Spatial multi-omics detection method for biological sample

Abstract

The invention discloses a spatial multi-omics detection method for a biological sample. For the same biological sample, protein detection is performed by reacting with the first antibody and the target protein second antibody with the horseradish peroxidase marker, and then gene detection is performed by adopting the space transcriptome probe method sequencing technology, so that both proteome information and transcriptome information can be obtained. Compared with the existing in-situ sequencing technology and single cell sequencing technology, the spatial multi-omics technology provided by the invention can achieve the purpose of spatial multi-omics analysis, is more favorable for researching the correlation between organism genes and proteins, and can better meet the use requirements.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a spatial multi-omics detection method for biological samples. Background Art

[0002] Cells are the most basic functional units of organisms. Due to different expression levels of biomarkers (e.g., gene and / or protein expression) in different cells, there are differences in morphology and / or function between cells. The specific position of cells in tissues (e.g., the position of cells relative to adjacent cells, the position of cells relative to the tissue microenvironment) may affect, for example, the morphology, differentiation, proliferation, behavior of cells, and signal transduction with other cells in the tissue. Therefore, in situ detection of biomarker expression levels within tissue cells, between cells, and between cells and tissue microenvironment has important application value for understanding the growth, development, disease, aging, etc. of organisms.

[0003] Existing technologies such as in situ sequencing and single-cell sequencing technology have problems such as tissue sample destruction and biomarker degradation, and cannot meet the needs of in situ detection of nucleic acids and proteins in the same biological sample. Patent CN115210760B proposes a method for in situ detection of proteins in biological samples using immunofluorescence (IF) method, and then using the biological sample for spatial transcriptome sequencing analysis. However, when the IF method is used to detect multiple proteins on the same sample, the primary antibody and secondary antibody corresponding to each protein must be of different species, and the emission wavelength of the fluorescent dye used to label the secondary antibody must be different. Therefore, due to the limitations of antibody species and fluorescent dyes, the IF method generally detects at most two proteins on the same biological sample at the same time (excluding DAPI nuclear fluorescence staining); at the same time, the fluorescence signal of the IF method is weak and the background fluorescence is strong, making it difficult to meet the detection needs of low abundance such as single cell protein levels.

[0004] Patent CN117441028A proposes a method for in situ detection of proteins and nucleic acids in the same biological sample using sequencing technology. This protein detection method does not limit the number of protein detections and has high sensitivity. However, the experimental process is non-visual and takes a long time, making it difficult for researchers to interpret the results quickly. Summary of the Invention

[0005] To address the shortcomings of existing detection technologies, the present invention proposes a spatial multi-omics detection method for biological samples. The objectives of the present invention are achieved through the following technical solutions:

[0006] A spatial multi-omics detection method for biological samples comprises the following steps:

[0007] Step 1) obtaining a biological sample to be tested for target protein and target nucleic acid detection;

[0008] Step 2) reacting the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then reacting the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected;

[0009] Step 3) reacting the biological sample to be detected with a tyramide fluorescein substrate to allow the target protein to bind to the tyramide fluorescein;

[0010] Step 4) detecting the target protein in the biological sample to be detected by fluorescence signal;

[0011] Step 5) reacting the biological sample to be detected, for which the fluorescence signal detection in step 4) has been completed, with a targeting probe for binding to the target nucleic acid to form a target nucleic acid-targeting probe complex; then attaching a biochip to the biological sample to be detected, wherein the surface of the biochip in contact with the biological sample to be detected is provided with an array of detection points; each detection point in the array of detection points contains a capture probe for detecting the target nucleic acid;

[0012] Step 6) The biological sample to be detected is permeabilized; the target probe conjugate is bound to the capture probe on the biochip; the target probe on the biochip generates a complementary nucleotide chain through a two-strand synthesis reaction; the complementary nucleotide chain is subjected to PCR to construct a sequencing library and sequence the target nucleic acid.

[0013] Optionally, the biochip includes a carrier; the carrier is provided with detection points evenly arranged in a rectangular array to form a detection point array; each detection point contains a capture probe for detecting the target nucleic acid;

[0014] Preferably, the carrier is glass or plastic.

[0015] Optionally, the capture probe is a nucleic acid fragment comprising a PCR primer sequence, a spatial position sequence, a unique molecular identification tag sequence and a nucleic acid capture sequence.

[0016] Optionally, there are multiple target proteins, and after the first target protein is bound to tyramide fluorescein in step 2) and step 3), the biological sample to be detected is subjected to antigen repair treatment; and the detection steps of step 2) and step 3) are repeated for another undetected target protein;

[0017] Repeat until each target protein is bound to tyramide fluorescein; then proceed to step 4) detecting the target protein in the biological sample to be detected by fluorescence signal;

[0018] Preferably, the antigen repair treatment is to heat the biological sample to be detected in a sodium citrate repair solution by microwave.

[0019] Optionally, different target proteins may be bound to tyramide fluorescein with different emission wavelengths;

[0020] Preferably, the tyramide fluorescein substrate is a fluorescent dye Alexa Fluor TM 488、AlexaFluor TM 546、Alexa Fluor TM Tyramide labeled with one of TYR-647, TYR-520, TYR-690, and TYR-780.

[0021] Optionally, in step 5), a clamp is installed on the biochip, the clamp surrounding the detection point array to form a reaction pool on the detection point array; then, a probe hybridization reaction solution containing a targeting probe is added to the reaction pool; the targeting probe includes a left probe and a right probe, and the left probe and the right probe bind to the target nucleic acid to form a target nucleic acid-targeting probe complex;

[0022] In step 6), the card holder is removed, and the biological sample to be detected is permeabilized to allow the nucleic acid-targeting probe complex to permeate out of the cell and bind to the capture probe on the biochip;

[0023] Then, a fixed clamp is installed on the biochip, and a two-strand synthesis reaction solution is added to the reaction pool to carry out a two-strand synthesis reaction, so that the targeting probe on the biochip generates a complementary nucleotide chain;

[0024] Preferably, the clip is made of plastic, metal and / or rubber.

[0025] Optionally, the target nucleic acid is whole transcriptome mRNA.

[0026] Optionally, the step 4) further includes performing DAPI staining on the biological sample to be detected, detecting DAPI in the biological sample to be detected, performing cell segmentation using the DAPI fluorescence signal, and determining the distribution of cells on the biological sample.

[0027] Optionally, the method further includes step 7): confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescence signal.

[0028] Optionally, based on the detection results of the target protein, target nucleic acid and DAPI fluorescence signal, confirming the correlation between the target protein and the target nucleic acid includes: using the DAPI fluorescence signal to perform cell segmentation to determine the distribution of cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; based on the relevant maker genes expressing the protein, marking on the target nucleic acid expression information map to obtain the spatial distribution map of the maker gene; comparing the spatial position information of the cells expressing the protein with the consistency of the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

[0029] Optionally, based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescence signal, confirming the correlation between the target protein and the target nucleic acid includes: performing cell segmentation using the DAPI fluorescence signal to determine the distribution of cells on the biological sample; superimposing the cell segmentation result with the protein fluorescence signal result to confirm the cells expressing the protein and their distribution on the biological sample;

[0030] Based on the results of target nucleic acid expression information, dimensionality reduction cluster analysis is performed to determine the differentially expressed genes in each cell population of the biological sample and classify and identify the cell type of each cell population; protein-related maker genes are searched for among all differentially expressed genes in each cell population, and the related idle position information is annotated; the spatial location information of cells expressing the protein is compared with the consistency of the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

[0031] Optionally, the target proteins include CD31, CD45 and CK-PAN.

