Biochips for spatial transcriptome analysis, their preparation and use - Patents.com

Abstract

The present invention provides a chip for analyzing nucleic acid information of a biological sample, and a method for manufacturing the chip. The chip is applied to analyze spatial transcriptome information of a biological tissue sample. The present invention further provides a method for analyzing spatial transcriptome information of a biological tissue sample. The method and device of the present invention can effectively obtain expression information (including spatial omics information) of nucleic acids in cells of a tissue sample.

Description

[Technical field]

[0001] This application claims priority to Chinese Application No. 202111601551.X (Title: Manufacturing Method of Biochip for Spatial Transcriptome Analysis and Use Thereof) filed on December 24, 2021 and Chinese Application No. 202210728404.7 (Title: Manufacturing Method of Biochip for Spatial Transcriptome Analysis and Use Thereof) filed on June 25, 2022, the entire contents of which are incorporated by reference into this application.

[0002] The present invention relates to the fields of biology and medical equipment. In particular, the present invention relates to a method for manufacturing a chip for analyzing nucleic acid information of cells in a biological sample, the chip being applied to analyze spatial transcriptome information of a biological tissue sample. [Background technology]

[0003] The analysis of cellular organization and expression patterns in biological tissues is a milestone in biomedical research and diagnostics. Cytological histology makes it possible to study protein distribution using various staining techniques, gene expression by immunohistochemistry and in situ hybridization.

[0004] New research promises to be able to characterize transcriptomic and / or genomic variation within tissues while preserving spatial information about the tissue.

[0005] In the prior art, chips are used to analyze nucleic acid information of cells in biological samples, and methods are provided for forming probes that identify and bind to the nucleic acids of biological samples on a substrate such as a glass sheet, as well as chip products that include these probes.

[0006] In the technical field, there is a need for a method for more convenient and efficient formation of probes capable of binding to target DNA or RNA molecules on a chip substrate in large-scale production of chips for analyzing nucleic acid information of cells in biological samples, to obtain chips with better quality, and to obtain manufacturing processes and products with more stable production rates. Summary of the Invention

[0007] The present invention provides a method for the large-scale production of biochips suitable for the analysis of cellular nucleic acid information of biological tissue samples, in particular spatial transcriptome information of biological tissue samples. Specifically, the present invention provides an improved method for producing a biochip having an array, the method comprising the steps of: Step 1: Provide a tip, Step 2: Immobilizing a chip surface linker nucleic acid on the chip surface, e.g., across the entire surface of the chip; Step 3: Add a first group of barcode nucleic acids to the chip surface through a plurality of microchannels arranged in parallel, and form a plurality of first barcode strips in a first direction under conditions in which the linker nucleic acid on the chip surface and the first barcode nucleic acid undergo a binding reaction, the first group of barcode nucleic acids includes a plurality of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is fixed to each first barcode strip, and the first barcode nucleic acids fixed to each first barcode strip have different barcode sequences; Step 4: Add a second group of barcode nucleic acids to the chip surface along a second direction through a plurality of parallel microchannels to form a plurality of second barcode strips, the second group of barcode nucleic acids including a plurality of second barcode nucleic acids having different barcode sequences, each second barcode strip has one type of second barcode nucleic acid, and the second barcode nucleic acids on each second barcode strip have different barcode sequences; Step 5: Under conditions in which the first barcode nucleic acid and the second barcode nucleic acid undergo a binding reaction, the second barcode nucleic acid and the first barcode nucleic acid are bound to each other at positions on the chip surface where the first barcode strips and the second barcode strips intersect to form probes, and the positions of the probes (i.e., the positions where the first barcode strips and the second barcode strips intersect) constitute features of the array, and each feature has probes whose sequences differ from each other.

[0008] In one aspect of the present invention, in the method, the first group of barcode nucleic acids or the second group of barcode nucleic acids are transported and fixed to the chip surface by a microchannel device having multiple microchannels arranged in parallel, and the surface of the microchannel that comes into contact with the chip surface is capable of passing through a solution or nucleic acids in a solution.

[0009] In one aspect of the present invention, in the method, a first group of barcode nucleic acids or a second group of barcode nucleic acids containing different barcode sequences are added to each microchannel of the microchannel device.

[0010] In one aspect of the present invention, the chip surface linker nucleic acid may be immobilized on the chip surface by chemical bonding. The chemical bonding may be any one selected from linking groups by amino group-aldehyde group reaction and covalent crosslinking. The surface of the chip may be coated with an activated group such as amino, aldehyde, epoxy, isothiocyanate, mercapto, or silyl by a surface chemical reaction. The chip surface linker nucleic acid has a group at one end (usually the 5' end) bonded to the chip surface that forms a chemical bond with the activated group to be coated.

[0011] In another embodiment of the present invention, the chip surface linker nucleic acid may be immobilized on the chip surface by physical adsorption such as hydrophobic action, electrostatic attraction, etc. For example, the chip surface may be modified with poly-L-lysine (PLL) or treated with a surfactant.

[0012] In another aspect of the present invention, the 3' end of the chip surface linker nucleic acid has a binding fragment that binds to a first barcode nucleic acid. In one embodiment of the present invention, the binding fragment of the chip surface linker nucleic acid is bound to the first barcode nucleic acid via a first single-stranded binding nucleic acid. The 5' end of the first barcode nucleic acid has a binding fragment for binding to the chip surface linker nucleic acid via the first single-stranded binding nucleic acid. The binding fragment at the 3' end of the chip surface linker nucleic acid and the binding fragment at the 5' end of the first barcode nucleic acid are reverse-complementary to the sequences at both ends of the first single-stranded binding nucleic acid, respectively.

[0013] In another embodiment of the invention, the chip surface linker nucleic acid has about 10-50 nucleotides, preferably less than 30 nucleotides, such as less than 25 nucleotides.

[0014] In one embodiment of the invention, in the method, a first barcode nucleic acid in the first group of barcode nucleic acids comprises a first barcode fragment. In another embodiment of the invention, a 5' end of a first barcode nucleic acid in the first group of barcode nucleic acids has a binding fragment for binding to the chip surface linker nucleic acid and a 3' end has a first binding fragment for binding to a second barcode nucleic acid.

[0015] In one embodiment of the present invention, the second barcode nucleic acid in the second group of barcode nucleic acids comprises, at its 3' end, a capture fragment for identifying and binding to a target nucleic acid in a biological sample (e.g., a fragment for identifying and binding to mRNA or cDNA, e.g., a poly-T sequence) and a second barcode fragment.

[0016] In one aspect of the invention, in the method, a second barcode nucleic acid in the second group of barcode nucleic acids further comprises a unique molecular identifier (UMI).

[0017] In one aspect of the present invention, in the method, the 3' end of the first barcode nucleic acid has a first binding fragment for binding to the second barcode nucleic acid via a second single-stranded binding nucleic acid, and the 5' end of the second barcode nucleic acid has a second binding fragment for binding to the first barcode nucleic acid via the second single-stranded binding nucleic acid, and the first binding fragment and the second binding fragment are each reverse complementary to the sequences at both ends of the second single-stranded linker nucleic acid.

[0018] In one aspect of the invention, the probe formed in step 5 of the method comprises a capture fragment for identifying and binding to a target nucleic acid in a biological sample at its 3' end, an optional unique molecular identifier (UMI), a second barcode fragment and a first barcode fragment, and a chip surface linker nucleic acid fragment at its 5' end. In another aspect of the invention, the 5' end of the chip surface linker nucleic acid fragment comprises a primer fragment for an amplification reaction.

[0019] In one aspect of the invention, in the method, the sequences of the first barcode fragments of the various first barcode nucleic acids in the first group of barcode nucleic acids are specified, and / or the sequences of the barcode fragments of the various second barcode nucleic acids in the second group of barcode nucleic acids are specified.

[0020] In one aspect of the invention, in the method, the sequences of the first barcode fragment and the second barcode fragment of the probe are designated.

