Bar code array renewable chip and preparation method and application thereof

Abstract

The invention discloses a bar code array renewable chip and a preparation method and application thereof, and belongs to the technical field of gene sequencing and tissue cell sample analysis. Comprising the following steps: coupling a bar code probe to a chip substrate through a chemical reaction to obtain a bar code chip; a nucleic acid probe containing a universal primer region, a strand displacement region and a capture region is hybridized to a barcode probe of a barcode chip, and a spatial position barcode is generated through polymerization and connection, so that spatial position information is obtained; capturing to-be-detected molecules, and releasing a nucleic acid probe for library establishment through a polymeric chain displacement reaction; and carrying out aggregation and connection operation on the bar code chip again to generate a spatial position bar code, namely obtaining a bar code array reproducible chip with spatial position information, and realizing bar code regeneration.

Description

Technical Field

[0001] The present invention belongs to the field of biochip and detection technology, and in particular to a barcode array regenerative chip and a preparation method and application thereof. Background Art

[0002] Spatial omics is a cutting-edge technology that is used to analyze the omics information of biological tissues or cells at a spatial scale. At present, there are many types of spatial omics, mainly including spatial transcriptomics, spatial proteomics, and spatial metabolomics. As an emerging hot technology, spatial transcriptomics combines spatial information with gene expression data, allowing researchers to accurately see the expression location of specific genes in tissues. This method provides insights into the spatial organization of tissues at the molecular level. Spatial transcriptomics maps the location of gene expression within tissues, which is essential for understanding complex biological processes, disease mechanisms, and tissue structures. Traditional transcriptomics (such as RNA sequencing) provides data on the expression of many genes, but loses their spatial context information. Spatial transcriptomics retains this spatial context information. Therefore, spatial transcriptomics technology represents a major advancement in the field of molecular biology, providing an unprecedented perspective on the spatial dynamics of gene expression. With the continuous advancement of technology, it is expected to play a key role in personalized medicine and provide new insights into the molecular basis of health and disease.

[0003] Spatial transcriptome analysis technology is mainly based on barcode arrays with spatial positions to achieve gene capture in tissues. It is mainly divided into four parts: microdissection, fluorescence in situ hybridization, fluorescence in situ sequencing, and in situ capture. Microdissection technology mainly relies on laser to obtain tissues in different parts, but its operation is complicated and requires high instrument accuracy. Fluorescence in situ hybridization mainly relies on targeted probes for in situ identification. In this process, the information obtained from the analysis may be lost, and it relies on multiple rounds of imaging, which is complicated to operate. Although fluorescence in situ sequencing uses non-targeted probes to identify targets, it is still complicated to operate and costly. In situ capture is based on the capture of mRNA information by polyT probes. It is simple to operate and combined with sequencing data analysis, it can accurately determine the distribution of genes in tissues and realize high-throughput transcriptomics research.

[0004] At present, the spatial transcriptomics sequencing analysis technologies mainly include the following: 1) 10x Genomics Visium, as the first company to invent spatial group technology, uses polyT capture probes modified on barcode arrays, and the mRNA in the cell is paired with the probes on the chip. In situ reverse transcription makes the information in the cell correspond to the spatial group position information on the chip. 2) Slide-seq technology, in order to improve the spatial resolution, this technology uses 10 μm barcode beads as the substrate, the bead interface is modified with polyT probes, and in situ reverse transcription obtains the distribution of tissue spatial information. However, sequencing is required to determine the barcode information carried by each bead, which requires multiple rounds of in situ imaging procedures, which takes a long time and the sequencing process is complicated and cumbersome. 3) Based on the barcode beads, BGI uses DNA nanoballs (DNBs) as capture molecules to increase the resolution to 220 nm, but sequencing is required to obtain the barcode information at each position, and alkali treatment is required to become a single strand, and the production method is cumbersome and costly.

