Preparation method of biological chip space bar code sequence and nucleic acid space omics detection method

Abstract

The invention discloses a preparation method of a biological chip space bar code sequence and a nucleic acid space omics detection method. The preparation method comprises the following steps: 1) connecting a silane group containing a silyl ether bond to a biological chip carrier; 2) establishing a plurality of parallel X-channel micro-channels on the biochip carrier, then performing DNA synthesis in each X-channel micro-channel by adopting a phosphoramidite synthesis method, and forming a first spatial position recognition DNA sequence on each detection point; and 3) establishing a plurality of parallel Y-channel micro-channels vertical to the X-channel on the biochip carrier, then performing DNA synthesis in each Y-channel micro-channel by adopting a phosphoramidite synthesis method, and forming a second spatial position recognition DNA sequence on the first spatial position recognition DNA sequence on each detection point.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a method for preparing a spatial barcode sequence for a biochip and a method for detecting nucleic acid spatial omics. Background Technology

[0002] Spatial omics aims to map the molecular biology information of cells in a 3D environment. Currently, the most widely used technique is spatial transcriptomics for RNA detection, with representative commercial products including 10X Genomics Visium and BGI Stereo-seq. Spatial transcriptomics links high-resolution spatial information with transcriptomic information, enabling researchers to reveal the spatial distribution of gene expression in tissue sections. This approach not only provides a detailed map of cellular activity within an anatomical context but also offers unique opportunities to interpret the molecular signatures behind cellular heterogeneity, tissue development, and disease progression. By preserving the complex relationships between neighboring cells and their surrounding microenvironment, spatial transcriptomics opens up new dimensions for biological research, becoming a key tool in fields such as cancer research, neuroscience, developmental biology, and drug development.

[0003] These commercial products typically utilize spatial barcode oligos on an coded chip, which capture the specific 5' end sequence of mRNA or mRNA reaction products released from the sample via 5'→3' end spatial barcodes. Complementary strands are then synthesized using DNA polymerases such as Bst DNA polymerase or Klenow Fragment (3'→5' exo-) polymerase. Spatial transcriptome detection is achieved through PCR amplification, library construction, and sequencing analysis. However, the fabrication of these coded chips usually requires the pre-synthesis of oligos with known base sequence structures to define the spatial barcode coordinates. These oligos are then linked together using DNA ligases to form a complete spatial barcode. This approach not only increases chip production costs but also limits the number of spatial barcode types. Furthermore, these methods require the use of DNA polymerases with 5'→3' end enzymatic activity, resulting in high reagent costs. Summary of the Invention

[0004] This invention provides a method for preparing spatial barcode sequences for biochips and a nucleic acid spatial omics detection method to address the shortcomings of existing detection technologies. The objective of this invention is achieved through the following technical solutions:

[0005] A method for preparing a spatial barcode sequence for a biochip includes the following steps:

[0006] Step 1) Connect silane groups containing silyl ether bonds to the biochip carrier;

[0007] Step 2) Multiple parallel X-channel microchannels are established on the biochip carrier, and then DNA is synthesized in each X-channel microchannel using the phosphoramide synthesis method to form a first spatial position recognition DNA sequence at each detection point; the first spatial position recognition DNA sequence synthesized in different X-channel microchannels is different.

[0008] Step 3) Multiple parallel Y-channel microchannels perpendicular to the X-channel are established on the biochip carrier. Then, DNA is synthesized in each Y-channel microchannel using the phosphoramide synthesis method. A second spatial position recognition DNA sequence is formed on the first spatial position recognition DNA sequence at each detection point. The second spatial position recognition DNA sequences synthesized in different Y-channel microchannels are different.

[0009] The nodes where each parallel X channel intersects perpendicularly with multiple parallel Y channels form a detection point array on the biochip.

[0010] Optionally, the biochip carrier in step one) is glass with a surface hydroxylation treatment.

[0011] Optionally, the hydroxylation treatment can be achieved by reacting the glass with a piranha solution or by plasma treatment of the glass surface.

[0012] Optionally, the silane group containing the silane bond is at least one of tert-butyldimethylchlorosilane, triisopropylchlorosilane, 3-aminopropyltrimethoxysilane, and 2-hydroxyethyl(methyl(3-trimethoxysilylpropyl)amino)propionate.

[0013] Optionally, the phosphorus amide synthesis method in steps three and four includes attaching a first phosphorus amide-protected nucleotide monomer to a coupling agent group; and then sequentially attaching nucleotides to the first nucleotide monomer through multiple cycles of deprotection, activation and coupling, capping, and oxidation.

[0014] The deprotection process includes: treating the biochip carrier with a trichloroacetic acid-dichloromethane solution to remove the protecting group at the 5' end of the nucleotides attached to the carrier;

[0015] The activation and coupling include: mixing phosphorus amide-protected nucleotide monomers with tetrazolium activator to form a phosphorus amide tetrazolium active intermediate, which then undergoes a condensation reaction with the deprotected nucleotides on the carrier, causing the nucleotide chain on the carrier to be extended by one base.

[0016] The capping includes: sealing the 5' end of the nucleotide chain on the carrier with an acetylation reagent;

[0017] The oxidation process involves converting phosphorous acyl into a triphosphate using a tetrahydrofuran solution of iodine to obtain a stable oligonucleotide.

[0018] Optionally, in step two: first, a phosphorus amide synthesis method is used to form PCR primer sequences at each detection point, and then a first spatial location recognition DNA sequence is formed on the PCR primer sequences using a phosphorus amide synthesis method.

[0019] Optionally, step three further includes: synthesizing a unique molecular recognition tag sequence and a first fixed nucleotide sequence on the DNA sequence identified at the second spatial location using a phosphoramide synthesis method.

[0020] Optionally, step one) further includes designing a spatial location identification sequence so that different detection points correspond to different spatial location identification sequences, and the GC content is 44% to 72%, hairpin structures are avoided, and there are no more than 5 consecutive identical bases; the spatial location identification sequence includes the first spatial location identification DNA sequence and the second spatial location identification DNA sequence.

[0021] In steps two and three, the first spatial location identification DNA sequence and the second spatial location identification DNA sequence are synthesized at each detection point according to the designed spatial location identification sequence.

[0022] Optionally, both the first spatial location identification DNA sequence and the second spatial location identification DNA sequence contain 8 nucleotides.

[0023] Optionally, the method further includes step four) using a protective nucleotide chain that is partially complementary to the bases of the intact oligonucleotide chain, and then using an exonuclease to cleave and remove the incomplete oligonucleotide chain.

