CN113684255A - Target positioning and quantitative detection method based on DNA spherical nanostructure imaging - Google Patents

Target positioning and quantitative detection method based on DNA spherical nanostructure imaging Download PDF

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CN113684255A
CN113684255A CN202111008239.XA CN202111008239A CN113684255A CN 113684255 A CN113684255 A CN 113684255A CN 202111008239 A CN202111008239 A CN 202111008239A CN 113684255 A CN113684255 A CN 113684255A
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邹秉杰
王琛
宋沁馨
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China Pharmaceutical University
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Abstract

The invention discloses a target positioning and quantitative detection method based on DNA spherical nanostructure imaging, which relates to signal amplification detection, in particular to high-sensitivity detection of low-abundance targets in samples; the multiple detection of the targets breaks through the limitation that a common fluorescence microscope can only detect 3-5 targets at the same time. The key content of the method relates to connecting a target specificity detector such as an antibody with a single-chain oligonucleotide label, then amplifying the single-chain oligonucleotide label by using DNA loop-mediated rolling circle amplification, wherein amplified nucleic acid can spontaneously form a spherical DNA nano structure under the action of intramolecular hybridization; by designing a ring probe sequence, a spherical DNA nano structure formed by rolling circle amplification products can be hybridized with a fluorescence labeling specificity nucleic acid probe, so that the fluorescent probe presents a bright spot under a fluorescence microscope, and the positioning and digital quantitative detection of a target to be detected are realized by positioning and counting the bright spot.

Description

Target positioning and quantitative detection method based on DNA spherical nanostructure imaging
Technical Field
The invention belongs to the field of positioning and quantitative detection, and particularly relates to a multi-target positioning and quantitative detection method in cells and tissues based on DNA spherical nanostructure imaging.
Background
Protein (Protein) and other biomolecules are important components of all cells and tissues of the human body and are important participants in the execution of physiological functions of the human body, and more researches show that the occurrence and development of diseases are closely related to the changes of some Protein and other biomolecules in tissue cells, so that the elucidation of the types and content changes of Protein and other biomolecules in tissue cells and other samples is very important for deeply understanding the occurrence and development of diseases.
It is noted that biomolecules such as proteins synthesized by cells need to be transported to specific subcellular locations to exert their functional effects, and many studies have shown that accurate analysis of the subcellular spatial distribution of biological material groups in human cells can greatly improve our understanding of diseases.
Therefore, it is necessary to perform biomolecule analysis such as protein analysis on samples such as tissues and cells while satisfying the requirements of type determination, quantification and localization, that is, information such as the type of a target, the content of the target, and the subcellular distribution position of the target. The in situ imaging technology is an ideal tool for in situ analysis of biomolecules, which satisfies all the above requirements, wherein the most common in situ imaging technology is immunofluorescence staining, and qualitative, quantitative and positioning analysis can be performed on the biomolecules respectively through the color, position and intensity of fluorescence in the detection process. The detection process is mainly completed by a fluorescent dye and a fluorescent microscope, however, the problem of spectral overlapping of excitation light or emission light among different fluorescent dyes limits the number of fluorescent detection channels which can be simultaneously utilized by the fluorescent microscope, and thus limits the number of targets which can be simultaneously detected and analyzed by the immunofluorescence staining method, so that the method is generally difficult to simultaneously detect more than 5 proteins (up to 7 to 9 if expensive and complicated instruments are used). When functioning, body cells are simultaneously acted by multiple biomolecules, and therefore, in-situ multiplex detection means are urgently needed to be developed. In addition, the immunofluorescence method has no signal amplification or limited signal amplification based on a secondary antibody method, so that the detection sensitivity is not high enough, and the low-abundance rare target cannot be effectively detected. In addition, when a biological sample is subjected to fluorescence detection, the sample has the problems of autofluorescence and light scattering, so that the immunofluorescence method using fluorescence intensity quantification is further limited when the low-abundance target is characterized.
Therefore, in situ signal amplification is needed to improve signal, detection throughput and sensitivity. In addition, in order to avoid the influence of autofluorescence and light scattering, a quantitative analysis method capable of independent quantification of fluorescence intensity should be used in the detection of low-abundance rare targets.
Disclosure of Invention
The purpose of the invention is as follows: the present invention provides a method for detecting a sample to be analyzed by binding potential targets to be analyzed in the sample to target-specific binding partners and determining the presence of such target-specific binding partners in an analytical order by repeatedly binding, imaging for detection and optionally removing fluorescently labeled probes (e.g., by using electrophoresis), which is a target localization and quantitative detection method based on DNA spherical nanostructure imaging.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the detection method, different oligonucleotide labels are obtained by directly or indirectly combining a sample to be detected with different target specificity binding couplers, DNA nanospheres with different target specificities are formed under the rolling circle replication action mediated by different oligonucleotide labels, and after the DNA nanospheres with different target specificities are combined with a specific fluorescence labeling probe, the target to be detected in the sample to be detected is positioned and quantitatively detected through different colors and different imaging results.
The preparation process of the DNA nanospheres with different target specificities is as follows: the method comprises the following steps of mediating different specific single-stranded DNA molecules corresponding to different oligonucleotide tags to perform cyclization reaction and rolling circle replication, adding a single-stranded DNA sequence unit which is completely complementary with the single-stranded DNA molecule sequence at the 3' tail end of the nucleic acid sequence of the oligonucleotide tags after each rolling circle replication by taking the single-stranded DNA molecules as templates, and finally forming a long single-stranded DNA with a plurality of repeated DNA sequence units after a plurality of rolling circle replications, wherein the long single-stranded DNA can spontaneously form a DNA nanosphere.
Wherein the target specific binding partner comprises a specific binding molecule for a target that interacts directly or indirectly with the target and an oligonucleotide tag fragment, and wherein the oligonucleotide tag sequences of target specific binding partner labels having different specificities are different; incubating the specific binding partner of the target and the assay sample under conditions that promote the binding of the specific binding molecule of the target to the target in the assay sample, optionally removing unbound specific binding partner of the target.
Wherein the oligonucleotide tag fragment in the target specific binding conjugate is contacted with one or more specific single-stranded DNA molecules, each of the two ends of the specific single-stranded DNA molecules comprises a portion complementary to the sequence of the oligonucleotide tag fragment, and the specific single-stranded DNA molecules further comprises at least two sequences identical to the specific fluorescently labeled probe.
