Biochip for spatial transcriptome analysis, method for manufacturing the same, and use

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, filed on December 24, 2021 (Title of Invention: Method for Manufacturing a Biochip for Spatial Transcriptome Analysis and Use Thereof) and Chinese Application No. 202210728404.7, filed on June 25, 2022 (Title of Invention: Method for Manufacturing a Biochip for Spatial Transcriptome Analysis and Use Thereof), the entire content of which is incorporated herein by reference.

[0002] The present invention relates to the fields of biology and medical devices. Specifically, the present invention relates to a method for manufacturing a chip for analyzing nucleic acid information of cells in a biological sample. The chip is applied to the analysis of spatial transcriptome information of a biological tissue sample.

Background Art

[0003] Analysis of the cellular organization and expression patterns of biological tissues is a milestone in biomedical research and diagnostics. Histology has made it possible to study protein distribution in gene expression by immunohistochemistry and in situ hybridization using various staining techniques.

[0004] New research expects that it will be possible to characterize transcriptome and / or genomic variations within tissues while preserving the spatial information about the tissues.

[0005] In the prior art, nucleic acid information of cells in a biological sample has been analyzed using a chip, and methods for forming probes that identify and bind nucleic acids of a biological sample on a substrate such as a glass sheet and chip products containing these probes have been provided.

[0006] In this technical field, in the large-scale production of chips for analyzing nucleic acid information of cells in biological samples, there is a need to more conveniently and efficiently form probes that can bind to target DNA or RNA molecules on the chip substrate, obtain chips of better quality, and acquire manufacturing processes and products with more stable production rates. [Overview of the project]

[0007] The present invention provides a method for large-scale production of biochips suitable for analyzing nucleic acid information of cells in biological tissue samples, particularly spatial transcriptome information of biological tissue samples. Specifically, the present invention provides an improved method for manufacturing a biochip having an array. This method includes the following steps: Step 1: Offer a tip, Step 2: Fix the chip surface linker nucleic acid to the chip surface, for example, the entire surface of the chip. Step 3: The first group of barcode nucleic acids is added to the chip surface by a plurality of parallel microchannels, and a plurality of first barcode strips are formed in a first direction under conditions in which a binding reaction occurs between the chip surface linker nucleic acid and the first barcode nucleic acid, wherein the first group of barcode nucleic acids includes a plurality of types of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is immobilized on each first barcode strip, and the first barcode nucleic acids immobilized on each first barcode strip have different barcode sequences. Step 4: Multiple parallel microchannels are used to add the second group of barcode nucleic acids to the chip surface along the second direction, forming multiple second barcode strips, wherein the second group of barcode nucleic acids includes multiple types 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 a binding reaction occurs 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 at a position on the chip surface where the plurality of first barcode strips and the plurality of second barcode strips intersect to form a probe, and the position of the probe (i.e., the position where the plurality of first barcode strips and the plurality of second barcode strips intersect) constitutes an array feature, and each feature has probes with different sequences from each other.

[0008] In one aspect of the present invention, in the method described above, the first group of barcode nucleic acids or the second group of barcode nucleic acids are transported to and fixed to the chip surface by a microfluidic device having a plurality of parallel microfluidic channels, and the surface of the microfluidic channel in contact with the chip surface is permeable to a solution or nucleic acids in a solution.

[0009] In one aspect of the present invention, in the method described above, 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 microfluidic device.

[0010] In one aspect of the present invention, the chip surface linker nucleic acid may be fixed to the chip surface by chemical bonding. Chemical bonding is selected from, for example, group linking by an amino group-aldehyde group reaction and covalent crosslinking. The chip surface may be coated with activating groups such as amino, aldehyde, epoxy, isothiocyanate, mercapto, and silyl by surface chemical reactions. The chip surface linker nucleic acid has a group at one end (usually the 5' end) attached to the chip surface that forms a chemical bond with the activating group being coated.

[0011] In another aspect of the present invention, the chip surface linker nucleic acid may be fixed to the chip surface by physical adsorption methods such as hydrophobicity or electrostatic attraction. 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 inversely complementary to the sequences at both ends of the first single-stranded binding nucleic acid.

[0013] In another aspect of the present invention, the chip surface linker nucleic acid has about 10-50 nucleotides, preferably fewer than 30 nucleotides, for example, fewer than 25 nucleotides.

[0014] In one aspect of the present invention, in the method, the first barcode nucleic acid in the first group of barcode nucleic acids includes a first barcode fragment. In another aspect of the present invention, the 5' end of the 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 the 3' end has a first binding fragment for binding to the second barcode nucleic acid.

[0015] In one aspect of the present invention, the second barcode nucleic acid in the second group of barcode nucleic acids includes 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 present invention, in the method described above, the 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 by the second single-stranded linker 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 by the second single-stranded linker nucleic acid, wherein the first and second binding fragments are inversely complementary to the sequences at both ends of the second single-stranded linker nucleic acid.

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

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

[0020] In one aspect of the present invention, in the method described above, the arrangement of the first barcode fragment and the second barcode fragment of the probe is specified.

[0021] In one aspect of the present 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 aspect of the present invention, in step 3 of the method, the nucleic acid concentration in the channel is approximately 0.1–100 μM, for example, approximately 1–20 μM.

[0023] In one aspect of the present invention, in step 4 of the method, the nucleic acid concentration in the channel is approximately 0.1–100 μM, for example, approximately 1–20 μM.