[0032] A spatial multi-omics detection system for biological samples, comprising:

[0033] The detection sample module is used to obtain the biological sample to be detected, which needs to detect the target protein and target nucleic acid;

[0034] An antibody binding module is used to react the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then react the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected;

[0035] A fluorescent labeling module, used for reacting the biological sample to be detected with a tyramide fluorescein substrate to bind the target protein to the tyramide fluorescein;

[0036] A fluorescence detection module, configured to detect the target protein in the biological sample to be detected through a fluorescence signal;

[0037] The probe binding module is used to react the biological sample to be detected after the fluorescent signal detection with the targeting probe used to bind to the target nucleic acid to form a target nucleic acid-targeting probe complex; a biochip is then attached to the biological sample to be detected, and the surface of the biochip that contacts the biological sample to be detected is provided with a detection point array; each detection point in the detection point array contains a capture probe for detecting the target nucleic acid;

[0038] The PCR detection module is used to permeabilize the biological sample to be detected; allow the nucleic acid-targeting probe complex to bind to the capture probe on the biochip; generate complementary nucleotide chains from the targeting probe on the biochip through a two-strand synthesis reaction; and then perform PCR on the complementary nucleotide chains to construct a sequencing library and sequence and detect the target nucleic acid.

[0039] Optionally, the fluorescence detection module is further used to perform DAPI staining on the biological sample to be detected, detect DAPI in the biological sample to be detected, perform cell segmentation using the DAPI fluorescence signal, and determine the distribution of cells on the biological sample.

[0040] Optionally, the system further includes a result analysis module for confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescence signal.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention uses multiple multiple immunohistochemistry (mIHC) methods for protein detection on the same biological sample, and then uses the spatial transcriptome probe sequencing technology for gene detection, which can obtain both proteome and transcriptome information. Compared with existing in situ sequencing and single-cell sequencing technologies, the spatial multi-omics technology proposed in this invention achieves the purpose of spatial multi-omics analysis, is more conducive to studying the correlation between organismal genes and proteins, and can better meet user needs.

[0043] The present invention proposes the use of multiple multiplex immunohistochemistry (mIHC) methods to detect proteins in biological samples and the use of spatial transcriptome sequencing methods to detect nucleic acids in the same biological sample. Because the mIHC method uses microwaves to treat the next round of protein detection reactions, the antibodies (primary and secondary antibodies) present in the previous round of protein detection reactions are removed. Therefore, compared with the IF method, this method has strong reaction specificity and weak background signals; each round of detection can use the same primary and secondary antibodies, which is not restricted by the antibody species and has lower costs. The mIHC method is also based on the signal of fluorescent dyes for imaging detection. However, since it is not restricted by the antibody species, this method can currently detect up to 6 proteins (excluding DAPI cell nuclear fluorescence staining), and the detection throughput is higher. In addition, the mIHC method uses the peroxidase reaction of tyramide to produce a large number of enzymatic products. This product can covalently bind to surrounding protein residues such as tyrosine residues. This will enrich a large number of fluorescent labeled molecules at the target detection protein, so that the fluorescent signal is effectively amplified, the detection sensitivity is higher, and it is more conducive to the detection of low-abundance proteins, especially at the single-cell level.

[0044] The mIHC method of the present invention uses imaging detection based on fluorescent dye signals, rather than sequencing detection, which allows for faster protein analysis results, making it easier for researchers to interpret results and determine whether to proceed to the next step of nucleic acid testing. This helps save experimental costs and shortens experimental cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation methods or the prior art. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 Schematic diagram of a carrier connected to a capture probe;

[0047] Figure 2 Schematic diagram of capture probe array on a carrier;

[0048] Figure 3 This is the HE staining result of human lung cancer tissue;

[0049] Figure 4 This is a diagram showing the results of triple protein (CD31, CD45, CK-PAN) and cell nucleus fluorescence detection in human lung cancer tissue;

[0050] Figure 5 This is the spatial distribution result of cells expressing CD31, CD45, and CK-PAN proteins in human lung cancer tissue;

[0051] Figure 6 This is the result of dimensionality reduction clustering analysis of human lung cancer tissue cells;

[0052] Figure 7 This is the first joint analysis process of protein and gene expression information;

[0053] Figure 8 The second is a joint analysis process of protein and gene expression information;

[0054] Figure 9 Schematic diagram of the clip structure;

[0055] Figure 10 This is the spatial distribution map of the maker gene PECAM1 corresponding to the CD31 protein;

[0056] Figure 11 This is the spatial distribution map of the maker gene PTPRC corresponding to the CD45 protein;

[0057] Figure 12 This is the spatial distribution map of the maker genes CSNK1A1, CSNK2A1, KRT5, KRT7, and KRT8 corresponding to the CK-PAN protein. DETAILED DESCRIPTION

[0058] Now, various exemplary embodiments of the present invention are described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0059] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0060] The present invention proposes a spatial multi-omics detection method for biological samples, comprising the following steps:

[0061] Step 1) attaching a biological sample to be tested, in which target proteins and target nucleic acids are to be detected, to a biochip, wherein a detection point array is provided on the surface of the biochip in contact with the biological sample to be tested; each detection point in the detection point array contains a capture probe for detecting the target nucleic acid;

[0062] Step 2) reacting the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then reacting the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected;

[0063] Step 3) reacting the biological sample to be detected with a tyramide fluorescein substrate to allow the target protein to bind to the tyramide fluorescein;

[0064] Step 4) detecting the target protein in the biological sample to be detected by fluorescence signal;

[0065] Step 5) reacting the biological sample to be tested, on which the fluorescence signal detection is completed in step 4), with a targeting probe for binding to the target nucleic acid to obtain a target nucleic acid-targeting probe complex;

[0066] Step 6) The biological sample to be detected is permeabilized; the nucleic acid-targeting probe complex is allowed to bind to the capture probe on the biochip; the targeting probe on the biochip generates a complementary nucleotide chain through a two-strand synthesis reaction; the complementary nucleotide chain is then subjected to PCR to construct a sequencing library and sequence the target nucleic acid.

[0067] The present invention utilizes horseradish peroxidase (HRP) to label a target protein (also referred to as an antigen) using an enzymatic detection method, also known as the tyramide signal amplification (TSA) method. During detection, an antigen-specific primary antibody is first bound to the antigen, and then an HRP-labeled secondary antibody is bound to the primary antibody. When a tyramide fluorescein substrate is added to the reaction system, HRP catalyzes the substrate to produce an activated fluorescent substrate. The activated substrate can covalently bind to residues such as tyrosine on the antigen, resulting in stable covalently bound tyramide fluorescein on the biological sample. Subsequently, the non-covalently bound primary antibody-secondary antibody-HRP complex is washed away using an antigen retrieval method, and the above-mentioned primary antibody-HRP-labeled secondary antibody-tyramide fluorescein substrate reaction step is repeated to label another target protein. Therefore, this reciprocating process can achieve multiple labeling.

[0068] The target protein refers to a protein to be detected in a biological sample, which may be located inside and / or outside the cell.