Wherein the detection method further comprises removing the bound fluorescently labeled probe from the DNA nanosphere structure by an applied electric field or a buffer solution.
Wherein the analysis sample is one or more of cells, frozen tissue or paraffin-embedded tissue; the target is one or more of nucleic acid, protein, polypeptide or proteoglycan; the specific binding partner of the target is a specific binding molecule that is non-covalently or covalently coupled or linked to an oligonucleotide tag.
Wherein, the specific binding molecule is an antibody, an antibody fragment, an aptamer, an oligonucleotide or a small molecule, and the oligonucleotide tag fragment comprises a nucleic acid sequence corresponding to the target, which can be hybridized with a single-stranded DNA molecule with matched sequence and used as a primer to extend along the hybridized circular DNA template under a proper extension condition.
Wherein, the specific single-stranded DNA molecule is a molecule which can be connected into circular DNA under the action of ligase after being hybridized with the oligonucleotide tag or a circular DNA molecule which is independently synthesized or engineered or a single-stranded rolling circle replication product.
Wherein the external electric field is electrophoresis, and the buffer solution is phosphate buffered saline solution or phosphate buffered saline solution containing surfactant Triton X-100 or Tween 20.
The target positioning and quantitative detection method based on the DNA spherical nano structure imaging specifically comprises the following steps:
1) contacting the specific binding partners for one or more targets with the assay sample, wherein the specific binding partners for each target comprise a specific binding molecule for one target that interacts directly or indirectly with the target and an oligonucleotide tag fragment, and wherein the oligonucleotide tag sequences of target specific binding partner labels having different specificities are different; incubating the specific binding partner of the target and the assay sample under conditions that promote the binding of the specific binding molecule of the target to the target in the assay sample, optionally removing unbound specific binding partner of the target;
2) contacting the oligonucleotide tag fragment of the specific binding partner of the target with one or more specific single-stranded DNA molecules comprising at each of its two ends a portion complementary to the sequence of the oligonucleotide tag fragment and at least two sequences identical to the specific fluorescently labeled probe before, simultaneously with, or after step 1); incubating said oligonucleotide tag fragment-specific single-stranded DNA molecule and target-specific binding partner under conditions promoting hybridization of the oligonucleotide tag fragment to its specific single-stranded DNA molecule, optionally removing unbound single-stranded DNA molecules;
3) after step 2), connecting the specific single-stranded DNA molecules hybridized with the oligonucleotide tag fragments into a ring under the condition of promoting the connection of the specific single-stranded DNA molecules into a ring;
4) after the step 3), extending the oligonucleotide tag fragment to form a long-chain DNA fragment with a repeated DNA sequence under the condition of promoting the rolling circle replication of the oligonucleotide tag fragment by using the single-stranded DNA molecules connected into a circle as a template, wherein the long-chain DNA fragment can spontaneously form a DNA nano spherical structure;
5) after step 4), contacting the assay sample with said specific fluorescently labeled probe by sequence complementary hybridization to produce fluorescently labeled probe stably bound to said DNA nanosphere structure, optionally removing unbound fluorescently labeled probe;
6) after step 5), imaging the assay sample to detect the location and number of DNA nanospheres stably bound to said fluorescently labeled probe, wherein detection of fluorescent DNA nanospheres indicates the presence of the corresponding target;
7) removing the combined fluorescent labeled probe from the DNA nanosphere structure by an external electric field or a buffer solution;
8) wherein, the step 5), the step 6) and the step 7) are repeated to remove the analysis sample of the fluorescence labeling probe, one or more fluorescence labeling probes with the specificity of the DNA nanospheres are used for combining the DNA nanospheres with the specificity of different targets each time, and different target specificity DNA nanospheres are distinguished by different colors of the fluorescence labeling probes and different imaging results in different rounds, thereby detecting various targets to be detected.
Wherein, the analysis sample in the step 1) is one or more of cells, frozen tissues or paraffin-embedded tissues; the target is one or more of nucleic acid, protein, polypeptide or proteoglycan; the specific binding partner of the target is a specific binding molecule that is non-covalently or covalently coupled or linked to an oligonucleotide tag.
Wherein, the specific binding molecule in step 1) is an antibody, an antibody fragment, an aptamer, an oligonucleotide or a small molecule, and the oligonucleotide tag fragment comprises a nucleic acid sequence corresponding to a target, which can be hybridized with a single-stranded DNA molecule with a matched sequence and used as a primer to extend along the hybridized circular DNA template under a proper extension condition.
Wherein, the specific single-stranded DNA molecule in the step 1) is a molecule which can be connected into circular DNA under the action of ligase after being hybridized with the oligonucleotide tag, or a circular DNA molecule which is independently synthesized or engineered, or a single-stranded rolling circle replication product.
Wherein, in step 1), each target specific binding couple and each oligonucleotide tag are different, the oligonucleotide tag sequences of different target specific binding couples are different, different oligonucleotide tags mediate different specific single-stranded DNA molecules (which can be used as template molecules for copying and extending corresponding oligonucleotide tags) corresponding to the different oligonucleotide tags to perform a ring-forming reaction and rolling-ring copying, each time the oligonucleotide tag is subjected to rolling circle replication by taking the single-stranded DNA molecule as a template, a single-stranded DNA sequence unit which is completely complementary to the single-stranded DNA molecule sequence is added at the 3' end of the nucleic acid sequence of the oligonucleotide tag, and finally, the oligonucleotide tag is subjected to rolling circle replication for multiple times, the oligonucleotide tag forms a long single-stranded DNA having a plurality of repeating DNA sequence units, the long single-strand DNA can be spontaneously curled under the action of intramolecular hybridization, and the long single-strand DNA is shrunk to form a DNA nanosphere with the size of hundreds of nanometers.
Wherein the DNA nanospheres are attached to the targets by oligonucleotide tags on target-specific binding conjugates, wherein each target-specific binding conjugate binds to one target, and each nanosphere with a repetitive DNA sequence corresponds to one target molecule, wherein the number and location of targets generated by the DNA nanospheres in different assay samples is indicative of the number and location of targets analyzed.
