A repeatable barcode array chip, its fabrication method and application

Abstract

This invention discloses a repeatable barcode array chip, its fabrication method, and its applications, belonging to the field of gene sequencing and tissue / cell sample analysis technology. The invention includes a chip substrate, universal primer probes, and barcode probes. The barcode probes contain four regions: a Nick region, a capture region, a positional barcode region, and a molecular barcode region, each performing a corresponding function. In particular, the design of the Nick region allows for the generation of a repeatable barcode chip through an SDA reaction. DNA probe hybridization, polymerization extension, and polymer chain replacement enable the barcode chip to be reused repeatedly. The chip structure is simple and easy to fabricate, and its usability allows for the fabrication of barcode chips at extremely low cost.

Description

Technical Field

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

[0002] Spatial omics is a rapidly evolving and dynamic field that combines the advantages of omics research and spatial resolution, aiming to reveal the three-dimensional spatial distribution and interactions of biomolecules in cells or tissues. Traditional omics techniques, such as transcriptomics, proteomics, and metabolomics, provide rich information but often lose the spatial information of the sample. Spatial omics aims to overcome this deficiency by combining microscopy techniques and molecular biology methods, allowing researchers to simultaneously detect multiple molecular markers within specific spatial regions of cell or tissue samples. Spatial transcriptomics, as a key technique in spatial omics, has been used to study processes such as organ growth, embryogenesis, and disease development. However, studying gene expression in space requires barcode arrays, and current methods suffer from the problem of one-time consumption of barcode arrays.

[0003] Space omics technologies mainly include: 1) space proteomics methods based on multiplex antibody detection, typically implemented using mass flow cytometry or multiplex circulating immunofluorescence; and 2) methods based on space transcriptomics and genomics. Barcode chips play a crucial role in space omics, used to label and track molecular information in samples. However, barcode chips are generally not reusable because they are designed to provide accurate and reliable molecular markers in a single experiment. These chips typically contain a series of unique barcode sequences to uniquely identify different samples or molecules during the experiment. Once these barcodes are used and read in the experiment, they lose their original identifying function. Furthermore, reusing barcode chips during space omics experiments can lead to barcode confusion, contamination, and cross-contamination. In practical applications, the use of barcode chips also faces several challenges. For example, as the amount of data increases, the barcode size and reading difficulty also increase, placing higher demands on the reading equipment. Additionally, different experimental conditions and sample types can affect the reading and performance of barcode chips.

[0004] Furthermore, existing technologies primarily rely on polyT probes on the chip to capture mRNA information within tissue cells. In situ reverse transcription extends the barcode probes, making reuse impossible. Moreover, current technologies involve hybridization between mRNA molecules within tissue cells and the capture probes on the barcode array surface, which can lead to the loss of target molecules. Therefore, there is an urgent need in this field to develop a simple, low-cost, and high-detection-rate reproducible barcode array chip, which is of great significance for spaceomics analysis. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention discloses a repeatable barcode array chip, comprising a chip substrate, a universal primer probe, and a barcode probe;

[0008] The barcode probe is coupled to the chip substrate via a chemical reaction;

[0009] The universal primer probe and barcode probe are complementary pairs for PCR amplification; the universal primer probe contains a Nick region and a universal primer sequence region for polymerization, extension and SDA reaction;

[0010] The barcode probe contains a Nick hybridization region, a capture region, a position barcode region, and a molecular barcode region. The Nick hybridization region and the Nick region are complementary. The capture region is used to capture the molecules to be detected, the position barcode region is used to determine the spatial location, and the molecular barcode region is used to count the molecules to be detected.

[0011] 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.

[0012] Preferably, the chip substrate surface is modified by a modifying group, which includes any one or more of aldehyde, epoxy, alkynyl, N-hydroxysuccinimide, carboxyl and streptavidin groups.

[0013] More preferably, nucleic acid molecules on the surface of the substrate chip are linked to modifying groups through specific groups, including amino, thiol, or epoxy groups.

[0014] Preferably, the barcode probe is a single probe.