[0069] The biological sample can be a tissue slice, organ slice, or cell aggregate slice; it can be a sample embedded using methods such as FFPE, FF, or FXF. During the experiment, the biological sample is first attached to a glass slide. Alternatively, the glass slide can be a standard glass slide.

[0070] In particular, when the biological sample is fixed with formaldehyde, paraformaldehyde, methanol, ethanol, or the like, such as FFPE samples fixed with paraformaldehyde, proteins cross-link with the paraformaldehyde to form a network structure. To achieve protein detection, the biological sample must first be subjected to antigen retrieval. This antigen retrieval is similar to the following method for removing antibodies using antigen retrieval.

[0071] The primary antibody is a protein or protein derivative that can specifically recognize and bind to the target protein. As a preferred embodiment, the primary antibody can be a protein modified by a chemical substance, such as a biotin-labeled primary antibody, an oligonucleotide-labeled primary antibody, etc.

[0072] The secondary antibody is a protein or protein derivative that can specifically recognize and bind to the primary antibody. As a preferred embodiment, the secondary antibody can be a protein modified by a chemical substance, such as an HRP-labeled secondary antibody, a poly-HRP-labeled secondary antibody, a streptavidin-HRP-labeled secondary antibody, etc.

[0073] As an optional solution, after the primary antibody specifically binds to the target protein, an HRP-labeled secondary antibody is used to bind to the primary antibody, ultimately forming a target protein-primary antibody-HRP-labeled secondary antibody complex.

[0074] As an optional solution, since one biotin molecule can bind to four streptavidin molecules, when the biotin-labeled primary antibody specifically binds to the target protein, multiple streptavidin-HRP-labeled secondary antibodies will be linked to the target protein, thereby providing more HRP reaction activity.

[0075] When a tyramide fluorescein substrate is added to the target protein-primary antibody-HRP-labeled secondary antibody complex system, due to the catalytic activity of HRP, it catalyzes the tyramide fluorescein substrate to produce an activated fluorescent substrate. The activated substrate can covalently bind to residues such as tyrosine on the target protein, thereby stably covalently binding tyramide fluorescein to the biological sample.

[0076] It is understood that when multiple protein detection is required for the same biological sample, such as duplex, triplex, quadruplex, quintuplex, sextuplex, etc., multiple rounds of target protein and primary antibody, primary antibody and HRP-labeled secondary antibody reaction are required to form a variety of target protein-primary antibody-HRP-labeled secondary antibody complexes. In addition, when each target protein-primary antibody-HRP-labeled secondary antibody complex is formed, a tyramide fluorescein substrate with a different fluorescence emission wavelength is added to the system, so that HRP catalyzes the tyramide fluorescein substrate to form an activated fluorescent substrate, and binds to the target protein, and finally each target protein is labeled with a different fluorescent substrate.

[0077] As an alternative approach, when triple protein detection is required on the same biological sample, the first primary antibody is first bound to the first target protein, followed by the first HRP-labeled secondary antibody, forming a first target protein-first primary antibody-first HRP-labeled secondary antibody complex (referred to as the first antigen-antibody complex). When a first tyramide fluorescein substrate is added to the reaction system, HRP catalyzes the substrate to produce an activated fluorescent substrate, which covalently binds to tyrosine residues, such as tyrosine residues, on the first target protein, resulting in a stable covalent binding of the first tyramide fluorescein to the biological sample. Subsequently, an antigen retrieval method is used to remove the non-covalently bound first primary antibody-first HRP-labeled secondary antibody complex. Similarly, a second primary antibody is bound to the second target protein, and a second HRP-labeled secondary antibody is bound to the second primary antibody, forming a second antigen-antibody complex. A second tyramide fluorescein substrate is added to the reaction system, and HRP catalyzes the production of an activated fluorescent substrate, which covalently binds to tyrosine residues, such as tyrosine residues, on the second target protein. Subsequently, an antigen retrieval method is used to remove the second primary antibody-second HRP-labeled secondary antibody complex. Similarly, a third primary antibody is used to bind to the third target protein, and a third HRP-labeled secondary antibody is used to bind to the third primary antibody to form a third antigen-antibody complex. A third tyramide fluorescein substrate is added to the reaction system, and an activated fluorescent substrate is generated under HRP catalysis to covalently bind to tyrosine residues on the third target protein. The third primary antibody-second HRP-labeled secondary antibody complex is then washed away using an antigen retrieval method. After three rounds of this reaction, the first target protein can be labeled with the first tyramide fluorescein, the second target protein can be labeled with the second tyramide fluorescein, and the third target protein can be labeled with the third tyramide fluorescein on the same biological sample. Therefore, when a fluorescence microscope is used to capture the fluorescent signal of the biological sample, triple protein detection can be achieved on the same biological sample.

[0078] The tyramide fluorescein substrate refers to tyramide labeled with a substance that can emit a fluorescent signal under irradiation with a specific wavelength of light, such as fluorescein or fluorescent dye. As an optional solution, the tyramide fluorescein substrate is a fluorescent dye AlexaFluor TM 488、Alexa Fluor TM 546、Alexa Fluor TM 647, TYR-520, TYR-690, TYR-780, etc. It is understood that when it is necessary to perform double protein detection on the same biological sample, the emission wavelengths of the fluorescent dyes used to label tyramide must be different to prevent the fluorescence signals from interfering with each other. As a solution for the above triple protein detection, the first tyramide fluorescein substrate, the second tyramide fluorescein substrate, and the third tyramide fluorescein substrate can be Alexa Fluor TM 488-labeled tyramide, Alexa Fluor TM 546 labeled with tyramide, Alexa FluorTM 647 labeled with tyramide.

[0079] The antigen repair refers to the process of re-exposing or correcting the antigen by using chemical reagents and / or heat. Since the primary antibody and the target protein are mainly bound by non-covalent means such as van der Waals forces and hydrogen bonds, and the tyramide fluorescein and the target protein are bound by covalent bonds, the antigen repair method can be used to remove the reacted antibody while retaining the tyramide fluorescein. As an optional solution, the antigen repair method can be one or more of microwave, boiling, and water bath. Since the antibodies bound to the previous round of reaction are washed and removed before each round of reaction, only a single antibody is incubated in each round of reaction system. There is no need to worry about antibody cross-reactions and species matching issues of primary and secondary antibodies, which greatly reduces the restrictions on the selection and matching of antibodies of different species during experimental design.

[0080] The fluorescence microscope can be used to capture fluorescent signals from a biological sample and generate visual data. As a preferred approach, once the visual data, such as image-formatted data, is obtained, bioinformatics tools can be used to further analyze the data to understand the distribution of cells expressing the target protein in the biological sample and the expression level of the target protein.

[0081] As an optional solution, after the protein is labeled with tyramide fluorescein, dyes can be used to label the cell nucleus, cytoplasm, cell membrane and / or its internal substances such as RNA in the cytoplasm, so that the spatial location of the cells in the biological sample can be distinguished during data analysis.

[0082] As a preferred method, DAPI fluorescent dye can be used to stain the cell nuclei of the biological sample.

[0083] On the one hand, biological samples, especially clinical FFPE embedded samples, are stored for a long time so that RNA is severely degraded; on the other hand, after the biological sample is detected by protein, RNA will also be further degraded. For example, the mRNA expressed by most eukaryotic organisms contains a poly A sequence, and then when the mRNA is degraded, the poly A sequence may be lost. Therefore, it is impossible to capture mRNA by conventional poly T in combination with poly A to achieve spatial transcriptome sequencing. For this reason, the present invention adopts spatial transcriptome probe method sequencing technology to detect the gene expression information of biological samples.