Wherein the number and position of the DNA nanospheres are generated by hybridizing the DNA nanospheres with corresponding complementary fluorescent labeled probes so that each nanosphere can emit specific fluorescence under specific excitation light and analyzing by using a fluorescence microscope or a fluorescence scanning instrument; the multiple DNA nanosphere structures corresponding to the multiple targets are distinguished by adopting fluorescence labeling probes with different excitation and emission wavelengths complementarily hybridized with the DNA nanospheres with specific sequences, and analysis of the DNA nanosphere structures corresponding to the more targets is realized by multiple rounds of hybridization imaging.
Wherein unbound target-specific binding partner, unbound oligonucleotide tag-specific single-stranded DNA molecules, unbound fluorescently labeled probes and bound fluorescently labeled probes are optionally removed in step 1), 2), 5) or 7) by using an applied electric field, which is electrophoresis, or a buffer solution, which is a phosphate buffered saline solution or phosphate buffered saline solution containing surfactant Triton X-100 or Tween 20.
Wherein, in the step 3), specific single-stranded DNA molecules are connected into a ring by using an enzyme having DNA connecting activity; and (4) extending the oligonucleotide tags to form DNA nano spherical structures with repeated sequences by using DNA polymerase with strand displacement activity.
The present invention provides methods for rapid multiplexed detection, localization and quantification of targets of interest (e.g., proteins), particularly digital quantification of low abundance targets. Some of the methods provided herein include (1) contacting a sample to be analyzed (e.g., a sample that may contain one or more targets to be analyzed) with a conjugate that specifically binds the targets (given a target-specific binding conjugate comprising a target-specific binding moiety and a target-specific oligonucleotide tag, where the oligonucleotide tag may serve as a downstream rolling circle replication primer), optionally removing excess unbound conjugate (e.g., by using phosphate buffered saline containing surfactants, Triton X-100, Tween20, etc., and phosphate buffered saline), (2) contacting one or more oligonucleotide tag-specific single-stranded DNA molecules with a target-specific binding conjugate that has bound to a target, the single-stranded DNA molecules being complementary to, and thus specific for, a nucleotide sequence of one of the oligonucleotide tags, optionally removing single-stranded DNA molecules specific to unbound oligonucleotide tags (e.g., by using electrophoresis, placing the assay sample in an electrophoresis buffer in an electrophoresis chamber, under the force of an applied electric field unless specifically bound), and (3) ligating specific single-stranded DNA molecules into a circle under conditions that promote ligation of the specific single-stranded DNA molecules ligated to the oligonucleotide tags into a circle (e.g., by using T4 DNA ligase), (4) extending the oligonucleotide tags into nanospheres having a repetitive DNA sequence under conditions that promote replication of the oligonucleotide tags using the ligated single-stranded DNA molecules as templates (e.g., by using phi29 DNA polymerase for a replication reaction time of preferably 70-100 minutes, more preferably 50-70 minutes), (5) contacting the assay sample with fluorescently labeled probes having nucleotide sequences complementary to the DNA nanospheres, to generate fluorescently labeled probes stably bound to the DNA nanospheres and remove non-specifically bound fluorescently labeled probes by an applied electric field (e.g., by using electrophoresis, placing the assay sample in an electrophoresis buffer in an electrophoresis tank, removing non-specifically bound fluorescently labeled probes in the sample under the force of the applied electric field), (6) imaging the assay sample to detect the location and number of DNA nanospheres stably bound with the fluorescently labeled probes. Wherein the detection of the DNA nanospheres stably bound with the fluorescently labeled probe indicates the presence of the corresponding target, (7) the specifically bound fluorescently labeled probe is removed from the DNA nanospheres by applying an electric field (e.g., by using electrophoresis, placing the analysis sample in an electrophoresis buffer in an electrophoresis tank, and removing the fluorescently labeled probe specifically bound to the DNA nanospheres under the action of the applied electric field), (8) the analysis sample from which the fluorescently labeled probe is removed can repeat the steps (5), (6), and (7), each time one or more of the DNA nanosphere-specific fluorescently labeled probes are bound to DNA nanospheres with different target specificities, and the different target-specific DNA nanospheres are distinguished by imaging with different colors of the fluorescently labeled probes, thereby finally completing the multiple target detection that breaks through the number of channels of fluorescence microscope detection.
The DNA nanospheres are attached to the targets by oligonucleotide tags on target-specific binding conjugates, wherein each target-specific binding conjugate binds to one target. Thus, each DNA nanosphere corresponds to one target molecule. Wherein the number and location of the targets generated by the nanospheres in the different assay samples is indicative of the number and location of the targets assayed. Therefore, the position information of the single target molecule is judged by imaging and counting the single DNA nanosphere, and the digital quantitative analysis is carried out.
Fluorescently labeled probes having different sequences can be the same label, including being labeled with the same fluorophore. This approach requires only a single excitation wavelength and detector. In other embodiments, fluorescently labeled probes having different sequences can be different labels. This method can be used with fluorescence microscopy with a variety of excitation wavelengths and multi-channel detectors.
It is understood that prior to analyzing the sample, its target is known, suspected, unknown or unsuspected. Whether the target-specific binding partner is capable of binding to the sample being analyzed depends on whether the given target is present in the sample being analyzed (e.g., the target-specific binding partner is capable of binding to the sample being analyzed when the given target is present on the sample being analyzed). "bound to the assay sample" means that the target-specific binding partner binds to its corresponding target.
The specific binding molecule on the target specific binding conjugate may be an antibody or antibody fragment. When the specific binding molecule is an antibody or antibody fragment, an oligonucleotide tag may be coupled to its constant region.
The oligonucleotide tag may be attached to the specific binding molecule via an intermediate linker. In some embodiments, one intermediate linker comprises streptavidin and/or biotin.
In some embodiments, the nucleic acid of the fluorescently labeled probe can be one, multiple, or more fluorophores.
The assay sample may be a cell or a lysate from a cell. The target may be a protein or a polypeptide.