[0024] The present invention also proposes a biochip prepared by the above preparation method, wherein the biochip includes a biochip carrier, the biochip carrier is provided with detection points, each detection point contains a spatial barcode oligonucleotide, and the spatial barcode oligonucleotide is connected to the biochip carrier through a silyl ether bond;

[0025] The spatial barcode oligonucleotide includes a PCR primer sequence, a first spatial location recognition DNA sequence, a second spatial location recognition DNA sequence, a unique molecular recognition tag sequence, and a first fixed nucleotide sequence.

[0026] A nucleic acid spatial omics detection method includes:

[0027] S1: The tissue slices are permeabilized and then reacted with the detection probe, allowing the detection probe to enter the tissue slices and specifically bind to the target sequence of the target nucleic acid to be tested, forming a target probe conjugate;

[0028] S2: The above-mentioned biochip is hybridized with the linker nucleic acid fragment and then attached to a tissue slice, so that the target probe conjugate is released from the tissue slice and linked with the spatial barcode oligonucleotide; then the target probe conjugate is linked with the spatial barcode oligonucleotide by a ligase to form a target probe template DNA.

[0029] S3: Treat the biochip with an alkaline solution to detach the target probe template DNA from the biochip carrier;

[0030] S4: Perform PCR on the target probe template DNA, sequence and analyze it to obtain the target nucleic acid detection results.

[0031] Optionally, the detection probe includes a targeting capture sequence, a sequencing adapter Read 2 sequence, and a second fixed nucleotide sequence;

[0032] The targeted capture sequence can specifically bind to the target sequence of the target nucleic acid to be tested in the biological sample;

[0033] In step S2, the linker nucleic acid fragment binds to the first fixed nucleotide sequence and the second fixed nucleotide sequence, respectively, so that the target probe ligand is linked to the spatial barcode oligonucleotide.

[0034] Optionally, the detection probe includes a first detection probe and a second detection probe, wherein the first detection probe includes a sequencing adapter sequence and a first targeting capture sequence; and the second detection probe includes a fixed nucleotide sequence and a second targeting capture sequence.

[0035] The target capture sequence is formed by combining the first target capture sequence and the second target capture sequence;

[0036] Step three also includes ligating the first target capture sequence and the second target capture sequence using a ligase.

[0037] Optionally, the 3-end of the sequencing adapter sequence in the first detection probe is connected to the 5-end of the first targeting capture sequence; the 3-end of the second targeting capture sequence in the second detection probe is connected to the 5-end of the fixed nucleotide sequence.

[0038] Optionally, the target probe ligand is released from the tissue slice and detached from the nucleic acid to be detected by at least one of the following methods: cell / nucleus lysis release, enzyme digestion release, and heat release.

[0039] Optionally, the method of treating the biochip with an alkaline solution in step S3 includes: treating it with a 10mM to 150mM sodium hydroxide or potassium hydroxide solution at a temperature of 4℃ to 50℃ for 1 to 10 minutes.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The method for preparing spatial barcode sequences for biochips according to the present invention eliminates the need for pre-synthesizing oligos with known base sequence structures to define spatial barcode coordinate information before fabricating the coded chip, significantly reducing chip production costs. Furthermore, the number of spatial barcode types is theoretically not limited by the number of synthesized oligos; the base sequence length and base types can be arbitrarily adjusted to meet the requirements for different numbers of spatial barcode types.

[0042] The nucleic acid spatial omics detection method of the present invention, after capturing RNA or RNA reaction products released from tissue sections, does not require the use of DNA polymerase with 5'→3' end enzyme activity to generate complementary strands. Instead, it can directly treat the DNA with alkaline solution to recover the DNA strand for subsequent PCR amplification and library construction, thereby reducing reagent costs and simplifying experimental operations. Attached Figure Description

[0043] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required for the specific embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram of the sequence characteristics of spatial barcode oligonucleotides on an encoding chip.

[0045] Figure 2 This is a schematic diagram of a spatial barcode oligonucleotide spot array on an encoding chip.

[0046] Figure 3 A schematic diagram of a single detection probe hybridizing with the target nucleic acid.

[0047] Figure 4 This is a schematic diagram illustrating the hybridization of dual detection probes with the target nucleic acid and the construction of sequencing libraries.

[0048] Figure 5 A schematic diagram of the clip structure for the encoding chip.

[0049] Figure 6 This is a schematic diagram of the encoding chip manufacturing process.

[0050] Figure 7 This is a schematic diagram showing the spatial location of mouse brain tissue transcriptome RNA sequencing data mapped to the corresponding spot points on the coding chip. Detailed Implementation

[0051] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is only for describing particular embodiments and is not intended to limit the present invention.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] Unlike existing technologies, this invention uses a phosphorus amide method for DNA chemical synthesis to create an encoded chip with spatial barcodes.

[0054] The method for fabricating the encoded chip includes: modifying the encoded chip carrier with a chemical coupling reagent to enrich its surface with specific chemical functional groups; coupling the specific chemical functional groups to the 3'-OH of a nucleotide whose 5'-OH is blocked and protected by a -DMT (dimethoxytriphenylmethyl) group through a chemical reaction; based on an orthogonal chemical reaction, through multiple rounds of deprotection-activation and coupling-capping-oxidation steps, sequentially coupling several nucleotides after the first nucleotide, and finally generating a complete spatial barcode oligonucleotide on the carrier.

[0055] The carrier refers to glass, which, after being treated with piranha solution and / or plasma, has a surface rich in hydroxyl groups.

[0056] The chemical coupling agent refers to a reactive reagent containing at least two chemical functional groups, one of which can react with chemical functional groups on the surface of the support, and the other of which can react with the 3'-OH group of the nucleotide. As one embodiment, the chemical coupling agent is a silanizing agent containing a silyl ether bond, such as tert-butyldimethylchlorosilane, triisopropylchlorosilane, 2-hydroxyethyl (methyl(3-trimethoxysilylpropyl)amino)propionate, etc. As one embodiment, when the chemical coupling agent is 2-hydroxyethyl (methyl(3-trimethoxysilylpropyl)amino)propionate, it can react with the hydroxyl groups on the surface of the support at one end to form a silyl ether bond, and react with the hydroxyl groups on the nucleoside at the other end to form an ester group, thereby generating the first base of the oligonucleotide chain on the support.

[0057] The orthogonal chemical reaction refers to using microchannels to form X-axis and Y-axis channels on the surface of the carrier, and then adding nucleotides to the surface of the carrier in the X-axis and Y-axis directions respectively, so that the oligonucleotide chains formed at the intersection of the X-axis and Y-axis channels are spatial barcode oligonucleotides.

