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0068] In some embodiments, target omics includes, but is not limited to, translatomics, three-dimensional genomics, whole-RNA transcriptomics, epigenomics, and proteomics. Transomemography, by capturing and sequencing ribosome-bound mRNA, reveals the actual protein translation activities occurring in cells at specific times, directly reflecting the synthetic state of functional proteins. Three-dimensional genomics analyzes the spatial conformation, chromatin loops, and topological association domains of chromatin to elucidate the organizational mechanisms of gene regulation in three-dimensional space. Whole-RNA transcriptomics comprehensively captures and sequences all RNA types in cells, including messenger RNA, long non-coding RNA, circular RNA, and small RNA, to fully reveal the expression dynamics and regulatory networks at the transcriptome level. Epigenomics covers analyses of DNA methylation, chromatin accessibility, and histone modifications, elucidating the regulatory mechanisms of gene expression at the epigenetic level. Proteomics, through oligonucleotide-tagged antibodies or active probe labeling technology, achieves indirect sequencing analysis of protein expression and modification states at the single-cell level.

[0069] Figure 1 This is a flowchart of a method for acquiring single-cell omics data provided in this specification. In practice, it may include the following steps:

[0070] S10: Separate multiple single cells from a cell suspension using a microwell array of microwell layers, wherein each microwell of the microwell array contains one single cell.

[0071] In some embodiments, the microfluidic chip may include a microwell layer, and the microwell array of the microwell layer may contain multiple microwells arranged in a row and column matrix. Multiple single cells in a cell suspension can be separated by the microwell array of the microwell layer, with each microwell of the microwell array containing one single cell.

[0072] The prepared single-cell suspension can be loaded onto a microwell array. Through gravity sedimentation and hydrodynamics, the distribution of cells in the microwells can be controlled, so that multiple single cells in the cell suspension are separated and confined in different microwells. The physical size of each microwell is constructed so that it can statistically accommodate only one single cell, thereby achieving high-throughput separation of single cells.

[0073] By isolating each cell in an independent physical microwell, it's equivalent to creating an independent "microreactor" for each cell. This ensures that the biomolecular information of each cell is strictly confined within its own microwell in subsequent steps, fundamentally preventing mixing between molecules from different cells—a prerequisite for obtaining truly pure single-cell data. Furthermore, the fixed spatial location of each microwell provides a defined, addressable physical anchor for the subsequently introduced nucleotide sequences, allowing the cell's "spatial address" to be translated into the "digital address" of its molecular data. Microwell arrays can contain thousands to tens of thousands of microwells, allowing for the simultaneous parallel processing of tens of thousands of single cells, meeting the statistical power requirements of single-cell research and effectively capturing the heterogeneity and rare cell types within cell populations. Once cells are immobilized in the microwells, the entire array can function as a stable platform, attaching to different functional layers to execute multi-step, continuous in-situ biochemical reactions. The fixed cell position guarantees the completion of these complex processes without sample contamination. Overall, using microwell arrays to isolate single cells is fundamental to achieving high-throughput, zero-contamination, addressable, and streamlined single-cell analysis.

[0074] S20: The fence layer is attached to the microwell layer to enable single cells in multiple microwells of the microwell array to undergo molecular reactions for targeted omics through reactive reagents.

[0075] In some embodiments, the microfluidic chip may include a fence layer. The fence layer may be attached to the microwell layer, and various reactive reagents may be sequentially added to multiple microwells of the microwell array to enable single cells in the multiple microwells to perform multi-step continuous molecular reactions for target omics.

[0076] Multiple different reaction reagents are sequentially added to a microwell array via reversible bonding or fluid injection, following a specific reaction sequence. The selection of reaction reagents is adapted to the type of target omics analysis, initiating and completing multi-step sequential molecular reactions within single cells in the microwells. These multi-step sequential molecular reactions occur in situ on the microwell array, and reaction intermediates are confined within their respective microwells.

[0077] The biochemical reaction steps, reagents, and enzymes required for different omics analyses, such as transcriptomics, epigenomics, and proteomics, are drastically different. For example, transcriptomics requires reverse transcription, while DNA methylomics requires bisulfite conversion. By sequentially replacing reagents with those matching the target omics, rather than using fixed reagents, the same hardware platform can flexibly adapt to the sample preprocessing needs of multiple omics.

[0078] Many omics preprocessing steps involve a series of sequential reactions that cannot be performed simultaneously, such as enzyme digestion followed by ligation. By adding reagents sequentially, these complex processes can be completed stepwise and in a controlled manner, ensuring that each reaction is carried out under optimal conditions, thereby guaranteeing the quality of the final data.

[0079] All reactions were carried out in situ within microwells where cells were immobilized and isolated. Intermediate and final products were always confined within their respective microwells, fundamentally preventing cross-contamination of molecular information between different single cells. This is a prerequisite for obtaining accurate and reliable single-cell data, rather than relying on pooled averages.

[0080] By adding the same reagent synchronously and uniformly to the entire array sequentially, it can be ensured that all cells are under the same reaction conditions at the same time, which greatly reduces the technical errors caused by reaction time differences or uneven conditions, and improves the repeatability of the experiment and the comparability of data between different batches.

[0081] S30: Adhere the coding layer to the microwell layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of single cells in multiple microwells.

[0082] In some embodiments, the microfluidic chip may have a coding layer. The coding layer may be bonded to the microwell layer to introduce multiple first nucleotide fluids into each row of microwells in the microwell array; each first nucleotide fluid is used to enter a single cell within a row of microwells in the microwell array and connect with nucleic acid molecules within the single cell; multiple second nucleotide fluids are introduced into each column of microwells in the microwell array; each second nucleotide fluid is used to enter a single cell within a column of microwells in the microwell array and pair with a pre-connected first nucleotide sequence within the single cell.

[0083] Multiple different first nucleotide barcode fluids can be introduced into the fluid channels of the coding layer aligned with the row direction of the microwell array. Each first nucleotide barcode fluid is configured to flow into all microwells in that row via its corresponding row channel and permeate into the single cells within each microwell in that row, where it covalently binds to the target nucleic acid molecule within the cell via an enzymatic ligation reaction. Subsequently, multiple different second nucleotide barcode fluids are introduced into the fluid channels of the coding layer aligned with the column direction of the microwell array. Each second nucleotide barcode fluid is configured to flow into all microwells in that column via its corresponding column channel and further into the single cells within each microwell in that column, where it pairs and ligates with the labeled first nucleotide barcode sequence within the cell via specific hybridization or enzymatic ligation, thereby forming a unique combined barcode with row and column two-dimensional identification on the nucleic acid molecule of each single cell.

[0084] By applying different nucleotide barcodes row by row and column by column, each single cell in a microwell acquires a unique row and column combination sequence identifier. This sequence serves as the cell's identity identifier in subsequent sequencing, ensuring that massive sequencing data can be accurately traced back to the original microwell spatial location and individual cells. Applying fluid independently row by row and column by column, and utilizing the physical isolation properties of the microfluidic structure, effectively prevents mixing between different barcode reagents, ensuring coding specificity and data reliability. Furthermore, by employing a strategy of first encoding the entire cell and then encoding the entire column, single-cell encoding of m×n microwells can be completed in only (m+n) operations, significantly improving coding efficiency.