[0021] In one embodiment of the invention, in step 2 of the method, the chip surface linker nucleic acid is added to the surface of the chip at a concentration of about 0.1-100 uM / L, for example, about 1-20 uM / L.

[0022] In one embodiment of the invention, in step 3 of the method, the concentration of nucleic acid in the flow path is about 0.1-100 uM, for example, about 1-20 uM.

[0023] In one embodiment of the invention, in step 4 of the method, the concentration of nucleic acid in the flow path is about 0.1-100 uM, for example, about 1-20 uM.

[0024] In one aspect of the present invention, in the method, in steps 3 and 4, the width of each microchannel of the parallel microchannels is about 2-200 μm, preferably about 5-50 μm, most preferably about 5-25 μm, for example about 5 μm, 10 μm or 50 μm.

[0025] In one aspect of the present invention, in steps 3 and 4 of the method, the parallel microchannels have a distance between adjacent microchannels of about 5-400 μm, preferably about 10-100 μm, most preferably about 10-50 μm, for example about 20 μm, 50 μm or 100 μm.

[0026] In one embodiment of the invention, the probe density in the features of the chip produced by the method is about 10 3 -10 5 pieces / μm 2 It is.

[0027] In one embodiment of the invention, chip features produced by the method have a probe uniformity deviation of less than 20%, preferably less than 10%, more preferably less than 6%.

[0028] In one embodiment of the invention, the feature size uniformity deviation of chips manufactured by the method is less than 10%, preferably less than 5%, more preferably less than 2%.

[0029] The present invention further provides a chip for analyzing nucleic acid information of a biological sample. In one aspect of the present invention, the chip for analyzing nucleic acid information of the biological sample is manufactured by the above method. In one aspect of the present invention, the surface of the chip for analyzing nucleic acid information of the biological sample has probes forming an array. The probe array includes rows and columns that are orthogonal to each other. The probes in the array each have a different barcode sequence, which can indicate the spatial position of the probe. In one aspect of the present invention, the probes include a first barcode and a second barcode. In another aspect of the present invention, the probes in each row of the probe array have the same first barcode, and the probes in each column have the same second barcode. The first barcodes of the probes in each row are different from each other, and the second barcodes of the probes in each column are different from each other. In one aspect of the present invention, the chip for analyzing nucleic acid information of the biological sample has a chip surface linker nucleic acid on its entire surface. In another aspect of the present invention, the 5' end of each probe in the probe array is the chip surface linker nucleic acid. In another aspect of the invention, the sequence of each probe in the probe array includes, from the 5' end to the 3' end, the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture fragment for identifying and binding to a target nucleic acid in a biological sample. In another aspect of the invention, the sequence of each probe in the probe array includes a primer fragment for an amplification reaction at the 5' end. In another aspect of the invention, each probe in the probe array further includes a unique molecular identifier (UMI).

[0030] In one aspect of the invention, the probe density in the features of the chip is about 10 3 -10 5 pieces / μm 2 In one embodiment of the invention, the uniformity deviation of the probes in the features of the chip is less than 20%, preferably less than 10%, more preferably less than 6%. In one embodiment of the invention, the uniformity deviation of the size of the features of the chip is less than 10%, preferably less than 5%, more preferably less than 2%.

[0031] The chip provided by the present invention or the chip produced by the method provided by the present invention can be applied to perform analysis of intracellular molecules (analysis of nucleic acids and proteins) on tissue samples, particularly thin tissue sections, and can be analyzed by, for example, PCR, mass spectrometry, next-generation sequencing, or ELISA to obtain expression and spatial information.

[0032] According to the present invention, there is provided a method for analyzing spatial transcriptome information of a biological tissue sample using a chip having an array. The method includes contacting the array of the chip with a tissue sample, and the probes on the array identify and bind to nucleic acids, particularly mRNA, of cells in the tissue. In one aspect of the invention, nucleic acids of cells in the tissue are separated from the tissue and bind to the probes on the chip. In another aspect of the invention, when the tissue is a fixed and embedded tissue, the nucleic acids are released from the cells by permeabilizing the tissue.

[0033] In another aspect of the invention, the method further comprises performing a reverse transcription reaction.

[0034] In another aspect of the invention, the method further comprises collecting the tissue / cells, isolating and purifying the cDNA, e.g., separating the tissue and the chip, dividing the tissue / cells in one container, and isolating and purifying the nucleic acid.

[0035] In another embodiment of the invention, the method further comprises amplifying the cDNA molecule.

[0036] In another aspect of the invention, the method further comprises performing library construction / sequencing on the amplified nucleic acid.

[0037] In one aspect of the present invention, the biological sample is a tissue sample from a subject, for example, a tissue sample excised during surgery, preferably a tissue thin section obtained by processing by microtomy.In one aspect of the present invention, the tissue sample is fixed and embedded (for example, embedded in paraffin), and attached to a support, for example, a glass sheet.In one aspect of the present invention, the nucleic acid of cells in the fixed tissue is separated from the tissue after permeabilization and binds to the probe on the chip.

[0038] In one embodiment of the present invention, the thin tissue sections can be subjected to morphological and / or histological analysis, and the histological analysis is performed by H&E staining, IHC staining, ISH staining and FISH staining.

[0039] In one aspect of the invention, the analysis for one or more biological molecules is performed by PCR, mass spectrometry, next generation sequencing or ELISA.

[0040] In one aspect of the invention, the subject is selected from an animal, a livestock, a pet, or a human subject.

[0041] In one aspect of the invention, the analyte further comprises one or more of a non-human cell, a human cell, a non-naturally occurring protein, a nucleic acid, a small molecule, a dye, a virus, a bacteria, a parasite, a protozoan, or a chemical. Small molecules include haptens, peptide tags, protein tags, fluorescent tags, nucleic acid tags, and combinations thereof.

[0042] In one aspect of the present invention, the chip is applicable to the analysis of quantitative and / or qualitative data of markers in samples. The markers include DNA, proteins, RNA, lipids, organelles, metabolites, or cells. The markers include genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids, and / or organelles. The markers include single base positions; intragenic or intergenic regions; exons, introns, or fragments thereof; coding or non-coding regions; promoters, enhancers, 5' untranslated regions (5'UTR), 3' untranslated regions (3'UTR) or fragments thereof; cDNAs or fragments; SNPs; somatic and / or germline mutations; point or single mutations; deletion mutations; in-frame deletions, intragenic deletions, whole gene deletions; insertion mutations; intragenic insertions; inversion mutations; intrachromosomal inversions, linked mutations, linked insertion mutations; inversion mutations; tandem duplications; intrachromosomal tandem duplications; translocations; chromosomal translocations, non-reciprocal translocations; rearrangements; genomic rearrangements; rearrangements of one or more introns or fragments thereof; rearranged introns; 5'-, 3'-UTRs, or combinations thereof. The markers can be measured by single cell sequencing, mononuclear sequencing, flow cytometry, immunohistochemical staining, hematoxylin and eosin staining, whole genome sequencing, high throughput sequencing, mass spectrometry, DNA microarrays, or a combination thereof.

[0043] The chip of the present invention can be applied to the analysis of tissue samples, including one or more pre-cancerous or malignant cells, cells from solid tumors, soft tissue tumors or metastases, tissues or cells from surgical margins, histologically normal tissues, one or more circulating tumor cells (CTCs), normal adjacent tissues (NATs), blood samples from the same subject suffering from or at risk of suffering from a tumor, and FFPE samples. [Brief description of the drawings]

[0044] In order to more clearly describe the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings necessary for the description of the embodiments or the prior art. It is obvious that the drawings in the following description are only some embodiments of the present invention.