[0058] The deprotection process involves treating the support with a dichloromethane solution of trichloroacetic acid (TCA) to remove the protecting group DMT of the 5'-OH nucleotide attached to the support, obtaining free 5'-OH for the next coupling reaction. As one embodiment, a 3% dichloromethane solution of trichloroacetic acid is used for the deprotection treatment.

[0059] The activation and coupling refer to the mixing of phosphorusamide-protected nucleotide monomers (dA, dC, dT, dG) with a tetrazolium activator, such as the acidic catalyst ETT [5-(ethylthio)-1H-tetrazole], which, after being "activated" (protonated), forms a phosphorusamide-tetrazole active intermediate. This intermediate immediately undergoes an affinity condensation reaction upon contact with the deprotected nucleotides on the carrier, at which point the oligonucleotide chain synthesized on the carrier is extended by one base. It can be understood that when the same base position (3'→5' end direction) of different spatial barcode oligonucleotides on the carrier is required to be identical, the phosphorusamide-protected nucleotide monomer used must be a specific one of dA, dC, dT, and dG. For example, in the PCR primer sequences described below, since the PCR primer sequences on each spatial barcode oligonucleotide are identical and the base sequences are fixed, the phosphorusamide-protected nucleotide monomer used in each reaction can only be a specific one of dA, dC, dT, and dG. When it is required that the bases at the same base position (e.g., the 3'→5' direction) of different spatial barcode oligonucleotides on the carrier be different, such as the unique molecular recognition tag sequence described below, the phosphoramidite-protected nucleotide monomers used are a mixture of dA, dC, dT, and dG.

[0060] The capping refers to the process of blocking the 5'-OH group on the support with an acetylation agent (such as a mixture of acetic anhydride and N-methylimidazole) after the activation and coupling reactions to prevent the unreacted 5'-OH group on the support from being extended in subsequent cycles.

[0061] The oxidation refers to the conversion of phosphorous acyl into a phosphate triester using a tetrahydrofuran solution of iodine in the coupling reaction, resulting in a stable oligonucleotide.

[0062] It is understood that the deprotection-activation and coupling-capping-oxidation steps, after multiple rounds of repeated reactions, will generate an oligonucleotide with a specific base sequence length on the support, with its 3' end close to the support surface and its 5' end far from the support surface. Finally, the deprotection step removes the protecting group DMT located at the end of the oligonucleotide chain.

[0063] Since chemical reactions are never 100% efficient, the resulting oligonucleotide chains will inevitably include complete chains of the predetermined base length and incomplete chains shorter than the predetermined base length. Therefore, as a preferred method, after generating the oligonucleotide chains, the incomplete oligonucleotide chains can be removed by partially base complementation between the protective nucleotide chain and the complete oligonucleotide chain using an exonuclease.

[0064] The sequence characterization results of spatial barcode oligonucleotides on an encoding chip are as follows: Figure 1 As shown, its 3' end is close to the carrier surface, and its 5' end is away from the carrier surface. The encoding chip 1000 contains a plurality of spatial barcode oligonucleotides, which are distributed on the carrier surface in a specific geometric array. The geometric array can be a spot array, and there may be no gaps or gaps between each spot. As one implementation, such as Figure 2 As shown, the area of ​​the geometric array is 7.5mm × 7.5mm, each spot point is 0.05mm × 0.05mm, and the center distance of each spot point is 0.05mm.

[0065] The spatial barcode oligonucleotide is an oligonucleotide fragment containing at least a PCR primer sequence 1001 and a first fixed nucleotide sequence 1004. Further, the spatial barcode oligonucleotide is an oligonucleotide fragment containing at least a PCR primer sequence 1001, a spatial location identification tag 1002, a unique molecular recognition tag sequence 1003, and a first fixed nucleotide sequence 1004. The PCR primer sequence 1001 is an oligonucleotide with a known base sequence, and the PCR primer sequence for each spatial barcode oligonucleotide can be the same or different. As one embodiment, the PCR primer sequence 1001 is an oligonucleotide sequence that is partially or entirely complementary to or identical to the sequencing primer of the sequencing instrument. For example, the PCR primer sequence 1001 is 3'-CACGACGCTCTTCCGATCT-5'. The spatial location identification tag 1002 is an oligonucleotide fragment composed of any combination of the four bases A, T, C, and G. For example, the spatial location identification tag 1002 is obtained by any combination of 1, 2, 3, 4, 5, 10, 20, 30, 60, 70, or 80 bases. In one embodiment, the spatial location identification tag 1002 contains 16 consecutive bases, denoted as JJJJJJJJJJJJJJJJ. All spatial location identification tags 1002 at each spot point are identical, while the spatial location identification tags 1002 between each spot point are different. To determine the base information of the spatial location identification tag 1002 at each spot point so that it can be used to define the spatial distribution of the analyte during subsequent biological sample detection, the four bases A, T, C, and G can be arranged and combined in a specific order before fabricating the coding chip, so that they ultimately form a nucleic acid fragment of 16 consecutive bases in length. Then, during the fabrication of the coding chip, a base coupling reaction is performed according to a preset order. When designing the spatial location identification tag 1002, the following principles are followed, including but not limited to: 1. GC content: 44%–72%; 2. Avoid hairpin structures; 3. Do not contain low-complexity sequences, such as AAAAA, TTTTT, CCCCC, GGGGG. The unique molecular identification tag sequence 1003 is an oligonucleotide fragment composed of any combination of the four bases A, T, C, and G. For example, the unique molecular identification tag sequence 1003 is obtained by any combination of 1, 2, 3, 4, 5, 8, 10, or 20 bases. As one embodiment, the unique molecular identification tag sequence 1003 contains 8 consecutive bases, represented by NNNNNNNNN. The first fixed nucleotide sequence 1004 is an oligonucleotide sequence, and the first fixed nucleotide sequence 1004 is the same on each of the spatial barcode oligonucleotides. As one embodiment, the first fixed nucleotide sequence 1004 is 3'-GCGTACGAC-5'.Therefore, as an example, the sequence characteristics of the spatial barcode oligonucleotide are: 3'-CACGACGCTCTTCCGATCT-JJJJJJJJJJJJJJJ-NNNNNNNN-GCGTACGAC-5', where J and N represent any one of the four bases A, T, C, and G, and the J base at each spot is predetermined.

[0066] Therefore, this method generates the spatial barcode oligonucleotides on the encoding chip by synthesizing bases one by one. Thus, before fabricating the encoding chip, it is unnecessary to pre-synthesize large quantities of oligos with known base sequence structures to define the spatial barcode coordinate information, which significantly reduces chip production costs. Furthermore, the number of spatial barcode types is theoretically not limited by the number of synthesized oligos; the base sequence length and base types can be arbitrarily adjusted to meet the requirements for different numbers of spatial barcode types.