[0085] S40: Nucleic acid molecules with nucleotide sequences are released by cleaving single cells in multiple microwells through chemical or electrolytic cleavage.

[0086] In some embodiments, single cells in multiple microwells are cleaved by chemical lysis to release nucleic acid molecules with nucleotide sequences.

[0087] A lysis buffer can be added to the microwell array, acting in parallel on multiple microwells to rupture the immobilized single-cell membrane structures, thereby releasing nucleotide-tagged nucleic acid molecules from within the cells. The lysed nucleic acid molecules remain spatially located within their original microwells or can be uniformly recovered, and their covalently linked barcode sequences remain intact, preparing them for subsequent molecular recognition and sequencing analysis based on single-cell origin.

[0088] By performing in-situ lysis after encoding, the mixing of contents from different single cells can be avoided, effectively preventing cross-well contamination. This ensures that the nucleic acid molecules of each cell, after release, still correspond to their original microwell positions via their associated barcodes, maintaining single-cell-level data resolution. Furthermore, the lysis buffer can process all cells in the entire microwell array simultaneously, guaranteeing lysis efficiency and consistency, and simplifying the operational steps. The released nucleic acid molecules can be uniformly recovered and used for library construction and sequencing, compatible with high-throughput sequencing workflows, improving experimental throughput and data analysis efficiency.

[0089] In some embodiments, single cells in multiple microwells are lysed by electrolysis to release nucleic acid molecules with nucleotide sequences.

[0090] A controllable electric field system can be constructed on a microwell array. By adjusting the electrical pulse parameters, a critical transmembrane potential can be formed across the cell membrane, inducing electroporation or dielectric breakdown in the lipid bilayer, thereby achieving millisecond-level rapid lysis. This electrolysis process can be simultaneously applied to multiple microwells in the array using a specifically designed electrode configuration, ensuring that single cells within each microwell undergo membrane structure disruption simultaneously in a uniform electric field environment, releasing their spatially encoded nucleic acid molecules. This lysis method based on physical field interaction completely avoids the use of chemical lysis reagents, eliminating the need for temperature-dependent long-term incubation reactions and omitting cumbersome subsequent washing steps. This maximizes the preservation of the integrity and bioactivity of nucleic acid molecules, providing a high-quality material basis for downstream molecular analysis. Compared to the incubation process of traditional chemical lysis, which requires tens of minutes to hours, electrolysis can be completed in milliseconds, significantly shortening the experimental cycle and increasing overall analytical throughput.

[0091] S50: Collect nucleic acid molecules with nucleotide sequences and perform sequencing analysis for the target omics.

[0092] In some embodiments, nucleic acid molecules carrying nucleotide sequences released by lysis can be extracted from the microwell and a library can be constructed; the library can be sequenced to obtain sequencing analysis data of the target omics.

[0093] The lysed nucleic acid molecules carrying nucleotide sequences can be processed as follows: a microfluidic collection system can be used to recover the lysate containing the target nucleic acid molecules from the microwell array; proteins, cell debris, and other impurities can be removed by magnetic bead purification to obtain a high-purity nucleic acid sample; subsequently, library construction can be performed, including end-flattening of nucleic acid fragments using end-repair enzymes, ligation of sequencing aptamers at both ends of the fragments, and pre-amplification using specific primers to obtain a sufficient quantity of sequencing library; finally, the constructed library is subjected to quality testing, including detecting fragment distribution using a bioanalyzer and accurately quantifying the library concentration using qPCR to ensure that the library quality meets sequencing requirements.

[0094] High-throughput sequencing analysis can be performed on qualified libraries, including using a paired-end sequencing strategy to simultaneously read sample indexes, cell barcodes, molecular tags, and target gene sequences during sequencing. By performing quality filtering, barcode identification, and sequence alignment on the raw sequencing data, sequencing reads are accurately classified to their corresponding single-cell origins, thereby generating an expression matrix containing multi-omics data such as gene expression levels, mutation information, and epigenetic characteristics, providing a complete and reliable data foundation for subsequent single-cell multi-omics analysis.

[0095] S60: Determine the single-cell spatial coding data of each nucleic acid molecule based on the nucleotide sequence, and obtain the target omics data for each single cell.

[0096] In some embodiments, single-cell spatial coding data of each nucleic acid molecule is determined based on the nucleotide sequence, and target omics data of each single cell are obtained.

[0097] Based on the barcode information in the nucleotide sequence, bioinformatics methods are used to decode the spatial coding data carried by each nucleic acid molecule, thereby accurately tracing the sequencing reads back to the single cell of origin. Subsequently, quantitative and omics characterization analysis can be performed on all nucleic acid molecules belonging to the same single cell, thereby obtaining the molecular expression profile or other omics parameters of each single cell at the target omics level.

[0098] In some embodiments, step S60 may further include: determining the spatial coding data of the nucleic acid molecules based on the nucleotide data of each nucleic acid molecule; determining the single cell to which the nucleic acid molecule belongs based on the spatial coding data of the nucleic acid molecule; and obtaining the molecular analysis results of the target omics of the single cell.

[0099] Based on the coding sequence regions in the sequencing reads of each nucleic acid molecule, bioinformatics algorithms can be used to analyze and extract the spatial coding data contained therein. Using predefined mapping relationships, the spatial coding data is matched with the physical coordinates of the microwell array, thereby uniquely determining the single-cell origin of each nucleic acid molecule.

[0100] Specifically, the massive reads generated by sequencing can be demultiplexed. By identifying the spatial coding segments embedded in the nucleotide sequence of each read and mapping them back to the two-dimensional physical coordinates of the microwell array, the origin of each molecule can be uniquely determined as a single cell. Based on this, cross-population integration analysis can be performed on the dynamic trajectories of single cells corresponding to different spatial codes: by constructing a cell state evolution model, the molecular changes of cells in different spatial locations over time can be compared to reveal the spatiotemporal characteristics of cell heterogeneity; at the same time, a spatial distribution map of cell population differentiation pathways can be established to identify the influence of the microenvironment on cell fate determination.

[0101] In some embodiments, the microfluidic chip may further include a conductive capping layer; the microwell layer further includes a conductive substrate layer disposed below and bonded to the microwell array. Based on this, step S40 may specifically include: aligning and bonding the conductive capping layer to the microwell array; connecting the conductive capping layer and the conductive substrate layer to two power supply terminals respectively, to form an independent electric field in each microwell of the microwell array; applying continuous electrical pulses to cause the single cells in each microwell to lyse synchronously under the action of the electric field, releasing nucleic acid molecules with nucleotide sequences.

[0102] The conductive capping layer can be precisely aligned and bonded to the microwell array carrying the single cells, ensuring that the lower surface of the conductive capping layer and the upper surface of the microwell array form a completely sealed contact, thereby constructing a closed electric field interaction space.

[0103] After precise alignment, the conductive capping layer and the conductive substrate layer located below the microwell array can be connected to the positive and negative electrodes of the pulse power supply, respectively. When energized, an independent parallel electric field is formed inside each microwell, and this electric field distribution has high uniformity and spatial confinement.

