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WO2022135598A1 - 用于空间转录组学分析的生物芯片及其制备方法和应用 - Google Patents

用于空间转录组学分析的生物芯片及其制备方法和应用 Download PDF

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Publication number
WO2022135598A1
WO2022135598A1 PCT/CN2021/141391 CN2021141391W WO2022135598A1 WO 2022135598 A1 WO2022135598 A1 WO 2022135598A1 CN 2021141391 W CN2021141391 W CN 2021141391W WO 2022135598 A1 WO2022135598 A1 WO 2022135598A1
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Prior art keywords
barcode
nucleic acid
nucleic acids
chip
fragment
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English (en)
French (fr)
Inventor
赵海峰
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Medspring International Ltd
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Medspring International Ltd
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Priority to CN202180087220.0A priority Critical patent/CN117460840A/zh
Priority to JP2023563145A priority patent/JP2024508042A/ja
Priority to US18/269,625 priority patent/US20240279643A1/en
Priority to EP21909593.2A priority patent/EP4269615A4/en
Publication of WO2022135598A1 publication Critical patent/WO2022135598A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00547—Bar codes
    • B01J2219/00549—2-dimensional
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips

Definitions

  • the present invention relates to the field of biology and medical instruments. Specifically, the present invention relates to a method for preparing a chip for analyzing nucleic acid information of cells of a biological sample, the chip being suitable for analyzing the spatial transcriptomic information of a biological tissue sample.
  • the new study hopes to characterize transcriptome and/or genomic variation in tissues while preserving spatial information about the tissue.
  • transcriptome analysis is performed on mRNA extracted from whole tissues (or even whole organisms). But collecting smaller tissue regions or individual cells for transcriptome analysis is typically laborious, expensive, and low in precision.
  • the present invention provides a method for preparing a biochip suitable for analyzing the nucleic acid information of cells of a biological sample, especially for analyzing the spatial transcriptomic information of a biological tissue sample. Specifically, the present invention provides a method for preparing a biochip with an array, comprising the following steps:
  • the first group of barcode nucleic acids is immobilized on the surface of the chip through a plurality of parallel microfluidic channels to form a plurality of first barcode strips in the first direction, and the first group of barcode nucleic acids includes a variety of barcodes with different barcode sequences.
  • a first barcode nucleic acid, one type of first barcode nucleic acid is fixed on each first barcode strip, and the first barcode nucleic acid fixed on each first barcode strip has different barcode sequences;
  • the second group of barcode nucleic acids is applied to the plurality of first barcode strips with the first direction on the chip surface along the second direction through a plurality of parallel microfluidic channels to form a plurality of second barcode strips, so
  • the second group of barcode nucleic acids includes a plurality of second barcode nucleic acids with different barcode sequences, each second barcode strip has one second barcode nucleic acid, and the second barcode nucleic acid fixed on each second barcode strip has different barcode sequences;
  • the second barcode nucleic acid and the A barcoded nucleic acid is ligated to form probes that make up spots of the array, each spot having a probe that differs in sequence from one another.
  • a microfluidic device having a plurality of microfluidic channels arranged in parallel is used to transport and immobilize the first group of barcoded nucleic acids or the second set of barcoded nucleic acids on the chip surface, wherein the The side of the microfluidic channel in contact with the chip surface can accommodate the solution or the nucleic acid in the solution to pass through.
  • a first group of barcode nucleic acids or a second group of barcode nucleic acids containing different barcode sequences are added to each microfluidic channel of the microfluidic device.
  • the first barcode nucleic acid in the first group of barcode nucleic acids includes a first barcode fragment; preferably, the first barcode nucleic acid also has a 5' end for amplification Reacted primer fragments.
  • the first barcode nucleic acid in the first set of barcode nucleic acids has a group at the 5' end for attaching to the chip surface.
  • the second barcode nucleic acid in the second set of barcode nucleic acids comprises a capture fragment at the 3' end for recognizing and binding target nucleic acid in a biological sample (eg, for recognizing and binding mRNA or cDNA) fragment, eg a poly-T sequence) and a second barcode fragment.
  • a biological sample eg, for recognizing and binding mRNA or cDNA
  • a poly-T sequence eg.g., a poly-T sequence
  • the second barcode nucleic acid in the second set of barcode nucleic acids in the method further has a Unique Molecular Identifier (UMI).
  • UMI Unique Molecular Identifier
  • the 3' end of the first barcode nucleic acid has a first linker fragment for linking with the second barcode nucleic acid through a single-stranded linker nucleic acid
  • the second barcode nucleic acid has a 3' end.
  • the 5' end has a second linker segment for linking with the first barcode nucleic acid through the single-stranded linker nucleic acid, and the first linker segment and the second linker segment are respectively reversed to the sequences at both ends of the single-stranded linker nucleic acid. to complement each other.
  • the probe formed in step C of the method includes a capture fragment at the 3' end for identifying and binding target nucleic acid in a biological sample, and a first barcode fragment and a second barcode fragment.
  • the probe also has a primer fragment at the 5' end for amplification reaction.
  • the method in which the sequence of the barcode fragment of each first barcode nucleic acid in the first set of barcode nucleic acids is specified, and/or the sequence of each barcode fragment in the second set of barcode nucleic acids is specified The sequence of the barcode fragment of a second barcode nucleic acid is specified.
  • the method in which the sequences of the first barcode fragment and the second barcode fragment of the probe are specified.
  • the nucleic acid concentration in the flow channel in step A of the method is about 0.1-100 uM, for example, about 1-20 uM.
  • the nucleic acid concentration in the flow channel in step B of the method is about 0.1-100 uM, for example, about 1-20 uM.
  • the nucleic acids of the first group of barcode nucleic acids are immobilized on the surface of the chip.
  • the nucleic acids of the first set of barcoded nucleic acids are preferably immobilized on the surface of the chip by chemical bonding.
  • the chemical bonding method is, for example, any one selected from the group consisting of amino-aldehyde group reaction and the like, and covalent cross-linking.
  • the surface of the chip can be coated with active groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, sulfhydryl groups, silanes, etc. through surface chemical reactions; the nucleic acids of the first group of barcode nucleic acids are connected to the chip surface.
  • One end (usually the 5' end) has a group that forms a chemical bond with the coated reactive group.
  • the width of each micro-channel of the parallel-arranged micro-channels in steps A and B of the method is about 2-200 ⁇ m, preferably about 5-50 ⁇ m, and most preferably About 5-25 ⁇ m, for example about 5 ⁇ m, 10 ⁇ m or 50 ⁇ m.
  • the distance between each adjacent microfluidic channel of the parallelly arranged microfluidic channels in step A and step B is about 5-400 ⁇ m, preferably about 10-100 ⁇ m , most preferably about 10-50 ⁇ m, for example about 20 ⁇ m, 50 ⁇ m or 100 ⁇ m.
  • the prepared chip of the present invention can be used to analyze tissue samples, especially tissue thin sections, for analysis of intracellular molecules, including analysis of nucleic acids and proteins, such as analysis by PCR, mass spectrometry, next-generation sequencing, or ELISA, Obtain its expressive and spatial information.
  • the biological sample is a tissue sample from a subject, such as a surgically resected tissue sample, preferably a tissue thin section processed by microsection.
  • the tissue sample is fixed and embedded (eg, in paraffin), and attached to a support such as a glass slide.
  • the tissue thin sections may be subjected to morphological and/or histological analysis by H&E staining, IHC staining, ISH staining, and FISH staining.
  • the one or more biomolecules are analyzed by PCR, mass spectrometry, next generation sequencing, or ELISA.
  • the subject is selected from animals, farm animals, pets, human subjects.
  • the analyte further comprises one or more of non-human cells, human cells, non-native proteins, nucleic acids, or small molecules, dyes, viruses, bacteria, parasites, protozoa, or chemicals .
  • Small molecules include haptens, peptide tags, protein tags, fluorescent tags, nucleic acid tags, and combinations thereof.
  • the chip can be used to analyze quantitative and/or qualitative data of markers in a sample.
  • the markers include DNA, protein, RNA, lipids, organelles, metabolites, or cells.
  • the protein may comprise a modification selected from the group consisting of acetylation, ADP-ribosylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, Covalent attachment of heme, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide Bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamic acid formation, formylation, ⁇ -carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation , myristoylation, oxidation, proteolytic processing, phosphorylation, pren
  • SNP genomic SNPs
  • somatic polymorphisms differential expression of proteins, lipids, and/or organelles.
  • the markers include single nucleotide positions; intragenic or intergenic regions; exons or introns, or fragments thereof; coding or noncoding regions; promoters, enhancers, 5' untranslated regions (5 'UTR), or 3' untranslated region (3'UTR), or fragments thereof; cDNA or fragments thereof; SNPs; somatic mutations, germline mutations, or both; point mutations or single mutations; deletion mutations; in-frame Deletion, Intragenic Deletion, Whole-Gene Deletion; Insertion Mutation; Intragenic Insertion; Inversion Mutation; Intrachromosomal Inversion; Linked Mutation; Translocation, non-reciprocal translocation; rearrangement; genomic rearrangement; rearrangement of one or more introns, or fragments thereof; rearranged introns; 5'- or 3'-UTR, or a
  • the marker includes altered nucleotide sequences encoding altered amino acid sequences, chromosomal translocations, intrachromosomal inversions, copy number changes, Changes in expression levels, changes in protein levels, changes in protein activity, or changes in methylation status.
  • this can be done by single-cell sequencing, single-nucleus sequencing, flow cytometry, immunohistochemical staining, hematoxylin and eosin staining, whole genome sequencing, high-throughput sequencing, mass spectrometry, DNA microarrays, or a combination thereof markers are measured.
  • the chip of the present invention can be used to analyze tissue samples.
  • the tissue sample includes a sample selected from the group consisting of one or more pre-malignant or malignant cells, cells from solid tumors, soft tissue tumors or metastases, tissue or cells from surgical margins, histologically normal tissue, a One or more circulating tumor cells (CTCs), normal adjacent tissue (NAT), blood samples from the same subject with a tumor or at risk of developing a tumor, or a FFPE sample.
  • FIG. 1 is a schematic diagram of the steps of the method for preparing a chip provided by the present invention.
  • FIG. 2 is an exemplary embodiment of an apparatus having a plurality of microfluidic channels arranged in parallel in the method of the present invention.
  • FIG. 3 is a schematic diagram of the steps of the method for preparing a chip provided by the present invention.
  • FIG. 4 is a schematic flowchart of an exemplary embodiment of the method for preparing a biochip provided by the present invention.
  • FIG. 5 is a schematic flowchart of another exemplary embodiment of the method for preparing a biochip provided by the present invention.
  • FIG. 6 is the observation of the barcode nucleic acid ligation reaction in the preparation process of the biochip provided by the present invention.
  • FIG. 7 is an image after HE staining of tissue sections in the biospatial group study of sample tissue by using the prepared biochip provided by the present invention.
  • the present invention provides a method for preparing a biochip suitable for analyzing nucleic acid information of cells of a biological sample. More specifically, the present invention provides a method for preparing a biochip having an array. In one aspect of the present invention, the chip provided by the present invention is suitable for analyzing spatial transcriptomic information of biological tissue samples.