[0024] In one aspect of the present invention, in the said method, in step 3 and step 4, the width of each microchannel of the microchannels provided in parallel 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 the said method, in step 3 and step 4, the distance between adjacent microchannels of the microchannels provided in parallel is 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 aspect of the present invention, the probe density at the feature of the chip manufactured by the said method is about 10 3 -10 5 probes / μm 2 is.

[0027] In one aspect of the present invention, the uniformity deviation of the probes of the feature of the chip manufactured by the said method is less than 20%, preferably less than 10%, more preferably less than 6%.

[0028] In one aspect of the present invention, the uniformity deviation of the size of the feature of the chip manufactured by the said 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 method described above. 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 mutually orthogonal rows and columns. Each probe in the array has a different barcode sequence that can indicate the spatial position of the probe. In one aspect of the present invention, the probe includes a first barcode and a second barcode. In another aspect of the present invention, each probe in each row of the probe array has the same first barcode, and each probe in each column has 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 across 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 present invention, the sequence of each probe in the probe array includes the chip surface linker nucleic acid from the 5' end to the 3' end, a first barcode, a second barcode, and a capture fragment for identifying and binding the target nucleic acid in the biological sample. In another aspect of the present invention, the sequence of each probe in the probe array includes a primer fragment for amplification reaction at the 5' end. In another aspect of the present invention, each probe in the probe array further includes a unique molecular identifier (UMI).

[0030] In one aspect of the present invention, the probe density at the chip feature is approximately 10 3 -10 5 pieces / μm 2 In one aspect of the present invention, the uniformity deviation of the probes on the chip features is less than 20%, preferably less than 10%, and more preferably less than 6%. In one aspect of the present invention, the uniformity deviation of the size of the chip features is less than 10%, preferably less than 5%, and more preferably less than 2%.

[0031] The chip provided in the present invention or the chip manufactured by the method provided in the present invention is applicable to the analysis of intracellular molecules (analysis of nucleic acids and proteins) in tissue samples, particularly tissue sections, and expression and spatial information can be obtained by analysis by methods such as PCR, mass spectrometry, next-generation sequencing, or ELISA.

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

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

[0034] In another aspect of the present invention, the method further includes, for example, separating the tissue / cells after collecting them, separating and purifying the cDNA, and then, after separating the tissue from the chip, dividing the tissue / cells in one container and separating and purifying the nucleic acids.

[0035] In another aspect of the present invention, the method further includes amplifying the cDNA molecule.

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

[0037] In one aspect of the present invention, the biological sample is a tissue sample derived from a subject, for example, a tissue sample excised during surgery, preferably a thin tissue section obtained by microsectioning. 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 acids of cells in the fixed tissue are permeabilized and then separated from the tissue and bound to a probe on a chip.

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

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

[0040] In one aspect of the present invention, the test subject is selected from animals, livestock, pets, and human test subjects.

[0041] In one aspect of the present invention, the analyte further comprises one or more of the following: non-human cells, human cells, non-natural proteins, nucleic acids, small molecules, dyes, viruses, bacteria, parasites, protozoa, or chemical substances. 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 can be applied to the analysis of quantitative and / or qualitative data of markers in a sample. These markers include DNA, proteins, RNA, lipids, organelles, metabolites, or cells. These 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 nucleotide positions; intragenetic or intergenetic 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; cDNA or fragments; SNPs; somatic mutations and / or germline mutations; punctate mutations or single mutations; deletion mutations; in-frame deletions, intragenetic deletions, whole gene deletions; insertion mutations; intragenetic insertions; inversion mutations; intrachromosomal inversions, linkage mutations, linkage insertion mutations; inverted 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. This marker 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. These tissue samples include one or more types of precancerous or malignant cells, cells derived from solid tumors, soft tissue tumors or metastatic lesions, tissue or cells derived from surgical margins, histologically normal tissue, one or more types of circulating tumor cells (CTCs), normal adjacent tissue (NAT), blood samples from the same subject who is affected by or at risk of developing a tumor, and FFPE samples. [Brief explanation of the drawing]

[0044] To more clearly explain the embodiments of the present invention or the technical means in the prior art, the following briefly introduces the drawings necessary for describing the embodiments or the prior art. As is clear from the following description, the drawings are only those of some embodiments of the present invention.

[0045] [Figure 1] This is a flowchart of the procedure for manufacturing the chip provided in the present invention. [Figure 2] This is one exemplary embodiment of a facility having a plurality of parallel microchannels used in the method of the present invention. [Figure 3] This is an observation diagram of the fluorescence signal of the probe array of the biochip provided in the present invention. [Figure 4] This figure shows the measurement of an array of biochips provided in the present invention based on probe fluorescence signals. Figures 4A and 4B show the measurement results for array size and fluorescence intensity, respectively. [Figure 5] This is a tissue HE staining image obtained by spatial transcriptome analysis of mouse brain tissue using the biochip provided in the present invention. [Figure 6] This is a quality inspection analysis diagram of the process of processing and analyzing nucleic acids in mouse brain tissue using the biochip provided in the present invention. [Figure 7A] This diagram shows the number of genes that can be detected by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided in the present invention. [Figure 7B] This figure shows the number of UMIs that can be detected by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided in the present invention. [Figure 7C] This is a distribution map showing the number of genes actually detected and the number of captured transcripts (UMIs) obtained by performing spatial transcriptome analysis on mouse brain tissue using the biochip provided in the present invention. [Figure 8] This figure shows the results of gene cluster analysis performed on mouse brain tissue using the biochip provided in the present invention, based on spatial transcriptome analysis. [Modes for carrying out the invention]

[0046] To further clarify the object, technical means, and advantages of the embodiments of the present invention, the technical means in the embodiments of the present invention will be described clearly and completely below with reference to the drawings of the embodiments. It will be clear that the following embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art without creative effort based on the embodiments of the present invention are all within the scope of protection of the present invention.