[0005] During the preparation of barcode chips, DNA barcode probes need to be at a high concentration and require specific instruments or microfluidic devices, which are complex to operate. In existing spatial transcriptome analysis methods, barcode probes will be polymerized and extended during in situ reverse transcription, making it impossible to reuse the barcodes. Therefore, developing a simple, low-cost, reproducible barcode array chip is of great significance for spatial group analysis. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a barcode array renewable chip and its preparation method and application, so as to solve the technical problems that the existing barcode chips are all disposable products, cannot be reused, and the preparation equipment is complicated.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide a barcode array regenerable chip, comprising: a chip substrate, a barcode probe, a universal primer probe, a capture probe and a strand displacement probe; wherein: The barcode probe is coupled to the chip substrate, and a position barcode region is provided on the barcode chip for determining the spatial position; The universal primer probe is used for PCR amplification; The capture probe is used to capture mRNA in the tissue; The strand displacement probe is used to hybridize with its complementary sequence, and releases a probe with a spatial position barcode, a molecular barcode and a universal primer through a polymerized strand displacement reaction.

[0008] A second object of the present invention is to provide a method for preparing the above-mentioned barcode array regenerable chip, comprising the following steps: 1) The barcode probe is coupled to the chip substrate through a chemical reaction to obtain a barcode chip; 2) Hybridizing the nucleic acid probe containing the universal primer region, the strand displacement region, and the capture region to the barcode probe of the barcode chip, and generating a spatial position barcode through polymerization and connection, that is, obtaining the spatial position information; 3) using the barcode chip processed in step 2) to capture the molecule to be detected, and releasing the nucleic acid probe for library construction through polymerase chain displacement reaction; 4) The barcode chips processed in step 3) are aggregated and connected again to generate a spatial position barcode, that is, a barcode array regenerable chip with spatial position information is obtained to achieve barcode regeneration.

[0009] Preferably, the barcode probes are spotted on the chip substrate using a spotter.

[0010] Preferably, the chip substrate is selected from a plastic sheet, a silicon chip, a glass slide or a polymethyl methacrylate sheet; the chip substrate has a length of 1-100 cm, a width of 1-100 cm, and a thickness of 0.1-10 mm.

[0011] Preferably, the surface of the chip substrate is modified by a modifying group (the chip is chemically modified and then a barcode probe with a specific group is spotted thereon), and the modifying group is usually an aldehyde group, an epoxy group, an alkynyl group, an N-hydroxysuccinimide group, a carboxyl group, a streptavidin group, a group known in the art or any combination thereof.

[0012] Further preferably, the nucleic acid probe is linked to the modification group via a specific group on the nucleic acid molecule; the specific group includes an amino group, a thiol group, an epoxy group or other methods known in the art or any combination thereof.

[0013] Preferably, the barcode probe is a single probe; it can be generated by a split-pool method, or other materials known in the art or any combination thereof.

[0014] The core idea of ​​the "Split-pool method" described in the present invention is to divide the sample or reactant into multiple parts (split), perform different treatments or labels on each part, and then merge the parts (pool) for the next round of processing or analysis. In molecular biology, the split-pool method is often used for labeling and sequencing of DNA, RNA and proteins.

[0015] Further preferably, the barcode probe comprises a molecular barcode region, a PCR amplification primer region, a strand displacement region and a capture probe region; Furthermore, the spatial position barcode between the universal primer probe and the capture probe is completed by DNA polymerase and ligase or other methods known in the art or any combination thereof.

[0016] The third object of the present invention is to provide the application of the above-mentioned barcode array regenerable chip in spatial omics analysis, including: The tissue sections are attached to the barcode array regenerable chip, and then reverse transcription is performed in situ to reverse transcribe the mRNA into cDNA; the tissue is then removed, and the cDNA sequence with the barcode is bound to the chip, and then a polymerase chain displacement reaction is performed to complete the spatial encoding of the transcriptome in the tissue cells to obtain a library.

[0017] Preferably, the thickness of the tissue slice is 0.1-1000 μm; and the tissue is permeabilized using a surfactant before use.

[0018] Preferably, tissue permeabilization uses a surfactant such as pepsin, trionX-100, NP-40, etc., preferably pepsin.

[0019] Preferably, the reaction temperature of the in situ reverse transcription treatment is 37-56°C; more preferably 42°C; The reaction time is 1-24 h; more preferably 6 h.

[0020] Preferably, the reagent used to remove tissue includes proteinase K or other methods known in the art or any other combination thereof.