  • FIG. 1 is a schematic flowchart of an exemplary method for preparing a biochip with an array provided by the present invention.
  • the method mainly includes the following steps:
  • Step 1 Provide chip substrate
  • Step 2 The first group of barcode nucleic acids (which includes a plurality of first barcode nucleic acids, wherein each first barcode nucleic acid has a different barcode sequence (Barcode A in the figure)) and
  • the chip surface is contacted and fixed on the chip surface, and a plurality of first barcode strips are formed on the chip surface in a first direction, wherein the first barcode nucleic acid fixed on each first barcode strip has a different first barcode sequence.
  • FIG. 2 is an exemplary embodiment of contacting and immobilizing a plurality of barcode nucleic acids on the chip surface through a plurality of parallel microfluidic channels.
  • the chip Below the left image of Figure 2 is the chip.
  • the middle of the left image of FIG. 2 shows a microfluidic device having a plurality of microfluidic channels (microfluidic channel 1 to microfluidic channel n) arranged in parallel, wherein the side of the microfluidic channel in contact with the surface of the chip is shown in the figure
  • the bottom of the microfluidic channel can accommodate solution or nucleic acid in solution to pass (permeate).
  • the side of the microfluidic channel in contact with the chip surface does not have a microfluidic channel wall.
  • the microfluidic device is covered on the surface of the chip along the first direction, and then a designated solution, such as a solution containing barcode nucleic acid, is passed into the microfluidic channel.
  • a designated solution such as a solution containing barcode nucleic acid
  • the upper part of the left image of FIG. 2 is an exemplary device for assisting the introduction of the solution, such as a vacuum suction device using negative pressure, which can be arranged at the outlet of the microfluidic channel.
  • the right panel of FIG. 2 shows that barcode nucleic acids containing different barcode sequences (barcode nucleic acids 1-n in the figure) are added through the inlet in each microfluidic channel.
  • the barcode sequence of the barcode nucleic acid passed into each microfluidic channel has a known or specified nucleotide sequence.
  • Step 3 The microfluidic channel in step 2 is removed, and the second group of barcoded nucleic acids (which includes a plurality of second barcoded nucleic acids, wherein each second barcoded nucleic acid has a Different barcode sequences (Barcode B in the figure) are in contact with the chip surface along a second direction (usually perpendicular to the first direction) that is different from the direction of the first barcode strip, forming a plurality of second barcode strips, each of which is The second barcode nucleic acid immobilized on the second barcode strip has a different second barcode sequence;
  • the second barcode nucleic acid is ligated with the first barcode on the surface of the chip where the plurality of first barcode strips and the plurality of second barcode strips intersect.
  • the barcoded nucleic acids are ligated to form probes.
  • Step 4 Remove the microfluidic channel in step 3 to obtain a biochip with a probe array on the surface.
  • Each dot on the probe array corresponds to a position where the plurality of first barcode strips and the plurality of second barcode strips intersect.
  • Each spot has a probe molecule that includes a first barcode sequence and a second barcode sequence.
  • the probe molecules of each array spot include different combinations of the first barcode sequence and the second barcode sequence.
  • the first barcode sequence and the second barcode sequence of the barcode nucleic acid passed into each microfluidic channel are known or specified, so that the probe molecules of each array point include First barcode sequences and second barcode sequences and combinations thereof are also known. Thereby, the spatial position of the probe molecules in the array on the chip surface can be known through the first barcode sequence and the second barcode sequence of the probe molecules of each array point.
  • a chip generally refers to a solid substrate on which chemical, biological, biophysical or biochemical processes and the like can be implemented.
  • Chips can have microstructures or microscale structures such as channels and wells, electrode elements, electromagnetic elements, etc. that facilitate chemical, biological, biophysical or biochemical processes that take place on the chip.
  • the chip surface can be flat or uneven. Chips with uneven surfaces may include channels or holes formed in the surface.
  • the chip can be made of any suitable material, exemplary types of chip materials include glass, modified glass, functionalized glass, inorganic glass, microspheres (including inert and/or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose Elements, ceramics, resins, silica, silica-based materials, carbon, fibers or fiber optic bundles, various polymers other than those exemplified above, and porous microtiter plates.
  • Specific types of exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and TeflonTM.
  • Specific types of exemplary silica-based materials include various forms of silicon and modified silicon. Chip surfaces often require deposition of biopolymers (including nucleic acids, polypeptides, and/or other polymers). The surface of the chip can be modified to accommodate the attachment of the target biopolymer by a variety of methods known to those skilled in the
  • the prepared array on the surface of the chip has probes (or called capture probes).
  • a probe is a single-stranded nucleotide molecule that can gene-specifically or target-specifically recognize and bind a target nucleic acid, such as nucleic acid from a tissue sample, that has a specific nucleotide sequence, i.e., is capable of selectively annealing to The nucleotide sequence of the target nucleic acid, usually the complementary nucleotide sequence.
  • tissue samples examples include genomic DNA, methylated DNA, specific methylated DNA sequences, messenger RNA (mRNA), poly A mRNA, fragmented mRNA, fragmented DNA, mitochondrial DNA, viral RNA , microRNAs, in situ synthesized PCR products, RNA/DNA hybrids, lipids, carbohydrates, proteins, glycoproteins, lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, or viral capsid proteins .
  • the capture probes may be gene-specific capture probes that hybridize, for example, to specifically targeted mRNA or cDNA in the sample.
  • the probes have barcode sequences and are used in subsequent high-throughput next-generation sequencing (NGS) or sequencing-by-synthesis (SBS) applications, such as high-throughput sequencing analysis.
  • NGS next-generation sequencing
  • SBS sequencing-by-synthesis
  • barcode sequences are employed to mark and identify the source of nucleic acid for the nucleic acid sequence obtained by sequencing.
  • Barcode molecules are used to barcode nucleic acid molecules (eg, RNA molecules) from biological particles (eg, cells) to generate sequencing libraries, which are subsequently sequenced to generate multiple sequencing reads. Some or all of the plurality of sequencing reads include barcode sequences.
  • cellular nucleic acids are typically amplified until barcoded overlapping fragments in the subject constitute at least IX coverage, at least 2X, at least 3X, at least 4X, at least 5X, at least 10X, At least 20X, at least 40X or higher coverage.
  • barcoded fragments Once barcoded fragments are generated, they can be sequenced directly on a suitable sequencing system, such as an Illumina system. The presence of the same barcode on multiple sequences can provide information about the origin of that sequence.
  • barcode sequences can be relied upon to identify the origin of nucleic acid fragments and, for example, to assemble larger sequences from sequenced fragments. Barcoding can allow identification and/or quantification of individual polynucleotide fragments during the sequencing process.
  • two barcode sequences are contained in the prepared probe.
  • the two barcode sequences can help determine the position of the probes in the array on the chip surface (position in the X and Y dimensions, respectively), and thus also function as position labels.
  • the barcode sequence on the probe can correspond to a feature in the array on the chip, and can also indicate the location of the cells on the tissue it identifies, including individual cells, in the tissue sample. Examples of other molecules that can be conjugated to nucleic acid tags include antibodies, antigen binding domains, proteins, peptides, receptors, haptens, and the like.
  • the probe further comprises one or more Unique Molecular Identifiers (UMI).
  • UMI Unique Molecular Identifiers
  • Unique molecular identifiers are contiguous nucleic acid fragments or two or more non-contiguous nucleic acid fragments that serve as labels or identifiers for a specific analyte or capture probe that binds to a specific analyte.
  • UMIs are nucleic acid sequences that do not substantially hybridize to analyte nucleic acid molecules in a biological sample. The UMI may comprise from about 6 to about 20 or more nucleotides within the sequence of the capture probe.
  • the immobilization of the first group of barcode nucleic acids to the chip can be performed by various methods known in the art. Immobilization of nucleic acids refers to direct or indirect attachment to a chip by covalent or non-covalent bonds. In one aspect of the invention, immobilization refers to remaining stationary or attached to a chip during reactions such as nucleic acid amplification and/or sequencing.
  • non-covalent linkages include, but are not limited to, non-specific interactions (eg, hydrogen bonding, ionic bonding, van der Waals interactions, etc.) or specific interactions (eg, affinity interactions, receptor-ligand interactions, etc.) effect, antibody-epitope interaction, avidin-biotin interaction, streptavidin-biotin interaction, lectin-carbohydrate interaction, etc.).
  • non-specific interactions eg, hydrogen bonding, ionic bonding, van der Waals interactions, etc.
  • specific interactions eg, affinity interactions, receptor-ligand interactions, etc.
  • the ligation of the first barcode nucleic acid and the second barcode nucleic acid can be performed by various methods known in the art. For example, three nucleic acid fragments (a first barcode nucleic acid, a second barcode nucleic acid, and a linker nucleic acid) are formed under conditions that allow a ligation reaction by each being complementary to sequences at different ends of another single-stranded nucleic acid fragment (linker nucleic acid). ) to achieve the purpose of connection.
  • the first barcode nucleic acid comprises a first barcode fragment.
  • the first barcode nucleic acid has a primer fragment at the 5' end for subsequent amplification reactions, such as a universal primer sequence used in known sequencing methods.
  • the first barcode nucleic acid has a group or sequence at the 5' end for linking to the chip surface. For example, if the surface of the chip is modified with an aldehyde group, the first barcode nucleic acid has an amino group at the 5' end.
  • the first barcode nucleic acid has a gamete that can specifically interact with a modified factor on the chip, for example, the factor and gamete are antibody-epitope, avidin-biotin, respectively , streptavidin-biotin, lectin-carbohydrate.
  • the second barcode nucleic acid comprises a second barcode fragment.
  • the second barcode nucleic acid has a capture fragment at the 3' end for recognizing and binding a target in a biological sample, such as a fragment that recognizes and binds mRNA or cDNA, such as a polynucleotide that recognizes mRNA -T sequence.
  • the 3' end of the first barcode nucleic acid has a first linker fragment (3' linker fragment) for ligation with the second barcode nucleic acid.
  • the 5' end of the second barcode nucleic acid has a second ligation fragment (5' ligation fragment) for ligation with the first barcode nucleic acid.
  • the first linker segment and the second linker segment are complementary to one end of the linker nucleic acid, respectively, and under ligable conditions (eg, in the presence of T4 ligase, etc.), the first linker The fragment and the second ligation fragment form a combination with the linker nucleic acid to achieve ligation of the first barcode nucleic acid and the second barcode nucleic acid.
  • the chip prepared by the method provided by the present invention can be used to analyze the intracellular molecules of tissue samples, especially tissue thin sections, including the analysis of nucleic acids and proteins, such as analysis by PCR, mass spectrometry, next-generation sequencing, or ELISA , to obtain its expression and spatial information.
  • Sequequencing generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides.
  • a polynucleotide can be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (eg, single-stranded DNA). Sequencing can be performed by various systems currently available, such as, but not limited to, sequencing systems by Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies.
  • sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (eg, digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification.
  • PCR polymerase chain reaction
  • Such systems can provide a plurality of raw genetic data corresponding to the genetic information of a subject (eg, a human), as generated by the system from a sample provided by the subject.