Has the advantages that: compared with the prior art, the invention has the following advantages: the methods of the invention can detect, locate and quantify one or more targets in a sample to be analyzed. The DNA nanospheres copied by the rolling rings can form dispersed bright spots obviously distinguished from the background under a conventional fluorescence microscope after being hybridized with the fluorescence labeling probe, and are particularly suitable for detecting low-abundance targets, especially for performing single-molecule imaging detection on the targets. Furthermore, regardless of the position of the targets, including how close the different targets are to each other, the method can be distinguished by the different fluorescently labeled probes, and thus the spatial distance between the targets can be smaller than the resolving distance of the imaging system. Therefore, the detection mode does not depend on a high-resolution microscope for imaging positioning analysis. The method can be used for characterizing various targets, target-specific DNA nanospheres are formed in situ, one or more (not more than the number of detection channels of a microscope detector) target-specific DNA nanospheres are subjected to detection analysis based on the fluorescence labeling probe in each round, then the fluorescence labeling probe is removed by an external electric field, and the next round of detection analysis of other target-specific DNA nanospheres is carried out. Detection of a wider variety of targets can ultimately be achieved through multiple rounds of imaging analysis. Thus, this approach breaks the limit of the detection multiplicity of the microscope imaging system.
Drawings
FIG. 1 is a schematic diagram of the method of the present invention for detecting a plurality of targets (e.g., proteins) in a sample (e.g., a biological sample) to be tested.
FIG. 2 is a schematic diagram of the principle of high throughput and essentially highly multiplexed imaging detection based on an applied electric field.
FIG. 3 is a schematic diagram of the design principle of an oligonucleotide tag and a corresponding single-stranded DNA molecule (Padlock molecule in the figure) for forming a circular template according to the present invention.
FIG. 4 shows the result of detecting glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) protein.
Fig. 5 shows the signal amplification efficiency for target detection.
FIG. 6 shows the result of detecting the protein of Epithelial cell adhesion molecule (Epithelial cell adhesion molecule) expressed in the human breast cancer cell line MCF-7 after the Epithelial-mesenchymal transition process.
Detailed Description
The invention provides, inter alia, methods for single molecule imaging or high-throughput, rapid multiplex imaging of one or more targets in an in situ environment, e.g., nucleic acid-based rolling circle amplification and labeled probe-based cells. The method involves analyzing one or more targets (e.g., proteins) in one or more specific samples (e.g., biological samples). In some cases, it is unknown whether a target is present in a sample, and a sample may contain one or more given targets to be analyzed. Thus, the methods of the invention can be used to determine whether one or more given targets are present in a particular sample.
Thus, the present invention provides a method for nucleic acid-based in situ rolling circle amplification and targeted single molecule imaging or high-throughput, rapid multiplex imaging of labeled probes. The method relies on the use of an orthogonal DNA tag that can be stably bound to a target-specific binding molecule (e.g., an antibody), followed by the formation of a stable partially double-stranded hybrid structure by the addition of nucleic acids complementary to the DNA tags, with the concomitant generation of a loop reaction and a subsequent in situ rolling circle replication reaction, followed by the formation of a DNA nanosphere with a repetitive DNA sequence. The DNA nanospheres are attached to the targets by oligonucleotide tags on target-specific binding conjugates, wherein each target-specific binding conjugate binds to one target. Thus, each DNA nanosphere corresponds to one target molecule. The sample is then imaged and the method achieves single molecule imaging of the target (see figure 1).
Furthermore, in one embodiment, the methods can ultimately allow the simultaneous formation of multiple target-specific DNA nanospheres by simultaneously contacting multiple target-specific binding conjugates (e.g., DNA-tagged antibodies) with multiple targets. After the target-specific fluorescently labeled probe is hybridized to a DNA nanosphere of a target and imaged, removal of the fluorescently labeled probe (e.g., applied electric field) is performed to eliminate fluorescence from the fluorescently labeled probe. The hybridization and imaging process of the fluorescently labeled probe is then repeated to perform imaging of another one or more DNA nanospheres to achieve imaging of multiple targets (see fig. 2).
The rapid multiplex detection disclosed by the invention is not limited by the limited number of detection channels or spectral overlap in the traditional imaging method. Orthogonal DNA labels are easy to design, and the method can realize detection and analysis of theoretically infinite targets through repeated steps and multiple rounds of imaging.
These methods have applicability, for example, in medical diagnostics (e.g., detection of malignancy and tumor typing, detection and characterization of circulating tumor cells).
The methods disclosed herein can also be used to analyze one or more targets and to absolutely quantify and image the targets in situ through the formation of target-specific DNA nanospheres; or to analyze the relative content between targets in the same sample or in different samples.
In the present invention, the term "target" is any biological component that is desired to be observed or quantified for analysis and for which a specific binding partner exists. In some embodiments, the target may be an engineered or non-naturally occurring biomolecule. The "biomolecule" is any molecule produced by a living organism, including large molecules such as proteins, proteoglycans, lipids and nucleic acids, and small molecules such as metabolites and natural products. Examples of biomolecules include, but are not limited to: DNA, RNA, cDNA, or the DNA product of RNA that undergoes reverse transcription.
In some embodiments, a target can be a protein target, for example, a protein of a cellular environment (e.g., a cytoplasmic protein, a cell membrane protein, or a nuclear protein). Examples of proteins include, but are not limited to: fibrous proteins such as cell scaffold proteins (e.g., actin, arp2/3, coronin, dystrophin, FtsZ, keratin, myosin, chaperonin, spectrin, tau, adiponectin, tropomyosin, tubulin, and collagen), and extracellular matrix proteins (e.g., collagen, elastin, baslate response protein, picatin, and fibronectin); globular proteins such as plasma proteins (e.g., serum amyloid P component and serum albumin), coagulation factors (e.g., complement proteins, C1-inhibitors and C3-convertase, factor VIII, factor XIII, fibrin, protein C, protein S, protein Z-related protease inhibitors, thrombin, von willebrand factor), and acute phase proteins such as C-reactive protein; a heme protein; cell adhesion proteins (e.g., cadherins, ependymal proteins, integrins, Ncam, and selectins); transmembrane transport proteins (e.g., CFTR, glycophorin D, and promiscuous enzymes), such as ion channels (e.g., ligand-gated ion channels, such as nicotinic acetylcholine receptors and GABAa receptors, and voltage-gated ion channels, such as potassium, calcium, and sodium channels), co-transport/reverse transport proteins (e.g., glucose transporters); hormones and growth factors (e.g., Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), peptide hormones such as insulin, insulin-like growth factor, and oxytocin, and steroid hormones such as androgens, estrogens, and progestins), receptors such as transmembrane receptors (e.g., G protein-coupled receptor, rhodopsin) and intracellular receptors (e.g., estrogen receptor), DNA binding proteins (e.g., histones, protamines, CI proteins), transcriptional modulators (e.g., c-myc, FOXP2, FOXP3, MyoD, and P53), immune system proteins (e.g., immunoglobulins, major histocompatibility antigens, and T cell receptors), nutrient storage/transport proteins (e.g., ferritin), and enzymes.