[0021] The invention also discloses the application of the above-mentioned barcode array regenerable chip in preparing a DNA library.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of the renewable barcode array chip disclosed in the present invention enables the barcode chip to be reused through DNA probe hybridization, polymerization extension, and polymer chain displacement, and can realize the spatial distribution of genetic information in tissue cells. Specifically, the present application hybridizes a nucleic acid probe comprising a universal primer region, a chain displacement region, and a capture region to a barcode probe, and obtains a capture probe with spatial position information through polymerization and connection; releases the nucleic acid probe for library construction and detection through polymer chain displacement, and regains the chip with the barcode probe; uses polymer connection again to obtain a capture probe with spatial position information, thereby realizing the regeneration of the barcode. Therefore, the method of the present invention can effectively solve the problem that the prior art mainly captures mRNA information in tissue cells based on the polyT probe on the chip, and the in situ reverse transcription allows the barcode probe to be extended, so it cannot be reused. The present invention is low in cost (only requires the use of a spotting machine, and no additional microfluidic device needs to be built), simple to operate, and has a low threshold. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the first barcode array regenerable chip for spatial transcriptome sequencing; Figure 2 Schematic diagram of the second type of barcode array regenerable chip used for spatial transcriptome sequencing; Figure 3 Schematic diagram of the third type of barcode array regenerable chip used for spatial transcriptome sequencing; Figure 4 Schematic diagram of the principle of verifying the reproducibility of the standard chain; Figure 5 This is a schematic diagram of the structure of a library containing spatial tags in an embodiment of the present invention; Figure 6 Data analysis diagram for verifying the reproducibility of the standard chain; Figure 7 Gray value map for verifying reproducibility based on tissue sections; Figure 8 This is the cDNA quality control data diagram; Fig. 9 It is the size distribution diagram of DNA sequencing library; Fig.10 A UMI and Gene data diagram of a spatial position barcode. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0025] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0026] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0027] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0028] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The present invention discloses a barcode array regenerable chip, comprising: a chip substrate, a barcode probe, a universal primer probe, a capture probe and a strand displacement probe; wherein: The barcode probe is coupled to the chip substrate, and a position barcode region is provided on the barcode probe for determining the spatial position; The universal primer probe is used for PCR amplification; The capture probe is used to capture mRNA in the tissue; The strand displacement probe is used to hybridize with its complementary sequence, and releases a probe with a spatial position barcode, a molecular barcode and a universal primer through a polymerized strand displacement reaction.

[0031] The "position barcode region of the barcode probe" described above is a specific sequence or identifier on the barcode probe, which encodes the exact position information of the probe in the detection platform or array. The position barcode region can ensure the accuracy of the data. Because each probe has a unique position identifier, data errors caused by position confusion can be avoided.

[0032] The "universal primer probe" described above is a primer probe combination designed for the detection of multiple target nucleic acid sequences. They usually have a wide range of applications and high specificity and can be reused under different experimental conditions. On barcode renewable chips, universal primer probes are used to capture and amplify target nucleic acid sequences, thereby achieving rapid and accurate detection of samples. The design of universal primer probes on barcode renewable chips (usually refers to reusable chips used for biological detection or analysis) is a complex and delicate process aimed at improving the sensitivity and specificity of detection while ensuring the repeatability and reliability of the chip.

[0033] The "capture probe" described above is a key component in barcode array chip technology, which is used to specifically identify and capture target molecules (such as DNA, RNA or protein, etc.). In the barcode regenerative chip, the capture probe is fixed on the chip surface to capture the target molecule in the sample flowing through the chip. When the target molecule binds to the capture probe, a stable complex is formed, thereby achieving the separation and enrichment of the target molecule.

[0034] The "strand displacement probe" described above is a probe designed based on the principle of strand displacement reaction, which is used to achieve high-sensitivity and high-specificity detection of target molecules in biological detection and analysis. In the barcode regenerative chip, the strand displacement probe is used to capture and detect the target nucleic acid sequence. When the target nucleic acid sequence flows through the surface of the chip, it will be captured by the capture probe fixed on the surface of the chip. Subsequently, the strand displacement probe specifically hybridizes with the target nucleic acid sequence through the toehold region, triggering the strand displacement reaction, causing the target nucleic acid sequence to dissociate from the capture probe and form a stable complex with the strand displacement probe. This complex can then be detected and analyzed, thereby achieving high-sensitivity and high-specificity detection of the target nucleic acid sequence.