[0045] [Figure 1] 1 is a flow chart of the steps of a method for manufacturing a chip provided by the present invention. [Diagram 2] 1 is an exemplary embodiment of a device having multiple microchannels arranged in parallel for use in the method of the present invention. [Diagram 3] FIG. 2 is an observation diagram of a probe array fluorescent signal of the biochip provided by the present invention. [Figure 4] 4A and 4B are diagrams showing the measurement results of the array of the biochip provided by the present invention based on the probe fluorescent signal, respectively showing the measurement results for the array size and the fluorescent intensity. [Diagram 5] FIG. 13 shows HE stained tissue images of mouse brain tissues subjected to spatial transcriptome analysis using the biochip provided by the present invention. [Figure 6] FIG. 2 is a quality inspection analysis diagram of the process of processing and analyzing nucleic acid of mouse brain tissue using the biochip provided by the present invention. [Figure 7A] FIG. 1 shows the number of genes that can be detected by spatial transcriptome analysis of mouse brain tissue using the biochip provided by the present invention. [Figure 7B] FIG. 1 shows the number of UMIs detectable by spatial transcriptome analysis of mouse brain tissue using the biochip provided by the present invention. [Figure 7C] FIG. 1 is a distribution diagram of the number of genes actually detected and the number of captured transcripts (UMIs) by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided by the present invention. [Figure 8] FIG. 1 shows the results of gene cluster analysis performed on mouse brain tissue using the biochip provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In order to make the purpose, technical means and advantages of the embodiments of the present invention clearer, the technical means in the embodiments of the present invention will be described clearly and completely below with reference to the drawings in the embodiments of the present invention. It is obvious that the following embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts are all included in the protection scope of the present invention.

[0047] Example 1 The present invention provides a method for producing a biochip suitable for analyzing nucleic acid information of cells in a biological sample. More specifically, the present invention provides a method for producing a biochip having an array. In one aspect of the present invention, the chip provided by the present invention is suitable for analyzing spatial transcriptome information of a biological tissue sample.

[0048] FIG. 1 is a flow chart of an exemplary method for fabricating a biochip having an array provided by the present invention.

[0049] As shown in FIG. 1, the method mainly includes the following steps: Step 1: Provide a chip (or "substrate") In order to immobilize the chip surface linker nucleic acid on the chip surface in the next step, the surface of the chip can be coated with activated groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, silyl groups, etc., by surface chemical reaction. Step 2: Immobilize a chip surface linker nucleic acid on the chip surface, for example, across the entire chip surface. The chip surface linker nucleic acid may have a binding fragment at its 3' end for binding to the first barcode nucleic acid and a primer fragment at its 5' end for use in a subsequent amplification reaction. Step 3: A first group of barcode nucleic acids is added to the chip surface through multiple microchannels arranged in parallel, and multiple first barcode strips in a first direction are formed under conditions in which the chip surface linker nucleic acid and the first barcode nucleic acid bind and react with each other, the first group of barcode nucleic acids includes multiple types of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is fixed to each first barcode strip, and the first barcode nucleic acids fixed to each first barcode strip have different barcode sequences.

[0050] FIG. 2 shows an exemplary embodiment in which a plurality of barcode nucleic acids are added to a chip surface through a plurality of microchannels arranged in parallel, and are immobilized on the chip surface by binding reaction with the chip surface linker nucleic acid on the chip surface. The lower part of the left diagram in FIG. 2 is a chip. The center of the left diagram in FIG. 2 shows a microchannel device having a plurality of microchannels (microchannel 1 to microchannel n) arranged in parallel, and the surface of the microchannel that contacts the chip surface, i.e., the bottom part of the illustrated microchannel, is one through which a solution or a nucleic acid in the solution can pass (permeate). For example, there is no microchannel wall on the surface of the microchannel that contacts the chip surface. This microchannel device is coated on the chip surface along the first direction, and then a predetermined solution, for example, a solution containing barcode nucleic acid, is introduced into the microchannel. The upper part of the left diagram in FIG. 2 shows an exemplary device for assisting in the introduction of a solution, for example, a vacuum suction device using negative pressure provided at the outlet of the microchannel. The right diagram in FIG. 2 shows the introduction of barcode nucleic acids (barcode nucleic acids 1-n in the diagram) containing different barcode sequences through the inlet into each microchannel. In another aspect of the invention, the barcode sequence of the barcode nucleic acid introduced into each of the microchannels has a known or predetermined nucleotide sequence.

[0051] As shown in Figure 1, the 5' end of the first barcode nucleic acid has a binding fragment for binding to the chip surface linker nucleic acid via one single-stranded binding nucleic acid (first linker). The binding fragment at the 3' end of the chip surface linker nucleic acid and the binding fragment at the 5' end of the first barcode nucleic acid are reverse-complementary to the sequences at both ends of the first linker, respectively.

[0052] Step 4: Remove the microchannel in step 3, and add a second group of barcode nucleic acids to the first barcode strips having a first direction on the chip surface along a second direction (usually perpendicular to the first direction) through another plurality of parallel microchannels to form a plurality of second barcode strips, the second group of barcode nucleic acids including a plurality of second barcode nucleic acids having different barcode sequences, each of the second barcode strips having one type of second barcode nucleic acid, and the second barcode nucleic acids on each of the second barcode strips having different barcode sequences.

[0053] The exemplary second barcode nucleic acid shown includes a poly-T sequence at the 3' end that identifies and binds to the mRNA, a unique molecular identifier (UMI), and a second barcode fragment.

[0054] In the illustrated example, the 3' end of the first barcode nucleic acid has a first binding fragment for binding to the second barcode nucleic acid via one single-stranded binding nucleic acid (second linker), and the 5' end of the second barcode nucleic acid has a second binding fragment for binding to the first barcode nucleic acid via the second linker, and the first binding fragment and the second binding fragment are reverse-complementary to the sequences at both ends of the second linker nucleic acid, respectively. Under conditions that allow a binding reaction between the first barcode nucleic acid and the second barcode nucleic acid, the second barcode nucleic acid and the first barcode nucleic acid are bound to each other on the chip surface where the multiple first barcode strips and the multiple second barcode strips cross each other to form a probe.

[0055] Step 5: Remove the microchannel in step 4 to obtain a biochip having a probe array on its surface. Each feature on the probe array corresponds to a position where the first barcode strips and the second barcode strips cross each other. Each feature has one type of probe molecule, and includes a first barcode sequence and a second barcode sequence. The combination of the first barcode sequence and the second barcode sequence contained in the probe molecule of each feature is different from each other. In one aspect of the present invention, the first barcode sequence and the second barcode sequence of the barcode nucleic acid introduced into each microchannel are known or predetermined, so that the first barcode sequence, the second barcode sequence and the combination thereof contained in the probe molecule of each feature are also known. This allows the spatial position in the array on the chip surface to be determined based on the first barcode sequence and the second barcode sequence of the probe molecule of each feature.

[0056] The chip, which is the substrate in step 1, generally refers to a solid substrate on which chemical, biological, biophysical or biochemical processes can be carried out. The chip may have microstructures or microscale structures such as passages and wells, electrode elements, electromagnetic elements, etc., to favor the chemical, biological, biophysical or biochemical processes occurring on the chip. The chip surface may be flat or uneven. A chip with an uneven surface may include passages or wells constructed on the surface.

[0057] The chip can be made of any suitable material. Exemplary types of chip materials include glass, modified glass, functionalized glass, inorganic glass, microspheres (containing inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, fibers or fiber bundles, various other polymers, and multi-well microtiter plates. Exemplary specific types of plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon. TM Exemplary specific types of silica-based materials include various forms of silicon and modified silicon. The chip surface can be modified for attachment of biopolymers of interest by a variety of methods known to those skilled in the art.

[0058] In the present invention, the array on the chip surface produced has probes (also called capture probes). A probe refers to a single-stranded nucleotide molecule of a target nucleic acid, e.g., a nucleic acid from a tissue sample, that can be recognized and bound in a gene-specific or target-specific manner and has a specific nucleotide sequence, i.e., can selectively anneal to the nucleotide sequence of the target nucleic acid, usually a complementary nucleotide sequence. Examples of analytes in tissue samples include genomic DNA, methylated DNA, specific methylated DNA sequences, messenger RNA (mRNA), polyA mRNA, mitochondrial DNA, viral RNA, microRNA, in situ synthesized PCR products, RNA / DNA hybrids, lipids, carbohydrates, and proteins. The capture probe may be a gene-specific capture probe, which hybridizes to a specific target mRNA or cDNA in the sample.

