A barcode array regenerable chip, its fabrication method and application

Abstract

The application discloses a bar code array regenerable chip and a preparation method and application thereof, and belongs to the technical field of gene sequencing and tissue cell sample analysis. The method comprises the following steps: coupling bar code probes to a chip substrate through a chemical reaction to obtain a bar code chip; hybridizing nucleic acid probes containing a universal primer region, a strand displacement region and a capture region to the bar code probes on the bar code chip; generating spatial position bar codes through polymerization and connection, so that spatial position information is obtained; capturing to-be-detected molecules, and releasing nucleic acid probes for library construction through a polymerization strand displacement reaction; performing polymerization and connection operations on the bar code chip again, generating spatial position bar codes, so that the bar code array regenerable chip with spatial position information is obtained, and bar code regeneration is realized.

Description

Technical Field

[0001] This invention belongs to the field of biochip and its detection technology, and specifically relates to a barcode array regenerable chip, its preparation method and application. Background Technology

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

[0003] Spatial transcriptomics analysis primarily relies on barcode arrays with spatial locations to capture genes within tissues. It mainly consists of four parts: microdissection, fluorescence in situ hybridization (FISH), fluorescence in situ sequencing (FISH), and in situ capture. Microdissection mainly uses lasers to acquire tissue samples from different sites, but it is complex and requires high instrument precision. FISH primarily uses targeted probes for in situ identification, which may lead to missing information and requires multiple imaging rounds, making it complex. While FISH utilizes non-target probes to identify target molecules, it remains complex and costly. In situ capture uses polyT probes to capture mRNA information; it is simple to operate, and combined with sequencing data analysis, it can accurately determine gene distribution within tissues, enabling high-throughput transcriptomics research.

[0004] Currently, spatial transcriptomics sequencing analysis technologies mainly include the following: 1) 10x Genomics Visium, as the company that first invented spatial genome technology, uses barcode arrays modified with polyT capture probes. Intracellular mRNA pairs with probes on the chip, and in situ reverse transcription allows intracellular information to correspond to spatial genome location information on the chip. 2) Slide-seq technology, to improve spatial resolution, uses 10 μm barcode beads as a substrate. The bead interface is modified with polyT probes, and in situ reverse transcription is used to obtain the spatial distribution of tissue information. However, sequencing is required to determine the barcode information carried by each bead, requiring multiple rounds of in situ imaging, which is time-consuming and complex. 3) BGI Genomics, based on barcode beads, uses DNA nanoballs (DNB) as capture molecules, improving the resolution to 220 nm. However, sequencing is required to obtain the barcode information at each location, and converting to single strands requires alkali treatment, making the fabrication method cumbersome and costly.

[0005] In the fabrication of barcode chips, DNA barcode probes require high concentrations and necessitate specific instruments or microfluidic devices, making the process complex. In existing spatial transcriptomics analysis methods, barcode probes are polymerized and extended during in situ reverse transcription, preventing their reuse. Therefore, developing a simple, low-cost, regenerable barcode array is of great significance for spatial genome analysis. Summary of the Invention

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

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] One objective of this invention is to provide a barcode array regenerable chip, comprising: a chip substrate, barcode probes, universal primer probes, capture probes, and chain displacement probes; wherein:

[0009] The barcode probe is coupled to the chip substrate, and the barcode chip has a position barcode area for determining spatial location;

[0010] The universal primers and probes are used for PCR amplification;

[0011] The capture probe is used to capture mRNA within the tissue;

[0012] The chain displacement probe is used to hybridize with its complementary sequence, releasing a probe with spatial location barcode, molecular barcode and universal primer through a polymer chain displacement reaction.

[0013] A second objective of this invention is to provide a method for fabricating the aforementioned barcode array regenerable chip, comprising the following steps:

[0014] 1) The barcode probe is coupled to the chip substrate through a chemical reaction to obtain a barcode chip;

[0015] 2) Hybridize nucleic acid probes containing universal primer regions, strand substitution regions, and capture regions onto barcode probes of barcode chips, and generate spatial location barcodes through aggregation and connection, thereby obtaining spatial location information;

[0016] 3) The barcode chip processed in step 2) is used to capture the molecules to be detected, and the nucleic acid probes for library construction are released through a polymerase chain displacement reaction;

[0017] 4) Perform aggregation and connection operations on the barcode chip processed in step 3) again to generate a spatial location barcode, that is, obtain a barcode array regenerable chip with spatial location information, and realize barcode regeneration.