[0035] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a method for preparing a barcode array regenerable chip, such as Figure 1 As shown, the steps are as follows: 1) Design a barcode array reproducible chip to identify probe sequences of mRNA in tissue cells, including: A barcode probe (i.e., probe 1) is designed for chip modification. The 3' end of probe 1 has an amino group, which is used to bind to the aldehyde chip. From the 5' end of probe 1, 15 bases are used as a hybridization region (hybird 1) for hybridization probe 3, 19 bases are used as a molecular barcode region, 33 bases are used for hybridization probe 2, and 10 bases are used as a spacer. The chip modification includes but is not limited to aldehyde and carboxyl groups; Design a universal primer probe R1 sequence (i.e., probe 2) for PCR amplification, wherein probe 2 consists of a 33-base universal primer probe R1 sequence for PCR amplification; A capture probe sequence (i.e., probe 3) is designed for capturing mRNA. The 5' end of probe 3 is phosphate-modified and the 3' end is thio-modified. Probe 3, starting from the 5' end, is composed of 15 bases of hybridization probe 1, 15 bases of hybridization polymer chain displacement probe 4 (Hybrid 2), 10 bases of a unique molecular identifier (UMI) for molecular counting, and 30 bases of hybridization mRNA sequence (Oligo-dT), a total of four parts; Design a probe sequence for polymerase chain displacement (i.e., probe 4), which consists of 15 bases and is used to hybridize with probe 3; Design an oligo-dA probe sequence (i.e., probe 5) for standard chain verification. Probe 5 consists of two parts: a 20-base polyA at the 3' end and a 15-base universal primer probe R2 sequence at the 5' end for PCR amplification; Design a probe sequence for template switching in tissue sections (i.e., probe 6), which consists of two parts: an R2 sequence at the 5' end for PCR amplification and an LNA modification at the 3' end; 2) Probe 1 at a final concentration of 10 μM is incubated with the functionalized chip, and probes 2 and 3 are incubated with the chip modified with probe 1 for later use.

[0036] 3) Generate spatial position barcodes using DNA polymerase and DNA ligase to form a complete capture probe for standard chain and tissue section verification.

[0037] 4) If Figure 4 As shown, the barcode chip functionally modified in step 3) is used for standard chain verification. In the presence of probe 5, probe 3 will bind to it and stably hybridize. In the presence of DNA polymerase, the probe in step 3) will dissociate from the barcode chip, allowing it to be regenerated and used. The dissociated probe is amplified by PCR, and the size of its electrophoresis band is verified by agarose gel electrophoresis. After completing the first round of verification, continue to repeat steps 2) to 4) to complete the verification of the regenerative ability of the barcode chip.

[0038] like Figure 1 As shown, the barcode chip that has been functionally modified in step 3) is used for tissue section verification. During the permeabilization process of the tissue section, the probe 5 sequence on the chip will capture the mRNA molecule, and during the in situ reverse transcription, the chip will capture the genetic information in the tissue cells. In the presence of probe 4, probe 3 will bind to it and stably hybridize with it. In the presence of DNA polymerase, the probe in step 3) will dissociate from the barcode chip, allowing it to be regenerated and used.

[0039] The dissociated probe is subjected to PCR amplification, such as Figure 5 As shown, library construction and sequencing are used to analyze the spatial distribution of genetic information within tissue cells.

[0040] also, Figure 2 and Figure 3 The figure schematically shows two other implementations of the preparation method and application of a barcode array regenerable chip according to the core innovative idea of ​​the present invention, which realize the analysis of spatial transcription through barcode beads or RCA barcode balls.

[0041] The present invention will be further described below in conjunction with a specific embodiment. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0042] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" represents mass percentage and ratio represents mass ratio.

[0043] 1. Design of nucleic acid probe molecules and their simulation In order to realize the use of barcode array regenerable chips for spatial group analysis, corresponding nucleic acid probes were designed.