[0059] In the present invention, the probes have barcode sequences and are used in subsequent high-throughput next-generation sequencing (NGS) or sequencing-by-synthesis (SBS) analytical applications, such as high-throughput sequencing analyses. In these sequencings, barcode sequences are used to mark and identify the nucleic acid source of the nucleic acid sequences obtained by sequencing. Barcode molecules are used to barcode nucleic acid molecules (e.g., RNA molecules) derived from biological particles (e.g., cells) to generate a sequencing library, which is then sequenced to produce a plurality of sequencing read lengths. Some or all of the plurality of sequencing read lengths include the barcode sequence. In these sequencing applications, cellular nucleic acids are typically amplified until the barcoded overlapping fragments in a subject constitute at least 1X coverage, at least 2X, at least 3X, at least 4X, at least 5X, at least 10X, at least 20X, at least 40X, or more coverage of a particular portion or all of a cellular genome. Once the barcoded fragments are produced, they can be directly sequenced on an appropriate sequencing system (e.g., an Illumina system). The presence of the same barcode on multiple sequences can provide information about the origin of this sequence.

[0060] In the present invention, the manufactured probe contains two barcode sequences. The two barcode sequences serve to identify the position of the probe in the array on the chip surface (identifying the X and Y coordinate positions, respectively), and therefore also act as position tags. The barcode sequence on the probe can correspond to a feature in the array on the chip, and can also indicate the location in this tissue sample of the cell (including a single cell) in the tissue it identifies. Examples of other molecules that can be bound to the nucleic acid tag include antibodies, antigen binding domains, proteins, peptides, receptors, haptens, etc.

[0061] In the present invention, the probe further comprises one or more unique molecular identifiers (UMIs). Unique molecular identifiers are contiguous nucleic acid fragments or two or more discontinuous nucleic acid fragments that act as markers or identifiers for a particular analyte or a capture probe that binds to a particular analyte. A UMI is a nucleic acid sequence that does not essentially hybridize with the nucleic acid molecules of the analyte in a biological sample. A UMI may be from about 6 to about 20 or more nucleotides contained within the sequence of the capture probe.

[0062] In step 2 of the method of this embodiment, the immobilization of the chip surface linker nucleic acid to the chip can be performed by various methods known in the art. The immobilization of the nucleic acid refers to direct or indirect attachment to the chip by covalent or non-covalent bonding. In one embodiment of the present invention, the immobilization refers to immobilization or attachment to the chip in a reaction requiring nucleic acid amplification and / or sequencing. In one invention of the present invention, the immobilization may refer to the ability of the nucleic acid immobilized or attached to the chip to be detached from the chip surface under a predetermined condition in a subsequent reaction such as nucleic acid amplification and / or sequencing. Exemplary non-covalent bonds include, but are not limited to, non-specific interactions (e.g., hydrogen bonds, ionic bonds, van der Waals interactions, etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). The chip surface linker nucleic acid may be immobilized to the chip surface by a physical adsorption method such as hydrophobic action and electrostatic attraction.

[0063] In steps 3 and 4 of the method of this embodiment, the connection between the chip surface linker nucleic acid and the first barcode nucleic acid, and the connection between the first barcode nucleic acid and the second barcode nucleic acid can be performed by various methods known in the art. For example, by each being complementary to the sequence at the different ends of another single-stranded nucleic acid fragment (linker nucleic acid), the combination of three nucleic acid fragments (first barcode nucleic acid, second barcode nucleic acid, and linker nucleic acid) can be formed under conditions allowing a binding reaction, and then the purpose of binding can be achieved.

[0064] In one embodiment of the present invention, the chip surface linker nucleic acid has a primer fragment (eg, a universal primer sequence used in known sequencing methods) at the 5' end for use in a subsequent amplification reaction.

[0065] In one aspect of the present invention, the chip surface linker nucleic acid has a group or sequence at the 5' end for binding to the chip surface. For example, the chip surface is modified with an aldehyde, and the chip surface linker nucleic acid has an amino group at the 5' end. In another aspect of the present invention, the chip surface linker nucleic acid has a ligand that forms a specific interaction with a factor to be modified on the chip. For example, the factor and ligand are antibody-epitope, avidin-biotin, streptavidin-biotin, and lectin-carbohydrate, respectively.

[0066] In one embodiment of the invention, the first barcode nucleic acid encompasses a first barcode fragment.

[0067] In one embodiment of the present invention, the second barcode nucleic acid includes a second barcode fragment. In one embodiment of the present invention, the second barcode nucleic acid has a capture fragment for identifying and binding to a target in a biological sample, such as a fragment for identifying and binding to mRNA or cDNA, such as a poly-T sequence for identifying mRNA, at the 3' end.

[0068] In one aspect of the invention, the 3' end of the first barcode nucleic acid has a first binding fragment for binding to a second barcode nucleic acid. In another aspect of the invention, the 5' end of the second barcode nucleic acid has a second binding fragment for binding to the first barcode nucleic acid. In another aspect of the invention, the first binding fragment and the second binding fragment are each complementary to one end of a linker nucleic acid, and under conditions allowing binding (e.g., in the presence of T4 ligase, etc.), the first binding fragment and the second binding fragment combine with the linker nucleic acid to achieve binding between the first barcode nucleic acid and the second barcode nucleic acid.

[0069] The chip prepared by the method provided in the present invention can be applied to analyze intracellular molecules (including nucleic acids and proteins) in tissue samples, particularly in tissue thin sections, for example, by PCR, mass spectrometry, first generation sequencing, or ELISA to obtain their expression and spatial information.

[0070] The use of the chip of the present invention involves contacting a tissue section with an array. The probes on the array can identify and bind to the nucleic acid, particularly mRNA, of cells in the tissue. Subsequent analysis includes reverse transcription and amplification, and can be analyzed by high-throughput next-generation sequencing (NGS) or sequencing by synthesis (SBS).

[0071] According to the method for manufacturing a chip with a probe array provided by the present invention, it is possible to realize the parallel synthesis of chips with multiple arrays of the same code regions on the same substrate. As shown in Fig. 3, multiple groups of the same first barcode strips and second barcode strips can be formed in the first and second directions of the chip substrate as required. This results in multiple chips with probes defined by the same first and second barcode sequences in corresponding features.

[0072] WO / 2022 / 135598 discloses a method for producing a biochip with an array. In this method, a first group of barcode nucleic acids is directly fixed to a glass sheet modified with optical epoxy by a plurality of microchannel devices arranged in parallel. In laboratory and large-scale industrial production environments, the inventors found that when nucleic acid molecules were attached to a modified chip by a microchannel, the special hydrodynamic characteristics of the microchannel had extremely high requirements for the conditions (including reaction temperature, reactant concentration, environmental humidity, pressure, etc.) of the reaction between the nucleic acid occurring in the channel and the modified group on the chip surface. In the reaction process, if the reaction conditions fluctuate, it may have a significant impact on the reaction result between the nucleic acid and the modified group on the chip surface, causing serious quality control defects in the product. For example, defects such as a weak probe signal over a large area on the chip and even a chip "plaque" (no probe signal on the chip).