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

[0048] Figure 1 is a flowchart illustrating an exemplary method for manufacturing a biochip having the array provided in the present invention.

[0049] As shown in Figure 1, the method mainly includes the following steps. Step 1: Provide a chip (or "substrate"). In the next step, to fix the chip surface linker nucleic acid to the chip surface, the chip surface can be coated with activating groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, and silyl groups by surface chemical reactions. Step 2: The chip surface linker nucleic acid is immobilized 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 later amplification reaction. Step 3: The first group of barcode nucleic acids is added to the chip surface by a plurality of parallel microchannels, and a plurality of first barcode strips are formed in a first direction under conditions in which the chip surface linker nucleic acid and the first barcode nucleic acid bind together. The first group of barcode nucleic acids includes a plurality of types of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is immobilized on each first barcode strip, and the first barcode nucleic acids immobilized on each first barcode strip have different barcode sequences.

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

[0052] Step 4: Remove the microchannels in Step 3 and, using a plurality of other parallel microchannels, form a plurality of second barcode strips in addition to the plurality of first barcode strips having a first direction on the chip surface along a second direction (usually perpendicular to the first direction) with the second group of barcode nucleic acids, wherein the second group of barcode nucleic acids comprises a plurality of types 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.

[0053] The illustrated exemplary second barcode nucleic acid includes a poly-T sequence at the 3' end for identifying and binding 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 a 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, wherein the first and second binding fragments are inversely complementary to the sequences at both ends of the second linker nucleic acid. Under conditions in which the first and second barcode nucleic acids can generate a binding reaction, the second barcode nucleic acid and the first barcode nucleic acid are bound at the chip surface where the plurality of first barcode strips and the plurality of second barcode strips intersect to form a probe.

[0055] Step 5: Remove the microchannels from Step 4 to obtain a biochip having a probe array on its surface. Each feature on the probe array corresponds to the intersection of the plurality of first barcode strips and the plurality of second barcode strips. Each feature has one type of probe molecule, which includes a first barcode sequence and a second barcode sequence. The combinations of the first barcode sequence and the second barcode sequence contained in the probe molecule of each feature are different from each other. In one aspect of the present invention, since the first barcode sequence and the second barcode sequence of the barcode nucleic acid introduced into each microchannel are known or predetermined, the first barcode sequence, the second barcode sequence and their combinations contained in the probe molecule of each feature are also known. This makes it possible to determine the spatial position on the array on the chip surface based on the first barcode sequence and the second barcode sequence of the probe molecule of each feature.

[0056] In Step 1, the substrate chip typically 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, and electromagnetic elements to facilitate the chemical, biological, biophysical, or biochemical processes that occur 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] Chips can be made from any suitable material. Exemplary types of chip materials include glass, modified glass, functional glass, inorganic glass, microspheres (including inert particles and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, fibers or fiber bundles, various polymers other than those listed above, and multiwell microtiter plates. Specific types of exemplary plastics include acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon. TM This includes. Specific types of exemplary silica-based materials include various forms of silicon and modified silicon. The chip surface can be modified for the adhesion of the target biopolymer by various methods known to those skilled in the art.

[0058] In the present invention, the array on the surface of the manufactured chip has probes (or called capture probes). A probe refers to a probe that specifically or specifically identifies and binds to a single-stranded nucleotide molecule of a target nucleic acid, such as a nucleic acid from a tissue sample, and has a specific nucleotide sequence, i.e., it can selectively anneal to the nucleotide sequence of the target nucleic acid, and is usually a complementary nucleotide sequence. Examples of analytes in a tissue sample include genomic DNA, methylated DNA, specific methylated DNA sequences, messenger RNA (mRNA), poly(A) 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 and is hybridized with a specific target mRNA or cDNA in the sample.

[0059] In the present invention, the probe has a barcode sequence and is used in subsequent high-throughput next-generation sequencing (NGS) or synthetic sequencing (SBS) analysis applications, such as high-throughput sequencing analysis. In these sequencing applications, the barcode sequence is used to mark and identify the nucleic acid source of the nucleic acid sequence obtained by sequencing. The barcode molecule barcodes nucleic acid molecules (e.g., RNA molecules) derived from biological particles (e.g., cells) to generate a sequencing library, and then the sequencing library is 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 the target constitute at least 1X, at least 2X, at least 3X, at least 4X, at least 5X, at least 10X, at least 20X, at least 40X or higher coverage of a specific portion or all of the cellular genome. Once barcoded fragments are generated, they can be directly sequenced on a suitable sequencing system (e.g., an Illumina system). The presence of the same barcode on multiple sequences can provide information about the origin of that sequence.

[0060] In this invention, the manufactured probe contains two barcode sequences. The two barcode sequences also function as a position tag, as they help to identify the probe's position on the array on the chip surface (identifying the X and Y coordinates, respectively). The barcode sequences on the probe can correspond to features on the array on the chip and can also indicate the position of cells (including single cells) in the tissue they identify within this tissue sample. Examples of other molecules that can bind to nucleic acid tags include antibodies, antigen-binding domains, proteins, peptides, receptors, and haptens.