[0084] Spatial transcriptome probe sequencing involves designing a set of probes that bind to transcriptome RNA related to protein expression and then bind to target genes in a biological sample. The probes are then permeabilized to penetrate tissue cells and captured by a biochip equipped with capture probes. A sequencing library is then constructed using DNA synthesis, and gene expression information is obtained through sequencing and bioinformatics analysis.

[0085] The probe is a group of oligonucleotides designed based on the RNA of an organism, including humans, mice, monkeys, fish, plants, etc. It is understandable that since different organisms express different RNAs, a group of probes needs to be designed for each organism. As an optional solution, the probe is a group of oligonucleotides designed based on the whole transcriptome mRNA. As a preferred solution, the probe is an oligonucleotide designed based on the target gene expressed by protein on mRNA, and each target gene contains at least a pair of targeting probes. The targeting probe includes a left-end probe and a right-end probe, which, after binding to the target gene, form a sequence that is completely complementary to the target gene under the action of DNA ligase.

[0086] The left probe consists of a nucleotide sequence 1 complementary to the targeted gene and a Read 2 nucleotide sequence; the right probe consists of a nucleotide sequence 2 complementary to the targeted gene and a poly A nucleotide sequence. When binding to the targeted gene, nucleotide sequence 1 of the left probe binds to the left side of the targeted gene, and nucleotide sequence 2 of the right probe binds to the right side of the targeted gene. As a preferred embodiment, nucleotide sequence 2 is phosphorylated at the 5' end so that, under the action of DNA ligase, nucleotide sequence 1 and nucleotide sequence 2 form a complete complementary sequence of the targeted gene.

[0087] The Read 2 nucleotide sequence is a base sequence that can be compatible with a sequencer, and the number of bases can be 10 to 50. As an example, the Read 2 nucleotide sequence can be compatible with an Illumina sequencer, and its sequence is: 5'-GAGTTCCTTGGCACCCGAGAATTCCA-3' (SEQ ID NO: 25).

[0088] The nucleotide sequence 1 and the nucleotide sequence 2 can be complementary to the bases of the targeted gene sequence, and the number of bases can be 10 to 80. As a preferred embodiment, the number of bases can be 20 to 40. For each targeted gene sequence, one or more pairs of the nucleotide sequence 1 and the nucleotide sequence 2 can be designed. When multiple pairs of the nucleotide sequence 1 and the nucleotide sequence 2 are designed, different positions on a gene can be paired with different probes, further improving the detection ability of the targeted gene. As an embodiment, for the genes corresponding to the proteins CD31, CD45, and CK-PAN to be tested, the following nucleotide sequences (sequences 1 to 24) can be designed, as shown in Table 1.

[0089] Table 1

[0090]

[0091] The poly A nucleotide sequence is a sequence containing multiple A bases. As a preferred embodiment, the poly A nucleotide sequence is a sequence containing 30 consecutive A bases.

[0092] The DNA ligase ligates the 3'-terminal hydroxyl group of the DNA chain and the adjacent 5'-terminal phosphate group to form a phosphodiester bond, thereby connecting two adjacent DNA chains. As an optional solution, the DNA ligase can be one or more of T4 ligase, Splint R ligase, Taq enzyme, etc.

[0093] The biochip comprises a carrier and a capture probe located on the carrier.

[0094] The carrier can be made of glass, plastic, etc., and its external dimensions can be customized according to experimental needs. As an embodiment, the carrier is glass, and its external dimensions are the size of a conventional glass slide 25mm×75mm, so as to have good compatibility with microscopes and other photographic equipment.

[0095] The capture probe is a nucleic acid fragment containing at least a spatial position sequence and a nucleic acid capture sequence, which can be directly or indirectly bound to the carrier in a certain arrangement order. As an embodiment, the capture probe is connected to the surface of the carrier by chemical coupling and forms a capture probe sopt point array. Each spot point on the point array is composed of capture probes with the same spatial position sequence, and the spot point can be square or circular. As an embodiment, when the spot point can be square, its size can be any one of 50um×50um, 30um×30um, 20um×20um, 15um×15um, 10um×10um, 8um×8um, 5um×5um, 2um×2um, etc. The spatial position sequence is known and can be used to define the plane coordinate (X-axis, Y-axis) position of each spot point on the point array, so the spatial position sequence of the capture probe of each spot point is different. The nucleic acid capture sequence can undergo base complementary pairing with the nucleic acid released from the cell, thereby enabling the capture probe to capture the nucleic acid.

[0096] As a preferred embodiment, the capture probe is a nucleic acid fragment containing at least a PCR primer sequence, a spatial position sequence, a unique molecular identification tag sequence, and a nucleic acid capture sequence, which can be directly or indirectly bound to the carrier in a certain arrangement order. The PCR primer sequence is a known sequence, and the PCR primer sequence of each capture probe can be the same or different. The unique molecular identification tag sequence is used to count the gene expression information of the cell during bioinformatics analysis, and the unique molecular identification tag sequence of each capture probe is different.

[0097] As an alternative, the capture probe comprises a nucleic acid fragment consisting of a PCR primer sequence, a spatial position sequence, a unique molecular identification tag sequence, and a nucleic acid capture sequence, connected sequentially from the 5' end to the 3' end. The nucleic acid capture sequence is a sequence containing multiple T bases that can capture mRNA or mRNA reaction products containing multiple A bases released from cells through complementary base pairing. As a preferred embodiment, the nucleic acid capture sequence is a sequence containing 30 consecutive T bases.

[0098] As an optional solution, after the biological sample undergoes multiple (eg, triple) protein detection through the above steps, the following spatial transcriptome probe sequencing technology can be used to complete gene expression information detection for the same biological sample.

[0099] The biological sample is stained with HE staining, and then photographed and imaged under a microscope under bright field. The glass slide is installed in a supporting card holder so that the front of the biological sample faces upward and is surrounded by the silicone pad of the supporting card holder to form a chamber that can accommodate reaction reagents. Probe hybridization reaction solution is added to the chamber so that the left probe and the right probe are combined with the target gene. Under the action of DNA ligase, the 3' end of the left probe and the 5' end of the right probe are connected together to form a complete target probe. Subsequently, on a DynaBlot instrument (produced by Suzhou Deyun Kangrui Biotechnology Co., Ltd.), the target probe in the biological sample is permeated from the cell by permeabilization and captured by the biochip with capture probe. The biochip is installed in a supporting card holder so that the front of the biochip faces upward and is surrounded by the silicone pad of the supporting card holder to form a chamber that can accommodate reaction reagents. Two-strand synthesis reaction solution is added to the chamber so that the target probe captured by the biochip generates a complementary oligonucleotide chain. Oligonucleotide chains are recovered from the biochip, a sequencing library is constructed by PCR, and gene expression information can be obtained by sequencing and bioinformatics analysis.

[0100] The matching card holder is a fixture made of plastic, metal and / or rubber, with a cavity inside to allow the glass sheet or biochip to be installed and fixed, and a space is left above the biological sample to allow reagents to be added to the card holder for reaction.

[0101] The probe hybridization reaction solution at least includes the left end probe, the right end probe, a salt solution, an organic reagent, etc. The two-strand synthesis reaction solution at least includes enzyme-free water, dNTP, two-strand synthase, primers, salt ions, etc.