[0104] A series of continuous and controllable electrical pulses can be applied. Under the precise control of these pulses, single cells in each microwell synchronously undergo electroporation and dielectric breakdown of the membrane structure. First, the cell membrane polarizes under a strong electric field, causing molecules in the phospholipid bilayer to rearrange and form nanoscale hydrophilic channels. As the electric field continues, these channels gradually expand and connect, ultimately leading to irreversible damage to the cell membrane structure and releasing nucleic acid molecules with labeled nucleotide sequences from within the cell.

[0105] The entire lysis process requires no chemical lysis reagents, which not only avoids potential damage to nucleic acid molecules from chemicals but also eliminates the inhibitory effect on downstream molecular biological reactions. This parallel electric field-based electrolysis technique provides reliable technical support for high-quality single-cell multi-omics research. Its highly synchronized and standardized characteristics make it particularly suitable for large-scale single-cell analysis projects. By establishing independent parallel electric fields in each microwell, all single cells are ensured to lyse synchronously under the same electric field strength, effectively eliminating the differences in lysis efficiency caused by uneven diffusion or temperature gradients in traditional chemical lysis. This electrolysis scheme eliminates the need to prepare complex lysis reagents, saving temperature control, long incubation times, and subsequent washing steps, shortening the lysis process from several hours to milliseconds, significantly improving experimental efficiency and reducing operational complexity. Furthermore, this electrolysis scheme avoids potential damage and modification to nucleic acid molecules by chemical lysis agents, maximizing the integrity and authenticity of nucleic acid sequences, and providing a high-quality sample basis for downstream high-precision omics analysis.

[0106] In some embodiments, the microfluidic chip may further include an interdigitated electrode capping layer; the interdigitated electrodes in the interdigitated electrode capping layer include multiple parallel electrodes. Based on this, step S40 may specifically include: aligning and attaching the interdigitated electrode capping layer to the microwell array, so that the multiple parallel electrodes of the interdigitated electrode capping layer are interlaced between multiple rows of microwells in the microwell array; connecting the two ends of the interdigitated electrodes to two power supply poles respectively, so as to form an electric field between adjacent parallel electrode fingers; applying continuous electrical pulses, so that the single cells in each row of microwells are synchronously lysed under the action of the electric field, releasing nucleic acid molecules with nucleotide sequences.

[0107] The interdigitated electrode capping layer can be precisely aligned and bonded to the microwell array carrying the single cells in three-dimensional space. Specifically, the parallel electrode array on the lower surface of the interdigitated electrode capping layer can be precisely interlaced with the interrow gaps of the microwell array, ensuring that each electrode finger can be accurately embedded in the gap area between two adjacent rows of microwells.

[0108] After completing the three-dimensional alignment and bonding, the output interfaces at both ends of the interdigital electrodes can be connected to the positive and negative output terminals of a high-frequency pulse power supply, respectively. Upon power-up, an electric field is generated between adjacent electrode fingers. Furthermore, by optimizing the electrode geometry, including electrode width, spacing, and finger length, the electric field intensity can be optimally distributed within the microwell region, ensuring that each single cell in the microwell receives a sufficient and uniform electric field.

[0109] A series of continuously controllable electrical pulses can be applied. Under the action of this pulse sequence, the phospholipid bilayer of the cell membrane undergoes polarization and reorganization under the influence of a strong electric field, forming nanoscale transient hydrophilic channels. This process is known as field-mediated electroporation of the cell membrane. With the continued action of the electroporation effect, the integrity of the cell membrane structure is disrupted, leading to irreversible membrane perforation and the failure of the intracellular and extracellular material exchange barrier. Under the influence of the osmotic pressure difference, cytoplasmic contents begin to leak out, while external buffer solutions enter the cell, ultimately causing complete rupture of the cell membrane.

[0110] This interdigitated electrode-based electrolysis technology not only achieves efficient and synchronous lysis of single cells, but also ensures high consistency and reproducibility of the lysis process due to its unique electric field distribution characteristics, providing strong technical support for large-scale single-cell research. The alternating electric field formed between rows of microwells by the interdigitated electrode structure has high uniformity and spatial concentration, enabling each single cell to obtain optimal electric field strength and high lysis efficiency. Furthermore, this electrolysis scheme solves the dependence of traditional electrolysis on conductive substrates, and can be directly applied to microwell layers made of non-conductive materials such as PDMS, greatly expanding the range of chip materials and reducing manufacturing costs. In addition, through synchronous activation of the inter-row electric field, parallel lysis of entire rows of cells can be achieved, increasing the throughput by orders of magnitude while maintaining single-cell accuracy.

[0111] In some embodiments, step S60 may further include: determining single-cell heterogeneity data based on the molecular analysis results of the target omics for each single cell.

[0112] Based on multi-omics features such as gene expression levels, chromatin accessibility, and protein expression levels at single-cell resolution, a molecular feature vector is constructed for each cell. By calculating the degree of similarity of molecular features among cells, cells with highly similar molecular patterns are divided into several populations, achieving preliminary structured classification of cell populations.

[0113] Furthermore, nonlinear transformation methods can be used to project high-dimensional molecular features into a two-dimensional or three-dimensional visualization space. Based on the distribution pattern of cells in the low-dimensional space, the natural aggregation state and transition region of cell populations can be presented, thereby characterizing potential cell subpopulations and their continuous change trends.

[0114] Furthermore, by statistically comparing the expression levels of molecular characteristics among different cell populations, key molecular markers that are significantly overexpressed or underexpressed in specific cell groups can be identified. These specific markers can then be used to functionally annotate cell subtypes, distinguishing biological state differences between cell groups, such as metabolic activity, stress response, or signaling pathway activation status. Additionally, by constructing trajectory models of cell state transitions, the dynamic pathways of cell evolution from initial to terminal states can be inferred, key molecular events at branching nodes can be analyzed, and the evolutionary processes of cell differentiation, activation, or malignant transformation can be reconstructed.

[0115] Based on the above analysis, rare cell subpopulations with extremely low frequency but specific molecular characteristics can be identified, their molecular characteristics can be quantified to determine the degree of difference between them and the mainstream population, and a single-cell heterogeneity data system can be constructed, including fine classification of cell subtypes, discrete and continuous variation spectrum of functional states, developmental trajectory branching path selection mechanism, and qualitative and quantitative characteristics of rare cell populations.

[0116] This integrated analytical process enables the transformation from molecular characteristics to biological meaning, providing systematic data support for understanding the diversity, dynamics, and functionality of cell populations.

[0117] In some embodiments, the above-described method for acquiring single-cell omics data can be applied to a microfluidic chip; the microfluidic chip includes a microwell layer, a fence layer, and a coding layer. Specifically, the method for acquiring single-cell omics data may include: controlling a microwell array of the microwell layer to capture multiple single cells in a cell suspension, each microwell of the microwell array capturing one single cell; controlling the fence layer to adhere to the microwell layer to induce molecular reactions of the single cells in the multiple microwells of the microwell array using reaction reagents; controlling the coding layer to adhere to the microwell layer to insert spatial coding information into the nucleic acid sequences of the single cells in the multiple microwells using nucleotide sequences; controlling the fence layer to adhere to the microwell layer to lyse the single cells in the multiple microwells through chemical lysis, or controlling the conductive capping layer to adhere to the microwell layer to lyse the single cells in the multiple microwells through electrolysis; and determining the molecular analysis results of the target omics of the corresponding single cell based on the spatial coding information of the nucleic acid sequences released from the lysed single cells.