[0061] In the present invention, the probe further comprises one or more unique molecular identifiers (UMIs). The unique molecular identifiers are contiguous nucleic acid fragments or two or more discontinuous nucleic acid fragments, which function as markers or identifiers for a specific analyte, or for a capture probe that binds to a specific analyte. The UMI is essentially a nucleic acid sequence that does not hybridize with the nucleic acid molecules of the analyte in the biological sample. The UMI may consist of about 6 to about 20 or more nucleotides 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 carried out by various known methods in the art. Nucleic acid immobilization means direct or indirect attachment to the chip by covalent or non-covalent bonds. In one embodiment of the present invention, immobilization means that the nucleic acid is stationary or attached to the chip in reactions requiring nucleic acid amplification and / or sequencing. In one invention of the present invention, immobilization may also mean that the nucleic acid stationary or attached to the chip can be detached from the chip surface under predetermined conditions in subsequent reactions such as nucleic acid amplification and / or sequencing. Exemplary non-covalent bonds include, but are not limited to, nonspecific 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 on the chip surface by physical adsorption methods such as hydrophobicity or 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 carried out by various known methods in the art. For example, the goal of binding can be achieved after forming a combination of three nucleic acid fragments (first barcode nucleic acid, second barcode nucleic acid and linker nucleic acid) under conditions that allow for a binding reaction, by each being complementary to the different terminal sequences of another single-stranded nucleic acid fragment (linker nucleic acid).

[0064] In one aspect of the present invention, the chip surface linker nucleic acid has a primer fragment at its 5' end (for example, a general-purpose primer sequence used in known sequencing methods) to be used 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 its 5' end for binding to the chip surface. For example, if the chip surface is modified with an aldehyde, the chip surface linker nucleic acid has an amino group at its 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 that modifies the chip. For example, the factor and ligand are antibody-epitope, avidin-biotin, streptavidin-biotin, and lectin-carbohydrate, respectively.

[0066] In one aspect of the present invention, the first barcode nucleic acid-containing first barcode fragment.

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

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

[0069] The chips produced by the method provided in this invention can be applied to the analysis of intracellular molecules (including nucleic acids and proteins) in tissue samples, particularly tissue sections. For example, their expression and spatial information can be obtained by analysis using PCR, mass spectrometry, neograde sequencing, or ELISA.

[0070] The present invention involves contacting a tissue section with an array when using the chip. Probes on the array can identify and bind to nucleic acids, particularly mRNA, of cells in the tissue. Subsequent analyses, including reverse transcription and amplification, can be performed by high-throughput next-generation sequencing (NGS) or synthetic sequencing (SBS).

[0071] The method for manufacturing a chip having a probe array provided in the present invention enables the parallel synthesis of multiple chips having the same code region array on the same substrate. As shown in Figure 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 needed. This results in multiple chips having probes defined by the same first barcode and second barcode arrays for corresponding features.

[0072] WO / 2022 / 135598 discloses a method for manufacturing a biochip having an array. In this method, a first group of barcode nucleic acids are directly immobilized onto a glass sheet modified with optical epoxy using a plurality of parallel-arranged microfluidic devices. In laboratory and large-scale industrial production environments, the inventors found that when nucleic acid molecules are bound to a chip modified by microfluidics, the special hydrodynamic properties of the microfluidics require extremely high conditions for the reaction between the nucleic acids generated in the channels and the modifying groups on the chip surface (including reaction temperature, reactant concentration, ambient humidity, pressure, etc.). If the reaction conditions fluctuate during the reaction process, it can significantly affect the reaction results between the nucleic acids and the modifying groups on the chip surface, potentially causing serious quality control defects in the product. For example, defects such as weak probe signals over large areas on the chip, and even chip "plaque" (no probe signal on the chip).

[0073] The inventors have unexpectedly found that, in the improved manufacturing process provided in the present invention, when immobilizing the nucleic acids of the first group of barcode nucleic acids onto the chip surface, the nucleic acids of the first group of barcode nucleic acids and nucleic acids immobilized on the chip surface (including the entire chip surface or a portion of the chip surface located in the same position as the first group of barcodes) (i.e., chip surface linker nucleic acids or chip surface linkers as defined herein) are linked by a nucleic acid-to-nucleic acid binding reaction (e.g., nucleic acid ligase and nucleic acid linker fragments that link the two nucleic acids), thereby significantly improving the efficiency of immobilization of the first group of barcode nucleic acids onto the chip, significantly increasing the density of probes on each probe array feature on the chip, and significantly improving uniformity. Not limited to this theory, the inventors believe that the efficiency and stability of the nucleic acid-to-nucleic acid binding reaction are stronger than other immobilization methods between nucleic acids and the chip surface (e.g., covalent bonding), which reduces the length of each nucleic acid fragment on the probe and improves the binding efficiency of each fragment. Furthermore, the length of the chip surface linker nucleic acid or chip surface linker may be relatively short. In this case, the efficiency of fixing to the chip by methods such as chemical bonding is higher, and the number of bonds can be increased. This is clearly advantageous for improving the size uniformity of the probe features of the manufactured chip, the amount of probe contained in the probe features, and the uniformity of the probe amount (in the manufactured chip, the intensity of the fluorescent signal supported on the probe is detected by a fluorescence microscope and camera. In applications, this is embodied in identifying the number of nucleic acid transductors or genes in samples such as tissues and cells).

[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 method described above. 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. Each probe in the array has a different barcode sequence that 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, each probe in each row of the probe array has the same first barcode, and each probe in each column has 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 chip surface linker nucleic acid across 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 present invention, the sequence of each probe in the probe array includes the chip surface linker nucleic acid from the 5' end to the 3' end, a first barcode, a second barcode, and a capture fragment for identifying and binding the target nucleic acid in the biological sample. In another aspect of the present invention, the sequence of each probe in the probe array includes a primer fragment for amplification reaction at the 5' end. In another aspect of the present invention, the sequence of each probe in the probe array further includes a unique molecular identifier (UMI).