[0073] The inventor unexpectedly found that in the improved manufacturing process provided by the present invention, when the nucleic acids of the first group of barcode nucleic acids are immobilized on the chip surface, the nucleic acids of the first group of barcode nucleic acids are bound to the nucleic acids (i.e., the chip surface linker nucleic acid or chip surface linker in this specification) immobilized on the chip surface (including the entire chip surface or the chip surface portion whose position is the same as that of the first group of barcodes) by a nucleic acid-nucleic acid binding reaction (e.g., a nucleic acid ligase and a nucleic acid linker fragment that binds the two nucleic acids), thereby significantly improving the immobilization efficiency of the first group of barcode nucleic acids on the chip, significantly increasing the density of the probes on the features of each probe array on the chip, and significantly improving the uniformity. Without being bound by this theory, the inventor believes that this is because the efficiency and stability of the nucleic acid-nucleic acid binding reaction are stronger than other immobilization methods (e.g., covalent bonds, etc.) between nucleic acids and the chip surface, thereby reducing the length of each nucleic acid fragment on the probe and improving the binding efficiency of each fragment. In addition, the length of the chip surface linker nucleic acid or chip surface linker may be relatively short. In this case, the efficiency of immobilization to the chip by a method such as chemical bonding is higher, and the number of bonds is also larger, which is obviously advantageous for improving the size uniformity of the probe features of the manufactured chip, the probe amount of the probe features, and the uniformity of the probe amount (in the manufactured chip, the intensity of the fluorescent signal carried by the probe is detected by a fluorescent microscope and a camera. In application, this is embodied to identify the number of transductors or genes of nucleic acid in a sample such as tissue or cell).

[0074] The present invention further provides a chip for analyzing nucleic acid information of a biological sample. In one aspect of the present invention, the chip for analyzing nucleic acid information of the biological sample is manufactured by the above method. In one aspect of the present invention, the surface of the chip for analyzing nucleic acid information of the biological sample has probes formed in an array. The probe array includes orthogonal rows and columns. The probes in the array each have a different barcode sequence, which can indicate the spatial position of the probe. In one aspect of the present invention, the probes include a first barcode and a second barcode. In another aspect of the present invention, the probes in each row of the probe array have the same first barcode, and the probes in each column have the same second barcode. The first barcodes of the probes in each row are different from each other, and the second barcodes of the probes in each column are different from each other. In one aspect of the present invention, the chip for analyzing nucleic acid information of the biological sample has a chip surface linker nucleic acid on its entire surface. In another aspect of the present invention, the 5' end of each probe in the probe array is the chip surface linker nucleic acid. In another aspect of the invention, the sequence of each probe in the probe array comprises, from the 5' end to the 3' end, the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture fragment for identifying and binding to a target nucleic acid in a biological sample. In another aspect of the invention, the sequence of each probe in the probe array comprises a primer fragment for an amplification reaction at the 5' end. In another aspect of the invention, the sequence of each probe in the probe array further comprises a unique molecular identifier (UMI).

[0075] The chips produced by the methods provided by the present invention can be applied to perform intracellular molecular analysis (nucleic acid and protein analysis) on tissue samples, particularly thin tissue sections, and can be analyzed by, for example, PCR, mass spectrometry, next-generation sequencing, or ELISA to obtain expression and spatial information.

[0076] The present invention further provides a method for analyzing spatial transcriptome information of a biological tissue sample. The method includes contacting the array with a tissue sample. A "tissue sample" suitable for the present invention includes tissue obtained from a subject, fixed, sectioned and mounted on a planar surface. The tissue sample may be a formalin-fixed paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or a frozen tissue sample. The method of the present invention can be performed before or after staining of the tissue sample. For example, after hematoxylin-eosin staining, spatial analysis can be performed on the tissue sample by the methods provided herein. The method includes spatial analysis of the tissue after histological analysis of the sample (e.g., using hematoxylin-eosin staining). Fixing tissue sections in formalin and embedding them in paraffin (FFPE) typically involves fixing tissue obtained from a subject in formaldehyde (e.g., 3%-5% formaldehyde in phosphate-buffered saline) or Bouin's solution, embedding it in paraffin, slicing it into thin sections, and then mounting it on a flat surface (e.g., a biopsy on a microscope slide). In the method of the present invention, the tissue section is contacted with a probe array on a chip. The probes on the array can identify and bind to the nucleic acids, particularly mRNA, of cells in the tissue. Subsequent analysis includes reverse transcription and amplification, and can be analyzed by high-throughput next-generation sequencing (NGS) or sequencing-by-synthesis (SBS).

[0077] "Sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. A polynucleotide may be, for example, a nucleic acid molecule, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including modifications or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by a variety of currently available systems, such as, but not limited to, Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies sequencing systems. Alternatively, sequencing can be performed by nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such systems can provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a human). For example, a sample provided from the subject is generated by the system. In some embodiments, such systems provide a sequencing read length (also referred to herein as "read length"). The read length may include a series of nucleic acid bases corresponding to the sequence of the sequenced nucleic acid molecule. In some cases, the systems and methods provided herein can be used in conjunction with proteomic information.

[0078] In some embodiments, nucleic acids in tissue sections (e.g., formalin-fixed paraffin-embedded (FFPE) tissue sections) are transferred to an array and captured on the array by hybridization with a capture probe. In some embodiments, the capture probe may be a universal capture probe, e.g., hybridizing to an adapter region in a nucleic acid sequencing library or a polyA tail of an mRNA. In some embodiments, the capture probe may be a gene-specific capture probe, e.g., hybridizing to a specifically targeted mRNA or cDNA in a sample.

[0079] In some embodiments, the nucleic acid in the tissue section (e.g., FFPE section) is transferred to an array and captured on the array by binding to a single strand of a universal adaptor oligonucleotide. In other embodiments, the nucleic acid on the chip may be transferred to the tissue section (e.g., FFPE section). The probe bound to the chip can be allowed to enter the cells on the tissue that contacts it after falling off in solution by a method known in the art. For example, a photodegradable linker or the like is added to the binding site between the nucleic acid probe and the chip, or the nucleic acid probe is bound to the chip by a pH-sensitive linker, and then the pH value of the solution is changed to separate the nucleic acid probe from the chip.

[0080] With regard to the chip of the present invention and its use, the spatial position in the array on the chip surface can be known based on the first barcode sequence and the second barcode sequence of the probe molecule carried by each feature, thereby also making it possible to know the positional information of the cell in which the nucleic acid molecule is located in the tissue.

[0081] In one embodiment of the invention, the method further comprises subjecting the tissue thin sections to morphological and / or histological analysis, the histological analysis being performed by H&E staining, IHC staining, ISH staining and FISH staining.

[0082] In one aspect of the invention, the method may involve analysing one or more biological molecules in the tissue sample, for example by PCR, mass spectrometry, next generation sequencing or ELISA.

[0083] In one aspect of the invention, in said method, the tissue sample is derived from an animal, livestock, pet or human subject.

[0084] In one aspect of the invention, in the method, the biological molecule comprises one or more of a non-human cell, a human cell, a non-naturally occurring protein, a nucleic acid, a small molecule, a dye, a virus, a bacterium, a parasite, a protozoan, or a chemical.

[0085] In one aspect of the invention, in the method, the small molecule comprises a hapten, a peptide tag, a protein tag, a fluorescent tag, a nucleic acid tag, and combinations thereof.

[0086] In one aspect of the invention, in said method, said analyzing comprises generating quantitative and / or qualitative data for the markers.

[0087] In one aspect of the invention, in the method, the marker comprises DNA, a protein, an RNA, a lipid, a cell organelle, a metabolite, or a cell.

[0088] In one aspect of the invention, in said method, said markers comprise genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids and / or organelles.

[0089] In one aspect of the invention, the marker comprises an altered nucleotide sequence encoding an altered amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, an altered copy number, an altered expression level, an altered protein level, an altered protein activity, or an altered methylation status in cancer tissue or cells compared to normal healthy tissue or cells.

[0090] In one aspect of the invention, the method measures the markers by single cell sequencing, mononuclear sequencing, flow cytometry, immunohistochemical staining, hematoxylin and eosin staining, whole genome sequencing, high throughput sequencing, mass spectrometry, DNA microarray, or a combination thereof.

[0091] In one embodiment of the invention, the method further comprises subjecting the tissue thin sections to morphological and / or histological analysis, the histological analysis being performed by H&E staining, IHC staining, ISH staining and FISH staining.

[0092] In one aspect of the invention, in said method, the analysis of one or more biological molecules is performed by PCR, mass spectrometry, next generation sequencing or ELISA.