[0067] A spatial omics method for biological sample detection includes: attaching a tissue slice to a glass slide; permeating the tissue slice to allow a detection probe to enter the tissue slice and specifically bind to the target sequence of a target nucleic acid, forming a target probe conjugate. After hybridizing spatial barcode oligonucleotides on an encoding chip with a linker nucleic acid fragment, the encoding chip is attached above the tissue slice to be tested. The surface of the encoding chip in contact with the tissue slice is provided with a spot array; each spot in the spot array contains a spatial barcode oligonucleotide for detecting the target nucleic acid. After permeating the tissue slice, the target probe conjugate is released and binds to the spatial barcode oligonucleotides on the encoding chip via DNA ligase to obtain a target probe template DNA; the target probe template DNA is detached from the encoding chip by alkaline solution treatment; PCR is then performed on the target probe template DNA to construct a sequencing library, and the target nucleic acid is sequenced and detected.

[0068] The tissue section refers to fresh frozen OCT-embedded tissue, formalin-fixed paraffin-embedded tissue, or formalin-fixed OCT-embedded tissue, which is cut into tissue sections of a specific thickness, such as 10 micrometers or 5 micrometers, by a tissue slicer.

[0069] Specifically, if the biological sample tissue has been fixed in formalin, it undergoes decrosslinking treatment before reacting with the detection probe, followed by permeabilization treatment. Decrosslinking refers to partially or completely reversing the fixation process of the fixed biological sample using physical or chemical methods. The physical methods can be a combination of one or more of the following: thermal lysis, photolysis, and acoustic lysis. The chemical methods refer to a combination of one or more of the following: reducing agents (e.g., DTT, TCEP), surfactants (e.g., Tween-20, Triton X-100, SDS), salt ion solutions (e.g., PBS, TE, citrate), and biological enzymes, to partially or completely reverse the crosslinking bonds in the fixed biological sample. As one embodiment, the fixed biological sample is decrosslinked using TE buffer at 90°C. As another embodiment, the fixed biological sample is decrosslinked using TE buffer containing Tween-20 at 85°C. In particular, when the fixed biological sample is a paraffin-embedded sample, the fixed biological sample needs to be dewaxed using chemical reagents (e.g., xylene, ethanol, isopropanol, etc.) before decrosslinking to remove the paraffin components from the sample surface.

[0070] The permeation refers to making the cell membrane and / or nuclear membrane of the biological sample permeable through physical or chemical methods, allowing small molecules (e.g., water molecules, Tris-HCl, short nucleotide fragments, etc.) and large molecules (e.g., enzymes, proteins, large nucleotide fragments) to enter the cell / nucleus. The physical methods can be a combination of one or more of the following: heating, light irradiation, and acoustic oscillation. The chemical methods refer to a combination of one or more of the following: surfactants (e.g., Tween-20, Triton X-100, SDS, etc.), salt ion solutions (e.g., PBS, TE, citrate, etc.), and biological enzymes (e.g., proteinase K, pepsin, etc.), to promote the permeability of the cell membrane and / or nuclear membrane in the biological sample. As one embodiment, the biological sample is permeated using a citrate solution containing a surfactant. The concentration of the surfactant can be 0.005% to 1%. The citrate solution can be 1×SSC, 2×SSC, 3×SSC, 4×SSC, 5×SSC, etc. As a preferred method, the biological sample is permeabilized using 2×SSC containing 0.01% Triton X-100.

[0071] The detection probe is an oligonucleotide fragment composed of several nucleotides. The detection probe consists of 10 to 150 bases; more specifically, it consists of 30, 40, 50, 60, 70, or 80 bases. As one embodiment, the detection probe consists of 60 bases. The detection probe includes at least a targeting capture sequence that specifically binds to the target sequence of the target nucleic acid in the biological sample, completing base complementary pairing. The number of bases in the targeting capture sequence can be 0, 5, 8, 10, 20, 30, 50, 100, or 120 fewer bases than the detection probe. For example, 25 bases of the 60-base detection probe are the targeting capture sequence. Each detection probe can only bind to one target sequence, while each target sequence can bind to one, two, three, four, or five detection probes. This not only ensures targeted measurement of the target nucleic acid, improving the pass rate of valid reads in the final sequencing data, but also helps to improve the detection efficiency and sensitivity of the target nucleic acid. For example: Figure 3 As shown, the detection probe 2000 includes a targeting capture sequence 2001, a sequencing adapter Read 2 sequence 2002, and a second fixed nucleotide sequence 2004. The targeting capture sequence 2001 specifically recognizes and binds to the targeting sequence 2006 of the target nucleic acid 2005. The characteristics of the sequencing adapter Read 2 sequence 2002 and the second fixed nucleotide sequence 2004 are described below.

[0072] In one implementation, the targeting sequence includes at least a first targeting sequence and a second targeting sequence, and the detection probe consists of at least a first detection probe and a second detection probe. The first detection probe specifically binds to the first targeting sequence, and the second detection probe specifically binds to the second targeting sequence. As a specific method, after the first detection probe binds to the first targeting sequence and the second detection probe binds to the second targeting sequence, one or a combination of methods such as enzymes or oligonucleotide fragments can be used to link the first detection probe and the second detection probe together, forming a complete complementary sequence to the targeting sequence. Therefore, the targeting sequence binds to a pair of detection probes.

[0073] In one implementation, when the target sequence binds to a pair of detection probes, the first detection probe pairs complementaryly with a base near the 3' end of the target sequence, and the second detection probe pairs complementaryly with a base near the 5' end of the target sequence. After binding to the target sequence, they can link together to form a sequence that is completely complementary to the target sequence. The first detection probe consists of a first target capture sequence complementary to the target sequence and a sequencing adapter Read 2 sequence; the second detection probe consists of a second target complement sequence complementary to the target sequence and a fixed nucleotide sequence. The first target capture sequence has a free hydroxyl group at its 3' end, and the second target capture sequence has a phosphorylation modification at its 5' end, so that the first and second target capture sequences can be linked to form a complete complementary sequence of the target gene.

[0074] The number of bases in the first and second targeted capture sequences can be 10–120; further, it can be 20–60; further still, it can be 20–30. The number of bases in the first and second targeted capture sequences can be the same or different. As one embodiment, the number of bases in the first and second targeted capture sequences is 25 each. For example, the first and second targeted capture sequences designed for the genes of PECAM1, PTPRC, CSNK1A1, FTH1, and CD55 target nucleic acids are shown in Table 1 (Sequence 1–Sequence 12).