[0075] The chips produced by the method provided in this invention are applicable to the analysis of intracellular molecules (analysis of nucleic acids and proteins) in tissue samples, particularly tissue sections, and can be analyzed by methods such as 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 the 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 the tissue sample. For example, after hematoxylin-eosin staining, spatial analysis can be performed on the tissue sample by the method 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 solution, embedding it in paraffin, slicing it into thin sections, and then mounting them on a flat surface (e.g., biopsy on a microscope slide). In the method of the present invention, the tissue section is brought into contact with a probe array on a chip. The probes on the array can identify and bind nucleic acids, particularly mRNA, from cells in the tissue. Subsequent analyses, including reverse transcription and amplification, can be performed by high-throughput next-generation sequencing (NGS) or synthetic sequencing (SBS).

[0077] "Sequencing" typically refers to methods and techniques for determining the nucleotide base sequence in one or more polynucleotides. Polynucleotides may be, for example, nucleic acid molecules, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) (including their variations or derivatives, such as single-stranded DNA). Sequencing can be performed using various currently available systems, such as (but not limited to) sequencing systems from Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies. 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 multiple raw genetic data corresponding to the genetic information of a subject (e.g., a human). For example, a sample provided by a subject is produced by the system. In some embodiments, such systems provide sequencing read lengths (also referred to herein as "read lengths"). Read lengths may contain a set 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 a tissue section (e.g., formalin-fixed paraffin-embedded (FFPE) tissue section) 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 general-purpose capture probe, for example, hybridized with an adapter region in a nucleic acid sequencing library or the poly-A tail of mRNA. In some embodiments, the capture probe may be a gene-specific capture probe, for example, hybridized with a specifically targeted mRNA or cDNA in the sample.

[0079] In some embodiments, nucleic acids in tissue sections (e.g., FFPE sections) are transferred to an array and captured by the array by binding to a single strand of a general-purpose adapter oligonucleotide. In other embodiments, nucleic acids on a chip may be transferred to tissue sections (e.g., FFPE sections). By methods known in the art, the probe bound to the chip can be introduced into cells on tissue that come into contact with it after it has detached in solution. For example, a photodegradable linker may be added to the binding site between the nucleic acid probe and the chip, or the nucleic acid probe may be bound to the chip using a pH-sensitive linker, and then the nucleic acid probe may be separated from the chip by changing the pH value of the solution.

[0080] The chip of the present invention and its use allow for the determination of the spatial position in the array on the chip surface based on the first and second barcode sequences of the probe molecules possessed by each feature, thereby enabling the determination of the positional information of the cell in which the nucleic acid molecule is located within the tissue.

[0081] In one aspect of the present invention, the method further comprises performing morphological and / or histological analysis on the tissue sections. This histological analysis is performed by H&E staining, IHC staining, ISH staining, and FISH staining.

[0082] In one aspect of the present invention, the method described above can analyze one or more types of biomolecules in a tissue sample, for example, by PCR, mass spectrometry, next-generation sequencing, or ELISA.

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

[0084] In one aspect of the present invention, in the method, the biomolecule includes one or more of the following: non-human cells, human cells, non-natural proteins, nucleic acids, small molecules, dyes, viruses, bacteria, parasites, protozoa, or chemical substances.

[0085] In one aspect of the present invention, the small molecule in the method includes haptens, peptide tags, protein tags, fluorescent tags, nucleic acid tags, and combinations thereof.

[0086] In one aspect of the present invention, the method includes generating quantitative and / or qualitative data of the marker.

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

[0088] In one aspect of the present invention, the marker in the method includes 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 present invention, compared to normal healthy tissue or cells, the marker in cancer tissue or cancer cells includes a modified nucleotide sequence encoding a modified amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, a change in copy number, a change in expression level, a change in protein level, a change in protein activity, or a change in methylation status.

[0090] In one aspect of the present invention, the marker is 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.

[0091] In one aspect of the present invention, the method further comprises performing morphological and / or histological analysis on the tissue sections. This histological analysis is performed by H&E staining, IHC staining, ISH staining, and FISH staining.

[0092] In one aspect of the present invention, in the method described above, the analysis of one or more types of biomolecules is performed by PCR, mass spectrometry, next-generation sequencing, or ELISA.

[0093] Example 2: Chip Manufacturing Figure 5 is a flowchart of an exemplary embodiment of the method for producing 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 surface chemical reactions with activating groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, and silane groups.

[0095] In this embodiment, a purchased optical epoxy-modified glass sheet (Nexterion® SlideE) was used as the chip substrate.

[0096] A general-purpose chip surface linker nucleic acid with the following sequence, modified with an amino group at its 5' end, was synthesized. Underlined T bases are FITC modifications. 5'-amino-CTACACGACGC T CTTCCGATC-3'

[0097] One hundred samples of the first group of barcode nucleic acids, having the following sequence and with phosphorylation modification at the 5' end, were synthesized. 5'-phosphorylated CTCTTTCCC T AC12345678 ACGACGCTCTTC-3' "12345678" represents a barcode fragment having eight nucleotides, the sequence of the eight nucleotides being known (specified). The sequences of the barcode fragments (referred to as the first barcodes) of the 100 barcode nucleic acids of the first group are distinct from each other, and the sequence of the first barcode of each barcode nucleic acid of the first group is known (specified). Underlined T bases are FITC modifications. In this embodiment, the barcode fragments were fluorescently modified and a fluorescent signal for production detection was used to observe or control production quality at each step of adding the barcode fragments in chip synthesis. In other embodiments, the barcode fragments may not be fluorescently modified.