Example 1
(1) Experimental materials and reagents:
the MDA-MB-231 cell line was purchased from Shanghai ATCC cell bank; cell culture grade phosphate buffered saline (PBS solution, without calcium chloride, magnesium chloride, 1 XPBS pH 7.4) was purchased from USA
Figure BDA0003236377120000101
DMEM medium (containing penicillin-streptomycin double antibody) was purchased from triumphant organisms; trypsin (Trypsin) was purchased from USA
Figure BDA0003236377120000102
Sterile Fetal Bovine Serum (FBS) was purchased from Argentina Natocor-Industria
Figure BDA0003236377120000103
Bovine Serum Albumin (BSA) was purchased from Amresco, usa; glass Bottom Cell Culture Dish purchased from Wuxi Kangsi Biotech limited; biotin, formamide, dimethyl sulfoxide (DMSO) and polyethylene glycol tert-octylphenylEther (Triton X-100) was purchased from Sigma-Aldrich, USA; 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Boster, Wuhan doctor; all the oligonucleotide probes, the biotinylated oligonucleotide probe, the phosphorylated oligonucleotide probe and the fluorophore modified oligonucleotide probe are prepared by the Shanghai Biotechnology Limited company, and the purification level is HPLC level; dNTPs were purchased from Baisheng Biotech, Inc. of Beijing Sai; salmon Sperm DNA Solution (Salmon Sperm DNA Solution) was purchased from Saimer Feishel technologies (China) Ltd; human TGF-beta factor was purchased from Peprotech; biotinylated antibodies Anti-EpCAM (Biotin), Anti-GAPDH (Biotin) were purchased from Abcam; t4 DNA ligase, phi29 DNA polymerase was purchased from New England Biolabs; ammonium chloride and sodium hydroxide were purchased from chemical reagents of national drug group, ltd; experimental deionized Water (18.2 M.OMEGA.) from a Water Purification apparatus, Explorer series Water Purification System, available from U.S. Blue
Figure BDA0003236377120000104
The cell grade experimental water was from autoclaved analytical grade experimental water; other molecular biology experiment water is purchased from Chechen distilled water; other reagents are analytically pure; the fluorescence microscope was a Nikon ECLIPSE Ni microscope, available from Nikon, Japan.
Oligonucleotide tag 1: Biotin-AAAAA AAAAA AAAAA GAGAG CGACA CTATG AGACA GGTGA TCCCA TCCTG AGC
Oligonucleotide tag 2: Biotin-AAAAA AAAAA AAAAA CCTGA GACAT CATAA TAGCG GACGA TCATC CAGCA CTAG
Single-stranded DNA molecule (Padlock molecule) 1: PO (PO)4-GTCTC ATAGT GTCGC TCTCT GA TTC GCGCC GAGGT TGTCT CAGCT TTAGT TTAAT ACGCG CCGAG GTAGG GCTCA GGATG GGATC ACCT
Single-stranded DNA molecule (Padlock molecule) 2: PO (PO)4-GCTAT TATGA TGTCT CAGGT AGATG GACGC GGAGT TGTCT CAGCT TTAGT TTAAT AGGAC GCGGA GTACC TAGTG CTGGA TGATC GTCC
Single-stranded DNA molecule (Padlock molecule) 3: alexa Fluor 488-GTCTC ATAGT GTCGC TCTCT GA TTC GCGCC GAGGT TGTCT CAGCT TTAGT TTAAT ACGCG CCGAG GTAGG GCTCA GGATG GGATC ACCT
Fluorescent probe 1: alexa Fluor 488-CGCGC CGAGG T
And (3) fluorescent probe 2: cy5-GGACG CGGAG T
(2) Cell culture experiment procedure, contents and conditions:
the MDA-MB-231 cell line was cultured in DMEM medium containing 10% FBS and a mixed solution of 50U/mL penicillin, 50. mu.g/mL streptomycin double antibody. The cell culture conditions were: 95% relative humidity, 5% carbon dioxide gas, 37 ℃. And (3) starting passage when the cells grow to about 90% fusion degree, wherein in order to ensure that the cells grow in the logarithmic growth phase, the inoculation density of the cells needs to be controlled, and the cell passage period is preferably 2-3 days. At passage, adherent cells were digested with 0.25% pancreatin-EDTA at 37 ℃ and when cells appeared to shed as a quicksand, digestion was immediately stopped using medium containing 10% FBS. After the cell suspension after being resuspended is counted by a blood ball counting plate, cells with proper density are taken according to the cell size and the growth speed to be inoculated in a cell culture bottle or a culture dish for continuous culture.
(3) Oligonucleotide tag labeled antibody modification experiment steps, contents and conditions:
a25. mu.L volume of 2.5. mu.M oligonucleotide tag 1 or 2 was mixed well with a 25. mu.L volume of 2.5. mu.M streptavidin and incubated at 37 ℃ for 30 minutes. Then, 50. mu.L of 1.25. mu.M biotinylated antibody Anti-GAPDH (Biotin) (oligonucleotide tag 1) or biotinylated antibody Anti-EpCAM (Biotin) (oligonucleotide tag 2) was added to the reaction mixture, mixed well, and incubated at 25 ℃ for 30 minutes. Finally, 1mM biotin was added and incubated at 25 ℃ for 20 minutes. The reaction diluent in this process was Assay Buffer (8mM Na)2HPO4，2mM NaH2PO4150mM NaCl, 0.1% BSA, 0.025% Tween20, pH 7.4). The concentration of the oligonucleotide labeled antibody finally used for sample detection is in the range of 0.5-2.5 mu g/100 mu L calculated by the equivalent concentration of the unmodified biotinylated antibody. The diluted solution of the oligonucleotide tag-labeled antibody was an Assay Buffer (8mM Na) containing salmon sperm DNA2HPO4，2mM NaH2PO4，150mM NaCl，01% BSA, 0.025% Tween20, pH 7.4, 0.5mg/mL salmon sperm DNA).