[0102] The gene expression information includes but is not limited to gene types, gene expression abundance, cell types and / or their spatial location information on the biological sample.

[0103] For the same biological sample, when protein detection and gene detection are completed, in addition to the above data analysis, a computer algorithm can also be used to jointly analyze the results of protein and gene detection. The computer method includes but is not limited to the following process (such as Figure 7As shown): Use DAPI fluorescence signal to perform cell segmentation to determine the distribution of cells on the biological sample; superimpose the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; based on the related maker genes expressing the protein, mark them on the spatial transcriptome gene expression information map to obtain the spatial distribution map of the maker gene; compare the spatial location information of the cells expressing the protein with the consistency of the spatial distribution of the maker gene to confirm the correlation between the protein and the gene. As another solution, the computer method includes but is not limited to the following process (as shown Figure 8 (as shown): using DAPI fluorescence signals to perform cell segmentation and determine the distribution of cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; performing dimensionality reduction cluster analysis based on the results of spatial transcriptome gene expression information to determine the differentially expressed genes of each cell population (Cluster) of the biological sample, and classifying and identifying the cell type of each cell population (Cluster); searching for protein-related maker genes among all differentially expressed genes in each Cluster, and marking the related idle position information; comparing the consistency of the spatial position information of the cells expressing the protein with the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

[0104] For the same biological sample, protein detection using the mIHC method is followed by gene detection using the spatial transcriptome probe sequencing technology, providing both proteomic and transcriptomic information. Compared to existing in situ sequencing and single-cell sequencing technologies, the spatial multi-omics technology proposed in this invention achieves the purpose of spatial multi-omics analysis, is more conducive to studying the correlation between genes and proteins in an organism, and is more responsive to user needs.

[0105] The present invention also proposes a spatial multi-omics detection system for biological samples developed based on the above method, comprising:

[0106] The detection sample module is used to obtain the biological sample to be detected, which needs to detect the target protein and target nucleic acid;

[0107] An antibody binding module is used to react the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then react the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected;

[0108] A fluorescent labeling module, used for reacting the biological sample to be detected with a tyramide fluorescein substrate to bind the target protein to the tyramide fluorescein;

[0109] A fluorescence detection module, configured to detect the target protein in the biological sample to be detected through a fluorescence signal;

[0110] The probe binding module is used to react the biological sample to be detected after the fluorescent signal detection with the targeting probe used to bind to the target nucleic acid to form a target nucleic acid-targeting probe complex; a biochip is then attached to the biological sample to be detected, and the surface of the biochip that contacts the biological sample to be detected is provided with a detection point array; each detection point in the detection point array contains a capture probe for detecting the target nucleic acid;

[0111] The PCR detection module is used to permeabilize the biological sample to be detected; allow the nucleic acid-targeting probe complex to bind to the capture probe on the biochip; generate complementary nucleotide chains from the targeting probe on the biochip through a two-strand synthesis reaction; and then perform PCR on the complementary nucleotide chains to construct a sequencing library and sequence and detect the target nucleic acid.

[0112] The fluorescence detection module is further used to perform DAPI staining on the biological sample to be detected, detect DAPI in the biological sample to be detected, perform cell segmentation using the DAPI fluorescence signal, and determine the distribution of cells on the biological sample.

[0113] The system also includes a result analysis module for confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, target nucleic acid and DAPI fluorescence signal

[0114] Example 1

[0115] As a specific embodiment, the present invention uses multiple immunohistochemistry to perform triple protein (CD31, CD45, CK-PAN) detection on formalin-fixed paraffin-embedded (FFPE) human lung cancer tissue sections, and then uses spatial transcriptome probe sequencing technology to detect the transcriptome gene expression information of the same tissue section. In medicine, CD31 protein is mainly used to prove the presence of endothelial cell tissue and to evaluate tumor angiogenesis; CD45 protein is expressed by white blood cells such as T cells and is of great significance in measuring the effect of tumor immunotherapy; CK-PAN protein belongs to cytokeratin and can be used to detect whether there are epithelial cell markers on the surface of lung cancer cells, thereby determining whether the tumor is epithelial. Therefore, it is of great significance to jointly detect the three proteins CD31, CD45, and CK-PAN and related transcriptome information. The primary and secondary antibodies used in the examples are from Thermo Fisher or Biolegend.

[0116] A schematic cross-sectional view of a biochip with capture probes is shown in FIG. Figure 1 As shown. The carrier 1000 of the biochip is a transparent glass with carboxyl chemical functional groups on the surface, and its size is 25mm×75mm. Using micro-nano processing technology and DNA probe connection technology, a patterned capture probe spot array can be formed on the surface of the carrier 1000, such as Figure 2 As shown. The capture probe is a nucleic acid fragment formed by sequentially connecting a PCR primer sequence 1001, spatial position sequences 1002 and 1003, a unique molecular identification tag sequence 1004, and a nucleic acid capture sequence 1005 from the 5' end to the 3' end. The spot formed by the capture probe is 15um × 15um square, and the spacing between adjacent spots is 15um. Each spot contains tens of thousands of capture probes, and the plane coordinate position of each spot is defined by the spatial position sequences 1002 and 1003. Therefore, it can be understood that one of the spatial position sequences 1002 and 1003 is used to define the X-axis coordinate, and the other sequence defines the Y-axis coordinate. For ease of understanding, the spatial position sequence 1002 is used to define the X-axis coordinate, and the spatial position sequence 1003 is used to define the Y-axis coordinate. Therefore, the plane coordinates of each spot point are (Xi, Yi), where i is a natural number, which can be 0, 1, 2, 3, 4, 5, ..., 50, 60, ..., 74, 75. In this embodiment, the spatial position sequence 1002 and the spatial position sequence 1003 each contain 34 bases. The PCR primer sequence 1001 in this embodiment is CTACACGACGCTCTTCCGATCT. The nucleic acid capture sequence 1005 in this embodiment: 30 T bases.

[0117] FFPE-embedded human lung cancer tissue was sectioned using a rotary microtome to a thickness of 5 μm. The sections were then attached to standard glass slides, centered as closely as possible on the slide's geometric center. The sections were dewaxed and hydrated at room temperature in the following sequence: xylene I for 15 minutes, xylene II for 10 minutes, anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, 70% ethanol for 2 minutes, and sterile, enzyme-free water for 5 minutes. The sections were placed in a glass jar filled with sodium citrate repair solution (pH 6.0) and heated in a microwave oven on medium heat for 8 minutes until the solution boiled. The solution was then kept warm for 8 minutes and then heated on low heat for 7 minutes.