[0093] Example 2: Chip fabrication FIG. 5 is a flow chart of an exemplary embodiment of a method for manufacturing a biochip provided by the present invention.

[0094] First, a glass sheet was used as a chip substrate, and the surface of the chip was modified by a surface chemical reaction with activating groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, and silane groups.

[0095] In this example, a purchased optical epoxy modified glass sheet (Nexterion® Slide E) served as the chip substrate.

[0096] A universal chip surface linker nucleic acid having the following sequence, whose 5' end was modified with an amino group, was synthesized. The underlined T base is modified with FITC. 5'Amino-CTACACGACGC T CTTCCGATC-3'

[0097] A first group of 100 barcode nucleic acids having the following sequences, each of which was phosphorylated at the 5' end, was synthesized. 5' phosphorylation - CTCTTTCCC T AC12345678 ACGACGCTCTTC-3' "12345678" indicates a barcode fragment having eight nucleotides, and the sequence of the eight nucleotides is known (specified). The sequences of the barcode fragments (called first barcodes) of the 100 first group of barcode nucleic acids are different from each other, and the sequence of the first barcode of each first group of barcode nucleic acid is known (specified). The underlined T bases are FITC modified. In this example, observation or production quality control was performed for each step of adding barcode fragments in chip synthesis using fluorescent signal that fluorescently modifies and produces and detects the barcode fragments. In other embodiments, the barcode fragments may not be fluorescently modified.

[0098] A second group of 100 barcode nucleic acids having the following sequences were synthesized. 5' phosphorylation -GAGTGATTGCT T GTGACGCCTT87654321NNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' "87654321" indicates a barcode fragment having eight nucleotides, the sequence of the eight nucleotides being known (specified). The sequences of the barcode fragments (referred to as second barcodes) of the 100 second group barcode nucleic acids are different from each other, and the sequence of the second barcode of each second group barcode nucleic acid is known (specified). The underlined T bases are Cy3 modified.

[0099] A first linker nucleic acid having the following sequence was synthesized. 5'-AGGGAAAGAGAGATCGGAAG-3'

[0100] A second linker nucleic acid having the following sequence was synthesized. 5'-GCAATCACTCGAAGAGCGT-3'

[0101] The cell culture cavity and the glass sheet were attached, and the cavity and the glass sheet were pressed together with a frame to improve the sealing. 10-20uM of general-purpose chip surface linker nucleic acid (dissolved in 300mM sodium phosphate buffer pH8.5) was added to the cavity using a pipette to cover the bottom of the cavity evenly, and then the glass sheet was placed in a thermomixer comfort and shaken at 40℃ and 800rpm to react uniformly for 3 hours. After the reaction was completed, the modified glass sheet was washed in order with 0.1% Triton X-100, 1mM HCl, and 100mM KCl, and then blocked at 50℃ using 0.1M Tris (pH9.0), 50mM ethanolamine, and 0.1% SDS. After blocking, the substrate was washed with deionized water for 1 minute, and then dried by blowing nitrogen gas onto the substrate. The FITC fluorescent signal of the universal nucleic acid was observed under a fluorescence microscope to confirm the complete reaction and the surface modification effect.

[0102] A device containing multiple microchannels arranged in parallel was fabricated using polydimethylsiloxane (PDMS) by a soft lithography process, as shown in Figure 2. The bottom of the microchannel was open.

[0103] The microchannel device comprises about 50-500 parallel microchannels, each of which has a width of about 2-200 μm, preferably about 5-50 μm, most preferably about 5-25 μm, such as about 5 μm, 10 μm or 50 μm, and the distance between adjacent microchannels is about 5-400 μm, preferably about 10-100 μm, most preferably about 10-50 μm, such as about 20 μm, 50 μm or 100 μm.

[0104] The PDMS microchannel device was bonded to a glass sheet to seal the channel. A clamping tool was used to press the top of the channel against the glass sheet substrate to improve sealing. One end of the microchannel was the solution inlet, and the other end was connected to a vacuum suction device via an interface.

[0105] After the microchannel device and the glass sheet were bonded together, a buffer solution was introduced into the channel to discharge the gas in the channel. Then, 10-20 uM of the first group of barcode nucleic acids was added into the channel, and the first barcode nucleic acid (the first barcode nucleic acid in each channel has a barcode sequence different from that of the first barcode nucleic acid in the other channels), the first linker nucleic acid, and T4 ligase were introduced into each channel. After filling the channel, the reaction was allowed to proceed at 37°C for 30 minutes. After the reaction was completed, the substrate was washed with deionized water for 1 minute, and then dried by blowing nitrogen gas onto the substrate. The FITC fluorescent signal of the first barcode nucleic acid was observed under a fluorescent microscope to confirm that the reaction had been completed, that the first group of barcode nucleic acids and the universal chip surface linker nucleic acid had undergone a binding reaction with the chip at the location where the channel was covered, and that the first group of barcode strips had been formed.

[0106] Another PDMS microchannel device was attached to a glass sheet along a direction perpendicular to the channels of the first PDMS microchannel device. After introducing a buffer solution into the channels and discharging the gas in the channels, 10-20 uM of the second group of barcode nucleic acids was introduced. One type of second barcode nucleic acid (the second barcode nucleic acid in each channel has a barcode sequence different from the second barcode nucleic acid in the other channels), a second linker nucleic acid, and T4 ligase were introduced into each channel. After the channels were filled, the mixture was left to react at 37°C for 30 minutes.

[0107] After the binding reaction was completed, 1x PBS buffer was introduced and the channel was washed with ultrapure water. Then, the channel was removed and the substrate was washed with deionized water for 1 minute, and then dried by blowing nitrogen gas onto the substrate. The Cy3 fluorescent signal of the second barcode nucleic acid was observed using a fluorescent microscope to determine that the reaction was complete, and that the binding reaction between the second barcode nucleic acid and the first group of barcode nucleic acids in the channel features was achieved, forming a barcode array, thereby completing the fabrication of the chip.

[0108] The fabricated chips were vacuum-packaged and stored in a dark place at room temperature or in a refrigerator at 4° C. The efficiency of the binding reaction of each barcode nucleic acid and the strength (density) and uniformity of the probes on the fabricated chips were evaluated by measuring the fluorescent signals on the fabricated chips.

[0109] Figure 3 shows an example of the detection of the fluorescent signal modified with Cy3 carried by the second group of barcode nucleic acid in the obtained chip. The image was photographed and measured using a fluorescent microscope (Olympus IX53) and a CCD camera (Olympus DP74).

[0110] 4A and 4B show the results of probe detection for chips (i.e., the sizes of the probe arrays on the chips are 50 μm×50 μm, 25 μm×25 μm, and 10 μm×10 μm, respectively) fabricated using microchannels with widths of 50 μm, 25 μm, and 10 μm, respectively, by the method described in Example 2. Images taken and measured by a fluorescence microscope (Olympus IX53) and a CCD camera (Olympus DP74) were analyzed, and the fluorescence intensity values ​​and boundaries were extracted by ImageJ, and the uniformity of the array intensity and the actual width of the array were calculated.

[0111] Figures 4A and 4B show the measurement results for array size and fluorescence intensity, respectively. As can be seen from the results of Figure 4A, the size uniformity deviation of each probe array on the chip manufactured by the present invention was less than 0.6% (50 μm), 1.2% (25 μm), and 1.3% (10 μm), respectively. As can be seen from the results of Figure 4B, the uniformity deviation of the probe modification was less than 5.5% (50 μm), 3.4% (25 μm), and 3.9% (10 μm), respectively. In other words, the chip manufactured by the present invention has extremely high array size uniformity and uniformity of the modified probes on each array.

[0112] Deionized water: tissue preparation and staining (1) Tissue OCT embedding Fresh mouse brain tissue samples were taken and the tissue surface was immediately washed with pre-cooled PBS solution or saline to remove any residue, then the liquid was blotted with clean absorbent paper. The tissue was placed in an embedding tank and OCT embedding medium was added until the tissue was completely covered. After making sure there were no air bubbles around the tissue, the embedding tank was placed on ice and left to stand until the OCT was completely frozen.