Example 2 oligonucleotide tags and corresponding design of Single-stranded DNA molecules for Forming circular templates
Referring to FIG. 3, in this example, specific experimental materials and reagents, see example 1, the 5' end of oligonucleotide tag 1 was biotinylated to bridge the tag to the target-specific binding molecule via the biotin-streptavidin system. The single-stranded DNA molecule (Padlock molecule 1) comprises a complementary portion to the nucleic acid tag to form a circular template and also comprises a template sequence for the generation of a sequence for hybridizing with the fluorescently labeled probe 1, so that only when the oligonucleotide tag 1 undergoes an amplification reaction, a sequence that complementarily hybridizes with the fluorescently labeled probe 1 is generated, and thus the specificity of the fluorescent signal is ensured.
Example 3 glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) protein assay
Referring to fig. 4, in this embodiment, specific experimental materials and reagents, cell culture experimental steps, contents and conditions, and oligonucleotide tag labeled antibody modification experimental steps, contents and conditions, referring to example 1, a MDA-MB-231 cell sample after being fixed with 4% paraformaldehyde is contacted with a target specific binding conjugate so as to be stably and specifically bound to the sample, and under the action of rolling circle replication mediated by a nucleic acid tag on the target specific binding conjugate, a target specific DNA nanosphere is formed and can be hybridized with a specific fluorescent labeled probe to form a hybrid, and a fluorescent spot clearly distinguished from the background is formed under a fluorescent microscope.
Target detection experiment steps, contents and conditions: MDA-MB-231 cells in the glass bottom culture dish grow to 50% -60%, 1 XPBS is fully rinsed for 3 times, serum, culture medium components and cell secretion are removed, 4% paraformaldehyde solution is incubated for 45 minutes at normal temperature, and then 100mM NH is added4The reaction was quenched in 1 XPBS solution of Cl for 20 minutes, washed in 1 XPBS for 5 minutes, permeabilized in 1 XPBS of 0.1% Triton X-100 for 10 minutes, rinsed three times in 1 XPBS solution, blocked in 5% BSA solution for 2 hours, then shaken at 37 deg.CShaking incubation in 0.05mg/mL RNase A, rinsing with 1 XPBS three times, adding 0.1mg/mL streptavidin (containing 0.5mg/mL Salmonon Sperm DNA) and shaking incubation at 37 ℃ for 30 minutes, discarding the solution, adding 1mM biotin and shaking incubation at 37 ℃ for 30 minutes, and rinsing with 1 XPBS 3 times. The samples were incubated with the oligonucleotide tag 1 labeled antibody Anti-GAPDH (Biotin) containing 0.5mg/mL Salmonon Sperm DNA at an antibody equivalent concentration of Anti-GAPDH (Biotin) of 0.5-2.5. mu.g/100 uL diluted in Assay Buffer at 4 ℃ overnight with shaking, and washed 3 times the next day with 1 XPBS containing 0.1% Triton X-100 and 2% BSA for 10 minutes each. The samples were then washed twice with 1 × PBS for 5 minutes each, and rinsed once with distilled water.
The samples were incubated with 100nM single-stranded DNA molecule (Padlock molecule 1) (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) at 37 ℃ with shaking for 15-30 minutes, rinsed with 1 XPBS three times, rinsed once with distilled water, aspirated off all the liquid and immediately added with T4 ligase system (50mM Tris-HCl, 10mM MgCl. sub.10 mM)210mM DTT, 1mM ATP, 0.1U/. mu. L T4 DNA ligase, pH7.5), L × PBS three rinses, one rinse with distilled water, pipetting all the liquid and adding the phi29 polymerase system (0.5mM dNTPs, 0.25U/. mu.L phi29 DNA polymerase, 0.2mg/mL BSA, 50mM Tris-HCl, L0mM MgCl2，10mM(NH4)2SO44mM DTT, pH7.5), incubated at 37 ℃ for 60 minutes with shaking, rinsed three times with 1 XPBS and once with distilled water. 0.5. mu.M of target-specific fluorescent probe 1 (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) was added and incubated at 37 ℃ for 20 to 30 minutes with shaking. Wash with 1 XPBS containing 0.1% Triton X-100 for 10 minutes, then wash twice with 1 XPBS for 5 minutes each. Mu.g/. mu.L DAPI staining for 5 min followed by 1 XPBS wash for 5 min. Imaging was then performed by fluorescence microscopy.
The experimental results are shown in fig. 4, the left side is the signal of protein target glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) detected in the MDA-MB-231 cell sample, and the result shows that the target signal presents one fluorescent bright spot clearly distinguished from the background, which indicates that the target signal is the result presented after the DNA nanospheres formed by the oligonucleotide tags after rolling circle amplification are hybridized with the target-specific fluorescent probe. The right image is the image after the fusion of the imaging image of the cell nucleus with the left image, and the increased central area image is the imaging result of the cell nucleus. The right graph results show that the signal of protein target glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) is mainly concentrated in cytoplasm, and the signal distribution of nuclear region is less, which is consistent with the actual position distribution of glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) in MDA-MB-231 cells, and these results show that the method has good target localization detection accuracy.
Example 4 comparison of Signal amplification efficiency for target detection
Referring to FIG. 5, in this example, the specific experimental materials and reagents, cell culture experimental procedures, contents and conditions, and oligonucleotide tag-labeled antibody modification experimental procedures, contents and conditions are described in example 1, which detects glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) protein in a single cell sample, and the sample is contacted with a target-specific binding conjugate so as to be specifically bound to the sample stably. Then, (1) hybridizing the fluorescent labeled probe complementary to the nucleic acid label on the target specific binding couple with the nucleic acid label to form a hybrid, and forming a target specific signal which is not amplified by the nucleic acid label under a fluorescent microscope, (2) forming a target specific DNA nanosphere under the action of rolling circle replication mediated by the nucleic acid label on the target specific binding couple, and hybridizing the target specific DNA nanosphere with the specific fluorescent labeled probe to form a hybrid, and forming a fluorescent signal which is obviously distinguished from the background and amplified by the rolling circle amplification under the fluorescent microscope. As can be seen from the calculation of the relative fluorescence quantification, in this example, the in situ target signal is amplified by at least 301 fold.