[0044] In the process of designing probe 1, according to the characteristics of rapid coupling between aldehyde and amino groups, the 3' amino group was combined with the aldehyde chip; in order to reduce steric hindrance, a spacer was designed between the chip and the functional area; 29 bases were designed for hybridization probe 2; 19 bases were designed as barcode regions for the determination of spatial position; and 15 bases were designed for hybridization probe 3. The sequence of probe 1 is shown in SEQ ID NO.1 in Table 1.

[0045] In the process of designing probe 2, the R1 region is designed according to the primers required in the PCR amplification process; the sequence of probe 2 is shown in SEQ ID NO.2 in Table 1.

[0046] In the process of designing probe 3, the 5' end is designed to be phosphate-modified to enable probe connection, the other end is designed as a capture region for hybridization with mRNA, and the 3' end is designed to be thio-modified to prevent enzyme cleavage; 10 bases are designed as UMI; and 15 bases are designed for hybridization probe 4. The sequence of probe 3 is shown in SEQ ID NO.3 in Table 1.

[0047] In the process of designing probe 4, 15 bases were designed as stable hybridization. The sequence of probe 4 is shown in SEQ ID NO. 4 in Table 1.

[0048] In the process of designing probe 5, the R2 region is designed according to the primers required in the PCR amplification process, and the polyA sequence is designed for hybridization probe 3. The sequence of probe 5 is shown in SEQ ID NO. 5 in Table 1.

[0049] In the process of designing probe 6, the R2 region is designed according to the primers required in the PCR amplification process. Probe 6 is a probe for template switching in tissue sections, and an LNA sequence is designed at the 3' end. The sequence of probe 6 is shown in SEQ ID NO. 6 in Table 1.

[0050] Table 1 Sequence Listing

[0051] In the probe 3 in the table, N represents a random base, any one of A / T / C / G, and V represents three bases.

[0052] The probes designed above were simulated using NUPACK software. The simulation results showed that none of the six probe simulations affected the formation of secondary structures that would affect hybridization. Meanwhile, the pairing hybridization efficiency of probe 1, probe 2, and probe 3 was high. The pairing hybridization efficiency of probe 3 with probes 4 and 5 was high.

[0053] Probe 1, probe 2, probe 3, probe 4, probe 5, and probe 6 in Table 1 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and used in the following experiments.

[0054] 2. Preparation of barcode array chip modified with probe 1, probe 2 and probe 3: The probe 1 at a concentration of 10 μM was combined with the aldehyde-based glass slide using a spotter and incubated overnight at room temperature. 0.25% NaBH4 was reacted with the chip at room temperature for 1 h to block the unreacted sites, and the chip surface was washed with 2×SSC solution (containing 0.1% SDS) to remove non-specifically adsorbed DNA. The final modified area of ​​DNA was 8×8 mm 2. 20 μL of 10 μM probes 2 and 3 were hybridized with probe 1 at 37°C for 30 min. The chip surface was washed with 2×SSC solution (containing 0.1% SDS) to remove unreacted and nonspecifically adsorbed DNA. Next, DNA ligase and DNA polymerase were incubated with the chip at 37°C for 2 h for gap-filling to connect probes 1 and 3 for reproducible use of the barcode slide.

[0055] 3. Barcode array reproducible chip for standard chain verification The probe 5 with a concentration of 1 μM was reacted with the barcode chip prepared in step 2. After reacting at 37°C for 2 h, the chip surface was washed with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. Finally, a standard chain with a barcode was obtained by DNA polymerase chain displacement, so that the chip can be reused. PCR amplification of the above products was repeated, such as Figure 6 As shown, 20 rounds of regeneration experiments were performed, and the gray value of agarose gel electrophoresis and the ratio of the number of barcode types in each round characterized its regeneration ability.