  • a subject eg, a human
  • sequencing reads also referred to herein as "reads”
  • a read length can include a string of nucleic acid bases that corresponds to the sequence of a nucleic acid molecule that has been sequenced.
  • the systems and methods provided herein can be used with proteomic information.
  • 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 or a fresh tissue sample or a frozen tissue sample and the like.
  • FFPE formalin-fixed paraffin-embedded
  • the methods disclosed herein can be performed before or after staining the tissue sample. For example, following hematoxylin and eosin staining, tissue samples can be spatially analyzed according to the methods provided herein.
  • Methods may include analyzing the histology of the sample (eg, using hematoxylin and eosin staining), followed by spatial analysis of the tissue.
  • Formalin fixation and paraffin embedding (FFPE) of tissue sections typically involves fixation of tissue obtained from a subject in formaldehyde (eg, 3%-5% formaldehyde in phosphate buffered saline) or Bouin's solution, embedding into wax, cut into thin sections, and mounted on a flat surface such as a microscope slide for biopsies.
  • formaldehyde eg, 3%-5% formaldehyde in phosphate buffered saline
  • Bouin's solution embedding into wax, cut into thin sections, and mounted on a flat surface such as a microscope slide for biopsies.
  • the tissue section is contacted with an array, and the probes on the array can identify and bind to nucleic acid, especially 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).
  • NGS next-generation sequencing
  • SBS sequencing-by-synthesis
  • nucleic acids in tissue sections are transferred to an array and captured onto the array by hybridization to capture probes.
  • the capture probe can be a universal capture probe that hybridizes, for example, to an adaptor region in a nucleic acid sequencing library, or to a poly-A tail of mRNA.
  • the capture probes may be gene-specific capture probes that hybridize, for example, to specifically targeted mRNA or cDNA in the sample.
  • nucleic acids in tissue sections are transferred to an array and captured onto the array by single-stranded ligation with a universal adaptor oligonucleotide.
  • the nucleic acids on the chip can be transferred into tissue sections (eg, FFPE sections).
  • the probes bound to the chip can be made to enter cells on the tissue in contact with them after being detached in solution by methods known in the art. For example, a photolytic linker can be added at the binding site of the nucleic acid probe and the chip, or the nucleic acid probe can be bound to the chip through a pH-sensitive linker, and then the nucleic acid probe can be separated from the chip by changing the pH value of the solution.
  • the spatial position of the probe molecule on the chip surface can be known through the first barcode sequence and the second barcode sequence of the probe molecules in each array point, and the location of the nucleic acid molecule can also be obtained. location information of cells in the tissue.
  • the method for preparing a chip with a probe array can realize parallel synthesis of a plurality of chips with an array of the same coding region on the same substrate. As shown in FIG. 3 , multiple groups of identical first barcode strips and second barcode strips can be formed in the first and second directions of the chip substrate as required, thereby obtaining multiple sets of identical A chip with probes defined by the first barcode and the second barcode sequence.
  • step 2 of the aforementioned method ie, step A of a method for preparing a biochip with an array provided by the present invention
  • step A of a method for preparing a biochip with an array provided by the present invention namely
  • the nucleic acid of the first group of barcode nucleic acids is immobilized on the chip surface (including the entire chip surface or the same part of the chip surface as the first group of barcodes).
  • nucleic acid (herein referred to as chip surface linker nucleic acid or chip surface linker) is connected by a ligation reaction of nucleic acid to nucleic acid (for example, by a nucleic acid ligase and a nucleic acid linker fragment bridging the two nucleic acids), which can significantly
  • the immobilization efficiency of the nucleic acids of the first group of barcode nucleic acids on the chip is significantly improved, so that the probe density on the array point where each probe of the chip constitutes the array is significantly increased, and the uniformity is significantly improved.
  • step A further includes a pre-step, which includes pre-immobilizing on the surface of a chip (eg, a glass slide) (eg, on the entire surface of the chip) for interacting with the first group of barcoded nucleic acids Attached chip surface linker nucleic acid.
  • a pre-step which includes pre-immobilizing on the surface of a chip (eg, a glass slide) (eg, on the entire surface of the chip) for interacting with the first group of barcoded nucleic acids Attached chip surface linker nucleic acid.
  • the chip surface linker nucleic acid can be immobilized on the chip surface by chemical bonding.
  • the chemical bonding method is, for example, any one selected from the group consisting of amino-aldehyde group reaction and the like, and covalent cross-linking.
  • the surface of the chip can be coated with reactive groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, and silanes through surface chemical reactions.
  • the end (usually the 5' end) of the chip surface linker nucleic acid connected to the chip surface has a group that forms a chemical bond with the coated active group.
  • the 3' end of the chip surface linker nucleic acid has a linker fragment for linking with the first barcode nucleic acid through a single-stranded linker nucleic acid.
  • the 5' end of the first barcode nucleic acid has a linker fragment for linking with the chip surface linker nucleic acid through a single-stranded linker nucleic acid
  • the chip surface linker The 3' end of the daughter nucleic acid has a linker fragment for linking with the first barcode nucleic acid through the single-stranded linker nucleic acid
  • the chip surface links the linker fragment at the 3' end of the child nucleic acid and the linker fragment at the 5' end of the first barcode nucleic acid They are respectively reverse complementary to the sequences at both ends of the single-stranded linking nucleic acid.
  • the 3' end of the first barcode nucleic acid has a linker segment for linking with a second barcode nucleic acid through a single-stranded linker nucleic acid
  • the 5' end of the second barcode nucleic acid has a linker for linking through the single-stranded nucleic acid
  • the nucleic acid is linked to the first barcode nucleic acid, and the linked fragment at the 3' end of the first barcode nucleic acid and the linked fragment at the 5' end of the second barcode nucleic acid are respectively reverse complementary to sequences at both ends of the single-stranded linked nucleic acid.
  • the chip surface linker nucleic acid has a primer fragment at the 5' end for subsequent amplification reaction, such as a general primer sequence used in known sequencing methods.
  • the present invention also provides a chip for analyzing nucleic acid information of biological samples.
  • the chip for analyzing nucleic acid information of biological samples is prepared by the aforementioned method.
  • the surface of the chip for analyzing nucleic acid information of biological samples has probes forming an array, the probe array includes orthogonal rows and columns, and the probes in the array are each There are different sequences that can be used to represent the spatial location of the probes.
  • the probe includes a first barcode and a second barcode.
  • probes in each row of the probe array have the same first barcode and probes in each column have the same second barcode; the probes in each row have different first barcodes And each column of probes has a different second barcode.
  • the chip for analyzing nucleic acid information of a biological sample has a chip surface linker nucleic acid on its entire surface.
  • the 5' end of each probe in the probe array is the chip surface linker nucleic acid.
  • the sequence of each probe in the probe array includes the chip surface linker nucleic acid, a first barcode, a second barcode for identification from the 5' end to the 3' end.
  • the sequence of each probe in the probe array includes a primer fragment at the 5' end for the amplification reaction.
  • the sequence of each probe in the probe array further includes a Unique Molecular Identifier (UMI).
  • UMI Unique Molecular Identifier
  • 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 or a fresh tissue sample or a frozen tissue sample and the like.
  • FFPE formalin-fixed paraffin-embedded
  • the methods of the present invention can be performed before or after staining the tissue sample. For example, following hematoxylin and eosin staining, tissue samples can be spatially analyzed according to the methods provided herein.
  • Methods may include analyzing the histology of the sample (eg, using hematoxylin and eosin staining), followed by spatial analysis of the tissue.
  • Formalin fixation and paraffin embedding (FFPE) of tissue sections typically involves fixation of tissue obtained from a subject in formaldehyde (eg, 3%-5% formaldehyde in phosphate buffered saline) or Bouin's solution, embedding into wax, cut into thin sections, and mounted on a flat surface such as a microscope slide for biopsies.
  • formaldehyde eg, 3%-5% formaldehyde in phosphate buffered saline
  • Bouin's solution embedding into wax, cut into thin sections, and mounted on a flat surface such as a microscope slide for biopsies.
  • a tissue section is contacted with an array of probes on a chip, and the probes on the array can recognize and bind nucleic acid, especially mRNA, of cells in the tissue.
  • Subsequent analysis includes reverse transcription and amplification, etc., and can be analyzed by high-throughput next-generation sequencing (NGS) or sequencing-by-synthesis (SBS).
  • NGS next-generation sequencing
  • SBS sequencing-by-synthesis
  • FIG. 4 is a schematic flowchart of an exemplary embodiment of the method for preparing a biochip provided by the present invention.
  • a glass sheet is used as the chip substrate.
  • PDMS polydimethylsiloxane
  • the microfluidic device includes about 50-500 microfluidic channels arranged in parallel.
  • the width of each microchannel of the parallel microchannels is about 2-200 ⁇ m, preferably about 5-50 ⁇ m, most preferably about 5-25 ⁇ m, for example about 5 ⁇ m, 10 ⁇ m or 50 ⁇ m.
  • the spacing between each adjacent microfluidic channel is about 5-400 ⁇ m, preferably about 10-100 ⁇ m, most preferably about 10-50 ⁇ m, for example about 20 ⁇ m, 50 ⁇ m or 100 ⁇ m.
  • “12345678” represents a barcode fragment with 8 nucleotides, wherein the sequence of the 8 nucleotides is known (specified), and the sequence of the first barcode of the first set of 100 barcode nucleic acids is each Are not the same.
  • the CTACACGACGCTCTTCCGATCT on the 5' side of the barcode fragment is the primer fragment used for the amplification reaction in the subsequent sequencing procedure.
  • CTCTTTCCCTACACGACGCTCTT on the 3' side of the barcode fragment is a ligation fragment for forming a ligation with the second barcode fragment.
  • 87654321 represents a barcode fragment with 8 nucleotides, wherein the sequence of the 8 nucleotides is known (specified), and the sequence of the first barcode of the 100 second set of barcode nucleic acids is each Are not the same. in one aspect of the present invention.
  • the barcode fragments of each barcode nucleic acid in the second set of barcode nucleic acids are identical to the barcode fragments of each barcode nucleic acid in the first set of barcode nucleic acids. the 5' side of the barcode fragment
  • GAGTGATTGCTTGTGACGCCTT is the ligation fragment used to form the ligation with the first barcode fragment.
  • This barcode fragment has a polyTVN sequence on the 3' side, which can be used to bind mRNA.
  • a linker nucleic acid for linking the first barcode nucleic acid and the second barcode nucleic acid is synthesized, the sequence of which is as follows:
  • the PDMS microfluidic device was attached to the glass slide to realize the closure of the flow channel. According to the interface characteristics, a clamping tool can be used to press the top of the flow channel and the base glass to improve the sealing.
  • One end of the microfluidic channel is the solution inlet, and the other end is connected to the vacuum suction device through an interface.
  • the flow channel was washed with PBS buffer, followed by streptavidin, and the flow channel was filled and allowed to stand for 10 minutes at room temperature. After the reaction is completed, local modification of the chip at the flow channel overlay is achieved.
  • the first set of biotinylated barcode nucleic acids were passed into the microfluidic channel after washing: 15uM of one first barcode nucleic acid was passed into each flow channel (the first barcode nucleic acid of each flow channel had the same number as the first barcode nucleic acid of the other flow channels. A barcoded nucleic acid with a different barcode sequence). After filling the channel, let it stand for 10 minutes at room temperature. After the reaction is completed, the immobilization of the first group of barcode nucleic acids on the chip at the cover of the flow channel is realized to form the first group of barcode strips.