[0018] Preferably, a barcode probe is applied to the chip substrate using a dotting machine.

[0019] Preferably, the chip substrate is selected from plastic sheet, silicon chip, glass slide or polymethyl methacrylate sheet; the length of the chip substrate is 1-100 cm, the width is 1-100 cm, and the thickness is 0.1-10 mm.

[0020] Preferably, the chip substrate surface is modified by a modifying group (the chip is chemically modified and then a barcode probe with a specific group is spotted onto it). The modifying group is usually an aldehyde group, an epoxy group, an alkynyl group, an N-hydroxysuccinimide group, a carboxyl group, a streptavidin group, or any combination thereof.

[0021] More preferably, the nucleic acid probe is linked to a modifying group via a specific group on the nucleic acid molecule; the specific group includes amino, thiol, epoxy, or other known methods in the art, or any combination thereof.

[0022] Preferably, the barcode probe is a single probe; it can be generated by the split-pool method, or by other materials known in the art or any combination thereof.

[0023] The core idea of ​​the "Split-pool method" described in this invention is to divide a sample or reactant into multiple parts (split), perform different treatments or labels on each part, and then pool these parts (pool) for the next round of processing or analysis. In molecular biology, the split-pool method is commonly used for labeling and sequencing DNA, RNA, and proteins.

[0024] More preferably, the barcode probe includes a molecular barcode region, a PCR amplification primer region, a strand displacement region, and a capture probe region;

[0025] Furthermore, the spatial location barcode between the universal primer probe and the capture probe is completed by DNA polymerase and ligase or by other methods known in the art or any combination thereof.

[0026] A third objective of this invention is to provide the application of the aforementioned barcode array regenerable chip in spatial omics analysis, including:

[0027] Tissue slices are attached to the barcode array regenerable chip and then subjected to in situ reverse transcription to convert mRNA into cDNA. Subsequently, the tissue is removed, allowing the cDNA sequence with barcodes to bind to the chip. After a polymerase chain displacement reaction, the transcriptome in the tissue cells is spatially encoded to obtain a library.

[0028] Preferably, the thickness of the tissue section is 0.1-1000 μm; the tissue is permeable with a surfactant before use.

[0029] Preferably, the tissue permeation treatment uses a surfactant such as pepsin, trionX-100, or NP-40, with pepsin being the most preferred.

[0030] Preferably, the in situ reverse transcription treatment reaction temperature is 37-56℃; more preferably 42℃;

[0031] The reaction time is 1-24 h; more preferably 6 h.

[0032] Preferably, the reagents used to remove tissue include proteinase K or other methods known in the art or any other combination thereof.

[0033] The present invention also discloses the application of the above-mentioned barcode array regenerable chip in the preparation of DNA libraries.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention discloses a method for preparing a reusable barcode array chip. Through DNA probe hybridization, polymerization extension, and polymer chain replacement, the barcode chip can be reused, enabling the spatial distribution of gene information within tissue cells. Specifically, this application hybridizes a nucleic acid probe containing a universal primer region, a chain replacement region, and a capture region to a barcode probe. Through polymerization and ligation, a capture probe with spatial location information is obtained. Polymer chain replacement releases the nucleic acid probe used for library construction and detection, and a chip with barcode probes is re-obtained. Polymerization and ligation are used again to obtain capture probes with spatial location information, achieving barcode regeneration. Therefore, this invention effectively solves the problem that existing technologies mainly rely on polyT probes on chips to capture mRNA information within tissue cells, and in situ reverse transcription extends the barcode probes, making them unusable. This invention is low-cost (requiring only a sample applicator, without the need for additional microfluidic devices), simple to operate, and has a low barrier to entry. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the first type of barcode array regenerative chip used for space transcriptome sequencing.