[0098] 100 samples of barcode nucleic acids from group 2 having the following sequence were synthesized. 5' Phosphorylated-GAGTGATTGCT T GTGACGCCTT87654321NNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' "87654321" represents a barcode fragment having eight nucleotides, the sequence of which is known (specified). The sequences of the barcode fragments (referred to as second barcodes) of the 100 second group barcode nucleic acids are distinct from each other, and the sequence of the second barcode of each second group barcode nucleic acid is known (specified). Underlined T bases are Cy3 modifications.

[0099] One 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] A cell culture cavity and a glass sheet were bonded together, and the cavity and glass sheet were pressed together with a frame to improve sealing. Using a pipette, 10-20 μM of general-purpose tip surface linker nucleic acid (dissolved in 300 mM sodium phosphate buffer pH 8.5) was added to the cavity, and the bottom surface of the cavity was evenly covered. Then the glass sheet was placed in a thermomixer comfort and shaken at 40°C and 800 rpm to allow for a uniform reaction for 3 hours. After the reaction was complete, the modified glass sheet was sequentially washed with 0.1% Triton X-100, 1 mM HCl, and 100 mM KCl, and then blocked at 50°C with 0.1 M Tris (pH 9.0), 50 mM 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 fluorescence signals of common nucleic acids were observed using a fluorescence microscope to confirm complete reaction and the surface modification effect.

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

[0103] The microfluidic apparatus includes approximately 50–500 parallel microfluidic channels. The width of each of the parallel microfluidic channels is approximately 2–200 μm, preferably approximately 5–50 μm, most preferably approximately 5–25 μm, for example, approximately 5 μm, 10 μm, or 50 μm. The distance between adjacent microfluidic channels is approximately 5–400 μm, preferably approximately 10–100 μm, most preferably approximately 10–50 μm, for example, approximately 20 μm, 50 μm, or 100 μm.

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

[0105] After bonding the microfluidic apparatus to the glass sheet, buffer solution was introduced into the channels to remove gases from the channels. Then, 10-20 μM of the first group of barcode nucleic acids was added to the channels, and the first group of barcode nucleic acids (each channel having a different barcode sequence from the first group of barcode nucleic acids in other channels), the first linker nucleic acid, and T4 ligase were introduced into each channel. After filling the channels, the reaction was allowed to proceed by standing at 37°C for 30 minutes. After the reaction was complete, the substrate was washed with deionized water for 1 minute, and then dried by blowing nitrogen gas onto the substrate. The FITC fluorescence signal of the first group of barcode nucleic acids was observed with a fluorescence microscope to confirm that the reaction was complete, and that the first group of barcode nucleic acids and the general-purpose chip surface linker nucleic acid achieved binding reactions with the chip at the areas covered by the channels, forming the first group of barcode strips.

[0106] Another PDMS microfluidic device was bonded to a glass sheet along a direction perpendicular to the flow channel of the first PDMS microfluidic device. After introducing buffer into the flow channels and removing gas from the channels, 10-20 μM of the second group of barcode nucleic acids were introduced. One type of second barcode nucleic acid (each second barcode nucleic acid having a different barcode sequence from the second barcode nucleic acids in other channels), a second linker nucleic acid, and T4 ligase were introduced into each flow channel. After the flow channels were filled, the mixture was allowed to react by standing at 37°C for 30 minutes.

[0107] After the binding reaction was complete, 1×PBS buffer was introduced and the channel was washed with ultrapure water. Then the channel was removed, the substrate was washed with deionized water for 1 minute, and then dried by blowing nitrogen gas onto the substrate. The Cy3 fluorescence signal of the second barcode nucleic acid was observed using a fluorescence microscope, confirming 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 feature was achieved, forming a barcode array. This completed the manufacturing of the chip.

[0108] The manufactured chips were vacuum-packed and stored in a light-shielded refrigerator at room temperature or 4°C. The efficiency of the binding reaction of each barcode nucleic acid and the intensity (density) and uniformity of the probes on the manufactured chips were evaluated by measuring the fluorescence signal on the chips.

[0109] Figure 3 illustrates the detection of the Cy3-modified fluorescence signal carried to the second group of barcode nucleic acids in the obtained chip. The images were captured and measured using a fluorescence microscope (Olympus IX53) and a CCD camera (Olympus DP74).

[0110] Figures 4A and 4B show the detection results of probes on chips manufactured using microchannels with widths of 50 μm, 25 μm, and 10 μm, respectively, according to the method described in Example 2 (i.e., the probe array sizes on the chips are 50 μm x 50 μm, 25 μm x 25 μm, and 10 μm x 10 μm, respectively). Images captured and measured using a fluorescence microscope (Olympus IX53) and a CCD camera (Olympus DP74) were analyzed, and fluorescence intensity values ​​and boundaries were extracted using ImageJ. 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 in Figure 4A, each probe array on the chip manufactured according to the present invention had a size uniformity deviation of less than 0.6% (50 μm), 1.2% (25 μm), and 1.3% (10 μm), respectively. As can be seen from the results in Figure 4B, the probe modification uniformity deviations were less than 5.5% (50 μm), 3.4% (25 μm), and 3.9% (10 μm), respectively. In other words, the chip manufactured according to the present invention has extremely high array size uniformity and uniformity of modified probes on each array.