[0118] Remove the tissue sections and allow them to cool naturally at room temperature. Wash the sections twice in 1× PBS (pH 7.4) on a decolorizing shaker for 5 minutes each, followed by a final wash in 1× PBST for 5 minutes. Incubate the sections in 3% hydrogen peroxide solution at room temperature in the dark for 60 minutes, then wash them three times in 1× PBS (pH 7.4) on a decolorizing shaker for 5 minutes each. After drying the sections with a slide centrifuge, draw a circle around the sections with a histochemical pen to prevent subsequent reaction solutions from escaping. Block the sections by adding 10% goat serum to the circle, evenly covering the tissue, and incubating at room temperature for 60 minutes. Remove the blocking solution, apply anti-CD31 rabbit primary antibody diluted in 10% goat serum to the sections, and incubate them in a light-proof, humidified chamber at room temperature for 60 minutes. Remove the reagents, and wash the sections three times in 1× PBS (pH 7.4) on a decolorizing shaker for 10 minutes each. After drying the tissue sections slightly, add HRP-labeled goat anti-rabbit secondary antibody that specifically binds to the anti-CD31 rabbit primary antibody to cover the tissue, incubate at room temperature in the dark for 60 minutes, then remove the reagent and wash three times with 1× PBS (pH = 7.4), each time for 10 minutes. TM 647-labeled tyramide fluorescein substrate was incubated at room temperature in the dark for 10 minutes. The reagent was then removed and the sections were washed three times with 1× PBS (pH = 7.4), each for 10 minutes. The tissue sections were placed in a glass jar filled with sodium citrate repair solution (pH = 6.0) and heated in a microwave oven on high heat for 2 minutes until the solution boiled, then reduced to low heat for 15 minutes.

[0119] Remove the tissue sections and, after cooling naturally at room temperature, place the tissue sections in 1×PBS (pH=7.4) and wash them twice with shaking on a decolorizing shaker for 5 minutes each time, and finally wash them with 1×PBST for 5 minutes. Place the tissue sections in 3% hydrogen peroxide solution and incubate them in the dark for 60 minutes at room temperature. Then, place them in 1×PBS (pH=7.4) and wash them three times with shaking on a decolorizing shaker for 5 minutes each time. After drying the water droplets on the tissue sections with a slide centrifuge, add 10% goat serum in the circle to evenly cover the tissue and incubate at room temperature for 60 minutes. Remove the blocking solution and add anti-CD45 rabbit primary antibody diluted with 10% goat serum to the tissue sections. Place them in a light-proof humidified box and incubate them at room temperature for 60 minutes. Remove the reagents and wash the tissue sections three times with shaking in 1×PBS (pH=7.4) on a decolorizing shaker for 10 minutes each time. After drying the tissue sections slightly, add HRP-labeled goat anti-rabbit secondary antibody that specifically binds to the anti-CD45 rabbit primary antibody to cover the tissue, incubate at room temperature in the dark for 60 minutes, then remove the reagent and wash three times with 1× PBS (pH = 7.4), each time for 10 minutes. TM 546-labeled tyramide fluorescein substrate was incubated at room temperature in the dark for 5 minutes. The reagent was then removed and the sections were washed three times with 1× PBS (pH = 7.4), each for 10 minutes. The tissue sections were placed in a glass jar filled with sodium citrate repair solution (pH = 6.0) and heated in a microwave oven on high heat for 2 minutes until the solution boiled, then reduced to low heat for 15 minutes.

[0120] Remove the slides and allow them to cool naturally at room temperature. Wash the sections twice with 1× PBS (pH 7.4) on a decolorizing shaker for 5 minutes each, followed by a final wash with 1× PBST for 5 minutes. Place the sections in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 60 minutes. Wash them three times with 1× PBS (pH 7.4) on a decolorizing shaker for 5 minutes each. Dry the sections with a slide centrifuge. Add 10% goat serum to the circle to evenly cover the tissue and incubate at room temperature for 60 minutes. Remove the blocking solution and add anti-CK-PAN rabbit primary antibody diluted in 10% goat serum to the sections. Incubate them in a light-proof, humidified chamber at room temperature for 60 minutes. Remove the reagents and wash the sections three times with 1× PBS (pH 7.4) on a decolorizing shaker for 10 minutes each. After drying the tissue sections slightly, add HRP-labeled goat anti-rabbit secondary antibody that specifically binds to the anti-CK-PAN rabbit primary antibody to cover the tissue, incubate at room temperature in the dark for 60 minutes, then remove the reagent and wash three times with 1× PBS (pH = 7.4), each time for 10 minutes. TM488 labeled tyramide fluorescein substrate, incubate at room temperature in the dark for 10 minutes, then remove the reagent and wash three times with 1×PBS (pH=7.4), each time for 10 minutes. After slightly drying the tissue slices, DAPI staining solution was added to the circle, incubated at room temperature in the dark for 10 minutes, then remove the reagent and wash three times with 1×PBS (pH=7.4), each time for 5 minutes. Then slightly dry the tissue slices, add SlowFade anti-fluorescence quenching sealing agent to the circle and seal the slices. Use a multi-channel 3D fluorescence scanner to observe and collect images, and obtain the fluorescence detection results of triple proteins (CD31, CD45, CK-PAN) in human lung cancer tissue slices, as shown below. Figure 4 As shown. Figure 4 In the tissue sections, DAPI-stained cell nuclei were detected using the DAPI fluorescence detection channel, and Alexa Fluor TM 647-labeled CD31 protein was detected using the CY5 fluorescence detection channel and Alexa Fluor TM 546-labeled CD45 protein was detected using the CY3 fluorescence detection channel and Alexa Fluor TM 488 labeled CK-PAN protein was detected using FITC fluorescence detection channel. In order to further determine the spatial distribution of cells expressing CD31, CD45, and CK-PAN proteins on tissue sections, bioinformatics was used to further analyze the data, and the following results were obtained: Figure 5 The results shown. Figure 5 In the analysis, the location of cells expressing related proteins on the tissue slice can be clearly known. Therefore, based on this information, the experimenter can select a specific target area (such as a region rich in cells expressing CK-PAN protein) or the entire tissue slice area for the next step of spatial transcriptome gene expression information detection. It is understandable that in some cases, when the experimenter finds that there are no cells expressing related proteins on the tissue slice, such as no cells expressing CK-PAN protein, the experimenter can determine in advance whether to continue with the next step of spatial transcriptome gene expression information detection.

[0121] After the protein detection is completed, the spatial transcriptome probe method is used to detect the transcriptome gene expression information of the tissue section. Unless otherwise specified, the relevant reagents are derived from the DynaSpatial FFPE Gene Expression Reagents Kit produced by Suzhou Deyun Kangrui Biotechnology Co., Ltd. The specific operation method is: first, wash the sealed tissue sections three times with 1×PBS (pH=7.4), each time for 5 minutes to remove the sealing coverslip and sealing agent. After drying, add Hematoxylin solution to the tissue sections to completely cover the tissue, incubate at room temperature for 3 minutes, wash with enzyme-free water 3 times, and then add Bluing Buffer solution and incubate at room temperature for 2 minutes. After washing with enzyme-free water twice, add freshly prepared alcohol-soluble eosin solution to the tissue sections, incubate at room temperature for 1 minute, then wash with enzyme-free water twice, and centrifuge on a slide centrifuge until there are no visible water droplets remaining on the surface of the tissue sections. Add 85% glycerol solution to the tissue sections to evenly cover the tissue and complete the sealing. Carefully place it under an optical microscope to complete the photographic imaging of the tissue morphology, and obtain the following Figure 3 The HE staining results are shown in the figure. To facilitate subsequent bioinformatics analysis, the images were taken with a 10x objective lens, and the saved images were at least 4000 × 4000 pixels in tif format.

