Bar code array repeatable chip and preparation method and application thereof

Abstract

The invention discloses a barcode array repeatable chip and a preparation method and application thereof, and belongs to the technical field of gene sequencing and tissue cell sample analysis. The chip comprises a chip substrate, a universal primer probe and a bar code probe, the bar code probe comprises a Nick region, a capture region, a position bar code region and a molecular bar code region which respectively execute corresponding functions, and particularly, the Nick region is arranged, so that the chip can generate a repeatable bar code chip through SDA reaction, and the chip can be used for detecting the bar code of the chip. The bar code chip can be repeatedly used through DNA probe hybridization, polymerization extension and polymerization strand displacement. The chip has a simple structure, is easy to process, and can be used, so that the preparation of the bar code chip is realized at extremely low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of biochips and detection thereof, and in particular relates to a barcode array repeatable chip and a preparation method and application thereof. Background Art

[0002] Spatial omics is a rapidly developing and dynamic field that combines the advantages of omics research and spatial resolution, aiming to reveal the three-dimensional spatial distribution and interactions of biological molecules in cells or tissues. Traditional omics technologies, such as transcriptomics, proteomics, and metabolomics, provide rich information, but often lose the spatial information of samples. Spatial omics aims to make up for this shortcoming. By combining microscopy technology and molecular biology methods, it allows researchers to simultaneously detect multiple molecular markers in specific spatial regions of cell or tissue samples. As a key technology in spatial omics, spatial transcriptomics technology has been used to study processes such as organ growth, embryo formation, and disease occurrence. However, barcode arrays are required to study the spatial expression of genes. In current methods, barcode arrays have the problem of one-time consumption.

[0003] Spatial omics technologies mainly include: 1) spatial proteomics methods based on multiple antibody detection, usually implemented by mass spectrometry flow cytometry or multiple cycle immunofluorescence technology; 2) methods based on spatial transcriptome and genomics. Barcode chips play a key role in spatial omics, used to mark and track molecular information in samples. However, barcode chips are usually not reusable, because the original design of barcode chips is to provide accurate and reliable molecular markers in an experiment. These chips usually contain a series of unique barcode sequences to uniquely identify different samples or molecules in the experiment. Once these barcodes are used and read in the experiment, they lose their original identification function. In addition, in the process of spatial omics experiments, the repeated use of barcode chips may cause problems such as barcode confusion, contamination and cross-contamination. At the same time, in practical applications, the use of barcode chips also faces some challenges. For example, as the amount of data increases, the barcode size and reading difficulty increase accordingly, and the requirements for reading equipment also increase simultaneously. For example, different experimental conditions and sample types may affect the reading and performance of barcode chips.

[0004] Furthermore, the existing technology mainly captures mRNA information in tissue cells based on polyT probes on the chip, and in situ reverse transcription allows the barcode probe to be extended, which cannot be reused. Moreover, the current technology is that the mRNA molecules in the tissue cells are hybridized with the capture probes on the surface of the barcode array, and there will be a situation where the target molecules are lost. Therefore, the field urgently needs to develop a barcode array repeatable chip with a simple method, low cost and high detection rate, which is of great significance for spatial group analysis. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a barcode array repeatable chip and a preparation method and application thereof, so as to solve the technical problem that the barcode chips prepared by the prior art are disposable and cannot be reused.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses a barcode array repeatable chip, comprising a chip substrate, a universal primer probe and a barcode probe; The barcode probe is coupled to the chip substrate through a chemical reaction; The universal primer probe and the barcode probe are complementary and paired for PCR amplification; The barcode probe contains a Nick region, a capture region, a position barcode region and a molecular barcode region. The Nick region is used for SDA reaction, the capture region is used to capture the molecules to be detected, the position barcode region is used to determine the spatial position, and the molecular barcode region is used to count the molecules to be detected.

[0007] 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.

[0008] Preferably, 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.

[0009] Further preferably, the nucleic acid molecules on the surface of the substrate chip are connected to the modification groups via specific groups, and the specific groups include amino groups, thiol groups or epoxy groups.

[0010] Preferably, the barcode probe is a single probe.