[0056] 4. Barcode array reproducible chips for tissue section verification Fresh brain tissue of mice was taken, embedded with OCT embedding reagent, quickly frozen in liquid nitrogen, and then placed in a -80℃ refrigerator for long-term storage. The tissue block stored at -80℃ was placed in a -20℃ cryostat and pre-equilibrated for 30min. The tissue block was sliced ​​using a cryostat with a rough thickness of 100 μm. After the rough adjustment to the tissue part, micro-cutting was performed to adjust the thickness to 10 μm, and attached to the barcode chip prepared in step 2. It was then placed at 37℃ for 1 min for drying, fixed with 4% paraformaldehyde, and the residual paraformaldehyde was washed away with 1×PBS. The tissue was permeabilized with a pre-permeabilization reagent and a permeabilization reagent, and washed with a 0.1×SSC solution. The reverse transcription reagent was then added for in situ reverse transcription, reacted at 42℃ for 6h, and the mRNA was reverse transcribed into cDNA. After completion, 0.1×SSC solution was used for washing. Tissue removal solution was added, and the cDNA sequence with the barcode was bound to the chip. Probe 4 at a concentration of 10 μM was added to hybridize with probe 3. Finally, the cDNA sequence is obtained by DNA polymerase chain displacement and PCR amplification. Figure 7 As shown, ten rounds of reproducibility verification were performed, PCR real-time curve and agarose gel electrophoresis were used to characterize its reproducibility, and second-generation sequencing was performed to determine its spatial location information. Finally, the library was obtained as shown in Figure 8 , 9 As shown in the figure, the cDNA library fragments are concentrated in the range of 500-2000 bp, and the sequenced DNA library fragments are concentrated around 400 bp, with no interference from other bands. Fig.10As shown, the sequencing results indicate that the UMI and Gene detected by the renewable barcode slide are 4245 and 10309.

[0057] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A barcode array regenerative chip, characterized in that: include: Chip substrate, barcode probe, universal primer probe, capture probe and strand displacement probe; wherein: The barcode probe is coupled to the chip substrate, and a position barcode region is provided on the barcode probe for determining the spatial position; The universal primer probe is used for PCR amplification; The capture probe is used to capture mRNA in the tissue; The strand displacement probe is used to hybridize with its complementary sequence, and releases a probe with a spatial position barcode, a molecular barcode and a universal primer through a polymerized strand displacement reaction.

2. The barcode array regenerative chip according to claim 1, characterized in that: The chip substrate is selected from a plastic sheet, a silicon chip, a glass slide or a polymethyl methacrylate sheet; the chip substrate has a length of 1-100 cm, a width of 1-100 cm and a thickness of 0.1-10 mm.

3. The barcode array regenerative chip according to claim 1, characterized in that: The surface of the chip substrate is modified by a modifying group, and the modifying group includes any one or more of an aldehyde group, an epoxy group, an alkynyl group, an N-hydroxysuccinimide group, a carboxyl group and a streptavidin group.

4. The barcode array regenerative chip according to claim 3, characterized in that: The nucleic acid probe is linked to the modification group via a specific group on the nucleic acid molecule; the specific group includes an amino group, a thiol group or an epoxy group.

5. The barcode array regenerative chip according to claim 1, characterized in that: The barcode probe is a single probe.

6. The method for preparing the barcode array regenerable chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) The barcode probe is coupled to the chip substrate through a chemical reaction to obtain a barcode chip; 2) Hybridizing the nucleic acid probe containing the universal primer region, the strand displacement region, and the capture region to the barcode probe of the barcode chip, and generating a spatial position barcode through polymerization and connection, that is, obtaining the spatial position information; 3) using the barcode chip processed in step 2) to capture the molecule to be detected, and releasing the nucleic acid probe for library construction through polymerase chain displacement reaction; 4) The barcode chips processed in step 3) are aggregated and connected again to generate a spatial position barcode, that is, a barcode array regenerable chip with spatial position information is obtained to achieve barcode regeneration.

7. Use of the barcode array regenerable chip according to any one of claims 1 to 5 in spatial omics analysis, characterized in that: include: attaching the tissue slice to the barcode array regenerable chip, and then performing in situ reverse transcription to reverse transcribe the mRNA into cDNA; The tissue is then removed, allowing the cDNA sequence with the barcode to bind to the chip, and then a polymerase chain displacement reaction is performed to complete the spatial encoding of the transcriptome in the tissue cells to obtain a library.

8. The use according to claim 7, characterized in that The thickness of the tissue slice is 0.1-1000 μm; a surfactant is used to perform tissue permeabilization treatment before use.

9. The use according to claim 7, characterized in that: The reaction temperature of in situ reverse transcription treatment is 37-56°C and the reaction time is 1-24 h.

10. Use of the barcode array regenerable chip according to claim 6 in preparing a DNA library.

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