[0118] In some embodiments, the fence layer includes vias corresponding to the microwell array; the control of the fence layer to adhere to the microwell layer to enable single cells in multiple microwells of the microwell array to perform molecular reactions of target omics using reaction reagents includes: controlling the fence layer to adhere to the microwell layer to sequentially add multiple reaction reagents into the vias; the multiple reaction reagents are used to enable single cells in multiple microwells of the microwell array to perform multi-step continuous molecular reactions of target omics.

[0119] In some embodiments, the coding layer includes a row coding layer and a column coding layer; the row coding layer includes multiple rows of parallel microchannels, each corresponding to a row of microwells in the microwell array; the column coding layer includes multiple columns of parallel microchannels, each corresponding to a column of microwells in the microwell array; the control coding layer is attached to the microwell layer to insert spatial coding information into the nucleic acid sequences of single cells in multiple microwells via nucleotide sequences, including: controlling the row coding layer to be attached to the microwell layer to introduce multiple first nucleotide fluids into the multiple rows of parallel microchannels; each first nucleotide fluid is used to enter the single cells of the multiple microwells in the corresponding row and connect with the nucleic acid sequences within the single cells; controlling the column coding layer to be attached to the microwell layer to introduce multiple second nucleotide fluids into the multiple columns of parallel microchannels; each second nucleotide fluid is used to enter the single cells of the multiple microwells in the corresponding column and pair with the already connected first nucleotides within the single cells.

[0120] In some embodiments, the above-described method for acquiring single-cell omics data can be based on Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 , Figure 17 , Figure 18 The microfluidic chip shown is used.

[0121] In some embodiments, before step S10, the following steps may be included: collecting cultured cells, staining them with Hochest staining agent, and fixing the cells with paraformaldehyde solution to cross-link intracellular proteins and maintain the cells' intact morphology. Staining with Hochest staining agent facilitates subsequent observation. For multi-omics analysis, cells may undergo special treatments, such as treating cells with actinomycete ketone solution to pause intracellular translation in translatomics analysis.

[0122] like Figure 2 As shown, in some embodiments, step S10 may include: adding a cell suspension above the microwell layer 1, allowing the cells to settle into the microwell array 6 due to gravity, with each second well 8 accommodating only one cell; after all cells have settled, rinsing away excess cells not in the microwell array 6 with PBS solution to achieve single-cell separation. The structure of the microwell layer 1 is as follows: Figure 6 As shown, a microwell array 6 is arranged in a neat pattern in the central region of the upper surface, and a first glass substrate 7 is placed on the lower surface. The microwell array has i*j vertically penetrating second through-holes 8, each through-hole being large enough to accommodate only one single cell for single-cell separation. Each through-hole can be represented by (i, j) coordinates, thereby marking the biological information of each single cell.

[0123] In some embodiments, step S20 may include: replacing the PBS solution on the surface of the microwell layer 1 with DI water by adding DI water with one end of a syringe and withdrawing the solution with a pipette at the other end. The solution is replaced in small amounts multiple times, leaving only a very thin water film on the surface of the microwell layer 1, which is then allowed to air dry. This reduces the amount of crystallization left by the PBS solution after drying, facilitating adhesion to the fence layer 5. The fence layer 5, used in conjunction with the microwell layer 1, is suitable for large-area synchronous cell reactions and can perform multi-step continuous molecular reactions, such as pore-forming reactions and enzymatic digestion reactions. Figure 3As shown, biochemical reaction reagents are added to the first well 15, ensuring full contact between the solution and the cells in the microwell array 6 below. This allows for the replacement of different multi-step reaction modules for various single-cell omics analyses, including but not limited to adding T4 PNK for RNA end-repair reactions after enzyme digestion in translatomics analysis; adding poly(A) polymerase reaction solution for polymerase ligation reactions and adding reverse transcription reaction solution for on-chip reverse transcription in whole RNA transcriptomics analysis. The structure of the fence layer 5 is as follows... Figure 7 As shown, there is a first through hole 15 running vertically through the middle, and the fence layer 5 can be attached to the micro well layer 1 to form a liquid storage pool.

[0124] like Figure 4 As shown, in some embodiments, step S30 may include: aligning and attaching the A-coded microchannel layer 2 with the microwell layer 1, so that the first parallel microchannel 10 in the middle i-row horizontal direction of the A-coded microchannel layer 2 corresponds one-to-one with the i-row microwell array 6 of the microwell layer 1, forming i independent closed fluid channels; introducing A-group nucleotide sequences (Barcode A1, A2, A3, ... Ai) at the inlet 9; and under negative pressure at the outlet 12, filling the i fluid channels with the A-group nucleotide sequences (Barcode A1, A2, A3, ... Ai), making full contact with the cells captured in the i-row second through-hole 8, allowing the A-group nucleotide sequences (Barcode A1, A2, A3, ... Ai) to enter the cells and bind to the target nucleic acid sequences within the cells, resulting in i rows of cells with different labels. Secondly, the microchannel layer 3 encoding B can be aligned and fitted with the microwell layer 1, so that the first parallel microchannel 10 in the vertical direction of the middle j column of the microchannel layer 3 encoding A corresponds one-to-one with the microwell array 6 in the j column of the microwell layer 1, forming j independent closed fluid channels. The B group nucleotide sequence (Barcode B1, B2, B3, ... Bj) is introduced at the inlet 9. Under negative pressure at the outlet 12, the B group nucleotide sequence (Barcode B1, B2, B3, ... Bj) will fill the j fluid channels and come into full contact with the cells captured in the second through-hole 8 in the j column. This allows the B group nucleotide sequence (Barcode B1, B2, B3, ... Bj) to enter the cell and pair and connect with the A group nucleotide sequence (Barcode A1, A2, A3, ... Ai) that has already bound to the target nucleic acid sequence, resulting in i×j cells with different two-dimensional labels, thus realizing the cross-coding of cells. Under the action of coding A microchannel layer 2 and coding B microchannel layer 3, the microwell array 6 in the middle of microwell layer 1 forms a mesh structure. Each cell in the second through-hole 8 has unique positional and coding information, which facilitates subsequent analysis of cell heterogeneity.

[0125] In some embodiments, for partial omics analysis, further biochemical reactions or processing steps are required after single-cell encoding. These include, but are not limited to, the ligation reaction of the target sequence palindrome structure in three-dimensional genomics analysis. The fence layer 5 can be used in conjunction with the microwell layer 1, and molecular reaction reagents can be added to the first through-hole 15 to perform the ligation reaction of the target sequence with the palindrome structure.

[0126] In some embodiments, step S40 may include: using the fence layer 5 in conjunction with the microwell layer 1 to perform a unified cell lysis reaction for various omics. This is chemical lysis, using a specific ratio of lysis buffer, and continuously reacting at a certain temperature to break the cell membrane, releasing the target nucleic acid molecules from the cell. These released target nucleic acid molecules are then collected for subsequent molecular experimental analysis of the target omics.