  • the channel was rinsed with PBS buffer and ultrapure water. After rinsing, the microchannel device was removed and the chip was air-dried at room temperature.
  • the other PDMS microfluidic device is attached to the glass slide in a direction orthogonal to the flow channel of the first PDMS microfluidic device.
  • pass 15uM of the second group of barcode nucleic acids pass a second barcode nucleic acid in each flow channel (the second barcode nucleic acid in each flow channel has the same Lane's second barcode nucleic acid with different barcode sequence) and linker nucleic acid and T4 ligase. After filling the runner, let it stand for 30 minutes at room temperature.
  • the buffer solution is passed through, and the flow channel is washed with ultrapure water. The runner is then removed and the substrate is allowed to dry. That is, the chip preparation is completed.
  • FIG. 5 is a schematic flowchart of another exemplary embodiment of the method for preparing a biochip provided by the present invention.
  • the glass sheet is used as the chip substrate, and the surface of the chip is modified with active groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, silanes and other active groups through surface chemical reactions.
  • active groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, mercapto groups, silanes and other active groups through surface chemical reactions.
  • a commercially available optical epoxy-modified glass sheet ( Slide E) is the chip substrate.
  • a first set of 100 barcoded nucleic acids with the following sequences were synthesized with the amino group at the 5' end modified:
  • “12345678” represents a barcode fragment having 8 nucleotides, wherein the sequence of the 8 nucleotides is known (assigned).
  • the sequences of the barcode fragments (referred to as first barcodes) of the 100 first set of barcode nucleic acids are different from each other, and the sequence of the first barcode of each of the first set of barcode nucleic acids is known (assigned).
  • the underlined T base is FITC modification.
  • the fluorescent signal generated by fluorescently modifying and detecting the barcode fragment is used to observe or control the production quality of each step of adding the barcode fragment in the chip synthesis.
  • the barcode fragments may not be fluorescently modified.
  • 87654321 represents a barcode fragment with 8 nucleotides, wherein the sequence of the 8 nucleotides is known (specified), the barcode fragment of the 100 second set of barcode nucleic acids (referred to as the first barcode fragment) The sequences of the two barcodes) vary, and the sequence of the second barcode of each second set of barcoded nucleic acids is known (assigned).
  • the underlined T base is Cy3 modification.
  • the PDMS microfluidic device described in Example 2 was attached to the glass slide to realize the closure of the flow channel. Use a clamping tool to press the top of the flow channel to the base glass to improve sealing.
  • One end of the microfluidic channel is the solution inlet, and the other end is connected to the vacuum suction device through an interface.
  • the first PDMS microfluidic device was attached to the glass slide, and ethanol was poured into it to remove air bubbles, and then the PBS buffer was changed to rinse once, and then 15uM of the first group of barcode nucleic acids (dissolved in 300mM sodium phosphate buffer) was added to the flow channel. , pH 8.5): pass a first barcode nucleic acid in each flow channel (the first barcode nucleic acid of each flow channel has a different barcode sequence from the first barcode nucleic acid of other flow channels). After the flow channel was filled, the slides were placed in a saturated sodium chloride wet box at 35°C and allowed to stand for three hours at room temperature. After the reaction is complete, rinse the flow channel with 1x PBS buffer for 1 min.
  • the PDMS microfluidic device was removed, and the modified slides were washed sequentially with 0.1% Triton X-100, 1 mM HCl, 100 mM KCl, and then blocked with 0.1 M Tris pH 9.0, 50 mM ethanolamine, and 0.1% SDS at 50 °C. After blocking, the substrates were rinsed with deionized water for 1 minute, and then blown dry with nitrogen gas. The FITC fluorescence signal of the first barcode nucleic acid is observed by a fluorescence microscope to confirm that the reaction is completed and to realize the immobilization of the first group of barcode nucleic acids on the chip covered by the flow channel to form the first group of barcode bands.
  • the other PDMS microfluidic device is attached to the glass slide in a direction orthogonal to the flow channel formation of the first PDMS microfluidic device.
  • pass 15uM of the second group of barcode nucleic acids pass a second barcode nucleic acid in each flow channel (the second barcode nucleic acid in each flow channel has the same Lane's second barcode nucleic acid with different barcode sequence) and linker nucleic acid and T4 ligase. After filling the flow channel, let it stand at 37°C for 30 minutes.
  • FIG. 6 is a graph showing the observation of each barcoded nucleic acid ligation reaction under a microscope (Olympus bx53). As shown in FIG. 6 , the FITC-modified fluorescent signal carried by the first group of barcode nucleic acids is displayed on the array in the horizontal direction of the chip; the fluorescent signal of Cy3 modification carried by the second group of barcode nucleic acids is displayed on the vertical array of the chip.
  • the temperature of the cryostat was set as the chamber temperature: -20°C, and the sample head temperature: -10°C. Before slicing, put the frozen tissue and substrate into the -20°C cryostat body for equilibration for more than 30 minutes, and then perform cryosectioning in the cryostat body with a thickness of 10 ⁇ m.
  • the cut tissue sections were attached to the barcode array-modified substrate prepared in Example 3, and then incubated at 37° C. for 1 minute.
  • the tissue-attached substrates were completely immersed in pre-cooled methanol and fixed at -20°C for 30 minutes. After the fixation, the substrate was taken out, the liquid on the back was wiped dry, 500 ⁇ l of isopropanol was added dropwise to the tissue section, and incubated at room temperature for 1 minute. After 1 minute, the isopropanol was removed and air dried at room temperature for 5-10 minutes.
  • the eosin was removed, the substrate was washed by immersing in RNase-free Water, and air-dried until the tissue was opaque. Brightfield imaging was performed after incubating the slides at 37°C for 5 min.
  • Figure 7 is the tissue image after HE staining.
  • permeabilase (0.1% pepsin diluted in 0.1N HCl) was added to the chamber to permeabilize the tissue at 37°C, and the permeabilase was removed and washed with 0.1 ⁇ SSC.
  • the reverse transcription mixture includes: 1x first-strand buffer, 5mM DTT, 500 ⁇ M dNTP, 0.19 ⁇ g/ ⁇ l BSA, 1% DMSO, 2.5 ⁇ M Template Switch Oligo, 20U/ ⁇ l Superscript III and 2U/ ⁇ l RNase inhibitor.
  • the Template Switch Oligo sequence is:
  • the penultimate bases 1, 2, and 3 are modified with riboguanosine.
  • the chamber was sealed with tape, it was placed on a temperature control plate, adjusted to about 50° C. for reverse transcription, and the reaction was performed for 16 hours.
  • the two-strand synthesis reaction solution includes: 1 ⁇ Kapa HiFi Hotstart ReadyMix, 0.8uM Second Strand Primer. After sealing the chamber with tape, it was placed on a temperature control plate, and the temperature was adjusted to about 37°C for cDNA double-strand synthesis, and the reaction was performed for 30 minutes.
  • the Second Strand Primer sequence is:
  • the two-strand synthesis reaction solution in the chamber was aspirated and discarded, and then 100ul of RNase-free Water was added to wash it once. Then 70 ⁇ l of 0.08M KOH was added to the chamber and incubated for 10 minutes at room temperature. Several new 1.5 ml centrifuge tubes were prepared and 10 ⁇ l of Tris (1 M, pH 7.0) was added to them. Transfer 70 ⁇ l of the sample in the chamber to the corresponding centrifuge tube containing Tris, mix well, and complete the preparation of the second strand of cDNA.
  • the PCR reaction solution includes: 1 ⁇ Kapa HiFi Hotstart ReadyMix, 0.8 ⁇ M cDNA Forward Primer, 0.8 ⁇ M cDNA Reverse Primer, 35 ⁇ l cDNA template, the total volume is 100 ⁇ l.
  • cDNA amplification was performed by the following protocol:
  • the cDNA Forward Primer sequence is:
  • the cDNA ReversePrimer sequence is:
  • the amplified products were selected for library construction/sequencing.