[0037] Figure 2 Schematic diagram of the second type of barcode array regenerative chip used for space transcriptome sequencing;

[0038] Figure 3 Schematic diagram of the third type of barcode array regenerative chip used for space transcriptome sequencing;

[0039] Figure 4 A schematic diagram illustrating the principle of verifying renewability in a standard chain;

[0040] Figure 5 This is a schematic diagram of a library structure with spatial tags in an embodiment of the present invention;

[0041] Figure 6 Data analysis diagram for verifying the reproducibility of the standard chain;

[0042] Figure 7 This is a grayscale image used to verify regeneration based on tissue sections;

[0043] Figure 8 This is a graph of cDNA quality control data;

[0044] Figure 9 This is a distribution map of DNA sequencing library sizes.

[0045] Figure 10 This is a UMI and Gene data diagram of a spatial location barcode. Detailed Implementation

[0046] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0047] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0048] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0049] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0050] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0051] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0052] This invention discloses a barcode array regenerable chip, comprising: a chip substrate, barcode probes, universal primer probes, capture probes, and chain displacement probes; wherein:

[0053] The barcode probe is coupled to the chip substrate, and the barcode probe has a position barcode area for determining spatial location;

[0054] The universal primers and probes are used for PCR amplification;

[0055] The capture probe is used to capture mRNA within the tissue;

[0056] The chain displacement probe is used to hybridize with its complementary sequence, releasing a probe with spatial location barcode, molecular barcode and universal primer through a polymer chain displacement reaction.

[0057] The "position barcode area" described above is a specific sequence or identifier on the barcode probe that encodes the probe's exact location information within the detection platform or array. This position barcode area ensures data accuracy. Because each probe has a unique location identifier, data errors caused by location confusion can be avoided.

[0058] The "universal primer probes" described above are primer probe combinations designed for the detection of multiple target nucleic acid sequences. They typically have a wide applicability and high specificity, and can be reused under different experimental conditions. On barcode-reproducible chips, universal primer probes are used to capture and amplify target nucleic acid sequences, thereby enabling rapid and accurate detection of samples. The design of universal primer probes on barcode-reproducible chips (generally referring to reusable chips used for biological detection or analysis) is a complex and meticulous process aimed at improving the sensitivity and specificity of detection while ensuring the reproducibility and reliability of the chip.

[0059] The "capture probes" described above are key components in barcode array chip technology, used to specifically identify and capture target molecules (such as DNA, RNA, or proteins). In barcode regenerable chips, capture probes are immobilized on the chip surface to capture target molecules in samples 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.

[0060] The "chain displacement probe" described above is a probe designed based on the principle of chain displacement reaction, used to achieve highly sensitive and specific detection of target molecules in biological detection and analysis. In barcode regenerable chips, chain displacement probes are used to capture and detect target nucleic acid sequences. When the target nucleic acid sequence flows across the chip surface, it is captured by the capture probe immobilized on the chip surface. Subsequently, the chain displacement probe specifically hybridizes with the target nucleic acid sequence through its anchorage region, triggering a chain displacement reaction, causing the target nucleic acid sequence to dissociate from the capture probe and form a stable complex with the chain displacement probe. This complex can then be detected and analyzed, thereby achieving highly sensitive and specific detection of the target nucleic acid sequence.

[0061] The present invention will now be described in further detail with reference to the accompanying drawings:

[0062] This invention provides a method for fabricating a barcode array regenerable chip, such as... Figure 1 As shown, the steps are as follows:

[0063] 1) Design a barcode array regenerable chip for identifying probe sequences of mRNA in tissue cells, including:

[0064] A barcode probe (i.e., probe 1) is designed for chip modification. Probe 1 has an amino group at its 3' end, which is used to bind aldehyde groups to the chip. Starting from its 5' end, probe 1 has 15 bases as a hybridization region (hybrid 1) for hybridizing probe 3, 19 bases as a molecular barcode region, 33 bases as a hybridization probe 2, and 10 bases as a spacer region. The chip modification includes, but is not limited to, aldehyde and carboxyl groups.