[0075] Table 1

[0076]

[0077] The sequencing adapter Read 2 sequence is a base sequence that can be adapted to a sequencer, and its number of bases can be 10 to 50. As one implementation, the sequencing adapter Read 2 sequence can be adapted to an Illumina next-generation sequencing instrument, and its sequence is: 5'-GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'.

[0078] The second fixed nucleotide sequence is an oligonucleotide fragment containing the same known base sequence information in each of the second detection probes. The number of bases in the fixed nucleotide sequence can be 5 to 100, 5 to 50, 5 to 10, etc. As one embodiment, the second fixed nucleotide sequence is 3'-GAATGCTCT-5'.

[0079] The first and second target capture sequences can be joined together using cross-linking agents, ligases (e.g., T4 ligase, Splint R ligase, etc.). As one embodiment, T4 ligase is used for ligation.

[0080] As one example, such as Figure 4 As shown, the target nucleic acid 2005 to be tested has a target sequence including a first target sequence 2016 and a second target sequence 2017, and the first target sequence 2016 and the second target sequence 2017 are adjacent. The detection probe includes a first detection probe 2018 and a second detection probe 2019. The first detection probe 2018 consists of a first target capture sequence 2001 complementary to the first target sequence 2016 and a sequencing adapter Read 2 sequence 2002; the second detection probe 2019 consists of a second target complementarity sequence 2003 complementary to the second target sequence 2017 and a second fixed nucleotide sequence 2004. When a pair of detection probes bind to the target sequence of the target nucleic acid 2005 to be tested, the first detection probe 2018 and the second detection probe 2019 are joined together using T4 ligase to form a structure as shown. Figure 4 The target probe conjugate shown is 2020.

[0081] The target nucleic acid can be DNA, RNA, or both. As one implementation, each target nucleic acid can contain one, two, three, or more target sequences and can bind to one or more pairs of detection probes; for example, each target nucleic acid can bind to two, three, four, or five pairs of detection probes. This approach improves the detection efficiency and sensitivity of the target nucleic acid. Therefore, when the whole transcriptome of the biological sample needs to be measured, the detection probes can be designed based on the whole transcriptome sequence information of the biological sample, ultimately resulting in thousands of pairs of detection probes.

[0082] The Linker nucleic acid fragment is an oligonucleotide fragment with known base sequence information. A portion of its bases are complementary to the first fixed nucleotide sequence (1004 bases) of the spatial barcode oligonucleotide on the encoding chip, and the remaining bases are complementary to the second fixed nucleotide sequence (2004 bases) of the second detection probe. The number of bases in the fixed nucleotide sequence can be 10–100, 10–30, 10–20, etc. As one embodiment, the Linker nucleic acid fragment is 3'-AGAGCATTCGTCGTACGC-5'.

[0083] The hybridization of the spatial barcode oligonucleotides on the encoding chip with the Linker nucleic acid fragments involves adding a Linker nucleic acid fragment solution to the encoding chip, then heating it to 95°C under sealed conditions, and subsequently slowly cooling it to room temperature at a rate of 0.1°C / s. As one embodiment, the reaction concentration of the Linker nucleic acid fragment solution is 10 μM.

[0084] The detection probe can be released from the tissue section in various ways, such as through cell / nuclear lysis, enzymatic digestion, heating, or any combination thereof. As one implementation, cells are lysed using chemical reagents such as surfactants (e.g., Tween-20, Triton X-100, SDS, etc.) to release the detection probe. The surfactant concentration can be 0.1%, 0.2%, 0.5%, 1%, 5%, or 10%. Alternatively, cells are lysed using a surfactant containing an enzyme (e.g., proteinase K, RNase, T4 ligase, etc.) to release the detection probe. The enzyme concentration can be 0.01 U / μL, 0.02 U / μL, 0.05 U / μL, 0.1 U / μL, 0.5 U / μL, 1 U / μL, 5 U / μL, 10 U / μL, 20 U / μL, 40 U / μL, or 100 U / μL. For example, a permeabilization mixed enzyme solution containing 1 U / μL RNase, 0.05 U / μL proteinase K, and 30 U / μL T4 ligase can be used.

[0085] As one implementation method, such as Figure 4The diagram illustrates the principle of sequencing library construction. When the tissue section to be tested is attached to a glass slide, the biological sample is stained using HE staining, and then imaged under a microscope in bright field. The glass slide is mounted in a matching clip with the tissue section facing upwards and surrounded by the silicone pad of the clip, forming a chamber to hold the reaction reagents. Probe hybridization reaction solution is added to the chamber, allowing the first detection probe 2018 and the second detection probe 2019 to bind to the target nucleic acid. Under the action of DNA ligase, the first detection probe 2018 and the second detection probe 2019 are linked together to form the target probe binding complex 2020. Subsequently, the glass slide is fixed on the stage of the DynaBlot instrument (manufactured by Suzhou Deyun Kangrui Biotechnology Co., Ltd.), and the encoding chip 1000 is fixed on the flip-top stage of the DynaBlot instrument. Before use, the encoding chip 1000 is hybridized with the Linker nucleic acid fragment 2008, so that the spatial barcode oligonucleotides on the encoding chip are partially complementary to the bases of the Linker nucleic acid fragment 2008. The permeabilization mixed enzyme solution is added dropwise to the tissue section area of ​​the slide, and then the flip-top stage of the DynaBlot instrument is immediately closed, so that the mixed enzyme permeabilization solution is between the slide and the encoding chip 1000, but the slide and the encoding chip 1000 are not in close contact. At this time (step 2007), the target probe binder 2020 in the tissue section permeates out of the cell, and its second fixed nucleotide sequence 2004 will perform base complementary pairing with the bases on the Linker nucleic acid fragment 2008 that are not bound to the spatial barcode oligonucleotides, thereby being captured. Simultaneously (step 2009), under the action of T4 ligase, the spatial barcode oligonucleotides and the target probe binder 2020 are linked together to form a complete DNA strand 2010. Subsequently, the coding chip is removed from the DynaBlot instrument and installed in a matching clip with the front side facing up and surrounded by the silicone pad of the clip, forming a chamber to hold the reaction reagents. An alkaline solution (step 2011) and / or heating to 95°C are added to the chamber, causing the silyl ether bonds between the spatial barcode oligonucleotides and the surface of the coding chip to break, thereby releasing DNA strands 2010. The alkaline solution containing DNA strands 2010 is aspirated and neutralized with Tris-HCl (pH = 7.0). Then, a large amount of DNA strands 2013 are enriched by PCR (step 2012). P5 and P7 end-connectors and indices are added to the DNA strands 2013 by PCR (step 2014), completing the construction of the sequencing library 2015. After sequencing and bioinformatics analysis, gene expression information can be obtained.