[0011] The present invention also discloses a method for preparing the above-mentioned barcode array repeatable chip, comprising the following steps: 1) The barcode probe containing the Nick region, the capture region, the position barcode region and the molecular barcode region is coupled to the chip substrate through chemical reaction; 2) Hybridizing the universal primer probe to the barcode probe, and obtaining a capture probe with spatial position information through polymerization and extension; 3) Through SDA reaction with the Nick region, a capture probe containing a molecular barcode region, a position barcode region and a capture region is released, and the capture probe is used to capture the molecule to be detected; 4) After the SDA reaction is completed, a capture probe with spatial position information is obtained, thereby realizing the reuse of the barcode array chip.

[0012] The present invention also discloses the application of the above-mentioned barcode array repeatable chip in spatial omics analysis, including: The tissue sections are attached to the barcode array regenerable chip, and then in situ reverse transcription is performed 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 gene library.

[0013] Preferably, the thickness of the tissue slice is 0.1-1000 μm; and the tissue is permeabilized with a surfactant before use.

[0014] Preferably, the reaction temperature of the in situ reverse transcription treatment is 37-56° C., and the reaction time is 1-24 h.

[0015] The invention also discloses the application of the above-mentioned barcode array repeatable chip in preparing a DNA library.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The barcode array reproducible chip disclosed in the present invention is designed with consideration of the problem that the existing chips mainly rely on polyT probes to capture mRNA information in tissue cells, and the barcode probes are extended through in situ reverse transcription, which makes it impossible to reuse. The present invention designs four regions on the barcode probe, each of which performs a corresponding function. In particular, the setting of the Nick region allows it to generate a reproducible barcode chip through the SDA reaction, and the barcode chip can be reused through DNA probe hybridization, polymerization extension, and polymer chain displacement. The chip has a simple structure and is easy to process. Since it can be used, the preparation of the barcode chip is achieved at a very low cost.

[0017] The barcode array repeatable chip of the present invention has a wide range of applications and can be used for detection of genomes, transcriptomes, proteomes, epigenomes, metabolomes, etc., providing very good support for gene library construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The first barcode array repeatable chip is used for spatial transcriptome sequencing principle diagram; A is the orthogonal encoding strategy assisted by the spotting machine; B is the repeatable chip used for spatial transcriptomics analysis; Figure 2 Schematic diagram of the principle of verifying the repeatability of the standard chain; Figure 3 This is a schematic diagram of the structure of a library containing spatial tags in one embodiment of the present invention; Figure 4 It is a characterization diagram of the modification of nucleic acid probe; Figure 5 Agarose gel electrophoresis diagram for standard strand verification of reproducibility; Figure 6 Grayscale value images showing the repeatability of the test based on tissue sections. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] 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).

[0022] 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.”

[0023] 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.

[0024] 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.

[0025] In the following embodiments of the present invention, the "Oligo(dT)" described is a special oligonucleotide sequence composed of consecutive thymine (T) bases, and the length is usually between 12 and 18 Ts, and sometimes it can be extended to 20 consecutive Ts. Oligo(dT), as a DNA fragment, has typical oligonucleotide chemical properties. Its 5' end is usually phosphorylated, which enables Oligo(dT) to connect with other DNA or RNA molecules through phosphodiester bonds to become a primer for DNA or RNA synthesis. Application-based principle: Oligo(dT) can specifically bind to the poly(A) tail at the 3' end of eukaryotic mRNA, because it is completely composed of T bases and forms a complementary pair with the A base on mRNA. It is commonly used for mRNA separation, mRNA purification and cDNA synthesis.

[0026] In the following embodiments of the present invention, the "Oligo-dA" described is an oligonucleotide sequence consisting of continuous adenine (A) bases. Oligo-dA has the property of complementary pairing with thymine (T) bases, so it can be used as a primer or probe in specific molecular biology experiments, such as PCR, sequencing, etc. In single-cell RNA sequencing (scRNA-seq) research, Oligo-dA is often used as part of the Barcode marker to distinguish individual cells or cell nuclei from different samples; in amplification reactions such as PCR, Oligo-dA can be used as a specific primer to amplify the target DNA sequence; in molecular hybridization experiments, Oligo-dA can be used as a probe to detect the presence or absence of the target DNA or RNA sequence. Qualitative and quantitative analysis of the target molecule can be achieved through specific binding to the target sequence.