[0122] Carefully wash the tissue sections in enzyme-free water, remove the coverslip and glycerol, and then mount the slide containing the tissue sections in the matching card holder, with the tissue sections facing up and centered in the card holder chamber. Figure 9 As shown, the matching card clip is a fixture made of plastic, metal and / or rubber material, with a cavity inside, which allows the glass slide or biochip to be installed and fixed, and leaves space above the biological sample to allow reagents to be added to the card clip for reaction. Add 200 μL of 0.1M HCl solution to the chamber to evenly cover the tissue and seal it with a sealing film, then place it on the PCR adapter in the PCR instrument and incubate at 37°C for 3 minutes. Tear off the sealing film, remove the solution, add 200 μL of 0.01M sodium citrate solution (pH = 6.0) and seal it with a sealing film, then place it on the PCR adapter in the PCR instrument, incubate at 95°C for 60 minutes and incubate at 22°C for 10 minutes.

[0123] After the reaction is complete, remove the sealing film, remove the solution, add 200 μL of 1× PBS-0.05% Tween 20 solution, and incubate at room temperature for 15 min. Remove the solution and wash once with 2× SSC solution. Then add 100 μL of probe hybridization reaction solution [10 μL Nuclease-free Water, 70 μL Probe-Hyb buffer, 10 μL LHS primer (Human), 10 μL LHS primer (Human)], apply the sealing film, place on the PCR adapter in the thermal cycler, and incubate at 50°C for 18 h.

[0124] After the reaction is complete, wash the tube three times with 200 μL of 2× SSC (50% formamide) solution, then add 200 μL of 2× SSC and incubate at room temperature for 3 minutes. Remove the solution and add 100 μL of probe ligation reaction solution [40 μL Nuclease-free Water, 50 μL 2× Ligase Buffer, 10 μL Ligase Enzyme]. Apply sealing film and place the tube on the PCR adapter in a thermal cycler. Incubate at 37°C for 1 hour.

[0125] After the reaction is completed, wash with 2×SSC (50% formamide) solution three times, 200 μL each time, incubate at 57°C for 5 minutes, and finally add 200 μL 2×SSC and incubate at room temperature for 5 minutes. Remove the solution, remove the slide from the card holder, add 150 μL 10% eosin solution to the tissue section, incubate at room temperature for 1 minute, then remove the solution and wash the tissue section with 1×PBS solution. According to the instruction manual of the DynaBlot instrument produced by Suzhou Deyun Kangrui Biotechnology Co., Ltd., the slide and biochip are mounted on the instrument, and then 30 μL probe transfer reaction solution [10 μL Nuclease-free water, 15 μL 2×Transfer buffer, 3 μL Transfer enzyme 1, 2 μL Transfer enzyme 2] is added to the tissue section to attach the biochip to the tissue section, and the instrument is started to start the reaction. After incubating at 37°C for 30 minutes, remove the biochip and carefully rinse the coding region three times with 1000 μL of 2× SSC. Shake dry and place in the included cartridge. Add 100 μL of probe extension reaction solution (40 μL Nuclease-free water, 50 μL 2× Extension buffer, 10 μL Transferase 2) to the biochip, apply a sealing film, and place it on the PCR adapter in a thermal cycler. Incubate at 37°C for 1 hour. Remove the solution, rinse three times with 200 μL of Buffer EB, and air-dry at room temperature until no visible liquid remains. Add 50 μL of 80 mM KOH to the biochip and incubate at room temperature for 10 minutes. Then, aspirate 48 μL of the solution into a centrifuge tube and add 7 μL of Tris-HCl (1 M, pH 7.0) to the tube. Mix thoroughly by pipetting and set aside.

[0126] Add 60uL of 2×PCR Mix and 5uL of PCR Primers to the above centrifuge tube, mix thoroughly by pipetting, and divide equally into two centrifuge tubes. Place the tubes on a PCR instrument for reaction. The reaction procedure is shown in Table 2.

[0127] Table 2

[0128]

[0129] After the reaction was completed, the PCR product was purified using 1.8× DNA purification magnetic beads, and the purified magnetic beads were eluted with 41 uL of enzyme-free water to obtain the target DNA solution.

[0130] To obtain library DNA, the target DNA solution was first measured using a qPCR instrument (SYBR Green assay). The target DNA solution was then amplified again using PCR according to the following protocol. The PCR reaction system consisted of 10 μL of the target DNA solution, 25 μL of 2× PCR Mix, and 15 μL of Index Primers. The reaction procedure is shown in Table 3.

[0131] Table 3

[0132]

[0133] After the reaction was completed, the PCR product was purified using 1.2× DNA purification magnetic beads, and the purified magnetic beads were eluted with 31 μL of enzyme-free water to obtain a library DNA solution.

[0134] When the library DNA was sequenced using an Illumina second-generation sequencing instrument and the sequencing results were analyzed using a bioinformatics analysis algorithm, the statistical results of the gene expression information of the tissue sections were obtained, as shown in Table 4. The results show that although the human lung cancer tissue samples used in this embodiment were processed by FFPE and had undergone multiple protein tests before spatial transcriptome detection, there is a possibility of high mRNA degradation, but from the indicators of Median_Genes_per_Spot and Reads_Mapped_to_Probe_Set, it can be seen that the technology of the present invention can still obtain better gene detection results, which will be of great significance in clinical and scientific research. Further, the gene expression information results can be subjected to dimensionality reduction clustering analysis, such as Figure 6 As shown, it can be seen that the sample is composed of five major cell types and their distribution in the tissue section area.

[0135] Table 4 Statistical results of gene expression information in human lung cancer tissues

[0136] Number_of_Reads 36,415,628 GC_Content 48.60％ Q20_Bases_in_RNA_Read 98.79％ Q30_Bases_in_RNA_Read 96.96％ Number_of_Spots_Under_Tissue 1,516 Fraction_Reads_in_Spots_Under_Tissue 50.47％ UMIs_in_Spots 2,312,729 Mean_Reads_per_Spot 24,020 Median_UMI_Counts_per_Spot 1,359 Median_Genes_per_Spot 1,013 Total_Genes_Detected 17,853 Reads_Mapped_to_Probe_Set 91.08％

[0137] In addition to the above data analysis, computer algorithms can also be used to jointly analyze the results of protein and gene detection. The computer method includes but is not limited to the following process (such as Figure 7 ): using DAPI fluorescence signals to perform cell segmentation and determine the distribution of cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; based on the maker genes related to the expressed proteins, annotating them on the spatial transcriptome gene expression information map to obtain a spatial distribution map of the maker genes; comparing the spatial location information of the cells expressing the protein with the consistency of the spatial distribution of the maker genes to confirm the correlation between the protein and the gene.

[0138] As another solution, the computer method includes but is not limited to the following process (such as Figure 8 (as shown): using DAPI fluorescence signals to perform cell segmentation and determine the distribution of cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; performing dimensionality reduction cluster analysis based on the results of spatial transcriptome gene expression information to determine the differentially expressed genes of each cell population (Cluster) of the biological sample, and classifying and identifying the cell type of each cell population (Cluster); searching for protein-related maker genes among all differentially expressed genes in each Cluster, and marking the related idle position information; comparing the consistency of the spatial position information of the cells expressing the protein with the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

[0139] Taking the first method as an example, the spatial distribution of the maker gene PECAM1 corresponding to CD31 protein, the spatial distribution of the maker gene PTPRC corresponding to CD45 protein, and the spatial distribution of the maker genes CSNK1A1, CSNK2A1, KRT5, KRT7, and KRT8 corresponding to CK-PAN protein were analyzed. Figures 10-12 It can be seen that the present invention can accurately detect the location of protein-related genes and can be used to confirm the correlation between proteins and genes.