[0086] The encoding chip comes with a matching clip (such as...) Figure 5 (As shown) is a fixture made of plastic, metal and / or rubber, with an internal cavity that allows the glass slide or biochip to be installed and fixed, and space above the biological sample to allow reagents to be added into the clip for reaction.

[0087] Compared to the original method, after capturing RNA or RNA reaction products released from tissue sections, it is not necessary to use DNA polymerase with 5'→3' end enzyme activity to generate complementary strands. Instead, the DNA strands can be recovered directly after treatment with alkaline solution for subsequent PCR amplification and library construction, reducing reagent costs and simplifying experimental operations.

[0088] Example 1: Fabrication of an Encoding Chip

[0089] The sequence characteristics of spatial barcode oligonucleotides on an encoding chip (e.g.) Figure 2 The sequence (shown) includes a PCR primer sequence 1001, a spatial location identification tag 1002, a unique molecular identification tag sequence 1003, and a first fixed nucleotide sequence 1004. Specifically, the PCR primer sequence 1001 is 3'-CACGACGCTCTTCCGATCT-5'; the spatial location identification tag 1002 is JJJJJJJJJJJJJJJJJ; the unique molecular identification tag sequence 1003 is NNNNNNNN; and the first fixed nucleotide sequence 1004 is 3'-GCGTACGAC-5'. Therefore, the complete spatial barcode oligonucleotide sequence is: 3'-CACGACGCTCTTCCGATCT-JJJJJJJJJJJJJJJ-NNNNNNNN-GCGTACGAC-5', where J and N represent any one of the four bases A, T, C, and G. In order to determine the base information of the spatial location identification tag 1002 at each spot point so that it can be used to define the spatial location distribution of the analyte when used for subsequent biological sample detection, the four bases A, T, C and G can be arranged and combined in a specific order before the coded chip is made to design a library of all spatial location identification tag 1002 sequences.

[0090] Subsequently, following an orthogonal chemical reaction method, spatial barcode oligonucleotides were synthesized on a glass slide using the phosphoramide method for DNA synthesis. The 3' end of the oligonucleotide was close to the surface of the glass slide, while the 5' end was far from the surface. The glass slides used were purchased from Jiangsu Shitai Experimental Equipment Co., Ltd., and the reagents used (unless otherwise specified) were purchased from Sigma.

[0091] After cleaning the slides with anhydrous ethanol, they were soaked overnight in a piranha solution to enrich the surface with hydroxyl groups. The slides were then thoroughly rinsed with purified water and dried under nitrogen. Next, the slides were immersed in a 3% aqueous solution of (3-trimethoxysilylpropyl)amino)hydroxyethyl propionate in ethanol (containing 5% water) for 4 hours. After thorough rinsing with purified water and drying under nitrogen, the phosphorus amide-protected nucleotide monomers were immediately subjected to a dC reaction, resulting in the formation of the first base C of a spatially barcoded oligonucleotide chain on the slide surface. Subsequently, based on the phosphorus amide method for DNA chemical synthesis, ACGACGCTCTTCCGATCT was ligated to the C base after multiple rounds of "deprotection-activation and coupling-capping-oxidation" steps, yielding the PCR primer sequence 1001: 3'-CACGACGCTCTTCCGATCT-5'.

[0092] Next, according to... Figure 6 As shown, the X-channel layer is directly attached to the top of the slide and secured with rivets, ensuring tight contact between the slide and the X-channel layer and preventing liquid leakage between the X-channels. The X-channel layer material is made of glass or high-temperature resistant polymer plastic and contains multiple parallel-spaced X-channels (X1, X2, X3, X4, etc.). The bottom of each X-channel penetrates the X-channel layer, while the top is located within the X-channel layer material. Each X-channel is 0.05 mm wide and 0.1 mm high, with a 0.05 mm gap between channels. Each X-channel has an inlet and an outlet at both ends, completely penetrating the X-channel layer material, allowing the introduction of reaction reagents into the channels after the X-channel layer is mounted on the slide. After installation and verification with enzyme-free water to ensure no liquid leakage, the first eight bases of the spatial location identification tag 1002 (JJJJJJJJ) are sequentially synthesized on specific X-channels using a phosphoramide DNA chemical synthesis method, based on a pre-designed spatial location identification tag 1002 sequence library.

[0093] The X channel was then removed, and the Y channel was installed in the same manner, orthogonal to the original X channel. The last eight bases of the spatial identification tag 1002, JJJJJJJJ, were synthesized sequentially on a specific Y channel. At this point, the oligonucleotide chain 3'-CACGACGCTCTTCCGATCT-JJJJJJJJJJJJ-5' at the orthogonal position of the X and Y channels was obtained as the target sequence.

[0094] It is understandable that, similar to the synthesis of PCR primer sequence 1001, the aforementioned slide can be further processed to add a unique molecular recognition tag sequence 1003 and a first fixed nucleotide sequence 1004 after the spatial recognition tag 1002. For a complete sequence, it should include PCR primer sequence 1001, spatial recognition tag 1002, unique molecular recognition tag sequence 1003, and first fixed nucleotide sequence 1004. If it is an incomplete sequence, such as lacking the first fixed nucleotide sequence 1004 as mentioned in this specification (3'-GCGTACGAC-5'), then when the designed protective nucleotide chain is 3'-GTCGTACGC-5', it can bind completely complementary to the first fixed nucleotide sequence on the complete sequence, forming double-stranded DNA. Because of the first fixed nucleotide sequence, the entire incomplete sequence will expose the 5' end of the single-stranded DNA. When a DNA exonuclease is present, this incomplete sequence is cleaved, while the complete sequence is not. After cleavage, heating or treatment with an alkaline solution can remove the protective nucleotide chain, leaving the complete sequence.

[0095] The final result is as follows Figure 2 The coded chip shown is composed of a spatial barcode oligonucleotide array with an area of ​​7.5 mm × 7.5 mm. Each spot (i.e., the intersection of the X and Y channels) is 0.05 mm × 0.05 mm, and the center-to-center distance between each spot is 0.05 mm.

[0096] Example 2: Transcriptome RNA detection in mouse brain tissue

[0097] Unless otherwise specified, the reagents are from the DynaSpatial FFPE Gene Expression Reagents Kit manufactured by Suzhou Deyun Kangrui Biotechnology Co., Ltd.