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Abstract

一种制备用于分析生物样品的核酸信息的芯片的方法，芯片适合用于分析生物组织样品的空间转录组学信息，还提供了用于分析生物样品的核酸信息的芯片和将芯片用于分析生物组织样品的空间转录组学信息的方法。能够有效地获得组织样品的细胞中的核酸表达信息，包括空间组学信息。

Description

用于空间转录组学分析的生物芯片及其制备方法和应用
本申请要求2020年12月25日提交的、申请号为202011568787.3、发明名称为“制备用于空间转录组学分析的生物芯片的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物学与医疗器械领域。具体的，本发明涉及制备用于分析生物样品的细胞的核酸信息的芯片的方法，所述芯片适合用于分析生物组织样品的空间转录组学信息。
背景技术
对生物组织的细胞组织和表达格局的分析，是生物医学研究和诊断学上的里程碑。细胞组织学运用各种染色技术，在一个多世纪前便首先确定了健康器官的基本结构机制和常见病理学变化。该领域的发展，带来了以免疫组织化学和原位杂交的基因表达来研究蛋白质分布的可能性。
新的研究希望可以在保存关于组织的空间信息的情况下对组织中的转录物组和/或基因组变异进行表征。
基因表达分析的最新发展，为利用微阵列或RNA测序评估组织的完整转录组提供了可能，然而，典型的转录组分析以从整块组织(或者甚至完整生物体)提取的mRNA为实施对象，但是要收集较小的组织区域或个体细胞用于转录组分析，却一般非常费力、费钱且精确度低。
发明内容
本发明提供了一种用于制备适于分析生物样品的细胞的核酸信息，特别是适于分析生物组织样品的空间转录组学信息的生物芯片的方法。具体的，本发明提供了一种用于制备具有阵列的生物芯片的方法，包括以下步 骤：
A.通过多条平行设置的微流道将第一组条形码核酸固定在芯片表面，形成第一方向的多条第一条形码带，所述第一组条形码核酸中包括多种具有不同条形码序列的第一条形码核酸，每条第一条形码带上固定一种第一条形码核酸，且每条第一条形码带上固定的第一条形码核酸具有不同的条形码序列；
B.通过多条平行设置的微流道将第二组条形码核酸沿第二方向施加到芯片表面的具有第一方向的所述多条第一条形码带上，形成多条第二条形码带，所述第二组条形码核酸中包括多种具有不同条形码序列的第二条形码核酸，每条第二条形码带上具有一种第二条形码核酸，且每条第二条形码带上固定的第二条形码核酸具有不同的条形码序列；
C.在使得第一条形码核酸和第二条形码核酸发生连接反应的条件下，在所述多条第一条形码带与所述多条第二条形码带产生交叉的芯片表面将第二条形码核酸与第一条形码核酸连接，形成探针，所述探针构成阵列的阵点，每个阵点具有一种序列相互不同的探针。
在本发明的其中一个方面，所述方法中采用具有多条平行设置的微流道的微流道装置将所述第一组条形码核酸或第二组条形码核酸输送和固定在芯片表面，其中所述微流道与芯片表面接触的一面可容溶液或溶液中的核酸通过。
在本发明的其中一个方面，所述方法中在所述微流道设备的每一条微流道中加入含有不同条形码序列的第一组条形码核酸或第二组条形码核酸。
在本发明的其中一个方面，所述方法中所述第一组条形码核酸中的第一条形码核酸包括第一条形码片段；优选的，所述第一条形码核酸在5’端还具有用于扩增反应的引物片段。
在本发明的其中一个方面，所述方法中所述第一组条形码核酸中的 第一条形码核酸在5’端具有用于与芯片表面连接的基团。
在本发明的其中一个方面，其中所述第二组条形码核酸中的第二条形码核酸包括3’端的用于识别和结合生物样品中的目标核酸的捕获片段(例如为识别和结合mRNA或cDNA的片段，例如为poly-T序列)和第二条形码片段。
在本发明的其中一个方面，所述方法中所述第二组条形码核酸中的第二条形码核酸还具有唯一分子标识符(UMI)。
在本发明的其中一个方面，所述方法中所述第一条形码核酸的3’端具有用于通过一个单链连接核酸与第二条形码核酸连接的第一连接片段，所述第二条形码核酸的5’端具有用于通过所述单链连接核酸与第一条形码核酸连接的第二连接片段，所述第一连接片段和第二连接片段分别与所述单链连接子核酸的两端的序列反向互补。
在本发明的其中一个方面，所述方法中步骤C中形成的探针包括3’端的用于识别和结合生物样品中的目标核酸的捕获片段，以及第一条形码片段和第二条形码片段。优选的，所述探针在5’端还具有用于扩增反应的引物片段。
在本发明的其中一个方面，所述方法中所述第一组条形码核酸中的每一种第一条形码核酸的条形码片段的序列是指定的，和/或所述第二组条形码核酸中的每一种第二条形码核酸的条形码片段的序列是指定的。
在本发明的其中一个方面，所述方法中所述探针的第一条形码片段和第二条形码片段的序列是指定的。
在本发明的其中一个方面，所述方法中步骤A中流道内的核酸浓度为约0.1-100uM，例如为约1-20uM。
在本发明的其中一个方面，所述方法中步骤B中流道内的核酸浓度为约0.1-100uM，例如为约1-20uM。
在本发明的其中一个方面，所述方法中步骤A中，所述第一组条形 码核酸的核酸固定在芯片表面上。第一组条形码核酸的核酸优选以化学键连接方式固定在芯片表面上。化学键连接方式例如为选自氨基-醛基反应等的基团连接、共价交联中的任意一种。芯片的表面可以通过表面化学反应用氨基、醛基、环氧基、异硫氰酸基、巯基、硅烷等活性基团等进行包被；所述第一组条形码核酸的核酸与芯片表面连接的一端(通常为5’端)则具有与包被的活性基团形成化学键的基团。
在本发明的其中一个方面，所述方法中步骤A和步骤B中所述平行设置的微流道的各条微流道的宽度为约2-200μm，优选为约5-50μm，最优选为约5-25μm，例如为约5μm，10μm或50μm。
在本发明的其中一个方面，所述方法中步骤A和步骤B中所述平行设置的微流道的各条相邻微流道之间的间距为约5-400μm，优选为约10-100μm，最优选为约10-50μm，例如为约20μm，50μm或100μm。
本发明的制备得到的芯片可用于对组织样品，特别是组织薄切片进行细胞内含分子的分析，包括对核酸和蛋白的分析，例如通过PCR、质谱法、新一代测序、或ELISA进行分析，获得其表达和空间信息。
在本发明的其中一个方面，所述生物样品为来自受试者的组织样品，例如为手术切除组织样品，优选为通过显微切片术加工得到的组织薄切片。在本发明的其中一个方面，所述组织样品经过固定和包埋(例如包埋在石蜡中)，以及附着在支持物例如玻片上。
在本发明的其中一个方面，可对所述组织薄切片进行形态分析和/或组织学分析，该组织学分析是通过H&E染色、IHC染色、ISH染色、以及FISH染色进行。
在本发明的其中一个方面，对该一种或多种生物分子进行分析是通过PCR、质谱法、新一代测序、或ELISA进行。
在本发明的其中一个方面，受试者选自动物、农场动物、宠物、人类受试者。
在本发明的其中一个方面，分析物进一步包括非人细胞、人细胞、非天然蛋白质、核酸、或小分子、染料、病毒、细菌、寄生虫、原生动物或化学物质中的一种或多种。小分子包括半抗原、肽标签、蛋白质标签、荧光标签、核酸标签、及其组合。
在本发明的其中一个方面，所述芯片可用于分析样品中标记物的定量和/或定性数据。该标记物包括DNA、蛋白质、RNA、脂质、细胞器、代谢物、或细胞。该蛋白质可包括修饰，所述修饰选自下组，该组由以下各项组成：乙酰化、ADP-核糖基化、酰化、ADP-核糖基化、酰胺化、共价附接黄素、共价附接血红素、共价附接核苷酸或核苷酸衍生物、共价附接脂质或脂质衍生物、共价附接磷脂酰肌醇、交联、环化、二硫键形成、脱甲基、形成共价交联、形成胱氨酸、形成焦谷氨酸、甲酰化、Y-羧化、糖基化、GPI锚形成、羟基化、碘化、甲基化、豆蔻酰化、氧化、蛋白水解加工、磷酸化、异戊烯化、外消旋化、硒化、硫酸化、精氨酰化、以及泛素化。该标记物包括基因组多态性，药物基因组学单核苷酸多态性
(SNP)，基因组SNP，体细胞多态性，以及蛋白质、脂质和/或细胞器的差异表达。该标记物包括单个核苷酸位置；基因内区域或基因间区域；外显子或内含子、或其片段；编码区或非编码区；启动子、增强子、5′非翻译区(5′UTR)、或3′非翻译区(3′UTR)、或其片段；cDNA或其片段；SNP；体细胞突变、种系突变、或两者；点突变或单一突变；缺失突变；框内缺失、基因内缺失、全基因缺失；插入突变；基因内插入；倒位突变；染色体内倒位；连锁突变；连锁插入突变；反转重复突变；串联重复；染色体内串联重复；易位；染色体易位，非相互易位；重排；基因组重排；一个或多个内含子、或其片段的重排；重排的内含子；5′-或3′-UTR、或其组合。其中与正常的健康组织或细胞相比，在癌组织或癌细胞中该标记物包括对改变的氨基酸序列进行编码的改变的核苷酸序列、染色体易位、染色体内倒位、拷贝数变化、表达水平变化、蛋白质水平变化、蛋白质活性变 化、或甲基化状态变化。其中可通过单细胞测序、单核测序、流式细胞术、免疫组织化学染色、苏木精和伊红染色、全基因组测序、高通量测序、质谱法、DNA微阵列、或其组合对该标记物进行测量。
本发明的芯片可用于分析组织样品。该组织样品包括选自以下项的组的样品：一种或多种恶变前或恶性细胞、来自实体瘤的细胞、软组织肿瘤或转移灶、来自手术边缘的组织或细胞、组织学正常组织、一种或多种循环肿瘤细胞(CTC)、正常邻近组织(NAT)、来自患肿瘤或处于患肿瘤风险的相同受试者的血液样品、或FFPE样品。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明提供的制备芯片的方法步骤的示意图。
图2为本发明的方法中采用具有多条平行设置的微流道的设备的一种示例性实施方式。
图3为本发明提供的制备芯片的方法步骤的示意图。
图4为本发明提供的制备生物芯片的方法的一种示例性实施方式的流程示意图。
图5为本发明提供的制备生物芯片的方法的又一种示例性实施方式的流程示意图。
图6为本发明提供的制备生物芯片的制备过程对其条形码核酸连接反应的观测。
图7为采用本发明提供的制备生物芯片对样品组织进行生物空间组研究中对组织切片进行HE染色后的图像。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的区间。
实施例1
本发明提供了一种制备适于分析生物样品的细胞的核酸信息的生物芯片的方法。更具体的，本发明提供了一种用于制备具有阵列的生物芯片的方法。在本发明的其中一个方面，本发明提供的芯片适于分析生物组织样品的空间转录组学信息。
图1为本发明提供的制备具有阵列的生物芯片的示例性方法的流程示意图。
如图1所示，所述方法主要包括以下步骤：
步骤1.提供芯片基底；
步骤2.通过多条平行设置的微流道将第一组条形码核酸(其包括多种第一条形码核酸，其中每一种第一条形码核酸具有不同的条形码序列(图中的Barcode A))与芯片表面接触和使其固定在芯片表面，在芯片表面形成走向为第一方向的多条第一条形码带，其中每条第一条形码带上固定的第一条形码核酸具有不同的第一条形码序列。