[0015] This invention also discloses a method for fabricating the above-mentioned repeatable barcode array chip, comprising the following steps:

[0016] 1) Coupling barcode probes containing Nick hybridization region, capture region, position barcode region and molecular barcode region to chip substrate via chemical reaction;

[0017] 2) Hybridize universal primers and probes to barcode probes, and obtain capture probes with spatial location information through polymerization and extension;

[0018] 3) An SDA reaction is performed through the Nick region on a universal primer probe to release a capture probe containing a molecular barcode region, a position barcode region, and a capture region. This capture probe is then used to capture the molecule to be detected.

[0019] 4) After the SDA reaction is completed, a capture probe with spatial location information is obtained, enabling the barcode array chip to be reused.

[0020] This invention also discloses the application of the above-mentioned repeatable barcode array chip in spatial omics analysis, including:

[0021] 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 gene library.

[0022] Preferably, the thickness of the tissue slice is 0.1-1000 μm; the tissue is permeable with a surfactant before use.

[0023] Preferably, the in situ reverse transcription treatment is performed at a temperature of 37-56°C for 1-24 hours.

[0024] This invention also discloses the application of the above-mentioned barcode array repeatable chip in the preparation of DNA libraries.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention discloses a reusable barcode array chip. In its design, it addresses the problem that existing chips primarily rely on polyT probes to capture mRNA information from tissue cells, extending the barcode probes through in situ reverse transcription, thus preventing reuse. This invention designs four regions on the barcode probes, each performing a corresponding function. Specifically, the inclusion of a Nick hybridization region, complementary to the Nick region on universal primer probes, allows for the generation of a reusable barcode chip via an SDA reaction. Through DNA probe hybridization, polymerization extension, and polymer chain replacement, the barcode chip can be reused repeatedly. The chip structure is simple and easy to fabricate, enabling barcode chip preparation at extremely low cost.

[0027] The barcode array repeatable chip of this invention has a wide range of applications, including detection of genomes, transcriptomes, proteomes, epigenetics, metabolomes, etc., providing excellent support for gene library construction. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the first type of barcode array reproducible chip used for spatial transcriptome sequencing; where A represents the orthogonal coding strategy assisted by the sampling machine; and B represents the reproducible chip used for spatial transcriptomics analysis.

[0029] Figure 2 A schematic diagram illustrating the principle of verifying repeatability in a standard chain;

[0030] Figure 3 This is a schematic diagram of a library structure with spatial tags in one embodiment of the present invention;

[0031] Figure 4 This is a characterization diagram of nucleic acid probe modification.

[0032] Figure 5 Agarose gel electrophoresis image for verifying reproducibility of the standard strand;

[0033] Figure 6 This is a grayscale image used to verify reproducibility based on tissue sections. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.”

[0038] 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.

[0039] 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.

[0040] In the following embodiments of the present invention, "Oligo(dT)" is a specific oligonucleotide sequence composed of consecutive thymine (T) bases, typically between 12 and 18 Ts in length, and sometimes extended to 20 consecutive Ts. As a DNA fragment, Oligo(dT) exhibits typical oligonucleotide chemistry. Its 5' end is usually phosphorylated, allowing Oligo(dT) to link with other DNA or RNA molecules via phosphodiester bonds, serving as a primer for DNA or RNA synthesis. Its application is based on the principle that Oligo(dT) specifically binds to the poly(A) tail at the 3' end of eukaryotic mRNA due to its composition entirely of T bases, forming a complementary pair with the A bases on the mRNA. It is commonly used for mRNA isolation, mRNA purification, and cDNA synthesis.

[0041] In the following embodiments of the present invention, "Oligo-dA" is described as an oligonucleotide sequence composed of consecutive adenine (A) bases. Oligo-dA has the property of complementary pairing with thymine (T) bases, and therefore can be used as a primer or probe in specific molecular biology experiments, such as PCR and sequencing. In single-cell RNA sequencing (scRNA-seq) studies, Oligo-dA is often used as part of a barcode marker to distinguish individual cells or nuclei from different samples; in amplification reactions such as PCR, Oligo-dA can be used as a specific primer to amplify target DNA sequences; in molecular hybridization experiments, Oligo-dA can be used as a probe to detect the presence or absence of target DNA or RNA sequences. Through specific binding to the target sequence, qualitative and quantitative analysis of the target molecule can be achieved.