Target detection experiment steps, contents and conditions: MDA-MB-231 cells in the glass bottom culture dish grow to 50% -60%, 1 XPBS is fully rinsed for 3 times, serum, culture medium components and cell secretion are removed, 4% paraformaldehyde solution is incubated for 45 minutes at normal temperature, and then 100mM NH is added41 of ClThe reaction was quenched in PBS solution for 20 minutes, washed with 1 XPBS for 5 minutes, permeabilized in 1 XPBS with 0.1% Triton X-100 for 10 minutes, rinsed three times with 1 XPBS solution, then blocked in 5% BSA solution for 2 hours, then incubated with shaking in 0.05mg/mL RNase A at 37 deg.C, rinsed three times with 1 XPBS, added with 0.1mg/mL streptavidin (containing 0.5mg/mL Salmonon Sperm DNA) for 30 minutes at 37 deg.C, the solution was discarded, incubated with shaking in 1mM biotin for 30 minutes at 37 deg.C, and rinsed 3 times with 1 XPBS. The samples were incubated with the oligonucleotide tag 1 labeled antibody Anti-GAPDH (Biotin) containing 0.5mg/mL Salmonon Sperm DNA at an antibody equivalent concentration of Anti-GAPDH (Biotin) of 0.5-2.5. mu.g/100 uL diluted in Assay Buffer at 4 ℃ overnight with shaking, and washed 3 times the next day with 1 XPBS containing 0.1% Triton X-100 and 2% BSA for 10 minutes each. The samples were then washed twice with 1 × PBS for 5 minutes each, and rinsed once with distilled water.
The samples were then divided into two groups, one for target-specific signal detection without nucleic acid tag amplification, i.e., the samples were incubated with 100nM Alexa Fluor 488 fluorophore modified single-stranded DNA molecule (Padlock molecule 3) (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) at 37 ℃ with shaking for 20 minutes, rinsed three times with 1 XPBS, rinsed once with distilled water, stained with 1 μ g/. mu.L of DAPI for 5 minutes, and then washed with 1 XPBS for 5 minutes. Another set of samples was amplified by amplifying the nucleic acid tags using the method of the present invention and then the specific signal enhancement efficiency was measured by incubating the samples with 100nM single-stranded DNA molecule (Padlock molecule 1) (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA), shaking at 37 ℃ for 20 minutes, rinsing three times with 1 XSSC, rinsing once with distilled water, and adding T4 ligase system (50mM Tris-HCl, 10mM MgCl. RTM.) immediately after aspirating all the liquid210mM DTT, 1mM ATP, 0.1U/. mu. L T4 DNA ligase, pH7.5), 1 XPBS rinsing three times, one rinsing with distilled water, pipetting all the liquid and adding the phi29 polymerase system (0.5mM dNTPs, 0.25U/. mu.L phi29 DNA polymerase, 0.2mg/mL BSA, 50mM Tris-HCl, 10mM MgCl2，10mM(NH4)2SO44mM DTT, pH7.5), incubated at 37 ℃ for 60 minutes with shaking, 1 XPBS rinsing for three times, and rinsing once with distilled water. 0.5. mu.M Alexa Fluor 488 fluorophore modified target specific fluorescent probe 1 (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) was added and incubated at 37 ℃ for 20-30 min with shaking. Wash with 1 XPBS containing 0.1% Triton X-100 for 10 minutes, then wash twice with 1 XPBS for 5 minutes each. Mu.g/. mu.L DAPI staining for 5 min followed by 1 XPBS wash for 5 min.
The two sets of samples were then subjected to imaging analysis using a fluorescence microscope under the same conditions.
Experimental results referring to fig. 5, in which the central region is the result of imaging the cell nucleus, the signal of the target protein glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase) is mainly concentrated in the region outside the cell nucleus. Quantitative statistical analysis of target signals was performed on the imaging results of one set of samples (without amplification of nucleic acid tags) and another set of samples (with amplification of nucleic acid tags using the method of the present invention) using the accepted Image processing software, Fiji Image J, and the fold of both signals after quantification was calculated. The result shows that compared with the target signal detected by the unamplified label, the target signal intensity detected by the method of the invention after the rolling circle amplification of the nucleic acid label is carried out by the method of the invention is improved by 301 times, namely, the sensitivity of the target detection is improved by the method of the invention, and the method is especially suitable for the detection of the low abundance target.
Example 5 detection of Epithelial cell adhesion molecule (Epithelial cell adhesion molecule) protein expressed in human breast cancer cell line MCF-7 undergoing Epithelial-mesenchymal transition Process
Referring to fig. 6, in this example, digital quantification of the target is achieved by counting target-specific DNA nanospheres.
Sample treatment and target detection experimental procedures, contents and conditions: culturing a human breast cancer cell line MCF-7 in a glass bottom culture dish, adding 10ng/mL of transforming growth factor TGF-beta1 into a culture medium after the cells grow adherently, changing the culture medium every 30 hours, adding 10ng/mL of transforming growth factor TGF-beta1, and culturing for 120 hours from the initial addition of TGF-beta 1.
Removing the glass-bottom culture dish containing cell sample, rinsing with 1 × PBS for 3 times, removing serum, culture medium components and cell secretion, incubating with 4% paraformaldehyde solution at room temperature for 45 min, and incubating with 100mM NH4The reaction was quenched in 1 XPBS solution of Cl for 20 minutes, washed in 1 XPBS for 5 minutes, permeabilized in l XPBS of 0.1% Triton X-100 for 2 minutes, rinsed three times in 1 XPBS solution, then blocked in 5% BSA solution for 2 hours, then incubated with shaking in 0.05mg/mL RNase A at 37 deg.C, rinsed three times in 1 XPBS, added with 0.1mg/mL streptavidin (containing 0.5mg/mL Salmonon Sperm DNA) for 30 minutes at 37 deg.C, the solution was discarded, incubated with shaking at 37 deg.C for 30 minutes with 1mM biotin and rinsed 3 times with 1 XPBS. Samples were incubated with the Assay Buffer diluted oligonucleotide tag 2 labeled antibody Anti-EpCAM (Biotin) (containing 0.5mg/mL Salmonon Sperm DNA, where Anti-EpCAM (Biotin) antibody equivalent concentration was 0.5-2.5. mu.g/100 uL) at 4 ℃ with shaking overnight, followed by 3 washes with 1 XPBS containing 0.1% Triton X-100 and 2% BSA the next day for 10 minutes each. The samples were then washed twice with 1 × PBS for 5 minutes each, and rinsed once with distilled water.