[0098] The specific procedure is as follows: Formalin-fixed paraffin-embedded mouse brain tissue was cut using a microtome. 5μm thick tissue sections were attached to clean adhesive slides. Hematoxylin solution (Sigma) was added to the tissue sections to completely cover them, and the sections were incubated at room temperature for 3 minutes. After washing three times with enzyme-free water, Bluing Buffer solution (Sigma) was added, and the sections were incubated at room temperature for 2 minutes. After washing twice with enzyme-free water, freshly prepared alcohol-soluble eosin solution (Sigma) was added to the tissue sections, and the sections were incubated at room temperature for 1 minute. The sections were then washed twice with enzyme-free water and centrifuged until no visible water droplets remained on the surface. 85% glycerol solution was added to the tissue sections to evenly cover them, and the sections were mounted. The sections were then carefully placed under an optical microscope for morphological imaging. For ease of subsequent bioinformatics analysis, images were taken using a 10x objective lens, and the saved images were at least 4000×4000 pixels in TIFF format.

[0099] Carefully wash the tissue section in enzyme-free water, remove the cover slip and glycerol, and then mount the slide containing the tissue section in the matching clip, with the tissue section facing upwards and centered in the clip chamber. Add 200 μL of 0.1M HCl solution to the chamber to evenly cover the tissue and seal with a sealing film. Then place it on the PCR adapter in the PCR instrument and incubate at 37°C for 3 min. Remove the sealing film, remove the solution, add 200 μL of 0.01M sodium citrate solution (pH = 6.0), seal with a sealing film, and then place it on the PCR adapter in the PCR instrument. Incubate at 95°C for 60 min and 22°C for 10 min.

[0100] After the reaction was complete, remove the sealing film, remove the solution, and add 200 μL of 1×PBS-0.05% Tween 20 solution. Incubate at room temperature for 15 min. Remove the solution and wash once with 2×SSC solution. Then add 100 μL of probe hybridization reaction solution [10 μL Nuclease-free Water, 70 μL Probe-Hyb buffer, 10 μL LHSprimer (Human), 10 μL LHSprimer (Human)]. Seal the container and place it on the PCR adapter in the PCR instrument. Incubate at 50 °C for 16 h.

[0101] After the reaction was complete, wash three times with 200 μL of 2×SSC (containing 50% formamide) solution each time. Finally, add 200 μL of 2×SSC and incubate at room temperature for 3 min. Remove the solution, add 100 μL of probe ligation reaction solution [40 μL Nuclease-free Water, 50 μL 2×Ligase buffer, 10 μL Ligase enzyme], seal with a sealing film, place on the PCR adapter in the PCR instrument, and incubate at 37°C for 1 h.

[0102] The coding chip produced in Example 1 was installed in the matching clip, with the side containing the spatial barcode oligonucleotide facing upwards and centered in the clip chamber. 200 μL of a 10 μM Linker nucleic acid fragment solution prepared with 1×Ligase buffer [Linker nucleic acid fragment sequence: 3'-AGAGCATTCGTCGTACGC-5'] was added to the chamber, evenly covering the spot array and sealed with a sealing film. The chamber was then placed on the PCR adapter in the PCR instrument and heated to 95°C, followed by cooling to 4°C at a rate of 0.2°C / s. When ready for use, the chip was removed from the instrument, carefully washed three times with 2×SSC solution, and after drying, mounted on the flip-top stage of the DynaBlot instrument described below.

[0103] After the reaction was complete, the tissue was washed three times with 200 μL of 2×SSC (containing 50% formamide) solution, incubated at 57°C for 5 min each time, and finally incubated at room temperature for 5 min with 200 μL of 2×SSC. The solution was removed, the slide was removed from the clip, and 150 μL of 10% eosin solution was added to the tissue section. The section was incubated at room temperature for 1 min, then the solution was removed, and the tissue section was washed with 1×PBS solution. Following the user manual for the DynaBlot instrument manufactured by Suzhou Deyun Kangrui Biotechnology Co., Ltd., the slide was fixed to the base stage of the DynaBlot instrument, and the coded chip was fixed to the flip-top stage of the DynaBlot instrument. Add 30 μL of permeabilization mixed enzyme solution [7 μL Nuclease-free water, 15 μL 2×Transferbuffer, 3 μL Transfer enzyme 1, 3 μL T4 Ligase enzyme, 2 μL Transfer enzyme 2] to the tissue section area. Immediately close the flip-top stage of the DynaBlot instrument, ensuring the mixed enzyme permeabilization solution is between the slide and the coded chip, but not in close contact. Start the instrument to begin the reaction. After reacting at 37°C for 60 min, remove the coded chip and carefully rinse the coded area of ​​the biochip three times with 1000 μL 2×SSC. Then, shake off the excess water and place the chip into the matching holder, ensuring the coded chip is face up and surrounded by the silicone pad of the matching holder, forming a chamber to accommodate the reaction reagents. Add 50 μL of 80 mM KOH to the chamber, seal the chamber, incubate at 95 °C for 3 min, cool, and then aspirate 48 μL of the solution into a centrifuge tube. Add 7 μL of Tris-HCl (1 M, pH = 7.0) to the tube and mix by pipetting.

[0104] Add 60 μL of 2×PCR Mix and 5 μL of PCR Primers to the centrifuge tubes, mix well by pipetting, divide into two portions and place on a PCR instrument for reaction. The reaction procedure is shown in Table 2.

[0105] Table 2

[0106]

[0107] After the reaction was completed, the PCR product was purified using 1.8× DNA purification magnetic beads, and the purified magnetic beads were eluted with 41 μL of enzyme-free water to obtain the target DNA solution.

[0108] To obtain the library DNA, the Cq value of the target DNA solution was first determined using a qPCR instrument (SYBR Green method). Then, the target DNA solution was amplified again by PCR according to the following procedure, as shown in Table 3. The PCR reaction system consisted of 10 μL of the target DNA solution, 25 μL of 2×PCR Mix, and 15 μL of Index Primers.

[0109] Table 3

[0110]

[0111]

[0112] After the reaction was completed, the PCR product was purified with 1.2× DNA purification magnetic beads and eluted with 31 μL of enzyme-free water to obtain the library DNA solution.

[0113] When the library DNA was sequenced using an Illumina next-generation sequencing instrument, and the sequencing results were analyzed using bioinformatics analysis algorithms, the statistical results of RNA expression information from the tissue sections were obtained, as shown in Table 4. Indicators such as Median_Genes_per_Spot and Reads_Mapped_to_Probe_Set suggest that the technology of this invention yields superior gene detection results, which will have significant clinical and research implications. Furthermore, the RNA expression information results can be mapped to coding chips, such as... Figure 7 As shown in the figure, cluster analysis revealed 14 cell types in the tissue section.