[0027] The barcode array repeatable chip disclosed in the present invention comprises a chip substrate, a universal primer probe and a barcode probe; The barcode probe is coupled to the chip substrate through a chemical reaction; The universal primer probe and the barcode probe are complementary and paired for PCR amplification; The barcode probe contains a Nick region, a capture region, a position barcode region and a molecular barcode region. The Nick region is used for SDA reaction, the capture region is used to capture the molecules to be detected, the position barcode region is used to determine the spatial position, and the molecular barcode region is used to count the molecules to be detected.

[0028] The "universal primer probe" described above can specifically bind to the target nucleic acid sequence and reflect the presence and quantity of the target nucleic acid sequence by detecting the signal intensity of the marker. By designing the sequence and detection conditions of the universal primer probe, parallel detection of a large number of biological samples can be achieved, thereby improving detection throughput, sensitivity and specificity.

[0029] The "nick region of the barcode probe" described above refers to the nicks made on the double-stranded DNA. These nicks are one of the key steps in the labeling process because they allow DNA polymerase to perform exonucleolytic and polymerizing activities at the nicks, thereby incorporating labeled nucleotides. When using barcode probes for nucleic acid detection or identification, techniques similar to nick translation may be used to label the probes.

[0030] The "capture region of the barcode probe" described above refers to a specific sequence on the barcode probe, which is designed based on the specificity of the target nucleic acid sequence (such as DNA or RNA) and can be complementary paired with the target nucleic acid sequence to achieve specific recognition and binding. The capture region is designed with high specificity and can accurately recognize and bind to the target nucleic acid sequence, avoiding the interference of non-specific binding. Through the complementary pairing of the capture region and the target nucleic acid sequence, the barcode probe can specifically capture the target nucleic acid sequence, providing a basis for subsequent detection and analysis.

[0031] The "position barcode area of ​​the barcode probe" described above is the area on the barcode probe used to encode and identify the position of the probe in the chip or array. In the chip or array, each probe has a unique position barcode to identify its position in the entire system. This helps to accurately track the detection results of each probe in subsequent data analysis. Through the encoding and identification of the position barcode, detection errors caused by confusion of probe positions can be avoided, and the accuracy and reliability of the detection results can be improved.

[0032] The "molecular barcode region of the barcode probe" described above is a unique sequence on the barcode probe, which is used to assign a unique identifier (UID) to each probe molecule. This identifier enables the signals from different probe molecules to be distinguished during the subsequent sequencing or detection process, thereby achieving accurate tracking and quantification of the target nucleic acid sequence. Through the molecular barcode region, the signal of each probe molecule can be accurately tracked and quantified, thereby improving the sensitivity and accuracy of the detection.

[0033] 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 repeatable chip and its application, which are as follows: 1. Design a barcode array reproducible chip to identify probe sequences of mRNA in tissue cells, see Figure 1 Medium A, including: The first group of barcode probes (i.e., probe 1) for chip modification is designed. Probe 1 is 20 to 100 bases. Among them, 15 bases are used for SDA reaction, 29 bases are complementary regions of universal primer sequences, 8 bases are the first group of spatial position barcode regions (Barcode X), and 15 bases are used to connect the second group of barcode probes, which are composed of four parts in total. The 5' end of probe 1 is phosphate-modified, and the 3' end is amino-modified. The amino group is used to bind to the aldehyde chip. The chip modification includes but is not limited to aldehyde and carboxyl groups; The second set of barcode probes (i.e., probe 2) designed for chip modification, probe 2 starting from 5' is composed of 30 bases forming the oligo-dA region, 10 bases as the unique molecular identifier (UMI) for molecular counting, 8 bases as the second set of spatial position barcodes (Barcode Y), and 15 bases for connecting to the first set of barcode probes, a total of four parts; Design a probe sequence for connecting the first group and the second group of barcode probes (i.e., probe 3), wherein probe 3 consists of two parts: 15 bases for connecting the first group of barcode probes and 15 bases for connecting the second group of barcode probes; Design a universal primer probe R1 sequence (i.e., probe 4) for PCR amplification (PCR handle, PH). Probe 4 consists of two parts: 29 bases of the universal primer probe R1 sequence for PCR amplification and 15 bases for SDA reaction; Design an oligo-dA probe sequence (i.e., probe 5) for standard chain verification. Probe 5 consists of two parts: a 20-base oligo-dA at the 3' end and a 15-base universal primer probe R2 sequence at the 5' end for PCR amplification. A probe sequence (ie, probe 6) was designed for template switching in tissue sections. Probe 6 consisted of two parts: an R2 sequence at the 5' end for PCR amplification and an LNA modification at the 3' end.