[0127] In some embodiments, step S50 may include: collecting the lysate in the first through-hole 15 of the fence layer 5 for subsequent molecular experimental analysis, such as extraction and purification, reverse transcription, PCR amplification, and library construction.

[0128] Specifically, in the cell lysis stage, in addition to the chemical lysis method in step S40 above, electrolysis can also be used for cell lysis. Firstly, electrolysis has the advantages of being fast and efficient, completing the process in milliseconds, and does not require complex reagent ratios or specific reaction temperature requirements. Figure 5 As shown, chemical lysis typically requires several tens of minutes to several hours under certain temperature conditions. Secondly, chemical lysis agents remain in the solution, affecting downstream reactions, while electrolysis eliminates the need for washing to remove chemical residues. Finally, electrolysis creates multiple localized micro-electric fields with a more uniform distribution, ensuring that each cell receives almost identical electrical stimulation.

[0129] One electrolysis method uses a conductive microwell layer and a conductive capping layer. Another method uses a non-conductive microwell layer and an interdigitated electrode capping layer. For example, when lysing cells using electrolysis, the two ends of the electric field are connected to the positive and negative terminals of a power source. After about 10 electrical pulses, the cells will lyse and release the target nucleic acids required for each omics study, all within tens of milliseconds. Silver-PDMS flexible conductive material, commonly used in electrolysis, offers good performance, high adhesion, and low cost. PDMS is a highly elastic silicone rubber that retains good tensile and bending properties even after the addition of silver conductive filler, exhibiting good stability and flexibility. This results in better adhesion and more uniform current distribution during use. Furthermore, compared to carbon-based conductive composites, silver-PDMS is less sensitive to environmental changes such as temperature and humidity, and its conductivity is more stable.

[0130] In some embodiments, the microwell layer 1 can be divided into a conductive microwell layer and a non-conductive microwell layer. The overall structure of the conductive microwell layer is as follows: Figure 12 As shown, the structure includes an upper microwell array 6, a middle conductive substrate layer 16, and a lower first glass substrate 7, which are tightly bonded together. The microwell array 6 is composed of i×j (i=80, j=80) vertically penetrating second through-holes 8 arranged in an orderly manner, which are used to capture and separate single cells. The conductive substrate layer 16 is made of ITO (indium tin oxide) and is used for subsequent electrolytic cell lysis reactions.

[0131] In some embodiments, the overall structure of the non-conductive microwell layer is as follows: Figure 13 As shown, it is made by molding process, with microwell array 6 on the upper surface for capturing and separating single cells, and the rest is made of PDMS material 21 cured.

[0132] In some embodiments, during an electrolytic cell lysis reaction, a conductive capping layer 17 can be aligned and bonded to the microwell layer 1, so that the conductive material layer 19 on the lower surface of the capping layer completely covers the microwell array. At this time, the bottom layer of each second via 8 of the microwell array 6 is an ITO conductive substrate layer 16, and the top layer is a conductive material layer 19. These two parts are connected to the positive and negative terminals of the power supply through wires, and each second via 8 forms an electric field. The cells captured in the second via 8 can be rapidly lysed under the action of the electric field.

[0133] In some embodiments, the structure of the silver-PDMS cover layer is as follows: Figure 15 As shown, silver is filled into PDMS and cured to form a silver-PDMS block 18. Silver-PDMS is a flexible conductive material with excellent conductivity and mechanical stability, and is widely used in flexible sensors, biosensors and other fields.

[0134] In some embodiments, the structure of the conductive cover layer is as follows: Figure 16 As shown, the upper surface is a second glass substrate 20, and the lower surface is a conductive material layer 19. The conductive material includes, but is not limited to, ITO, metal, etc.

[0135] like Figure 17 and Figure 18 As shown, in some embodiments, in another electrolytic cell lysis reaction, the interdigitated electrode capping layer 22 can be aligned and attached to the microwell layer 1, so that the interdigitated structures 24 on the lower surface of the interdigitated electrode capping layer 22 are interlaced in the middle of each row of second through holes 8 of the microwell array 6. The two ends of the interdigitated electrodes are connected to the positive and negative terminals of the power supply through wires, and an electric field is formed between each two adjacent interdigitated electrodes. The cells captured in each row of second through holes 8 between each electric field are lysed under the action of electricity.

[0136] In some embodiments, the structure of the interdigitated electrode cover layer 22 is as follows: Figure 13As shown, the lower surface has uniformly distributed interdigitated grooves 24 filled with silver, while the remaining portion is made of PDMS material 23. The interdigitated structures 24 result in a larger effective electrode area, a more concentrated electric field, and higher sensitivity. Meanwhile, the materials of the interdigitated electrode capping layer 22 include, but are not limited to, silver-PDMS, ITO glass, and metals, with silver-PDMS being preferred.

[0137] The following are three specific embodiments of this specification:

[0138] Example 1: Test of the effectiveness of single-cell translatomics analysis, the specific steps are as follows:

[0139] (1) Preparation of cell solution. Collect the cultured cells and treat them with 100 μg / mL actinomycin ketone solution for 5 min to pause the translation process. Then stain with Hochest staining agent for 15 min for easy observation. Centrifuge and wash the cells and fix them with 4% paraformaldehyde solution to maintain the integrity of the cell morphology. Centrifuge and wash the cells again and add a small amount of PBS solution to obtain a higher concentration of cell solution.

[0140] (2) Cell separation. The prepared cell solution is loaded onto the micro-well array 6 of micro-well layer 1, such as... Figure 2 As shown, due to surface tension, the cell suspension gradually spreads out, and the cells in the liquid gradually diffuse and settle into the microwell array 6 by their own gravity. Excess cells not in the microwell array 6 are washed away with PBS solution, completing the separation of single cells. The structure of microwell layer 1 is as follows: Figure 6 As shown, the structure includes a microwell array 6 on the upper surface and a first glass substrate 7 on the lower surface, which are tightly bonded together. The microwell array 6 is composed of i×j (i=120, j=120) vertically penetrating second through holes 8 arranged in an orderly manner, which is used to capture and separate single cells.

[0141] (3) Clean the surface of microwell layer 1. Replace the PBS solution on the surface of microwell layer 1 with DI water. Use a syringe to add DI water at one end and a pipette to draw the solution at the other end. Repeat the process in small amounts until only a very thin water film is left on the surface of microwell layer 1. Allow it to air dry. The purpose is to reduce the crystals left by the PBS solution after air drying, which will facilitate subsequent bonding with fence layer 5, coded A microchannel layer 2, and coded B microchannel layer 3.

[0142] (4) Secure the fence layer 5 and the microwell layer 1 tightly together, ensuring that the first through hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add 0.5% Triton X-100 solution to the first through hole 15 and culture at room temperature for 20 min to permeate and punch the cells.

[0143] (5) Replace the liquid in the fence layer 5 with RNase I enzyme digestion solution, and incubate at room temperature for 30 min to obtain mRNA fragments wrapped by ribosomes.

[0144] (6) Replace the liquid in the fence layer 5 with T4 PNK reaction solution and incubate at 37°C for 60 min to perform end repair on the ribosome-encapsulated mRNA fragments.