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

[0113] (2) Frozen section The temperature of the free sectioning machine was set to -20°C for the machine body and -10°C for the sample head. Before sectioning, the frozen tissue and substrate were placed in the machine body at -20°C and allowed to equilibrate for at least 30 minutes. After that, free sectioning was performed in the machine body to a thickness of 10 μm.

[0114] (3) Tissue fixation, HE staining The prepared tissue sections were mounted onto the barcode array-modified substrate prepared in Example 2, and then incubated at 37°C for 1 minute. The substrate with the mounted 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, and 500 μl of isopropanol was added dropwise to the tissue sections and incubated at room temperature for 1 minute. After 1 minute, the isopropanol was removed, and the sections were then dried at room temperature for 5-10 minutes.

[0115] 1 ml of hematoxylin was added and uniformly coated onto the tissue section on the substrate, and incubated at room temperature for 7 minutes. The hematoxylin reagent was removed, the substrate was washed by immersion in RNase-free water, and dried. 1 ml of blue coloring solution was added and incubated at room temperature for 2 minutes. The blue coloring solution was removed, the substrate was washed by immersion in RNase-free water, and the liquid on the back of the substrate was wiped off. 1 ml of eosin mixture was added and incubated at room temperature for 1 minute.

[0116] Eosin was removed, the substrate was washed by immersion in RNase-free water, and dried until the tissue became opaque. After incubation of the glass sheet at 37°C for 5 minutes, bright-field imaging was performed.

[0117] Figure 5 shows HE staining of a mouse brain tissue section.

[0118] (4) Tissue permeabilization The fixture cavities were attached to the manufactured tissue tips so that each tissue section was located inside the corresponding cavity. 70 µl of permeabilizing enzyme (0.1% pepsin diluted in 0.1 N HCl) was added to the cavities, and the tissue was permeabilized at 37°C. After removing the permeabilizing enzyme, the tissue was washed with 0.1 × SSC.

[0119] (5) Chip quality inspection The chips produced in Example 2 were stored for 6 months and 12 months, respectively, and then removed from the -20°C refrigerator. The reaction solution that had been tissue-fixed, HE-stained, and (iv) permeabilized in step (iii) was added to them, and after processing under the reaction conditions, the strength (density) and uniformity of the probes of the chips were examined using the method described in Example 2.

[0120] Example 4: A reverse transcription reaction was performed on tissue sample sections using a tip. 70 μl of reverse transcription mixture was added to the cavity washed with deionized water. The reverse transcription mixture contained 1x first-chain buffer, 5 mM DTT, 500 μM dNTP, 0.19 μg / μl BSA, 1% DMSO, 2.5 μM template switch oligo, 20 U / μl Superscript III, and 2 U / μl RNase inhibitor.

[0121] Array of template switch oligos: 5'Biotin-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3' In the template switch oligo, the last 1st, 2nd, and 3rd bases were modified with riboguanosine.

[0122] After sealing the cavity with tape, it was placed on a temperature-controlled board, adjusted to 50°C, and reverse transfer was performed, allowing the reaction to proceed for 16 hours.

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

[0124] The cDNA second-chain synthesis reaction solution was added to the cavity after washing. The second-chain synthesis reaction solution consisted of 1x first-chain buffer and 10U Klenow Exo. - The mixture contains a 2.5 μM second-strand primer. After sealing the cavity with tape, it was placed on a temperature-controlled board and adjusted to approximately 37°C for cDNA second-strand synthesis, with a reaction time of 1 hour.

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

[0126] After the reaction was complete, the second-strand synthesis reaction solution was aspirated and removed from the cavity. Then, the cavity was washed once with 100 μl of RNase-free water. 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 each. 35 μl of the sample from the cavity was transferred to the corresponding Tris-containing centrifuge tubes 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 mixture. The PCR reaction mixture contained 1 × Kapa HiFi Hotstart ReadyMix, 0.8 μM cDNA forward primer, 0.8 μM cDNA reverse primer, and 35 μl of cDNA template, for 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 amplification, the amplified product was purified using 0.6×AMpure XP Beads. The purified product was 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 the fragmentation reaction mixture was prepared. This mixture 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, for a total volume of 50 μl. The mixture was uniformly pipetted or shaken using a pipette, briefly centrifuged, and the reaction mixture was collected at the bottom of the tube. The PCR tube was placed in the PCR instrument, and the following process was performed.

[0130] [Table 2]

[0131] This process allows for DNA fragmentation, end-filling of the fragmented DNA, phosphorylation of the 5' end, and addition of a dA tail to the 3' end.

[0132] Adapter coupling A new PCR tube was placed on ice, and the adapter-conjugated reaction mixture was prepared. This mixture 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, and 5 μl of adapter (10 pM), for a total volume of 100 μl. The mixture was uniformly pipetted or shaken using a pipette, briefly centrifuged, and the reaction mixture was collected at the bottom of the tube. The PCR tube was placed in the PCR instrument, and the following process was performed.

[0133] [Table 3]

[0134] The adapter array is, 5'Phosphorylated-GATCGGAAGAGCACACGTCTGAACTCCAGTCA*C-3' 5'-GCTCTTCCGATC*T-3' That is the case. In the adapter sequence, the second to last base was thio-modified. After the binding reaction was complete, the conjugation 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. This mixture contained 25 μl of VAHTS HiFi Amplification Mix, 20 μl of adapter-bound DNA purified in the previous step, and 5 μl of Index PCR Primer Mix (10 pM each), for a total volume of 50 μl. The mixture was uniformly pipetted or shaken using a pipette, briefly centrifuged, and the reaction mixture was collected at the bottom of the tube. The PCR tube was placed in the PCR instrument, and the following process was performed.

[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' That is the case.