[0034] like Figure 1 As shown, during preparation, the following steps are included: 1) Probe 1 with a final concentration of 10 μM is incubated and combined with the functionalized chip, and probes 2 and 3 are gradient annealed and hybridized, and incubated with the chip modified with probe 1 for use. Specifically, the annealed probes 2 and 3 are spotted by a spotter, orthogonal to the direction of probe 1, but at the same spotting position.

[0035] 2) Generate capture probes with spatial position information through SDA reaction for standard chain and tissue section verification.

[0036] like Figure 2As shown, the barcode chip functionally modified in step 2) is used for standard chain verification. It can be seen that in the presence of probe 5, the capture probe generated by the SDA reaction will bind to it and stably hybridize, and the capture probe obtained in step 2) will dissociate from the barcode chip, making it reusable. 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 step 2) to complete the verification of the repeatability of the SDA barcode chip.

[0037] 2. Use functionally modified barcode chips for tissue section verification like Figure 1 As shown in B, first, the SDA reaction generates the capture probe, and then during the permeabilization process of the tissue section, the capture probe will bind to the mRNA molecules in the cell, and the genetic information in the tissue cell will be captured during the in situ reverse transcription process. The probe in step 2) above will be dissociated from the barcode chip, making it reusable. The probe obtained by reverse transcription is amplified by PCR, as shown in Figure 3 As shown, library construction and sequencing are used to analyze the spatial distribution of genetic information within tissue cells.

[0038] 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.

[0039] 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.

[0040] 1. Design of nucleic acid probe molecules and their simulation: In order to realize the use of barcode array reproducible chips for spatial group analysis, corresponding nucleic acid probes are designed.

[0041] In the process of designing probe 1, according to the characteristics of rapid coupling between aldehyde group and amino group, one end is modified with amino group, so that it can be combined with aldehyde group modified chip substrate; 16 bases are designed as position barcode area for determining spatial position; of course, in order to reduce steric hindrance, it is also possible to consider designing a spacer area between the chip and the functional area; at the same time, in order to achieve SDA reaction, Nick area is designed. The sequence of designed probe 1 is shown in SEQ ID NO.1~6 in Table 1, indicating that probe 1 can be any one of 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6 in Table 1.

[0042] In the process of designing probe 2, 30 oligo-dA regions were designed for generating oligo-dT according to the principle of base complementary pairing. The sequence of probe 2 is shown in SEQ ID NO. 7-12 in Table 2, indicating that probe 2 can be any one of 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6 in Table 1.

[0043] In the process of designing probe 3, 15 bases were designed to stably connect probe 1 and probe 2. The sequence of probe 3 is shown in SEQ ID NO. 13 in Table 3.

[0044] In the process of designing probe 4, the R1 region is designed according to the primers required in the PCR amplification process; the hybridization region is designed for the hybridization Nick region. The sequence of probe 4 is shown in SEQ ID NO. 14 in Table 3.

[0045] In the process of designing probe 5, the R2 region is designed according to the primers required in the PCR amplification process, and the oligo-dA sequence is designed for hybridization probe 2. The sequence of probe 4 is shown in Table 3 as SEQ ID NO. 15.

[0046] 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. 16 in Table 3.

[0047] Table 1 Part of the first group of barcode probe sequences

[0048] Table 2 Part of the second group of barcode probe sequences

[0049] Table 2 Some related sequences in the examples

[0050] The above probes 1, 2, 3, 4, 5 and 6 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and used in the following experiments. Note that probes 1 and 2 are shown in Tables 1 and 2 above, and various designs are possible.