[0145] (7) Remove the fence layer 5 from the microwell layer 1, and align and attach the coded A microchannel layer 2 to the microwell layer 1 under a microscope, as shown. Figure 8 As shown, a group A nucleotide sequence (Barcode A1, A2, A3, ..., Ai, i=120) is introduced at inlet 9. A vacuum cover 4 is placed above outlet 12, and negative pressure is created by evacuation, allowing (Barcode A1, A2, A3, ..., Ai, i=120) to enter the first parallel microchannel 10. T4 RNA ligase is used to ligate the ribosome-encapsulated mRNA fragments captured in each second well 8 of the microwell array 6. The mixture is then incubated at room temperature for 3 hours. The structure of the microchannel layer 2 encoding group A is shown below. Figure 10 As shown, it consists of three parts: inlet 9, first parallel microchannel 10, and outlet 12. Each inlet 9 and each outlet 12 is connected by a microchannel. Each microchannel is parallel to the others, forming an i-row horizontal first parallel microchannel 10 in the central region, corresponding to the i-row second through-hole 8 of the microwell array 6 in the microwell layer 1. The structure of the vacuum cover layer 4 is as follows. Figure 11 As shown, there is a small cube-shaped groove 13 on the lower surface and a vent 14 in the middle of the upper surface, which is connected to the small cube-shaped groove 13 on the lower surface. When used with an air extraction device, a negative pressure environment is formed inside the small cube-shaped groove 13.

[0146] (8) Remove the coded A microchannel layer 2 and clean the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, align and attach the coded B microchannel layer 3 to the microwell layer 1 under a microscope, as shown. Figure 9 As shown, a group B nucleotide sequence (Barcode B1, B2, B3, ..., Bj) is introduced at inlet 9. A vacuum hood 4 is placed over outlet 12, and negative pressure is created by evacuation, allowing the group B nucleotide sequence (Barcode B1, B2, B3, ..., Bj) to enter the second parallel microchannel 11. T4 RNA ligase is used to ligate the intracellular ribosome-encapsulated mRNA fragments captured in each second well 8 of the microwell array 6. The mixture is then cultured at 37°C for 2.5 hours. The structure of the microchannel layer 3 encoding group B is shown below. Figure 10As shown, it consists of three parts: inlet 9, middle second parallel microchannel 11, and outlet 12. Each inlet 9 and each outlet 12 are connected by a microchannel. Each microchannel is parallel to each other, forming j columns of vertical second parallel microchannels 11 in the central region, corresponding to j columns of second through holes 8 in the microwell array 6 in the microwell layer 1.

[0147] (9) Remove the microchannel layer 3 encoding B and wash the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, tightly attach and fix the fence layer 5 to the microwell layer 1, ensuring that the first through-hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add the prepared lysis buffer to the first through-hole 15 and incubate at 55°C for 2 hours to release the encoded ribosome-encapsulated mRNA fragment.

[0148] (10) Collect the lysate from the first well 15 of the fence layer 5 for subsequent extraction, purification, reverse transcription, PCR amplification and library construction.

[0149] Example 2: Test of the effectiveness of single-cell three-dimensional genomics analysis, the specific steps are as follows:

[0150] (1) Preparation of cell solution. Collect the cultured cells and stain them with Hochest staining agent for 15 min for easy observation. Centrifuge and wash the cells and fix them with 4% paraformaldehyde solution to achieve intracellular protein cross-linking and maintain the integrity of the cell morphology. After the reaction, centrifuge and wash the cells again and add a small amount of PBS solution to obtain a higher concentration of cell solution.

[0151] (2) Cell separation. The prepared cell solution is loaded onto the microwell array 6 of microwell layer 1. Due to the surface tension, the cell suspension will gradually spread out, and the cells in the liquid will gradually diffuse and settle into the microwell array 6 by their own gravity. The excess cells not in the microwell array 6 on the surface of microwell layer 1 are washed away with PBS solution, thus completing the separation of single cells.

[0152] (3) Clean the surface of microwell layer 1. Replace the PBS solution on the surface of microwell layer 1 with DI water. Use a syringe to add DI water at one end and a pipette to draw the solution at the other end. Repeat the process in small amounts until only a very thin water film is left on the surface of microwell layer 1. Allow it to air dry. The purpose is to reduce the crystals left by the PBS solution after air drying, which will facilitate subsequent bonding with fence layer 5, coded A microchannel layer 2, and coded B microchannel layer 3.

[0153] (4) Secure the fence layer 5 and the microwell layer 1 tightly together, ensuring that the first through hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add 0.5% Triton X-100 solution to the first through hole 15 and culture at room temperature for 20 min to permeate and punch the cells.

[0154] (5) Replace the liquid in the fence layer 5 with DpnII restriction endonuclease reaction solution and incubate at room temperature for 30 min to allow it to act on the open regions of chromatin in the cell nucleus and produce the sticky ends of GATC.

[0155] (6) Remove the fence layer 5 from the microwell layer 1, and align and attach the coded A microchannel layer 2 to the microwell layer 1 under a microscope, as shown. Figure 8 As shown, a group of nucleotide sequences (Barcode A1, A2, A3, ..., Ai, i=120) are introduced into the inlet 9. A vacuum cover 4 is placed over the outlet 12. By pumping air, a negative pressure is formed, allowing (Barcode A1, A2, A3, ..., Ai, i=120) to enter the first parallel microchannel 10 and ligate with the intracellular enzyme-digested DNA captured in each second well 8 of the microwell array 6. The mixture is then cultured at 37°C for 2 hours.

[0156] (7) Remove the coded A microchannel layer 2 and clean the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, align and attach the coded B microchannel layer 3 to the microwell layer 1 under a microscope, as shown. Figure 9 As shown, a group B nucleotide sequence (Barcode B1, B2, B3, ... Bj) is introduced into the inlet 9, and a vacuum cover 4 is placed over the outlet 12. By evacuating the air to create negative pressure, the group B nucleotide sequence (Barcode B1, B2, B3, ... Bj) enters the second parallel microchannel 11 and is covalently linked with the intracellular group A nucleotide sequence (Barcode A1, A2, A3, ... Ai, i=120) captured in each second through-hole 8 of the microwell array 6. The mixture is then cultured at 37°C for 2 hours.

[0157] (8) Remove the coded B microchannel layer 3 and clean the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, tightly attach and fix the fence layer 5 to the microwell layer 1, ensuring that the first through hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add a palindromic structure connection system to the first through hole 15 and incubate at 37°C for 2 hours.

[0158] (9) Replace the liquid in the fence layer 5 with the prepared lysis buffer, and incubate at 55°C for 2 hours to completely digest the cell membrane, nuclear membrane and proteins, and release the encoded circular DNA.

[0159] (10) Collect the lysate from the first well 15 of the fence layer 5 for subsequent extraction, purification, reverse transcription, PCR amplification and library construction.

[0160] Example 3: Test of the effectiveness of single-cell whole RNA transcriptomics analysis, the specific steps are as follows:

[0161] (1) Preparation of cell solution. Collect the cultured cells and stain them with Hochest staining agent for 15 min for easy observation. Centrifuge and wash the cells and fix them with 4% paraformaldehyde solution to maintain the integrity of the cell morphology. After the reaction, centrifuge and wash the cells again and add a small amount of PBS solution to obtain a higher concentration of cell solution.