[0114] Table 4 Results of RNA detection in mouse brain tissue transcriptome

[0115]

[0116]

[0117] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a spatial barcode sequence for a biochip, characterized in that, Includes the following steps: Step 1) Connect silane groups containing silyl ether bonds to the biochip carrier; Step 2) Multiple parallel X-channel microchannels are established on the biochip carrier, and then DNA is synthesized in each X-channel microchannel using the phosphoramide synthesis method to form a first spatial position recognition DNA sequence at each detection point; the first spatial position recognition DNA sequence synthesized in different X-channel microchannels is different. Step 3) Multiple parallel Y-channel microchannels perpendicular to the X-channel are established on the biochip carrier. Then, DNA is synthesized in each Y-channel microchannel using the phosphoramide synthesis method. A second spatial position recognition DNA sequence is formed on the first spatial position recognition DNA sequence at each detection point. The second spatial position recognition DNA sequences synthesized in different Y-channel microchannels are different. The nodes where each parallel X channel intersects perpendicularly with multiple parallel Y channels form a detection point array on the biochip.

2. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, The biochip carrier in step one) is glass with a surface hydroxylation treatment.

3. The method for preparing a spatial barcode sequence for a biochip according to claim 2, characterized in that, The hydroxylation treatment involves reacting the glass with a piranha solution or performing plasma treatment on the glass surface.

4. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, The silane group containing the silane bond is at least one of tert-butyldimethylchlorosilane, triisopropylchlorosilane, 3-aminopropyltrimethoxysilane, and 2-hydroxyethyl(methyl(3-trimethoxysilylpropyl)amino)propionate.

5. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, The phosphorus amide synthesis method in steps three and four involves attaching a first phosphorus amide-protected nucleotide monomer to a coupling agent group; and then sequentially attaching nucleotides to the first nucleotide monomer through multiple cycles of deprotection, activation and coupling, capping, and oxidation. The deprotection process includes: treating the biochip carrier with a trichloroacetic acid-dichloromethane solution to remove the protecting group at the 5' end of the nucleotides attached to the carrier; The activation and coupling include: mixing phosphorus amide-protected nucleotide monomers with tetrazolium activator to form a phosphorus amide tetrazolium active intermediate, which then undergoes a condensation reaction with the deprotected nucleotides on the carrier, causing the nucleotide chain on the carrier to be extended by one base. The capping includes: sealing the 5' end of the nucleotide chain on the carrier with an acetylation reagent; The oxidation process involves converting phosphorous acyl into a triphosphate using a tetrahydrofuran solution of iodine to obtain a stable oligonucleotide.

6. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, In step two): firstly, phosphoramide synthesis method is used to form PCR primer sequences at each detection point, and then phosphoramide synthesis method is used to form the first spatial position recognition DNA sequence on the PCR primer sequence.

7. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, Step 3 further includes: synthesizing a unique molecular recognition tag sequence and a first fixed nucleotide sequence on the DNA sequence at the second spatial location using a phosphoramide synthesis method.

8. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, Step one) further includes designing a spatial location identification sequence so that different detection points correspond to different spatial location identification sequences, and the GC content is 44% to 72%, hairpin structures are avoided, and there are no more than 5 consecutive identical bases; the spatial location identification sequence includes the first spatial location identification DNA sequence and the second spatial location identification DNA sequence. In steps two and three, the first spatial location identification DNA sequence and the second spatial location identification DNA sequence are synthesized at each detection point according to the designed spatial location identification sequence.

9. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, Both the first spatial location identification DNA sequence and the second spatial location identification DNA sequence contain 8 nucleotides.

10. The method for preparing a spatial barcode sequence for a biochip according to claim 1, characterized in that, It also includes step four) using a protective nucleotide chain to partially complement the bases of the intact oligonucleotide chain, and then using an exonuclease to cut and remove the incomplete oligonucleotide chain; Preferably, the nucleic acid sequence of the protective nucleotide chain is complementary to the first fixed nucleotide sequence.

11. The biochip prepared by the method according to any one of claims 1 to 10, characterized in that, The biochip includes a biochip carrier, on which detection points are provided. Each detection point contains a spatial barcode oligonucleotide, which is connected to the biochip carrier via a silyl ether bond. The spatial barcode oligonucleotide includes a PCR primer sequence, a first spatial location recognition DNA sequence, a second spatial location recognition DNA sequence, a unique molecular recognition tag sequence, and a first fixed nucleotide sequence.

12. A nucleic acid spatial omics detection method, characterized in that, include: S1: The tissue slices are permeabilized and then reacted with the detection probe, allowing the detection probe to enter the tissue slices and specifically bind to the target sequence of the target nucleic acid to be tested, forming a target probe conjugate; S2: Hybridize the biochip described in claim 11 with the linker nucleic acid fragment, then attach it to a tissue slice to release the target probe conjugate from the tissue slice and link it with the spatial barcode oligonucleotide; then use a ligase to bind the target probe conjugate to the spatial barcode oligonucleotide to form a target probe template DNA. S3: Treat the biochip with an alkaline solution to detach the target probe template DNA from the biochip carrier; S4: Perform PCR on the target probe template DNA, sequence and analyze it to obtain the target nucleic acid detection results.

13. The nucleic acid spatial omics detection method according to claim 12, characterized in that, The detection probe includes a targeted capture sequence, a sequencing adapter Read 2 sequence, and a second fixed nucleotide sequence; The targeted capture sequence can specifically bind to the target sequence of the target nucleic acid to be tested in the biological sample; In step S2, the linker nucleic acid fragment binds to the first fixed nucleotide sequence and the second fixed nucleotide sequence, respectively, so that the target probe ligand is linked to the spatial barcode oligonucleotide.

14. The nucleic acid spatial omics detection method according to claim 12, characterized in that, The detection probe includes a first detection probe and a second detection probe. The first detection probe includes a sequencing adapter sequence and a first target capture sequence. The second detection probe includes a fixed nucleotide sequence and a second target capture sequence. The target capture sequence is formed by combining the first target capture sequence and the second target capture sequence; Step three also includes ligating the first target capture sequence and the second target capture sequence using a ligase.

15. The nucleic acid spatial omics detection method according to claim 14, characterized in that, In the first detection probe, the 3-end of the sequencing adapter sequence is connected to the 5-end of the first targeting capture sequence; in the second detection probe, the 3-end of the second targeting capture sequence is connected to the 5-end of the fixed nucleotide sequence.

16. The nucleic acid spatial omics detection method according to claim 12, characterized in that, The targeted probe conjugate can be released from tissue sections in at least one of the following ways: cell / nuclear lysis release, enzyme digestion release, and heat release.

17. The nucleic acid spatial omics detection method according to claim 12, characterized in that, The method of treating the biochip with an alkaline solution in step S3 includes: treating it with a 10mM to 150mM sodium hydroxide or potassium hydroxide solution at a temperature of 4℃ to 50℃ for 1 to 10 minutes.

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