图2为通过多条平行设置的微流道将多种条形码核酸与芯片表面接触并固定在芯片表面的一种示例性实施方式。图2的左图的下方为芯片。图2的左图的中间显示一种具有多条平行设置的微流道(微流道1至微流道n)的微流道装置，其中微流道与芯片表面接触的一面，即图示的微流道的底部，可容溶液或溶液中的核酸通过(透过)。例如，所述微流道与 芯片表面接触的一面不存在微流道壁。将该微流道装置沿第一方向覆盖在芯片表面，然后在微流道内通入指定的溶液，例如含有条形码核酸的溶液。图2的左图的上方为示例性的协助通入溶液的装置，例如为利用负压的真空抽吸装置，其可设置在微流道的出口。图2右图显示在每一条微流道中通过入口加入含有不同条形码序列的条形码核酸(图中的条形码核酸1-n)。在本发明的其中一个方面，所述每一条微流道中通入的条形码核酸的条形码序列具有已知的或指定的核苷酸序列。
步骤3.移走步骤2中的微流道，通过另一组多条平行设置的微流道将第二组条形码核酸(其包括多种第二条形码核酸，其中每一种第二条形码核酸具有不同的条形码序列(图中的Barcode B))沿与第一条形码带的方向不同的第二方向(通常为与第一方向垂直)与芯片表面接触，形成多条第二条形码带，其中每条第二条形码带上固定的第二条形码核酸具有不同的第二条形码序列；
在使得第一条形码核酸和第二条形码核酸能够发生连接反应的条件下，在所述多条第一条形码带与所述多条第二条形码带产生交叉的芯片表面将第二条形码核酸与第一条形码核酸连接，形成探针。
步骤4.移走步骤3中的微流道，获得表面具有探针阵列的生物芯片。所述探针阵列上的每个阵点对应所述多条第一条形码带与所述多条第二条形码带产生交叉的位置。每个阵点具有一种探针分子，其包括第一条形码序列和第二条形码序列。各个阵点具有的探针分子包括的第一条形码序列和第二条形码序列的组合各不相同。在本发明的其中一个方面，所述每一条微流道中通入的条形码核酸的第一条形码序列和第二条形码序列是已知的或指定的，由此各个阵点具有的探针分子包括的第一条形码序列和第二条形码序列及其组合也是已知的。由此可通过各个阵点具有的探针分子的第一条形码序列和第二条形码序列获知其在芯片表面的阵列中的空间位置。
在本发明中，芯片通常是指固体基片，其上可以实施化学，生物，生物物理或生物化学过程等。芯片可具有微观结构或微尺度结构，如通道和孔井，电极元件，电磁元件等利于芯片上发生的化学，生物，生物物理或生物化学过程。芯片表面可以是平的，或不平的。表面不平的芯片可以包括构造在表面的通道或孔。
芯片可以由任何合适的材料制成，芯片材料的示例性类型包括玻璃、改性玻璃、功能化玻璃、无机玻璃、微球(包括惰性和/或磁性颗粒)、塑料、多糖、尼龙、硝酸纤维素、陶瓷、树脂、二氧化硅、基于二氧化硅的材料、碳、光纤或光纤束、除上文例示的材料以外的多种聚合物和多孔微量滴定板。示例性塑料的具体类型包括丙烯酸树脂(acrylics)、聚苯乙烯、苯乙烯和其它材料的共聚物、聚丙烯、聚乙烯、聚丁烯、聚氨基甲酸酯和TeflonTM。示例性的基于二氧化硅的材料的具体类型包括硅和改性硅的各种形式。芯片表面通常需要沉积生物聚合物(包括核酸、多肽和/或其它聚合物)。可通过对于本领域技术人员公知的多种方法，对芯片表面做修饰以适应目标生物聚合物的附着。
在本发明中，制备得到的所述芯片表面的阵列上具有探针(或称为捕捉探针)。探针是指可以基因特异性或靶物特异性地识别和结合目标核酸，例如来自组织样品的核酸的单链核苷酸分子，其具有特异性的核苷酸序列，即能够选择性退火到靶定核酸的核苷酸序列，通常为互补的核苷酸序列。组织样品中的分析物的实例包括基因组DNA、甲基化DNA、特定的甲基化DNA序列、信使RNA(mRNA)、多聚A mRNA、片段化mRNA、片段化的DNA、线粒体DNA、病毒RNA、微小RNA、原位合成的PCR产物、RNA/DNA杂合物、脂质、碳水化合物、蛋白质、糖蛋白、脂蛋白、磷蛋白、蛋白质的特定磷酸化或乙酰化变体或病毒壳体蛋白。捕捉探针可以是基因特异性捕捉探针，其例如与样品中特异性靶定的mRNA或cDNA杂交。
在本发明中，所述探针具有条形码序列，用于后续的高通量下一代测序(NGS)或合成测序(SBS)进行分析的应用中，例如在进行大通量的测序分析中。在这些测序中，采用条形码序列来标志和鉴定测序获得的核酸序列的核酸的来源。条形码分子用于将来自生物颗粒(例如，细胞)的核酸分子(例如，RNA分子)条形码化，以生成测序文库，随后对所述测序文库进行测序以产生多个测序读长。多个测序读长中的一些或全部包括条形码序列。在这些测序应用中，通常对细胞核酸进行扩增，直到对象中的条形码化重叠片段构成特定部分或全部细胞基因组的至少1X覆盖率、至少2X、至少3X、至少4X、至少5X、至少10X、至少20X、至少40X或更高覆盖率。一旦产生条形码化片段，就可以将它们直接在合适的测序系统上测序，例如Illumina系统。多个序列上相同条形码的存在可以提供关于该序列的起源的信息。
多核苷酸测序中，可以依赖于独特的条形码序列来鉴定核酸片段的起源，以及，例如，从经测序的片段装配较大的序列。条形码可以允许在测序过程中对单个多核苷酸片段进行鉴别和/或量化。
在本发明中，在制备得到的探针中含有两个条形码序列。两个条形码序列可以帮助确定探针在芯片表面的阵列中的位置(分别确定X维度和Y维度的位置)，因此还还具有位置标签的作用。探针上的条形码序列可与芯片上的阵列中的阵点(feature)对应，也可指示其识别的组织上的细胞，包括单个细胞，在该组织样品中的位置。可以与核酸标签偶联的其它分子的实例包括抗体、抗原结合结构域、蛋白质、肽、受体、半抗原等。
在本发明中，所述探针还包括一个或多个唯一分子标识符(UMI)。唯一分子标识符是连续的核酸片段或两个或多个非连续的核酸片段，它们充当特定分析物或结合特定分析物的捕获探针的标记或标识符。UMI是基本上不与生物样品中的分析物核酸分子杂交的核酸序列。UMI可为捕获探针的序列内包含约6至约20或更多个核苷酸。
在本方面的方法的步骤2中，第一组条形码核酸与芯片的固定可采用领域内各种已知的方法进行。核酸的固定指通过共价或非共价键直接或间接附着在芯片上。在本发明的其中一个方面，固定指在需要核酸扩增和/或测序的等反应中，保持静止或附着于芯片上。示例性的非共价连接包括但不限于非特异性相互作用(例如氢键键合、离子键键合、范德华相互作用等)或特异性相互作用(例如亲和相互作用、受体-配体相互作用、抗体-表位相互作用、抗生物素蛋白-生物素相互作用、链霉亲合素-生物素相互作用、凝集素-碳水化合物相互作用等)。
在本方面的方法的步骤3中，第一条形码核酸和第二条形码核酸的连接可通过本领域已知的各种方法进行。例如，通过各自与另一单链核酸片段(连接子核酸)的不同末端的序列互补，在可进行连接反应的条件下形成3个核酸片段(第一条形码核酸、第二条形码核酸和连接子核酸)的组合后达到连接的目的。
在本发明的其中一个方面，所述第一条形码核酸包括第一条形码片段。在本发明的其中一个方面，所述第一条形码核酸在5’端具有用于后续扩增反应的引物片段，例如为用于已知测序方法中通用引物序列。在本发明的其中一个方面，所述第一条形码核酸在5’端具有用于与芯片表面连接的基团或序列。例如，芯片表面为醛基修饰，则在第一条形码核酸在5’端具有氨基基团。在本发明的其中一个方面，所述第一条形码核酸具有可与芯片上修饰的因子形成特异性相互作用的配子，例如所述因子和配子分别为抗体-表位、抗生物素蛋白-生物素、链霉亲合素-生物素、凝集素-碳水化合物。
在本发明的其中一个方面，所述第二条形码核酸包括第二条形码片段。在本发明的其中一个方面，所述第二条形码核酸在3’端具有用于识别和结合生物样品中的目标的捕获片段，例如为识别和结合mRNA或cDNA的片段，例如为识别mRNA的poly-T序列。
在本发明的其中一个方面，所述第一条形码核酸的3’端具有用于与第二条形码核酸连接的第一连接片段(3’连接片段)。在本发明的其中另一个方面，所述第二条形码核酸的5’端具有用于与第一条形码核酸连接的第二连接片段(5’连接片段)。在本发明的其中又一个方面，所述第一连接片段和第二连接片段分别与连接子核酸的一端形成互补，在可连接的条件下(如T4连接酶等的存在下)，第一连接片段和第二连接片段与连接子核酸形成组合，达到第一条形码核酸和第二条形码核酸的连接。
本发明提供的方法制备的芯片可用于对组织样品，特别是组织薄切片进行细胞内含分子的分析，包括对核酸和蛋白的分析，例如通过PCR、质谱法、新一代测序、或ELISA进行分析，获得其表达和空间信息。
“测序”通常是指用于确定一种或多种多核苷酸中的核苷酸碱基序列的方法和技术。多核苷酸可以是例如核酸分子例如脱氧核糖核酸(DNA)或核糖核酸(RNA)，包括其变体或衍生物(例如，单链DNA)。测序可以通过目前可用的各种系统执行，例如但不限于通过Illumina、Pacific Biosciences、Oxford Nanopore或Life Technologies的测序系统。可替代地或另外地，可使用核酸扩增、聚合酶链式反应(PCR)(例如，数字PCR、定量PCR或实时PCR)或等温扩增来执行测序。此类系统可以提供对应于受试者(例如人)的遗传信息的多个原始遗传数据，如从受试者提供的样品由系统所产生。在一些实例中，此类系统提供测序读长(在本文中也称为“读长”)。读长可以包括一串核酸碱基，其对应于已经测序的核酸分子的序列。在一些情况下，本文提供的系统和方法可与蛋白质组信息一起使用。
本发明制备的芯片适合用于分析生物样品的细胞的核酸信息，特别是生物组织样品的空间转录组学信息。适于本发明的“组织样品”包括从受试者获得，固定，切片并且安装在平面表面的组织。组织样品可以是福尔马林固定的石蜡包埋(FFPE)组织样品或新鲜组织样品或冷冻组织样品等。本文公开的方法可以在染色组织样品之前或之后进行。例如，在苏木 精和曙红染色之后，组织样品可以按照本文提供的方法进行空间分析。方法可以包括分析样品的组织学(例如使用苏木精和曙红染色)，然后空间分析组织。组织切片用福尔马林固定和石蜡包埋(FFPE)通常包括将从受试者获得的组织在甲醛(例如磷酸盐缓冲盐水中的3％-5％甲醛)或Bouin溶液中固定，包埋到蜡中，切成薄切片，然后安装在平面表面，如显微镜载玻片中的生检。
本发明的芯片在使用中，包括将组织切片与阵列接触，阵列上的探针可识别和结合组织中的细胞的核酸，特别是mRNA。后续的分析包括逆转录和扩增等，并且可通过高通量下一代测序(NGS)或合成测序(SB S)进行分析。
在一些实施方案中，将组织切片(例如福尔马林固定的石蜡包埋(FFPE)组织切片)中的核酸转移到阵列，并通过与捕捉探针杂交捕捉到阵列上。在一些实施方案中，捕捉探针可以是通用捕捉探针，其例如与核酸测序文库中的衔接子区域，或mRNA的多聚-A尾杂交。在一些实施方案中，捕捉探针可以是基因特异性捕捉探针，其例如与样品中特异性靶定的mRNA或cDNA杂交。
在一些实施方案中，将组织切片(例如FFPE切片)中的核酸转移至阵列，并通过与通用衔接子寡核苷酸的单链连接捕捉到阵列上。在其它实施方案中，可将芯片上的核酸转移到组织切片(例如FFPE切片)中。可通过本领域已知的方法使得结合在芯片上的探针在溶液中脱落后进入与其接触的组织上的细胞。例如，可在核酸探针与芯片的结合处加入可光解的接头等，或者是通过pH敏感型接头将核酸探针与芯片结合，然后通过改变溶液的pH值使得核酸探针与芯片分离。