[0162] (2) Cell separation. The prepared cell solution is loaded onto the microwell array 6 of microwell layer 1. Due to the surface tension, the cell suspension will gradually spread out, and the cells in the liquid will gradually diffuse and settle into the microwell array 6 by their own gravity. The excess cells not in the microwell array 6 on the surface of microwell layer 1 are washed away with PBS solution, thus completing the cell separation.

[0163] (3) Clean the surface of microwell layer 1. Replace the PBS solution on the surface of microwell layer 1 with DI water. Use a syringe to add DI water at one end and a pipette to draw the solution at the other end. Repeat the process in small amounts until only a very thin water film is left on the surface of microwell layer 1. Allow it to air dry. The purpose is to reduce the crystals left by the PBS solution after air drying, which will facilitate subsequent bonding with fence layer 5, coded A microchannel layer 2, and coded B microchannel layer 3.

[0164] (4) Secure the fence layer 5 and the microwell layer 1 tightly together, ensuring that the first through hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add 0.5% Triton X-100 solution to the first through hole 15 and culture at room temperature for 20 min to permeate and punch the cells.

[0165] (5) Replace the liquid in the fence layer 5 with MgCl2 solution and incubate at 94℃ for 5 min to fragment all types of RNA in the cells.

[0166] (6) Replace the liquid in the fence layer 5 with T4 PNK reaction solution and incubate at 37°C for 30 min to perform end repair on the fragmented RNA.

[0167] (7) Replace the liquid in the fence layer 5 with poly(A) polymerase reaction solution, incubate at 37°C for 30 min, and ligate a poly(A) sequence to the 3' end of the fragmented RNA.

[0168] (8) Replace the liquid in the fence layer 5 with the reverse transcription reaction solution, incubate at room temperature for 30 min and at 42℃ for 130 min to achieve complementary pairing of the poly(A) sequence at the 3-end of the fragmented RNA.

[0169] (9) Remove the fence layer 5 from the microwell layer 1, and align and attach the coded A microchannel layer 2 to the microwell layer 1 under a microscope, as shown. Figure 8As shown, a group A nucleotide sequence (Barcode A1, A2, A3, ..., Ai, i=120) is introduced into the inlet 9. A vacuum cover 4 is placed over the outlet 12. By evacuating the air, a negative pressure is formed, allowing (Barcode A1, A2, A3, ..., Ai, i=120) to enter the first parallel microchannel 10. The fragmented RNA captured in each second well 8 of the microwell array 6 is covalently ligated using T4 DNA ligase. The mixture is then cultured at 37°C for 30 min.

[0170] (10) Remove the coded A microchannel layer 2 and clean the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, align and attach the coded B microchannel layer 3 to the microwell layer 1 under a microscope, as shown. Figure 9 As shown, a group B nucleotide sequence (Barcode B1, B2, B3, ... Bj) is introduced into the inlet 9, and a vacuum cover 4 is placed over the outlet 12. By evacuating the air to create negative pressure, the group B nucleotide sequence (Barcode B1, B2, B3, ... Bj) enters the second parallel microchannel 11. Using T4 DNA ligase, it is covalently ligated with the intracellular group A nucleotide sequence (Barcode A1, A2, A3, ... Ai, i=120) captured in each second well 8 of the microwell array 6. The mixture is then cultured at 37°C for 30 min.

[0171] (11) Remove the microchannel layer 3 encoding B and wash the surface of the microwell layer 1 with DI water. After the DI water on the surface of the microwell layer 1 has dried, tightly attach and fix the fence layer 5 to the microwell layer 1, ensuring that the first through-hole 15 in the middle of the fence layer 5 completely covers the microwell array 6 below. Add the prepared lysis buffer to the first through-hole 15 and incubate at 55°C for 2 hours to release the encoded fragmented RNA.

[0172] (12) Collect the lysate from the first well 15 of the fence layer 5 for subsequent extraction, purification, reverse transcription, PCR amplification and library construction.

[0173] Based on the above-described method for acquiring single-cell data, this specification also provides embodiments of a device for acquiring single-cell omics data. For example... Figure 19 As shown, the single-cell omics data acquisition device 1900 may specifically include the following modules:

[0174] The separation module 1901 is used to separate multiple single cells from a cell suspension through a microwell array of microwell layers, wherein each microwell of the microwell array contains one single cell.

[0175] Reaction module 1902 is used to attach the fence layer to the microwell layer so that single cells in multiple microwells of the microwell array can undergo molecular reactions for targeted omics through reaction reagents.

[0176] The coding module 1903 is used to attach the coding layer to the microwell layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of single cells in multiple microwells.

[0177] The lysis module 1904 is used to lyse single cells in multiple microwells via chemical or electrolysis to release nucleic acid molecules with nucleotide sequences.

[0178] Analysis module 1905 is used to collect nucleic acid molecules with nucleotide sequences and perform sequencing analysis for target omics.

[0179] The acquisition module 1906 is used to determine the single-cell spatial coding data of each nucleic acid molecule based on the nucleotide sequence and to acquire the target omics data of each single cell.

[0180] In some embodiments, the reaction module 1902 described above can be specifically used for:

[0181] Multiple reaction reagents are sequentially added to multiple microwells of the microwell array to enable single cells in the multiple microwells to perform multi-step continuous molecular reactions for target omics.

[0182] In some embodiments, the encoding module 1903 described above can be specifically used for:

[0183] Multiple first nucleotide fluids are introduced into each row of microwells in the microwell array; each first nucleotide fluid is used to enter a single cell in a row of microwells in the microwell array and connect with nucleic acid molecules within the single cell;

[0184] Multiple second nucleotide fluids are introduced into each column of microwells in the microwell array; each second nucleotide fluid is used to enter a single cell of a column of microwells in the microwell array and pair with the already linked first nucleotide sequence in the single cell.

[0185] In some embodiments, the microfluidic chip further includes a conductive capping layer; the microwell layer further includes a conductive substrate layer disposed below the microwell array and attached to the microwell array.

[0186] Based on this, the aforementioned pyrolysis module 1904 can be specifically used for:

[0187] Align and attach the conductive cap plate layer to the microwell array;

[0188] The conductive cover plate layer and the conductive substrate layer are respectively connected to the two poles of the power supply to form an independent electric field in each micro well of the micro well array;

[0189] Continuous electrical pulses are applied to cause the single cells in each microwell to lyse synchronously under the influence of the electric field, releasing nucleic acid molecules with nucleotide sequences.

[0190] In some embodiments, the microfluidic chip further includes an interdigitated electrode capping layer; the interdigitated electrodes in the interdigitated electrode capping layer include a plurality of parallel electrodes.

[0191] Based on this, the aforementioned pyrolysis module 1904 can be specifically used for:

[0192] The interdigitated electrode capping layer is aligned and attached to the microwell array so that multiple parallel electrodes of the interdigitated electrode capping layer are interspersed between multiple rows of microwells in the microwell array.

[0193] Connect the two ends of the interdigitated electrodes to the two poles of the power supply to create an electric field between adjacent parallel electrode fingers.

[0194] Continuous electrical pulses are applied to cause the single cells in each row of microwells to lyse synchronously under the influence of the electric field, releasing nucleic acid molecules with nucleotide sequences.