[0065] A universal primer probe R1 sequence (i.e. probe 2) for PCR amplification was designed. Probe 2 consists of a 33-base universal primer probe R1 sequence for PCR amplification.

[0066] The capture probe sequence designed to capture mRNA (i.e., probe 3) consists of four parts: a 5' phosphate-modified probe and a 3' thio-modified probe. Starting from the 5' end, probe 3 is composed of four parts: a 15-base hybridization probe 1, a 15-base hybridization polymerase chain substitution probe 4 (Hybrid 2), a 10-base unique molecular identifier (UMI) for molecule counting, and a 30-base hybridization mRNA sequence (Oligo-dT).

[0067] A probe sequence (i.e., probe 4) was designed for polymer chain substitution. Probe 4 consists of 15 bases and is used to hybridize probe 3.

[0068] The oligo-dA probe sequence (i.e. probe 5) designed for standard chain validation consists of two parts: a 20-base polyA sequence at the 3' end and a 15-base universal primer probe R2 sequence at the 5' end for PCR amplification.

[0069] The probe sequence designed for template conversion in tissue sections (i.e., probe 6) consists of two parts: an R2 sequence at the 5' end for PCR amplification and an LNA modification at the 3' end.

[0070] 2) Incubate probe 1 with the functionalized chip at a final concentration of 10 μM, and incubate probe 2 and probe 3 with the chip modified with probe 1 for later use.

[0071] 3) Use DNA polymerase and DNA ligase to generate spatial location barcodes, forming a complete capture probe for verification with standard strands and tissue sections.

[0072] 4) such as Figure 4As shown, the barcode chip functionalized in step 3) is used for standard chain verification. In the presence of probe 5, probe 3 binds to and stably hybridizes with it. In the presence of DNA polymerase, the probe from step 3) dissociates from the barcode chip, allowing it to be regenerated and reused. The dissociated probe is subjected to PCR amplification, and the size of the electrophoretic bands is verified by agarose gel electrophoresis. After completing the first round of verification, steps 2) to 4) are repeated to verify the regenerability of the barcode chip.

[0073] like Figure 1 As shown, the barcode chip functionalized in step 3) is used for tissue section verification. During the permeabilization process of the tissue section, probe 5 on the chip captures mRNA molecules. During in situ reverse transcription, the chip captures the genetic information within the tissue cells. In the presence of probe 4, probe 3 binds to it and hybridizes stably. In the presence of DNA polymerase, the probe from step 3) dissociates from the barcode chip, allowing it to be regenerated and reused.

[0074] The probe obtained from dissociation was 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.

[0075] also, Figure 2 and Figure 3 The illustrations schematically demonstrate two other embodiments of the preparation method and application of a barcode array regenerable chip based on the core innovative idea of ​​the present invention, which realizes the analysis of spatial transcription through a barcode bead or an RCA barcode sphere.

[0076] The present invention will be further illustrated below with reference to a specific embodiment. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0077] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" represents a percentage by mass, and "ratio" represents a mass ratio.

[0078] 1. Design of nucleic acid probe molecules and their simulation

[0079] To enable the use of barcode array regenerable chips for spatial group analysis, corresponding nucleic acid probes were designed.

[0080] In the design of probe 1, based on the rapid coupling properties of aldehyde and amino groups, the 3'-amino group is combined with the aldehyde chip. To reduce steric hindrance, a spacer region is designed between the chip and the functional region. 29 bases are designed for hybridization probe 2; 19 bases are designed as a barcode region for spatial positioning; and 15 bases are designed for hybridization probe 3. The sequence of probe 1 is shown in SEQ ID NO.1 in Table 1.

[0081] In the design of probe 2, the R1 region was designed based on the primers required for PCR amplification; the sequence of probe 2 is shown in SEQ ID NO.2 in Table 1.

[0082] In the design of probe 3, the 5' end was phosphate-modified to enable probe ligation, and the other end was designed as a capture region for hybridization with mRNA. To prevent enzyme cleavage, the 3' end was thio-modified. Ten bases were designed as the UMI, and 15 bases were designed for hybridization probe 4. The sequence of probe 3 is shown in SEQ ID NO.3 in Table 1.