[0042] The barcode array repeatable chip disclosed in this invention includes a chip substrate, a universal primer probe, and a barcode probe;

[0043] The barcode probe is coupled to the chip substrate via a chemical reaction;

[0044] The universal primer probe and barcode probe are complementary pairs for PCR amplification; the universal primer probe contains a Nick region and a universal primer sequence region for polymerization, extension and SDA reaction;

[0045] The barcode probe contains a Nick hybridization region, a capture region, a position barcode region, and a molecular barcode region. The Nick hybridization region and the Nick region are complementary. The capture region is used to capture the molecules to be detected, the position barcode region is used to determine the spatial location, and the molecular barcode region is used to count the molecules to be detected.

[0046] The "universal primer-probe" described above can specifically bind to target nucleic acid sequences 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, improving detection throughput, sensitivity, and specificity.

[0047] The "nick region" described above refers to a nick created on the DNA double helix. These nicks are a key step in the labeling process because they allow DNA polymerase to perform exonuclease and polymerization activities at the nick site, thereby incorporating labeled nucleotides. Similar nick translation techniques may be used to label probes during nucleic acid detection or recognition.

[0048] The "capture region" of the barcode probe described above refers to a specific sequence on the barcode probe. Designed specifically for the target nucleic acid sequence (such as DNA or RNA), it can complementaryly pair with the target nucleic acid sequence, thereby achieving specific recognition and binding. The capture region is designed with high specificity, accurately recognizing and binding to the target nucleic acid sequence, avoiding interference from non-specific binding. Through complementary pairing between the capture region and the target nucleic acid sequence, the barcode probe can specifically capture the target nucleic acid sequence, providing a foundation for subsequent detection and analysis.

[0049] The "position barcode area" described above is the region on the barcode probe used to encode and identify the probe's position within the chip or array. Within the chip or array, each probe has a unique position barcode to identify its location within the entire system. This helps in accurately tracking the detection results of each probe in subsequent data analysis. By encoding and recognizing position barcodes, detection errors caused by probe position confusion can be avoided, improving the accuracy and reliability of the detection results.

[0050] The "molecular barcode region" described above is a unique sequence on the barcode probe used to assign a unique identifier (UID) to each probe molecule. This identifier enables the differentiation of signals from different probe molecules during subsequent sequencing or detection, thereby achieving precise tracking and quantification of the target nucleic acid sequence. Through the molecular barcode region, the signal of each probe molecule can be precisely tracked and quantified, thus improving the sensitivity and accuracy of detection.

[0051] The present invention will now be described in further detail with reference to the accompanying drawings:

[0052] This invention provides a method for fabricating a repeatable barcode array chip and its application, as detailed below:

[0053] 1. Design a repeatable barcode array chip for identifying probe sequences of mRNA in tissue cells. See [link to relevant documentation]. Figure 1 A in the middle includes:

[0054] The first set of barcode probes (i.e., probe 1) for chip modification is designed, consisting of 20-100 bases. Of these, 15 bases are used for the SDA reaction, 29 bases are the complementary region of the universal primer sequence, 8 bases are the first set of spatial location barcode regions (Barcode X), and 15 bases are used to connect to the second set of barcode probes, making a total of four parts. Probe 1 is modified with a phosphate group at its 5' end and an amino group at its 3' end. The amino group is used to bind to the aldehyde chip. The chip modification includes, but is not limited to, aldehyde and carboxyl groups.

[0055] The second set of barcode probes (i.e., probe 2) is designed for chip modification. Probe 2 consists of four parts: starting from 5', 30 bases form an oligo-dA region, 10 bases form a unique molecular identifier (UMI) for molecular counting, 8 bases form a second set of spatial location barcodes (Barcode Y), and 15 bases are used to connect to the first set of barcode probes.