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

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

[0140] Sequencing PE150 sequencing was performed on the library using an Illumina NovaSeq 6000. Data processing and analysis a) Extract the UMI and Barcode from Read1 using data processing software such as umitools (version: 1.1.2). b) Align read2 to the mouse reference genome, 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) Compare barcode analysis and HE images using Adobe Illustrator.

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

[0142] The above description is merely a preferred embodiment of the present invention and does not limit it. All modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for manufacturing a biochip having an array, comprising the following steps 1 to 5, Step 1: Offer a tip, Step 2: Immobilize the chip surface linker nucleic acid onto the chip surface. Step 3: The first group of barcode nucleic acids is added to the chip surface by a plurality of parallel microchannels, and a plurality of first barcode strips are formed in a first direction under conditions in which a binding reaction occurs between the chip surface linker nucleic acid and the first barcode nucleic acid, wherein the first group of barcode nucleic acids includes a plurality of types of first barcode nucleic acids having different barcode sequences, one type of first barcode nucleic acid is immobilized on each first barcode strip, and the first barcode nucleic acids immobilized on each first barcode strip have different barcode sequences. Step 4: Multiple parallel microchannels are used to add the second group of barcode nucleic acids to the chip surface along the second direction, forming multiple second barcode strips, wherein the second group of barcode nucleic acids includes multiple types 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 a binding reaction occurs 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 at a position on the chip surface where the plurality of first barcode strips and the plurality of second barcode strips intersect, forming a probe. The position of the probe constitutes a feature of the array, and each feature has a position-specific barcode sequence probe. The 3' end of the chip surface linker nucleic acid has a binding fragment that binds to the first barcode nucleic acid by the 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 inversely complementary to the sequences at both ends of the first single-stranded binding nucleic acid, A method wherein the 3' end of the first barcode nucleic acid has a first binding fragment bound to the second barcode nucleic acid by the second single-stranded binding nucleic acid, and the 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, wherein the first and second binding fragments are inversely complementary to the sequences at both ends of the second single-stranded binding nucleic acid.

2. The method according to claim 1, wherein a microfluidic device having a plurality of parallel microfluidic channels transports and fixes the first group of barcode nucleic acids or the second group of barcode nucleic acids to the chip surface, and the surface of the microfluidic channel in contact with the chip surface is permeable to 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 microfluidic device, and the sequences of the barcode fragments of each first barcode nucleic acid in the first group of barcode nucleic acids and the sequences of the barcode fragments of each second barcode nucleic acid in the second group of barcode nucleic acids are specified.

4. The method according to claim 1, wherein the second barcode nucleic acid in the second group of barcode nucleic acids comprises a probe fragment and a second barcode fragment at its 3' end for identifying and binding to a target nucleic acid in a biological sample.

5. The method according to claim 4, wherein the second barcode nucleic acid in the second group of barcode nucleic acids further comprises a unique molecular identifier (UMI).

6. The method according to claim 1, wherein in step 2, the chip surface linker nucleic acid is fixed to the chip surface by chemical bonding.

7. The probe density on the features of the manufactured chip is approximately 10 ３ -10 ５ pieces / μm ２ The method according to claim 1.

8. The method according to claim 1, wherein the uniformity deviation of the probes of the features of the manufactured chip is less than 10%.

9. The method according to claim 1, wherein the size uniformity deviation of the features of the manufactured chip is less than 10%.

10. A chip for analyzing nucleic acid information of biological samples, The surface of the chip has probes forming an array, the probe array includes mutually orthogonal rows and columns, each probe of each feature in the array has a different barcode sequence, the probe includes a first barcode and a second barcode, each probe in each row of the probe array has the same first barcode, each probe in each column has 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, and the chip for analyzing nucleic acid information of the biological sample has chip surface linker nucleic acid across its entire surface. The 3' end of the chip surface linker nucleic acid has a binding fragment that binds to the first barcode nucleic acid by the 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 inversely complementary to the sequences at both ends of the first single-stranded binding nucleic acid, A chip in which the 3' end of the first barcode nucleic acid has a first binding fragment bound to the second barcode nucleic acid by the second single-stranded binding nucleic acid, and the 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, and the first and second binding fragments are inversely complementary to the sequences at both ends of the second single-stranded binding nucleic acid.

11. The chip according to claim 10, wherein the arrangement of each probe in the probe array includes, from the 5' end to the 3' end, a 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.

12. The probe density on the features of the manufactured chip is approximately 10 ３ -10 ５ pieces / μm ２ The chip according to claim 10.

13. The chip according to claim 10, wherein the probe uniformity deviation of the features of the manufactured chip is less than 10%.

14. A method for analyzing spatial transcriptome information of a biological tissue sample using a chip having the array described in Claim 10, The method comprises the steps of bringing the array of chips into contact with a tissue sample, and having probes in the array identify and bind to nucleic acids of cells in the tissue.

15. The method according to claim 14, further comprising the step of releasing nucleic acids from cells in a tissue and bringing them into contact with a probe in the chip.

16. The method according to claim 14, wherein the tissue is fixed and embedded, and nucleic acids are released from cells by permeabilization of the tissue.

17. The steps include performing a reverse transcription reaction, The steps involve amplifying the cDNA molecule and The steps include: building a library and sequencing the nucleic acids obtained through amplification, The method according to claim 14, further comprising:

18. The aforementioned biological sample is a tissue section derived from the subject, The method according to claim 14, further comprising the step of performing morphological and / or histological analysis on the tissue thin section, wherein the histological analysis is performed by H&E staining, IHC staining, ISH staining and FISH staining.

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