[0083] In designing probe 4, 15 bases were designed for stable hybridization. The sequence of probe 4 is shown in SEQ ID NO.4 in Table 1.

[0084] In designing probe 5, based on the primers required for PCR amplification, the R2 region was designed as hybridization probe 3, and a polyA sequence was designed. The sequence of probe 5 is shown in SEQ ID NO.5 in Table 1.

[0085] In designing probe 6, the R2 region was designed based on the primers required for PCR amplification. Probe 6 is a template conversion probe in tissue sections, and an LNA sequence was designed at the 3' end. The sequence of probe 6 is shown in SEQ ID NO.6 in Table 1.

[0086] Table 1 Sequence List

[0087]

[0088] In the table, N in probe 3 represents a random base, any one of A / T / C / G, and V represents three bases.

[0089] The designed probes were simulated using NUPACK software. The simulation results showed that none of the six probes affected the formation of secondary structures during hybridization. Furthermore, probes 1, 2, and 3 exhibited high hybridization efficiency. Probe 3 also showed high hybridization efficiency with probes 4 and 5.

[0090] Probes 1, 2, 3, 4, 5, and 6 in Table 1 above were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and used in the following experiments.

[0091] 2. Fabrication of a barcode array chip modified with probe 1, probe 2, and probe 3:

[0092] The probe 1 at a concentration of 10 μM was attached to an aldehyde-based glass slide using a spotting device and incubated overnight at room temperature. 0.25% NaBH4 was then reacted with the chip at room temperature for 1 h to block unreacted sites. The chip surface was then washed with 2×SSC solution (containing 0.1% SDS) to remove non-specifically adsorbed DNA. The final DNA-modified area 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 then washed with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. Next, DNA ligase and DNA polymerase were incubated with the chip at 37°C for 2 h for gap-filling, ligating probes 1 and 3 for reusable barcode slides.

[0093] 3. Barcode array regenerable chips are used for standard chain verification.

[0094] The 1 μM probe 5 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 strand with the barcode was obtained by DNA polymerase chain displacement, making the chip reusable. The above product was amplified by PCR, and this process was repeated as follows. Figure 6 As shown, 20 rounds of regenerability experiments were conducted, and the gray value of agarose gel electrophoresis and the proportion of barcode types in each round characterized its regenerability.

[0095] 4. Barcode array regenerable chips are used for tissue slide verification.

[0096] Fresh mouse brain tissue was embedded using OCT embedding reagent, rapidly frozen in liquid nitrogen, and then stored at -80°C for long-term preservation. The tissue blocks stored at -80°C were placed in a -20°C cryostat and pre-equilibrated for 30 min. Sections were prepared using the cryostat, with a coarse section thickness of 100 μm. After coarse sectioning to the tissue portion, microsections were performed to a thickness of 10 μm, and the sections were attached to the barcode chip prepared in step 2. The sections were then dried at 37°C for 1 min, fixed with 4% paraformaldehyde, and washed with 1×PBS to remove residual paraformaldehyde. The tissue was permeabilized using pre-permeabilization and permeabilization reagents, and washed with 0.1×SSC solution. In situ reverse transcription was then performed using reverse transcription reagent at 42°C for 6 h, converting mRNA to cDNA. After completion, the tissue was washed with 0.1×SSC solution. Tissue removal solution was added, and the barcode-bearing cDNA sequence bound to the chip. Probe 4 at a concentration of 10 μM was added to hybridize with probe 3. Finally, the cDNA sequence was obtained through DNA polymerase chain displacement, followed by PCR amplification. This process was repeated, as follows: Figure 7 As shown, ten rounds of regeneration capability verification were performed. PCR real-time curves and agarose gel electrophoresis were used to characterize its regeneration capability. Next-generation sequencing was performed to determine its spatial location information, ultimately yielding the library as shown. Figure 8 , 9 As shown, the cDNA library fragments are concentrated between 500-2000 bp, while the sequenced DNA library fragments are concentrated around 400 bp, with no interfering bands. Figure 10 As shown, the sequencing results indicate that the UMI and Gene detected on the regenerable barcode slide are 4245 and 10309, respectively.