The samples were incubated with 100nM single-stranded DNA molecules (Padlock molecule 2) (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) at 37 ℃ with shaking for 15-30 minutes, rinsed with 1 XPBS three times, rinsed once with distilled water, aspirated off all the liquid and immediately added with T4 ligase system (50mM Tris-HCl, 10mM MgCl. sub.10 mM)210mM DTT, 1mM ATP, 0.1U/. mu. L T4 DNA ligase, pH7.5), 1 XPBS rinsing three times, one rinsing with distilled water, pipetting all the liquid and adding the phi29 polymerase system (0.5mM dNTPs, 0.25U/. mu.L phi29 DNA polymerase, 0.2mg/mL BSA, 50mM Tris-HCl, 10mM MgCl2，10mM(NH4)2SO44mM DTT, pH7.5), incubated at 37 ℃ for 60 minutes with shaking, rinsed three times with 1 XPBS and once with distilled water. 0.5. mu.M of target-specific fluorescent probe 2 (diluted in a solution containing 2 XSSC, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) was added and incubated at 37 ℃ for 20-30 minutes with shaking. Washing with 1 XPBS containing 0.1% Triton X-100 for 10 minutes, and then washing twice with 1 XPBS, each timeFor 5 minutes. Mu.g/. mu.L DAPI staining for 5 min followed by 1 XPBS wash for 5 min. Imaging was then performed by fluorescence microscopy.
The experimental results are shown in fig. 6, the left side of the graph is that the cells are circled according to the actual positions of the cells in the bright field, the cells 1, 2 and 3 are circled respectively, the image of the central position of each cell area is the image of the cell nucleus, and the scattered fluorescent bright spots outside the cell nucleus are the signals of Epithelial cell adhesion molecule (Epithelial cell adhesion molecule) proteins detected by the method of the present invention. The tumor transforming growth factor TGF-beta1 can induce the breast cancer cell MCF-7 to undergo an epithelial-mesenchymal transition (EMT) process, and the expression level of the cell EpCAM is gradually reduced along with the extension of the stimulation time of the TGF-beta 1. The EpCAM protein with the reduced expression quantity can be effectively detected by a high-sensitivity method, and the traditional immunofluorescence method is difficult to detect, so the method is used for detection and analysis. The results in the figure show that although the expression level of EpCAM protein is reduced, the method of the invention can still detect dispersed target signals, the signals present fluorescent bright spots obviously distinguished from the background, the signals are easy to identify and judge, and the digital quantitative strategy of the invention can also be used for counting and analyzing the target signals in each cell, so that the method does not relate to the analysis of the fluorescent brightness of the bright spots, avoids the quantitative influence caused by the autofluorescence background, and breaks through the dependence of the traditional method on the fluorescence intensity quantitative method. In addition, the results in the figure also show that the digital quantitative results of the three cells are different, and reflect the inherent heterogeneity of the tumor cells to a certain extent.
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Claims (10)

1. The target positioning and quantitative detection method based on the DNA spherical nanostructure imaging is characterized in that different oligonucleotide labels are obtained by directly or indirectly combining a sample to be detected with different target specificity binding couplers, DNA nanospheres with different target specificities are formed under the rolling circle replication action mediated by different oligonucleotide labels, and after the DNA nanospheres with different target specificities are combined with a specific fluorescence labeling probe, the target to be detected in the sample to be detected is positioned and quantitatively detected through different colors and different imaging results.
2. The method for target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 1, characterized in that the DNA nanospheres with different target specificity are prepared as follows: the method comprises the following steps of mediating different specific single-stranded DNA molecules corresponding to different oligonucleotide tags to perform cyclization reaction and rolling circle replication, adding a single-stranded DNA sequence unit which is completely complementary with the single-stranded DNA molecule sequence at the 3' tail end of the nucleic acid sequence of the oligonucleotide tags after each rolling circle replication by taking the single-stranded DNA molecules as templates, and finally forming a long single-stranded DNA with a plurality of repeated DNA sequence units after a plurality of rolling circle replications, wherein the long single-stranded DNA can spontaneously form a DNA nanosphere.
3. The method for targeted localization and quantification of DNA spherical nanostructure imaging-based target according to claim 1, wherein the target-specific binding partner comprises a target-specific binding molecule that interacts directly or indirectly with the target and an oligonucleotide tag fragment, and wherein the oligonucleotide tag sequences of the target-specific binding partner labels with different specificities are different; incubating the specific binding partner of the target and the assay sample under conditions that promote the binding of the specific binding molecule of the target to the target in the assay sample, optionally removing unbound specific binding partner of the target.
4. The method for target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 1, characterized in that the oligonucleotide tag fragment in the target specific binding conjugate is contacted with one or more specific single-stranded DNA molecules, wherein both ends of the specific single-stranded DNA molecules each comprise a portion complementary to the sequence of the oligonucleotide tag fragment, and the specific single-stranded DNA molecules further comprise at least two sequences identical to the specific fluorescently labeled probe.
5. The method for target localization and quantification based on DNA globular nanostructure imaging according to claim 1, characterized in that the detection method further comprises removing the bound fluorescently labeled probe from the DNA globular structure by an applied electric field or a buffer solution.
6. The method for multi-target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 1, characterized in that the analysis sample is one or more of cells, frozen tissues or paraffin-embedded tissues; the target is one or more of nucleic acid, protein, polypeptide or proteoglycan; the specific binding partner of the target is a specific binding molecule that is non-covalently or covalently coupled or linked to an oligonucleotide tag.
7. The method for multi-target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 3, characterized in that the specific binding molecule is an antibody, an antibody fragment, an aptamer, an oligonucleotide or a small molecule, and the oligonucleotide tag fragment comprises a nucleic acid sequence corresponding to the target, which can be hybridized with a single-stranded DNA molecule with matched sequence and extended as a primer under suitable extension conditions along the hybridized circular DNA template.
8. The method for multi-target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 1, characterized in that the specific single-stranded DNA molecule is a molecule capable of being linked into circular DNA under the action of ligase after hybridization with the oligonucleotide tag or a separately synthesized or engineered circular DNA molecule or a single-stranded rolling circle replication product.
9. The method for multi-target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 5, characterized in that the applied electric field is electrophoresis, and the buffer solution is phosphate buffered saline solution or phosphate buffered saline solution containing surfactant Triton X-100 or Tween 20.
10. The method for multi-target localization and quantitative detection based on DNA spherical nanostructure imaging according to claim 1, characterized in that the method uses epi-fluorescence or confocal microscopy to image the sample.
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