[0051] 2. Prepare a barcode 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 . Perform gradient annealing on probe 2 and probe 3: cool from 95°C to 20°C, at a rate of 0.1°C per second. Then hybridize with probe 1 at 37°C for 30 min. Wash the chip surface with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. Use DNA ligase to connect probe 1 and probe 2, and use KOH solution to remove probe 3 for reusable SDA barcode array chips. The annealed probes 2 and 3 are spotted by a spotter, orthogonal to the direction of probe 1, but at the same spotting position, reducing the original 900 probes to 60 probes, greatly reducing the encoding cost. Some probe sequences are listed in Tables 1 and 2. Figure 4 As shown, fluorescence microscopy characterized the connection status of two sets of spatially located barcode probes.

[0052] 3. Barcode array repeatable chip for standard chain verification The probe 4 at 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. The DNA polymerase was incubated with the chip at 37°C for 2 h for SDA reaction to obtain a capture probe with a barcode. The capture probe was polymerized and extended with the probe 5 at a concentration of 1 μM, and the above product was amplified by PCR. The barcode chip continued to undergo polymerization extension and SDA reaction, and this was repeated so that the barcode chip can be reused. Figure 5 As shown, three rounds of reproducibility experiments were performed, and PCR real-time curves and agarose gel electrophoresis were used to characterize the reproducibility.

[0053] 4. Barcode array reproducible chips for tissue section verification Take fresh brain tissue from mice, embed it with OCT embedding reagent, freeze it quickly in liquid nitrogen, and then put it in a -80℃ refrigerator for long-term storage. Place the tissue block stored at -80℃ in a -20℃ freezing microtome and pre-balance the tissue block for 30 minutes. Use a freezing microtome to slice, the rough cutting thickness is 100 μm, and after the rough adjustment to the tissue part, make a micro-cut and adjust the thickness to 10 μm, and attach it to the barcode chip prepared in step 2. Then place it at 37℃ to dry for 1 min. Put it in 4% paraformaldehyde for fixation, and the residual paraformaldehyde in 1×PBS. Use pre-permeabilization reagent and permeabilization reagent to permeabilize the tissue, and wash it with 0.1×SSC solution. Then add reverse transcription reagent for in situ reverse transcription, react at 42℃ for 6 hours, and reverse transcribe mRNA into cDNA. After completion, wash with 0.1×SSC solution. Add tissue removal solution, the supernatant contains cDNA sequence with barcode, PCR amplification. Repeat this process, such as Figure 6 As shown, three rounds of reproducibility validation were performed, and PCR real-time curves and agarose gel electrophoresis were used to characterize its reproducibility.

[0054] 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 repeatable chip, characterized in that: It includes a chip substrate, a universal primer probe and a barcode probe; The barcode probe is coupled to the chip substrate through a chemical reaction; The universal primer probe and the barcode probe are complementary and paired for PCR amplification; The barcode probe contains a Nick region, a capture region, a position barcode region and a molecular barcode region. The Nick region is used for SDA reaction, the capture region is used to capture the molecules to be detected, the position barcode region is used to determine the spatial position, and the molecular barcode region is used to count the molecules to be detected.

2. The barcode array repeatable 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 repeatable 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 repeatable chip according to claim 3, characterized in that: The nucleic acid molecules on the surface of the substrate chip are connected to the modification groups through specific groups, and the specific groups include amino groups, thiol groups or epoxy groups.

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

6. The method for preparing a repeatable barcode array chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) The barcode probe containing the Nick region, the capture region, the position barcode region and the molecular barcode region is coupled to the chip substrate through chemical reaction; 2) Hybridizing the universal primer probe to the barcode probe, and obtaining a capture probe with spatial position information through polymerization and extension; 3) Through SDA reaction with the Nick region, a capture probe containing a molecular barcode region, a position barcode region and a capture region is released, and the capture probe is used to capture the molecule to be detected; 4) After the SDA reaction is completed, a capture probe with spatial position information is obtained, thereby realizing the reuse of the barcode array chip.

7. Use of the barcode array repeatable 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 gene library.

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

10. Use of the barcode array repeatable chip according to any one of claims 1 to 5 in preparing a DNA library.

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