[0097] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A bar code array reproducible chip, characterized by, The chip substrate, the barcode probe, the capture probe, the universal primer probe and the strand displacement probe; wherein: The barcode probe is coupled to the chip substrate; The barcode probe is provided with a universal primer hybridization region, a spatial position barcode region and a capture probe hybridization region; wherein the universal primer hybridization region is used for complementary pairing with the universal primer probe, the spatial position barcode region is used for determining the spatial position, and the capture probe hybridization region is used for complementary pairing with the capture probe; The capture probe is provided with a barcode probe hybridization region, a strand displacement probe hybridization region and a capture region; wherein the barcode probe hybridization region is used for complementary pairing with the capture probe hybridization region on the barcode probe, the strand displacement probe hybridization region is used for complementary pairing with the strand displacement probe, and the capture region is used for capturing mRNA in the tissue; The universal primer probe and the capture probe are used for generating a full-length capture probe containing a universal primer probe sequence, a spatial position barcode sequence and a capture probe sequence through hybridization to the barcode probe and then polymerization and ligation reaction; The strand displacement probe is used for complementary pairing with the strand displacement probe hybridization region on the capture probe and performing polymerization strand displacement reaction to displace and release the full-length capture probe. The chip substrate is selected from a plastic sheet, a silicon chip, a glass slide or a polymethyl methacrylate sheet; the length of the chip substrate is 1-100 cm, the width is 1-100 cm, and the thickness is 0.1-10 mm.

2. The bar code array reproducible chip of claim 1, wherein The chip substrate surface is modified by a modification group, and the modification group includes any one or more of an aldehyde group, an epoxy group, an alkyne group, an N-hydroxysuccinimide group, a carboxyl group and a streptavidin group.

3. The bar code array reproducible chip of claim 1 wherein, The nucleic acid molecule on the barcode probe is connected to the modification group on the chip substrate surface through a specific group; the specific group includes an amino group, a thiol group or an epoxy group.

4. The bar code array reproducible chip of claim 3, wherein, The steps include:

5. The method for preparing and regenerating the use of the bar code array regenerable chip according to any one of claims 1 to 4, characterized in that, 1) coupling the barcode probe to the chip substrate through a chemical reaction to obtain a barcode chip; 2) hybridizing the universal primer probe and the capture probe to the barcode probe to generate a full-length capture probe containing a universal primer probe sequence, a spatial position barcode sequence and a capture probe sequence through polymerization and ligation reaction; 3) capturing the molecules to be detected using the barcode chip obtained in step 2), hybridizing the strand displacement probe to the strand displacement probe hybridization region on the capture probe and performing polymerization strand displacement reaction to displace and release the full-length capture probe capturing the molecules to be detected for library construction; 4) performing the polymerization and ligation treatment of step 2) again on the barcode chip after step 3) to generate full-length capture probes again, thereby regenerating the barcode array regenerable chip. The steps include:

6. Use of the bar code array regenerable chip according to any one of claims 1 to 4 in spatial omics analysis, characterized in that, attaching a tissue section to the barcode array regenerable chip for mRNA capture in the cells of the tissue, and then performing in situ reverse transcription to convert mRNA into cDNA; ​ Subsequently, a tissue removal solution is added to make the cDNA sequences on the barcode array regenerable chip bindable, and after a polymer chain displacement reaction, full-length capture probes capturing the cDNA sequences are displaced and released, a sequencing library is amplified and constructed, and at this time, the barcode array regenerable chip can be reused; finally, sequencing and spatial omics analysis are performed.

7. Use according to claim 6, characterized in that, The thickness of the tissue section is 0.1-1000 μm; after the tissue section is attached to the barcode array regenerable chip, a surfactant is used for tissue permeation treatment.

8. Use according to claim 6, characterized in that, The reaction temperature of the in situ reverse transcription process is 37-56℃, and the reaction time is 1-24 h.

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