[0113] (2) Frozen section The temperatures of the frozen sectioning machine were set as follows: box temperature: -20° C., sample head temperature: -10° C. Before sectioning, the frozen tissue and substrate were placed in the box of the frozen sectioning machine at -20° C. and equilibrated for 30 minutes or more, and then frozen sections were taken at a thickness of 10 μm in the box of the frozen sectioning machine.

[0114] (3) Tissue fixation, HE staining The prepared tissue slice was attached to the barcode array modified substrate produced in Example 2, and then incubated at 37°C for 1 minute. The substrate with the attached tissue was completely immersed in pre-cooled methanol and fixed at -20°C for 30 minutes. After fixation, the substrate was removed, the liquid on the back was wiped off, 500μl of isopropanol was dropped onto the tissue slice, and incubated at room temperature for 1 minute. After 1 minute, the isopropanol was removed, and then the tissue slice was dried at room temperature for 5-10 minutes.

[0115] 1ml hematoxylin was added and evenly coated on the tissue sections on the substrate and incubated at room temperature for 7 minutes. The hematoxylin reagent was removed, and the substrate was immersed in RNase-free water to wash and then dried. 1ml blue solution was added and incubated at room temperature for 2 minutes. The blue solution was removed, and the substrate was immersed in RNase-free water to wash, and then the liquid on the backside of the substrate was wiped off. 1ml eosin mixture was added and incubated at room temperature for 1 minute.

[0116] The eosin was removed, and the substrate was washed by immersion in RNase-free water and dried until the tissue became opaque. The glass sheet was incubated at 37 °C for 5 min before bright field imaging.

[0117] FIG. 5 shows HE staining of mouse brain tissue sections.

[0118] (4) Tissue permeabilization The fixture cavities were attached to the fabricated tissue chip so that each tissue section was located inside the corresponding cavity. 70 ul of permeabilization enzyme (0.1% pepsin diluted with 0.1N HCl) was added to the cavity to permeabilize the tissue at 37°C, and the permeabilization enzyme was removed and then washed with 0.1x SSC.

[0119] (5) Chip quality inspection The chips prepared in Example 2 were stored for 6 and 12 months, respectively, and then taken out of the -20°C refrigerator. The reaction solutions for tissue fixation, HE staining, and tissue permeabilization in step (3) and (4) were added thereto, and the chips were treated under the reaction conditions. The strength (density) and uniformity of the probes on the chips were then inspected using the method described in Example 2.

[0120] Example 4: Reverse transcription reaction was carried out on tissue sample sections using a chip. 70 μl of reverse transcription mix was added to the deionized water-washed cavity, containing 1× first strand buffer, 5 mM DTT, 500 μM dNTPs, 0.19 μg / μl BSA, 1% DMSO, 2.5 μM template switch oligo, 20 U / μl SuperscriptIII and 2 U / μl RNase inhibitor.

[0121] Template switch oligo sequence: 5'Biotin-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3' In the template switch oligo, the penultimate bases, 1, 2, and 3, were modified with riboguanosine.

[0122] After sealing the cavity with tape, the plate was placed on a temperature-controlled board and the temperature was adjusted to 50° C. to carry out reverse transcription, followed by reaction for 16 hours.

[0123] After the reverse transcription was completed, the reverse transcription mixture in the cavity was aspirated and discarded. 70 μl of 0.08 M KOH was added to the cavity, and the cavity was incubated at room temperature for 5 minutes, and then washed once with 100 μl of RNase-free water.

[0124] The cDNA second strand synthesis reaction solution was added to the washed cavity. The second strand synthesis reaction solution consisted of 1x first strand buffer, 10U Klenow Exo - , containing 2.5 μM of second strand primer. After sealing the cavity with tape, it was placed on a temperature control board and adjusted to about 37° C. for cDNA second strand synthesis, and the reaction was allowed to proceed for 1 hour.

[0125] Sequence of the second strand primer: 5'-AAGCAGTGGTATCAACGCAGAGTACAT-3'

[0126] After the reaction was completed, the second strand synthesis reaction solution in the cavity was removed by aspirating. Then, 100 ul of RNase-free water was added to wash once. 35 μl of 0.08 M KOH was added to the cavity and incubated at room temperature for 10 minutes. Several new 1.5 ml centrifuge tubes were prepared and 10 μl of Tris (1 M, pH 7.0) was added to them. The 35 μl sample in the cavity was transferred to the corresponding Tris-containing centrifuge tube and mixed uniformly to produce the second strand of cDNA.

[0127] cDNA Amplification A new 1.5 ml centrifuge tube was taken and placed on ice to prepare the PCR amplification reaction solution. The PCR reaction solution contained 1x Kapa HiFi Hotstart ReadyMix, 0.8 μM cDNA forward primer, 0.8 μM cDNA reverse primer, 35 μl of cDNA template, and a total volume of 100 μl. cDNA amplification was performed according to the following scheme: [Table 1] cDNA forward primer sequence: 5'-CTACACGACGCTCTTCCGATC-3' cDNA reverse primer sequence: 5'-AAGCAGTGGTATCAACGCAGAG-3'

[0128] After the amplification was completed, the amplified products were purified using 0.6×AMpure XP Beads and used for library construction and sequencing.

[0129] Example 5: Library construction and sequencing Fragmentation, end repair, A addition A new PCR tube was placed on ice and a fragmentation reaction solution was prepared. Here, the fragmentation reaction solution contained 5 μl of FEA Buffer V2, 10 μl of DNA purified in the previous step, 25 μl of ddH2O, and 10 μl of FEA Enzyme Mix V2, with a total volume of 50 μl. The reaction solution was collected at the bottom of the tube by pipetting or shaking evenly with a pipette and centrifuging briefly. The PCR tube was placed in a PCR machine and the following process was carried out.

[0130] [Table 2]

[0131] This enabled DNA fragmentation and, at the same time, end-filling of the fragmented DNA, phosphorylation of the 5' end, and addition of a dA tail to the 3' end.

[0132] Adapter ligation A new PCR tube was placed on ice and the adapter ligation reaction solution was prepared. Here, the adapter ligation reaction solution contained 25 μl of Rapid Ligation Buffer V2, 50 μl of fragmented DNA purified in the previous step, 15 μl of ddH2O, 5 μl of Rapid DNA Ligase V2, 5 μl of adapter (10 pM), and a total volume of 100 μl. The reaction solution was collected at the bottom of the tube by pipetting or shaking evenly with a pipette and centrifuging briefly. The PCR tube was placed in the PCR machine and the following process was performed.

[0133] [Table 3]

[0134] The adapter sequence is 5' phosphorylation-GATCGGAAGAGCACACGTCTGAACTCCAGTCA*C-3' 5'-GCTCTTCCGATC*T-3' It is. In the adaptor sequence, the penultimate base was thio-modified. After the binding reaction was completed, the ligation product was purified using 0.6×XP SPRIselect Beads.

[0135] Library amplification A new PCR tube was placed on ice and the library amplification reaction mixture was prepared. Here, the library amplification reaction mixture contained 25 μl of VAHTS HiFi Amplification Mix, 20 μl of the adapter-ligated DNA purified in the previous step, and 5 μl of Index PCR Primer Mix (10 pM each), with a total volume of 50 μl. The mixture was pipetted or shaken evenly with a pipette, centrifuged briefly, and the reaction mixture was collected at the bottom of the tube. The PCR tube was placed in the PCR machine and the following process was carried out.

[0136] [Table 4]

[0137] Here, the sequence of the Index PCR Primer is i5 index primer:5'-AATGATACGGCGACCACCGAGATCTACAC-[i5 index]-ACACTCTTTCCCTACACGACGCTC-3' i7 index primer:5'-CAAGCAGAAGACGGCATACGAGAT-[i7 index]-GTGACTGGAGTTCAGACGTGT-3' It is.