[0140] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A spatial multi-omics detection method for biological samples, characterized in that: The steps include: Step 1) obtaining a biological sample to be tested for target protein and target nucleic acid detection; Step 2) reacting the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then reacting the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected; Step 3) reacting the biological sample to be detected with a tyramide fluorescein substrate to allow the target protein to bind to the tyramide fluorescein; Step 4) detecting the target protein in the biological sample to be detected by fluorescence signal; Step 5) reacting the biological sample to be detected, for which the fluorescent signal detection in step 4) has been completed, with a targeting probe for binding to the target nucleic acid to form a target nucleic acid-targeting probe complex; then attaching a biochip to the biological sample to be detected, wherein the surface of the biochip in contact with the biological sample to be detected is provided with an array of detection points; each detection point in the array of detection points contains a capture probe for detecting the target nucleic acid; Step 6) The biological sample to be detected is permeabilized; the target probe conjugate is bound to the capture probe on the biochip; the target probe on the biochip generates a complementary nucleotide chain through a two-strand synthesis reaction; the complementary nucleotide chain is subjected to PCR to construct a sequencing library and sequence the target nucleic acid.

2. The spatial multi-omics detection method of biological samples according to claim 1, characterized in that: The biochip includes a carrier; the carrier is provided with detection points evenly arranged in a rectangular array to form a detection point array; each detection point contains a capture probe for detecting the target nucleic acid; Preferably, the carrier is glass or plastic.

3. The spatial multi-omics detection method of biological samples according to claim 1, characterized in that: The capture probe is a nucleic acid fragment comprising a PCR primer sequence, a spatial position sequence, a unique molecular identification tag sequence and a nucleic acid capture sequence.

4. The spatial multi-omics detection method of biological samples according to claim 1, characterized in that: There are multiple target proteins, and after the first target protein is bound to tyramide fluorescein in step 2) and step 3), the biological sample to be detected is subjected to antigen repair treatment; and the detection steps of step 2) and step 3) are repeated for another undetected target protein; Repeat until each target protein is bound to tyramide fluorescein; then proceed to step 4) detecting the target protein in the biological sample to be detected by fluorescence signal; Preferably, the antigen repair treatment is to heat the biological sample to be detected in a sodium citrate repair solution by microwave.

5. The spatial multi-omics detection method of biological samples according to claim 4, characterized in that: Different target proteins bind to tyramide fluorescein with different emission wavelengths; Preferably, the tyramide fluorescein substrate is a fluorescent dye Alexa Fluor TM 488、Alexa Fluor TM 546、Alexa Fluor TM Tyramide labeled with one of TYR-647, TYR-520, TYR-690, and TYR-780.

6. The spatial multi-omics detection method of biological samples according to claim 1, characterized in that: In step 5), a clamp is installed on the biochip, the clamp surrounding the detection point array to form a reaction pool on the detection point array; then, a probe hybridization reaction solution containing a targeting probe is added to the reaction pool; the targeting probe includes a left probe and a right probe, and the left probe and the right probe bind to the target nucleic acid to form a target nucleic acid-targeting probe complex; In step 6), the card holder is removed, and the biological sample to be detected is permeabilized to allow the nucleic acid-targeting probe complex to permeate out of the cell and bind to the capture probe on the biochip; Then, a fixed clamp is installed on the biochip, and a two-strand synthesis reaction solution is added to the reaction pool to carry out a two-strand synthesis reaction, so that the targeting probe on the biochip generates a complementary nucleotide chain; Preferably, the clip is made of plastic, metal and / or rubber.

7. The spatial multi-omics detection method of biological samples according to claim 1, characterized in that: The target nucleic acid is the whole transcriptome mRNA.

8. The spatial multi-omics detection method for biological samples according to claim 1, characterized in that: The step 4) further includes performing DAPI staining on the biological sample to be detected, detecting DAPI in the biological sample to be detected, performing cell segmentation using the DAPI fluorescence signal, and determining the distribution of cells on the biological sample.

9. The spatial multi-omics detection method of biological samples according to claim 8, characterized in that: The method further includes step seven): confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescence signal.

10. The spatial multi-omics detection method of biological samples according to claim 9, characterized in that: Confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescence signal includes: using the DAPI fluorescence signal to perform cell segmentation to determine the distribution of the cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; based on the relevant maker genes expressing the protein, marking on the target nucleic acid expression information map to obtain the spatial distribution map of the maker gene; comparing the consistency of the spatial position information of the cells expressing the protein with the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

11. The spatial multi-omics detection method for biological samples according to claim 9, characterized in that: Confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid, and the DAPI fluorescence signal includes: performing cell segmentation using the DAPI fluorescence signal to determine the distribution of cells on the biological sample; superimposing the cell segmentation results with the protein fluorescence signal results to confirm the cells expressing the protein and their distribution on the biological sample; Based on the results of target nucleic acid expression information, dimensionality reduction cluster analysis is performed to determine the differentially expressed genes in each cell population of the biological sample and classify and identify the cell type of each cell population; protein-related maker genes are searched for among all differentially expressed genes in each cell population, and the related idle position information is annotated; the spatial location information of cells expressing the protein is compared with the consistency of the spatial distribution of the maker gene to confirm the correlation between the protein and the gene.

12. The spatial multi-omics detection method for biological samples according to claim 1, characterized in that: The target proteins include CD31, CD45 and CK-PAN.

13. A spatial multi-omics detection system for biological samples, characterized in that: include: The detection sample module is used to obtain the biological sample to be detected, which needs to detect the target protein and target nucleic acid; An antibody binding module is used to react the biological sample to be detected with a first target protein antibody for specifically binding to the target protein, and then react the biological sample to be detected with a second target protein antibody labeled with horseradish peroxidase for specifically binding to the first target protein antibody, thereby forming a target protein-primary antibody-secondary antibody complex labeled with horseradish peroxidase in the biological sample to be detected; A fluorescent labeling module, used for reacting the biological sample to be detected with a tyramide fluorescein substrate to bind the target protein to the tyramide fluorescein; A fluorescence detection module, configured to detect the target protein in the biological sample to be detected through a fluorescence signal; The probe binding module is used to react the biological sample to be detected after the fluorescent signal detection with the targeting probe used to bind to the target nucleic acid to form a target nucleic acid-targeting probe complex; a biochip is then attached to the biological sample to be detected, and the surface of the biochip that contacts the biological sample to be detected is provided with an array of detection points; each detection point in the detection point array contains a capture probe for detecting the target nucleic acid; The PCR detection module is used to permeabilize the biological sample to be detected; allow the nucleic acid-targeting probe complex to bind to the capture probe on the biochip; generate complementary nucleotide chains from the targeting probe on the biochip through a two-strand synthesis reaction; and then perform PCR on the complementary nucleotide chains to construct a sequencing library and sequence and detect the target nucleic acid.

14. The spatial multi-omics detection system for biological samples according to claim 13, characterized in that: The fluorescence detection module is further used to perform DAPI staining on the biological sample to be detected, detect DAPI in the biological sample to be detected, perform cell segmentation using the DAPI fluorescence signal, and determine the distribution of cells on the biological sample.

15. The spatial multi-omics detection system for biological samples according to claim 14, characterized in that: The system also includes a result analysis module for confirming the correlation between the target protein and the target nucleic acid based on the detection results of the target protein, the target nucleic acid and the DAPI fluorescent signal.

Images