[0056] The probe sequence (i.e., probe 3) is designed to connect the first group and the second group of barcode probes. 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.

[0057] The universal primer probe R1 sequence (i.e. probe 4) designed for PCR amplification (PCR handle, PH) consists of two parts: a 29-base universal primer probe R1 sequence for PCR amplification and a 15-base sequence for SDA reaction.

[0058] The Oligo-dA probe sequence (i.e. probe 5) designed for standard chain validation consists of two parts: a 20-base oligo-dA sequence at the 3' end and a 15-base universal primer probe R2 sequence at the 5' end for PCR amplification.

[0059] 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.

[0060] like Figure 1 As shown, the preparation process includes the following steps:

[0061] 1) Incubate probe 1 with a final concentration of 10 μM with the functionalized chip. Perform gradient annealing hybridization on probes 2 and 3 and incubate them with the chip modified with probe 1 for later use. Specifically, spot the annealed probes 2 and 3 with a spotting machine, orthogonal to the direction of probe 1, but at the same spotting position.

[0062] 2) Capture probes with spatial location information are generated through SDA reaction for verification on standard strands and tissue sections.

[0063] like Figure 2 As shown, the barcode chip functionalized in step 2) was used for standard chain verification. It can be seen that in the presence of probe 5, the capture probe generated by the SDA reaction binds to it and hybridizes stably. The capture probe obtained in step 2) dissociates from the barcode chip, allowing for reusability. The dissociated probe was subjected to PCR amplification, and the size of the electrophoretic bands was verified by agarose gel electrophoresis. After completing the first round of verification, step 2) was repeated to verify the reproducibility of the SDA barcode chip.

[0064] 2. Barcode chips that have undergone functional modifications are used for tissue slice verification.

[0065] like Figure 1 As shown in Figure B, the SDA reaction first generates a capture probe. Subsequently, during the permeabilization process of the tissue section, the capture probe binds to intracellular mRNA molecules, capturing genetic information from the tissue cells during in situ reverse transcription. The probe from step 2) above is then detached from the barcode chip, allowing for reuse. The probe obtained from reverse transcription is then subjected to PCR amplification, as shown in Figure B. Figure 3 As shown, library construction and sequencing are used to analyze the spatial distribution of genetic information within tissue cells.

[0066] 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.

[0067] 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.

[0068] 1. Design of nucleic acid probe molecules and their simulation:

[0069] To enable the use of repeatable barcode array chips for spatial group analysis, corresponding nucleic acid probes were designed.

[0070] In designing probe 1, based on the rapid coupling properties of aldehyde and amino groups, an amino group was modified at one end to allow it to bind to the aldehyde-modified chip substrate. A 16-base region was designed as a position barcode area for spatial location determination. To reduce steric hindrance, a spacer region could also be designed between the chip and the functional area. Simultaneously, a Nick hybridization region was designed to enable the SDA reaction. The sequence of the designed probe 1 is shown in Table 1 as SEQ ID NO. 1~6, 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.

[0071] In the design of probe 2, based on the principle of complementary base pairing, 30 oligo-dA regions were designed to generate oligo-dT. 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.

[0072] In the design of 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.

[0073] In designing probe 4, the R1 region was designed based on the primers required for PCR amplification. The sequence of probe 4 is shown in SEQ ID NO.14 in Table 3.

[0074] In designing probe 5, based on the primers required for PCR amplification, the R2 region was designed as hybridization probe 2, and the oligo-dA sequence was designed. The sequence of probe 4 is shown in SEQ ID NO.15 in Table 3.

[0075] 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.16 in Table 3.

[0076] Table 1. Partial Group 1 Barcode Probe Sequences

[0077]

[0078] Table 2 Partial Group 2 Barcode Probe Sequences

[0079]

[0080] Table 3. Some relevant sequences in the examples

[0081]

[0082] 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, as shown in Tables 1 and 2 above, can be designed in various ways.