[0138] After the amplification reaction was completed, the amplified product was purified using 0.9×AMpure XP Beads.

[0139] Library quality check The concentration and length distribution of the constructed library were detected using Qubit and Agilent Bioanalyzer High Sensitivity chip, respectively. As shown in Figure 6, the library concentration measured by Qubit was more than 20ng / μl. For length distribution detection, 1μl library sample was taken and fragment distribution was detected according to the instrument and kit instructions. The measured library fragments were distributed between 200-600bp.

[0140] Sequencing Libraries were subjected to PE150 sequencing using an Illumina NovaSeq 6000. Data processing and analysis a) Extract the UMI and barcode in Read1 using data processing software such as umitools (version: 1.1.2). b) Align read2 to the mouse reference genome, Mouse reference, mm10 (GENCODE vM23 / Ensembl 98) using data processing software such as STAR (version: 2.5.3a). featureCounts(Version2.0.3)assign gene. c) Generate an expression matrix using data processing software such as umitools (version: 1.1.2). d) Barcode analysis and HE images are compared using Adobe illustrator.

[0141] The results of transcriptome analysis of mouse olfactory bulb are shown in Figure 7. Figure 7A shows a diagram of the number of genes detectable by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided by the present invention. Figure 7B shows a diagram of the number of UMIs detectable by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided by the present invention. Figure 7C shows a distribution diagram of the number of genes actually detected and the number of captured transcripts (UMIs) by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided by the present invention. The results of unsupervised cluster analysis of the obtained data are shown in Figure 8.

[0142] The above description is only the preferred embodiment of the present invention, and does not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing a biochip having an array, comprising the following steps 1 to 5: Step 1: Give a tip Step 2: Immobilizing the chip surface linker nucleic acid on the chip surface; Step 3: Add a first group of barcode nucleic acids to the chip surface through a plurality of microchannels arranged in parallel, and form a plurality of first barcode strips in a first direction under conditions in which the linker nucleic acid on the chip surface and the first barcode nucleic acid undergo a binding reaction, the first group of barcode nucleic acids includes a plurality of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is fixed to each first barcode strip, and the first barcode nucleic acids fixed to each first barcode strip have different barcode sequences; Step 4: Add a second group of barcode nucleic acids to the chip surface along a second direction through a plurality of parallel microchannels to form a plurality of second barcode strips, the second group of barcode nucleic acids including a plurality of second barcode nucleic acids having different barcode sequences, each second barcode strip having one second barcode nucleic acid, and the second barcode nucleic acids on each second barcode strip having different barcode sequences; Step 5: Under conditions in which the first barcode nucleic acid and the second barcode nucleic acid undergo a binding reaction, the second barcode nucleic acid and the first barcode nucleic acid are bound to each other at positions on the chip surface where the first barcode strips and the second barcode strips intersect to form probes, the positions of the probes constituting features of an array, and each feature having a probe of a position-specific barcode sequence.

2. The method according to claim 1, wherein the first group of barcode nucleic acids or the second group of barcode nucleic acids are transported and fixed to a chip surface by a microchannel device having a plurality of microchannels arranged in parallel, and the surface of the microchannels that contacts the chip surface is capable of passing through a solution or nucleic acids in a solution.

3. The method according to claim 2, wherein a first group of barcode nucleic acids or a second group of barcode nucleic acids having different barcode sequences are added to each microchannel of the microchannel device, and preferably the sequence of the barcode fragment of each first barcode nucleic acid in the first group of barcode nucleic acids and the sequence of the barcode fragment of each second barcode nucleic acid in the second group of barcode nucleic acids are specified.

4. The method of claim 1, wherein the second barcode nucleic acid in the second group of barcode nucleic acids comprises, at its 3' end, a probe fragment (e.g., a fragment that identifies and binds to mRNA or cDNA, e.g., a poly-T sequence) for identifying and binding to a target nucleic acid in a biological sample and a second barcode fragment, and optionally, the second barcode nucleic acid in the second group of barcode nucleic acids further comprises a unique molecular identifier (UMI).

5. The method of claim 1, wherein the 3' end of the chip surface linker nucleic acid has a binding fragment that binds to a first barcode nucleic acid by a first single-stranded binding nucleic acid, and the 5' end of the first barcode nucleic acid has a binding fragment that binds to the chip surface linker nucleic acid by the first single-stranded binding nucleic acid, and the binding fragment at the 3' end of the chip surface linker nucleic acid and the binding fragment at the 5' end of the first barcode nucleic acid are each reverse complementary to the sequences at both ends of the first single-stranded binding nucleic acid.

6. 2. The method of claim 1, wherein a 3' end of the first barcode nucleic acid has a first binding fragment bound to a second barcode nucleic acid by a second single-stranded binding nucleic acid, and a 5' end of the second barcode nucleic acid has a second binding fragment bound to the first barcode nucleic acid by the second single-stranded binding nucleic acid, the first binding fragment and the second binding fragment being reverse-complementary to sequences at both ends of the second single-stranded binding nucleic acid, respectively.

7. The method according to claim 1, wherein in step 2, the chip surface linker nucleic acid is fixed to the chip surface by linkage through a chemical bond, and the chemical bond is, for example, any one selected from linkage of groups through an amino group-aldehyde group reaction and covalent crosslinking.

8. The probe density in the features of the fabricated chip is approximately 10 ３ -10 ５ pieces / μm ２ The method according to any one of claims 1 to 7, wherein

9. 8. The method according to any one of claims 1 to 7, wherein the manufactured chip features have a probe uniformity deviation of less than 20%, preferably less than 10%, more preferably less than 6%.

10. 8. The method according to claim 1, wherein the size uniformity deviation of the features of the manufactured chips is less than 10%, preferably less than 5%, more preferably less than 2%.

11. A chip for analyzing nucleic acid information of a biological sample, comprising: The surface of the chip has probes forming an array, the probe array includes mutually orthogonal rows and columns, the probes of each feature in the array each have a different barcode sequence, the probes include a first barcode and a second barcode, the probes in each row of the probe array have the same first barcode and the probes in each column have the same second barcode, the first barcodes carried by the probes in each row are different from each other, and the second barcodes carried by the probes in each column are different from each other, and preferably, the chip for analyzing nucleic acid information of the biological sample has a chip surface linker nucleic acid over its entire surface.

12. The chip of claim 11, wherein the sequence of each probe in the probe array comprises, from the 5' end to the 3' end, the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture fragment for identifying and binding to a target nucleic acid in a biological sample.

13. The probe density in the features of the fabricated chip is approximately 10 ３ -10 ５ pieces / μm ２ The chip of claim 11 .

14. 12. The chip of claim 11, wherein the features of the manufactured chip have a probe uniformity deviation of less than 20%, preferably less than 10%, more preferably less than 6%.

15. A chip according to any one of claims 11 to 14, produced by a method according to any one of claims 1 to 10.

16. A method for analyzing spatial transcriptome information of a biological tissue sample using a chip having an array according to any one of claims 11 to 15, comprising the steps of: The method includes the step of contacting an array on the chip with a tissue sample, such that the probes in the array identify and bind to nucleic acids, particularly mRNA, of cells in the tissue.

17. The method further comprises the step of releasing nucleic acids in a tissue from cells and contacting the probes in the chip, preferably, when the tissue is a fixed and embedded tissue, releasing nucleic acids from cells by permeabilizing the tissue; Optionally, further comprising the step of performing a reverse transcription reaction; Optionally, further comprising amplifying said cDNA molecule; 17. The method of claim 16, optionally further comprising a step of performing library construction / sequencing on the amplified nucleic acid.

18. 18. The method of claim 17, wherein the biological sample is a tissue sample from a subject, for example a tissue sample excised during surgery, preferably a thin tissue section obtained by processing by microsectioning.

19. 20. The method of claim 18, further comprising the step of performing morphological and / or histological analysis on the thin tissue sections, wherein the histological analysis is performed by H&E staining, IHC staining, ISH staining and FISH staining.

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