本发明的芯片和对其的使用，可通过各个阵点具有的探针分子的第一条形码序列和第二条形码序列获知其在芯片表面的阵列中的空间位置，也由此可获得核酸分子所在的细胞在所述组织中的位置信息。
本发明提供的制备具有探针阵列的芯片的方法，可以在同一基底上，实现多个具有相同的编码区域的阵列的芯片的并行合成。如图3所示，可以根据需要，在芯片基底的第一方向和第二方向上形成多组相同的第一条形码带和第二条形码带，由此得到多个在对应阵点上具有相同的第一条形码和第二条形码序列定义的探针的芯片。
在本发明的其中一种实施方式中，发明人出乎意料地发现，在前述方法的步骤2(即本发明提供的一种用于制备具有阵列的生物芯片的方法的步骤A中)，即将所述第一组条形码核酸的核酸固定在芯片表面上时，通过将所述第一组条形码核酸的核酸与已固定在芯片表面(包括整个芯片表面或与第一组条形码位置相同的芯片表面部分)的核酸(本文中称为芯片表面连接子核酸或芯片表面连接子)通过核酸与核酸的连接反应(例如通过核酸连接酶和桥接所述两个核酸的核酸连接子片段)进行相连，能够显著地改进第一组条形码核酸的核酸在芯片上的固定效率，使得在芯片的每个探针构成阵列的阵点上的探针密度显著增加，并且均匀性显著改善。在不受此理论限制的条件下，发明人认为这是由于核酸与核酸的连接反应效率和稳定性比其它核酸与芯片表面固定的方式(如共价键方式等)更强，以及由此可减小探针上每个核酸片段的长度，增加各片段的连接效率等。由此，在本发明的其中一种实施方式中，步骤A之前还包括预步骤，其包括在芯片(例如玻片)表面(例如在芯片的整个表面)预先固定用于与第一组条形码核酸连接的芯片表面连接子核酸。芯片表面连接子核酸可以以化学键连接方式固定在芯片表面上。化学键连接方式例如为选自氨基-醛基反应等的基团连接、共价交联中的任意一种。芯片的表面可以通过表面化学反应用氨基、醛基、环氧基、异硫氰酸基、巯基、硅烷等活性基团等进行包被。所述芯片表面连接子核酸与芯片表面连接的一端(通常为5’端)则具有与包被的活性基团形成化学键的基团。在本发明的其中又一种实施方式中，所述芯片表面连接子核酸的3’端具有用于通过一个单链连接核酸与第一条形码核酸连接的连接片段。在本发明的其中又一种实施方式中，其中所述第一条形码核酸的5’端具有用于通过一个单链连接核酸与 所述芯片表面连接子核酸连接的连接片段，所述芯片表面连接子核酸的3’端具有用于通过所述单链连接核酸与第一条形码核酸连接的连接片段，所述芯片表面连接子核酸的3’端的连接片段和第一条形码核酸的5’端的连接片段分别与所述单链连接核酸的两端的序列反向互补。以及，所述第一条形码核酸的3’端具有用于通过一个单链连接核酸与第二条形码核酸连接的连接片段，所述第二条形码核酸的5’端具有用于通过所述单链连接核酸与第一条形码核酸连接的连接片段，所述第一条形码核酸的3’端的连接片段和第二条形码核酸的5’端的连接片段分别与所述单链连接核酸的两端的序列反向互补。在本发明的其中又一种实施方式中，所述芯片表面连接子核酸在5’端具有用于后续扩增反应的引物片段，例如为用于已知测序方法中通用引物序列。
本发明还提供了用于分析生物样品的核酸信息的芯片。在本发明的其中一个方面，所述用于分析生物样品的核酸信息的芯片通过前述方法制备得到。在本发明的其中一个方面，所述用于分析生物样品的核酸信息的芯片的表面具有形成阵列的探针，所述探针阵列包括正交的行和列，所述阵列中的探针各具有不同的序列，可用于体现所述探针的空间位置。在本发明的其中一个方面，所述探针包括第一条形码和第二条形码。在本发明的其中又一个方面，所述探针阵列的每一行探针具有相同的第一条形码以及每一列的探针具有相同的第二条形码；每一行探针具有的第一条形码各不相同以及每一列探针具有的第二条形码各不相同。在本发明的其中一个方面，所述用于分析生物样品的核酸信息的芯片在其整个表面具有芯片表面连接子核酸。在本发明的其中又一个方面，所述探针阵列中的每个探针的5’端为所述芯片表面连接子核酸。在本发明的其中又一个方面，所述探针阵列中的每个探针的序列从5’端到3’端包括所述芯片表面连接子核酸、第一条形码、第二条形码、用于识别和结合生物样品中的目标核酸的捕获片段。在本发明的其中又一个方面，所述探针阵列中的每个探针的序列包括5’端的用于扩增反应的引物片段。在本发明的其中又一个方面， 所述探针阵列中的每个探针的序列还包括唯一分子标识符(UMI)。
本发明还提供了用于分析生物组织样品的空间转录组学信息的方法，所述方法包括将前述芯片的阵列与组织样品接触。适于本发明的“组织样品”包括从受试者获得，固定，切片并且安装在平面表面的组织。组织样品可以是福尔马林固定的石蜡包埋(FFPE)组织样品或新鲜组织样品或冷冻组织样品等。本发明的方法可以在染色组织样品之前或之后进行。例如，在苏木精和曙红染色之后，组织样品可以按照本文提供的方法进行空间分析。方法可以包括分析样品的组织学(例如使用苏木精和曙红染色)，然后空间分析组织。组织切片用福尔马林固定和石蜡包埋(FFPE)通常包括将从受试者获得的组织在甲醛(例如磷酸盐缓冲盐水中的3％-5％甲醛)或Bouin溶液中固定，包埋到蜡中，切成薄切片，然后安装在平面表面，如显微镜载玻片中的生检。本发明的方法在将组织切片与芯片上的探针阵列接触，阵列上的探针可识别和结合组织中的细胞的核酸，特别是mRNA。后续的分析包括逆转录和扩增等，并且可通过高通量下一代测序(NGS)或合成测序(SBS)进行分析。
实施例2芯片(表面局部修饰)的制备
图4为本发明提供的制备生物芯片的方法的示例性实施方式的流程示意图。
其中，以玻璃片为芯片基底。
通过软光刻工艺制备由聚二甲基硅氧烷(PDMS)制成如图2所示的包括多条平行设置的微流道的装置，微流道的底部开口。
微流道的装置包括约50-500条平行设置的微流道。所述平行设置的微流道的各条微流道的宽度为约2-200μm，优选为约5-50μm，最优选为约5-25μm，例如为约5μm，10μm或50μm。各条相邻微流道之间的间距为约5-400μm，优选为约10-100μm，最优选为约10-50μm，例如为约20μm，50μm或100μm。
合成PLL-PEG-生物素，用PBS配置成1mg/ml的PLL-PEG-生物素溶液。
合成100条5’端生物素修饰的具有以下序列的第一组条形码核酸：
5’生物素-CTACACGACGCTCTTCCGATCT 12345678 CTCTTTCCCTACACGACGCTCTT-3′
其中，“12345678”表示具有8个核苷酸的条形码片段，其中所述8个核苷酸的序列是已知(指定的)，所述100条第一组条形码核酸的第一条形码的序列各不相同。该条形码片段的5’侧的CTACACGACGCTCTTCCGATCT为后续测序程序中用于扩增反应的引物片段。该条形码片段的3’侧的CTCTTTCCCTACACGACGCTCTT为用于与第二条形码片段形成连接的连接片段。
合成100条具有以下序列的第二组条形码核酸：
5’磷酸化-GAGTGATTGCTTGTGACGCCTT 87654321 NNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′
其中，“87654321”表示具有8个核苷酸的条形码片段，其中所述8个核苷酸的序列是已知(指定的)，所述100条第二组条形码核酸的第一条形码的序列各不相同。在本发明的其中一个方面。第二组条形码核酸中的各条条形码核酸的条形码片段与第一组条形码核酸中的各条条形码核酸的条形码片段相同。该条形码片段的5’侧的
GAGTGATTGCTTGTGACGCCTT为用于与第一条形码片段形成连接的连接片段。该条形码片段的3’侧具有polyT VN序列，可用于结合mRNA。
合成用于连接第一条形码核酸和第二条形码核酸的连接子核酸，其序列如下：
5′-AAGGCGTCACAAGCAATCACTCAAGAGCGTCGTGTAGGGAAAGAG-3′
将PDMS微流道装置与玻片贴合，实现流道封闭。根据界面特性可以用夹持工具对流道顶部与基底玻片进行压合以提高密封性。微流道的一端为溶液入口，另一端通过接口与真空抽吸装置相连接。
将第一个PDMS微流道装置与玻片贴合，通入乙醇排除气泡，再换PBS缓冲液冲洗一次，然后在流道内加入1mg/ml的PLL-PEG-生物素溶液， 反应约30分钟，室温。保持低负压以维持流体低速流动，使得消耗的反应物得以补充。
反应完成后，通入PBS缓冲液清洗流道，接着通入链霉亲和素，充满流道后静置10分钟，室温。反应完成后，实现在流道覆盖处的芯片的局部修饰。
通入PBS缓冲液进行清洗。清洗之后在微流道内通入生物素化的第一组条形码核酸：在每一个流道通入15uM的一种第一条形码核酸(每个流道的第一条形码核酸具有与其它流道的第一条形码核酸不同的条形码序列)。充满流道后静置10分钟，室温。反应完成后，实现第一组条形码核酸在流道覆盖处的芯片的固定，形成第一组条形码带。
用PBS缓冲液和超纯水对流道进行冲洗，冲洗之后，移走微流道装置，对芯片进行室温晾干。
晾干之后，将另一个PDMS微流道装置与玻片沿与第一个PDMS微流道装置的流道形成正交的方向进行贴合。流道内通入缓冲液排出流道内的气体之后，通入15uM的第二组条形码核酸：在每一个流道通入一种第二条形码核酸(每个流道的第二条形码核酸具有与其它流道的第二条形码核酸不同的条形码序列)和连接子核酸以及T4连接酶。充满流道后静置30分钟，室温。
完成连接反应之后，通入缓冲液，超纯水对流道进行清洗。之后取下流道，对基底进行晾干。即完成芯片制备。
实施例3芯片(表面完全修饰)的制备
图5为本发明提供的制备生物芯片的方法的另一示例性实施方式的流程示意图。
其中，先以玻璃片为芯片基底，通过表面化学反应用氨基、醛基、环氧基、异硫氰酸基、巯基、硅烷等活性基团等芯片表面进行修饰。
在本实施例中，以商购的光学环氧基修饰的玻璃片(
Figure PCTCN2021141391-appb-000001
Slide E)为芯片基底。
合成100条5’端氨基修饰的具有以下序列的第一组条形码核酸：
5’氨基-CTACACGACGC TCTTCCGATCT 12345678 CTCTTTCCCTACACGACGCTCTT-3′
其中，“12345678”表示具有8个核苷酸的条形码片段，其中所述8个核苷酸的序列是已知(指定的)。所述100条第一组条形码核酸的所述条形码片段(称为第一条形码)的序列各不相同，且各条第一组条形码核酸的第一条形码的序列是已知(指定的)。其中带下划线的T碱基为FITC修饰。在本实施例中，采用对条形码片段进行荧光修饰和检测产生的荧光信号来对芯片合成中每个加入条形码片段的步骤进行观察或生产质控。在其它实施方式中，可以不对条形码片段进行荧光修饰。
合成100条具有以下序列的第二组条形码核酸：
5’磷酸化-GAGTGATTGCT TGTGACGCCTT 87654321 NNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′
其中，“87654321”表示具有8个核苷酸的条形码片段，其中所述8个核苷酸的序列是已知(指定的)，所述100条第二组条形码核酸的条形码片段(称为第二条形码)的序列各不相同，且各条第二组条形码核酸的第二条形码的序列是已知(指定的)。其中带下划线的T碱基为Cy3修饰。