[0195] In some embodiments, the analysis module 1905 described above can be specifically used for:

[0196] Nucleic acid molecules carrying nucleotide sequences released from the cleavage were extracted from the microwell and a library was constructed.

[0197] The library was sequenced to obtain sequencing analysis data for the target omics.

[0198] In some embodiments, the acquisition module 1906 described above can be specifically used for:

[0199] Based on the nucleotide data of each nucleic acid molecule, determine the spatial coding data of the nucleic acid molecule;

[0200] Based on the spatial coding data of the nucleic acid molecule, determine the single cell to which the nucleic acid molecule belongs;

[0201] Obtain the molecular analysis results of the target omics of this single cell.

[0202] As can be seen from the single-cell omics data acquisition device provided in the embodiments of this specification above, the embodiments of this specification can separate multiple single cells from a cell suspension through a micro-well array of micro-well layers, where each micro-well in the micro-well array contains one single cell; a fence layer is attached to the micro-well layer to allow the single cells in the multiple micro-wells of the micro-well array to undergo molecular reactions for target omics using reaction reagents; a coding layer is attached to the micro-well layer to insert nucleotide sequences carrying spatial coding data into the nucleic acid molecules of the single cells in the multiple micro-wells; the single cells in the multiple micro-wells are lysed by chemical cleavage or electrolysis to release nucleic acid molecules carrying nucleotide sequences; the nucleic acid molecules carrying nucleotide sequences are collected and subjected to sequencing analysis for target omics; the single-cell spatial coding data of each nucleic acid molecule is determined based on the nucleotide sequence, and the target omics data of each single cell is obtained. Compared with existing methods, the embodiments of this specification, through the micro-well array structure, utilize gravity sedimentation and hydrodynamics to achieve parallel capture and separation of single cells, with each micro-well confining one cell, allowing thousands to tens of thousands of cells to be processed simultaneously in a single experiment. By adding customizable reaction reagents in situ within microwells, various omics analysis needs can be supported, and different omics pretreatment types can be achieved simply by changing the reagents. Introducing spatially encoded nucleotide sequences ensures a unique spatial coding combination on the nucleic acid molecular markers of each single cell. This coding allows for accurate tracing of the cell origin of each sequencing read in subsequent mixed sequencing, preventing cross-contamination of molecular information between cells and ensuring the purity and reliability of single-cell omics data.

[0203] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for acquiring single-cell data, characterized by, The microfluidic chip is applied to a microfluidic chip comprising a micro-well layer, a fence layer and an encoding layer; The method comprises: separating a plurality of single cells in a cell suspension through a micro-well array of the micro-well layer, each micro-well of the micro-well array containing one single cell; adhering the fence layer to the micro-well layer to make the single cells in a plurality of micro-wells of the micro-well array perform molecular reactions of target omics through reaction reagents; adhering the encoding layer to the micro-well layer to chain nucleotide sequences carrying spatial encoding data into nucleic acid molecules of the single cells in the plurality of micro-wells; lysis of the single cells in the plurality of micro-wells to release nucleic acid molecules with nucleotide sequences through chemical lysis or electrical lysis; collecting the nucleic acid molecules with nucleotide sequences and performing sequencing analysis of target omics; determining the spatial encoding data of each nucleic acid molecule based on the nucleotide sequences and obtaining the omics data of each single cell.

2. The method of claim 1, wherein, The method of making the single cells in a plurality of micro-wells of the micro-well array perform molecular reactions of target omics through reaction reagents comprises: adding a plurality of reaction reagents to the plurality of micro-wells of the micro-well array in sequence to make the single cells in the plurality of micro-wells perform a plurality of continuous molecular reactions of target omics.

3. The method of claim 1, wherein, The method of chaining nucleotide sequences carrying spatial encoding data into nucleic acid molecules of the single cells in the plurality of micro-wells comprises: passing a plurality of first nucleotide fluids into each row of micro-wells of the micro-well array respectively; each first nucleotide fluid is used to enter the single cells of one row of micro-wells of the micro-well array and connect with the nucleic acid molecules in the single cells; passing a plurality of second nucleotide fluids into each column of micro-wells of the micro-well array respectively; each second nucleotide fluid is used to enter the single cells of one column of micro-wells of the micro-well array and pair-connect with the connected first nucleotide sequences in the single cells.

4. The method of claim 1, wherein, The microfluidic chip further comprises a conductive cover plate layer; the micro-well layer further comprises a conductive substrate layer arranged below the micro-well array and adhered to the micro-well array; The method of lysis of the single cells in the plurality of micro-wells to release nucleic acid molecules with nucleotide sequences through chemical lysis or electrical lysis comprises: aligning and adhering the conductive cover plate layer to the micro-well array; connecting the conductive cover plate layer and the conductive substrate layer to two poles of a power source respectively to form an independent electric field in each micro-well of the micro-well array; applying continuous electric pulses to make the single cells in each micro-well lyse synchronously under the action of the electric field to release nucleic acid molecules with nucleotide sequences.

5. The method of claim 1, wherein, The microfluidic chip further comprises an interdigital electrode cover plate layer; the interdigital electrodes in the interdigital electrode cover plate layer comprise a plurality of parallel electrodes; aligning and adhering the interdigital electrode cover plate layer to the micro-well array to make the plurality of parallel electrodes of the interdigital electrode cover plate layer interpenetrate between a plurality of rows of micro-wells of the micro-well array; connecting two ends of the interdigital electrodes to two poles of a power source respectively to form an electric field between adjacent parallel electrode fingers; applying continuous electric pulses to make the single cells in each row of micro-wells lyse synchronously under the action of the electric field to release nucleic acid molecules with nucleotide sequences.

6. The method of claim 1, wherein, The method of performing sequencing analysis of target omics comprises: extracting the released nucleic acid molecules carrying nucleotide sequences from the micro-wells and constructing a library; sequencing the library to obtain sequencing analysis data of target omics.

7. The method of claim 1, wherein, The obtaining of the target omics data of each single cell comprises: determining spatial coding data of each nucleic acid molecule according to nucleotide data of the nucleic acid molecule; determining a single cell to which the nucleic acid molecule belongs according to the spatial coding data of the nucleic acid molecule; obtaining a molecular analysis result of the target omics of the single cell.

8. An acquisition device of single-cell data, characterized by, The device comprises: a separation module configured to separate a plurality of single cells in a cell suspension through a micro-well array of a micro-well layer, each micro-well of the micro-well array containing a single cell; a reaction module configured to attach a fence layer to the micro-well layer to make the single cells in the plurality of micro-wells of the micro-well array perform a molecular reaction of target omics through a reaction reagent; a coding module configured to attach a coding layer to the micro-well layer to chain a nucleotide sequence carrying spatial coding data into nucleic acid molecules of the single cells in the plurality of micro-wells; a lysis module configured to lyse and release the nucleic acid molecules carrying the nucleotide sequence from the single cells in the plurality of micro-wells through chemical lysis or electrical lysis; an analysis module configured to collect the nucleic acid molecules carrying the nucleotide sequence and perform sequencing analysis of target omics; an obtaining module configured to determine single cell spatial coding data of each nucleic acid molecule based on the nucleotide sequence and obtain target omics data of each single cell.

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