[0083] 2. Fabrication of barcode chips modified with probes 1, 2, and 3:

[0084] The probe 1 at a concentration of 10 μM was bound 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 Probes 2 and 3 were subjected to gradient annealing: 95°C was cooled to 20°C at a rate of 0.1°C per second. They were then hybridized with probe 1 at 37°C for 30 min. The chip surface was cleaned with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. Probes 1 and 2 were ligated using DNA ligase, and probe 3 was removed with KOH solution for reusability in the SDA barcode array chip. The annealed probes 2 and 3 were spotted using a spotting machine, orthogonal to probe 1 but at the same spotting location, reducing the number of probes from 900 to 60, significantly reducing encoding costs. Some probe sequences are listed in Tables 1 and 2. Figure 4As shown, fluorescence microscopy characterizes the connection between the two sets of spatially positioned barcode probes.

[0085] 3. Repeatable barcode array chips are used for standard chain verification.

[0086] The barcode chip prepared in step 2 was reacted with 1 μM probe 4 at 37°C for 2 h. The chip surface was then washed with 2×SSC solution (containing 0.1% SDS) to remove unreacted and non-specifically adsorbed DNA. DNA polymerase was incubated with the chip at 37°C for 2 h to perform an SDA reaction, yielding a capture probe with a barcode. This capture probe was then polymerized and extended with 1 μM probe 5, and the product was amplified by PCR. The barcode chip underwent repeated polymerization extension and SDA reactions, allowing for reusability. Figure 5 As shown, three rounds of reproducibility experiments were conducted, and the reproducibility was characterized by real-time PCR curves and agarose gel electrophoresis.

[0087] 4. Barcode array repeatable chips are used for tissue slide verification.

[0088] 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 adjust the thickness to 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. The tissue was fixed in 4% paraformaldehyde, with residual paraformaldehyde removed by 1×PBS. 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 the reaction, the tissue was washed with 0.1×SSC solution. Tissue removal solution was added; the supernatant contained the barcode-containing cDNA sequence, which was then amplified by PCR. This process was repeated as follows. Figure 6 As shown, three rounds of reproducibility verification were performed, and the reproducibility was characterized by real-time PCR curves and agarose gel electrophoresis.

[0089] 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 re-usable chip, characterized by, The chip substrate, the barcode probe and the universal primer probe; The barcode probe is coupled to the chip substrate through a chemical reaction; The barcode probe contains a Nick hybridization region, a universal primer sequence complementary region, a capture region, a position barcode region and a molecular barcode region; The universal primer probe contains a Nick region and a universal primer sequence region, and is used for polymerization, extension and SDA reaction; The Nick hybridization region is used for complementary pairing with the Nick region, the universal primer sequence complementary region is used for complementary pairing with the universal primer sequence region, the capture region is used for generating a capture sequence after hybridization of the barcode probe and the universal primer probe through polymerization and extension, the position barcode region is used for spatial position determination, and the molecular barcode region is used for counting the to-be-detected molecules; The universal primer probe generates a capture probe containing a molecular barcode sequence, a position barcode sequence and a capture sequence after hybridization to the barcode probe through polymerization and extension; the Nick region is used for specific cutting by a nicking enzyme to generate a gap and perform SDA reaction, so as to release the capture probe, and the capture probe is used for capturing the to-be-detected molecules through the capture sequence.

2. The bar code array reprogrammable chip of claim 1 wherein, The chip substrate is selected from a plastic sheet, a silicon chip, a glass slide and 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.

3. The bar code array reprogrammable 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.

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

5. The method of making and reusing a bar code array reusable chip according to any one of claims 1 to 4, wherein, The method comprises the following steps: 1) coupling the barcode probe to the chip substrate through a chemical reaction; 2) hybridizing the universal primer probe to the barcode probe to obtain a capture probe containing a molecular barcode sequence, a position barcode sequence and a capture sequence through polymerization and extension reaction; 3) releasing the capture probe through specific cutting of the Nick region by a nicking enzyme and performing SDA reaction, and capturing the to-be-detected molecules by using the capture probe; 4) performing SDA reaction again in step 3) to obtain the capture probe again, so as to realize repeated use of the barcode array repeatable chip.

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