合成1条具有以下序列的连接子核酸：
5’-CAAGCAATCACTCGAAGAGCGTCGTGT-3’
将实施例2所描述的PDMS微流道装置与玻片贴合，实现流道封闭。用夹持工具对流道顶部与基底玻片进行压合以提高密封性。微流道的一端为溶液入口，另一端通过接口与真空抽吸装置相连接。
将第一个PDMS微流道装置与玻片贴合，通入乙醇排除气泡，再换PBS缓冲液冲洗一次，然后在流道内加入15uM的第一组条形码核酸(溶解于300mM磷酸钠缓冲液中，pH8.5)：在每一个流道通入一种第一条形码核酸(每个流道的第一条形码核酸具有与其它流道的第一条形码核酸不同的条形码序列)。充满流道后，将玻片置于35℃饱和氯化钠湿盒，静置三小时，室温。反应完成后，使用1×PBS缓冲液冲洗流道1分钟。然后拆下PDMS微流道装置，修饰的玻片依次使用0.1％Triton X-100、1mM HCl、100mM KCl清洗，然后使用0.1M Tris pH 9.0、50mM乙醇胺、0.1％ 的SDS于50℃进行封闭。封闭结束后使用去离子水冲洗基片1分钟，然后用氮气吹干基片。采用荧光显微镜观察第一条形码核酸的FITC荧光信号，确定反应完成和实现第一组条形码核酸在流道覆盖处的芯片的固定，形成第一组条形码带。
将另一个PDMS微流道装置与玻片沿与第一个PDMS微流道装置的流道形成正交的方向进行贴合。流道内通入缓冲液排出流道内的气体之后，通入15uM的第二组条形码核酸：在每一个流道通入一种第二条形码核酸(每个流道的第二条形码核酸具有与其它流道的第二条形码核酸不同的条形码序列)和连接子核酸以及T4连接酶。充满流道后37℃静置30分钟。
完成连接反应之后，通入1×PBS缓冲液，超纯水对流道进行清洗。之后取下流道，用去离子水冲洗基片1分钟，然后用氮气吹干基片。采用荧光显微镜观察第二条形码核酸的Cy3荧光信号，确定反应完成和实现第二条形码核酸与第一组条形码核酸在流道交叉点的连接反应，形成条形码阵列，由此完成芯片制备。
通过对制备得到的芯片上荧光信号的观测来评估各条形码核酸连接反应的效率以及制备得到的芯片的探针的强度(密度)和均匀性。图6显示在显微镜(奥林巴斯bx53)下对各条形码核酸连接反应的观测的图。如图6所示，在芯片横向的阵列上显示第一组条形码核酸的携带的FITC修饰的荧光信号；在芯片竖向的阵列上显示第二组条形码核酸的携带的Cy3修饰的荧光信号。
实施例4组织样品制备和染色
(一)组织OCT包埋
取新鲜小鼠脑组织样本，迅速用预冷的PBS溶液或者生理盐水冲洗组织表面残留，然后用干净的吸水纸吸干液体。将组织置于包埋槽，添加OCT包埋剂至完全覆盖组织。确认组织周围没有气泡，将包埋槽置于干冰上，直至OCT完全冻结。
(二)冷冻切片
冷冻切片机温度设定为箱体温度：-20℃，样本头温度：-10℃。切片之前将冷冻组织和基片先放入-20℃冷冻切片机箱体中平衡30分钟以上，然后在冷冻切片机箱体中进行冷冻切片，厚度为10μm。
(三)组织固定、HE染色
将切好的组织切片贴附于实施例3制备得到的条形码阵列修饰的基片上，然后置于37℃孵育1分钟。将贴附组织的基片完全浸入预冷的甲醇中，-20℃固定30分钟。固定结束后，取出基片，擦干背面液体，在组织切片上滴加500μl异丙醇，室温孵育1分钟。1分钟后，除去异丙醇，然后室温晾干5-10分钟。
加入1ml苏木精，均匀覆盖基片上的组织切片，室温孵育7分钟。去除苏木精试剂，将基片浸入RNase-free Water中清洗，晾干。加入1ml返蓝液，室温孵育2分钟。去除返蓝液，将基片浸入RNase-free Water清洗后，擦干基片背面液体。加入1ml伊红混合物，室温孵育1分钟。
去除伊红，将基片浸入RNase-free Water清洗，晾干直至组织不透明。在37℃下孵育玻片5分钟后，进行明场成像。
图7为HE染色后的组织图像。
(四)组织透化
将夹具腔室组装至制备得到的组织芯片上，确保组织切片位于对应的腔室内部。向腔室中加入70ul透化酶(0.1N HCl稀释的0.1％胃蛋白酶)37℃透化组织，移除透化酶后用0.1×SSC清洗。
实施例5组织样品切片通过芯片进行逆转录反应、建库和测序
向实施例4中清洗后的腔室中加入70μl反转录混合液。其中，反转录混合液包括：1x第一链缓冲液，5mM DTT，500μM dNTP，0.19μg/μl BSA，1％DMSO，2.5μM Template Switch Oligo，20U/μl Superscript III以及2U/μl RNase inhibitor。
其中Template Switch Oligo序列为：
5’Biotin-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3’
Template Switch Oligo中，倒数第1、2、3位碱基进行核糖鸟嘌呤核 苷修饰。
使用胶带将腔室密封之后，置于温控板上，调控至约50℃进行反转录，反应16小时。
反转录结束后，吸弃腔室中的反转录混合液。向腔室中加入70μl 0.08M KOH，室温孵育5分钟，然后加入100ul RNase-free Water清洗一次。
向清洗后的腔室中加入cDNA二链合成反应液。其中二链合成反应液包括：1×Kapa HiFi Hotstart ReadyMix，0.8uM Second Strand Primer。使用胶带将腔室密封之后，置于温控板上，调控至约37℃进行cDNA二链合成，反应30分钟。
其中Second Strand Primer序列为：
5’-AAGCAGTGGTATCAACGCAGAGTACAT-3’
反应结束后，吸弃腔室内二链合成反应液，然后加入100ul RNase-free Water清洗一次。接着向腔室中加入70μl 0.08M KOH，室温孵育10分钟。准备几个新的1.5ml离心管，向其中加入10μl Tris(1M，pH 7.0)。将腔室中的70μl样品转移至相应的含有Tris的离心管内，混匀，即完成cDNA的二链制备。
cDNA扩增
取新的1.5ml离心管置于冰上，配制PCR扩增反应液。其中PCR反应液包括：1×Kapa HiFi Hotstart ReadyMix，0.8μM cDNA Forward Primer，0.8μM cDNA Reverse Primer，35μl cDNA template，总体积100μl。通过以下方案进行cDNA扩增：
Figure PCTCN2021141391-appb-000002
其中cDNA Forward Primer序列为：
5’-CTACACGACGCTCTTCCGATC-3’
cDNA ReversePrimer序列为：
5’-AAGCAGTGGTATCAACGCAGAG-3’
结果显示CT值为约10.6。
扩增结束后，使用AMpure SPRIselect Beads纯化扩增产物。
将扩增后产物选行建库/测序。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (13)

  1. 制备具有阵列的生物芯片的方法，包括以下步骤：
    A.通过多条平行设置的微流道将第一组条形码核酸固定在芯片表面，形成第一方向的多条第一条形码带，所述第一组条形码核酸中包括多种具有不同条形码序列的第一条形码核酸，每条第一条形码带上固定一种第一条形码核酸，且每条第一条形码带上固定的第一条形码核酸具有不同的条形码序列；
    B.通过多条平行设置的微流道将第二组条形码核酸沿第二方向施加到芯片表面的具有第一方向的所述多条第一条形码带上，形成多条第二条形码带，所述第二组条形码核酸中包括多种具有不同条形码序列的第二条形码核酸，每条第二条形码带上具有一种第二条形码核酸，且每条第二条形码带上的第二条形码核酸具有不同的条形码序列；
    C.在使得第一条形码核酸和第二条形码核酸发生连接反应的条件下，在所述多条第一条形码带与所述多条第二条形码带产生交叉的芯片表面将第二条形码核酸与第一条形码核酸连接，形成探针，所述探针构成阵列的阵点，每个阵点具有一种序列相互不同的探针。
  2. 权利要求1的方法，其中采用具有多条平行设置的微流道的微流道装置将所述第一组条形码核酸或第二组条形码核酸输送和固定在芯片表面，其中所述微流道与芯片表面接触的一面可容溶液或溶液中的核酸通过，其中在所述微流道设备的每一条微流道中加入含有不同条形码序列的第一组条形码核酸或第二组条形码核酸。
  3. 权利要求1的方法，其中所述第一组条形码核酸中的第一条形码核酸包括第一条形码片段，以及在5’端具有用于扩增反应的引物片段。
  4. 权利要求3的方法，其中所述第一组条形码核酸中的第一条形码核酸在5’端具有用于与芯片表面连接的基团。
  5. 权利要求1的方法，其中所述第二组条形码核酸中的第二条形码核酸包括3’端的用于识别和结合生物样品中的目标核酸的探针片段(例如为识别和结合mRNA或cDNA的片段，例如为poly-T序列)和第二条形码片段。
  6. 权利要求5的方法，其中所述第二组条形码核酸中的第二条形码核酸还具有唯一分子标识符(UMI)。
  7. 权利要求1的方法，其中所述第一条形码核酸的3’端具有用于通过一个单链连接核酸与第二条形码核酸连接的第一连接片段，所述第二条形码核酸的5’端具有用于通过所述单链连接核酸与第一条形码核酸连接的第二连接片段，所述第一连接片段和第二连接片段分别与所述单链连接核酸的两端的序列反向互补。
  8. 权利要求1的方法，其中步骤C中形成的探针包括3’端的用于识别和结合生物样品中的目标核酸的捕获片段，以及第一条形码片段和第二条形码片段，优选的，所述探针在5’端还具有用于扩增反应的引物片段。
  9. 权利要求1的方法，其中所述第一组条形码核酸中的每一种第一条形码核酸的条形码片段的序列和所述第二组条形码核酸中的每一种第二条形码核酸的条形码片段的序列是指定的。
  10. 权利要求1的方法，其中步骤A或B中流道内的核酸浓度为约0.1-100uM，例如为约1-20uM。
  11. 权利要求1的方法，其中步骤A中，所述第一组条形码核酸的核酸以化学键连接方式固定在芯片表面上，所述化学键连接方式例如为选自氨基-醛基反应、静电吸附、共价交联中的任意一种。
  12. 权利要求1的方法，其中步骤A和步骤B中所述平行设置的微流道的各条微流道的宽度为约2-200μm，优选为约5-50μm，最优选为约5-25μm，例如为约5μm，10μm或50μm，
    以及，
    所述平行设置的微流道的各条相邻微流道之间的间距为约5-400μm，优选为约10-100μm，最优选为约10-50μm，例如为约20μm，50μm或100μm。
  13. 根据权利要求1-12中任一项所述的方法制备的具有阵列的生物芯片用于分析生物组织样品的空间转录组学信息的方法，所述方法包括将前述芯片的阵列与组织样品接触，阵列上的探针识别和结合组织中的细胞的核酸，特别是mRNA，然后进行逆转录和扩增等反应，任选的，可通过高通量下一代测序(NGS)或合成测序(SBS)进一步对获得的核酸进行分析。
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