CN113684256B

CN113684256B - Method for detecting multiple targets by multiple positioning based on green solvent and programmable oligonucleotide probe

Info

Publication number: CN113684256B
Application number: CN202111008240.2A
Authority: CN
Inventors: 王琛; 邹秉杰; 宋沁馨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2024-05-14
Anticipated expiration: 2041-08-30
Also published as: CN113684256A

Abstract

The invention discloses a method for detecting multiple targets (such as proteins in cells or other targets) based on multiple localization of green solvents and programmable oligonucleotide probes, which comprises the steps of directly or indirectly connecting target-specific oligonucleotides with the targets, specifically connecting oligonucleotides connected with one or more targets with corresponding fluorescent probes, and realizing localization analysis of the corresponding targets through localization detection of the fluorescent probes. In order to realize more multi-target localization analysis, the method uses a green solvent to remove the detected fluorescent probes, then the oligonucleotides on other unanalyzed targets are specifically connected with the corresponding fluorescent probes, and then localization analysis of other corresponding targets is realized through localization detection of the fluorescent probes. Therefore, the method is not affected by signal crossing of fluorescent probes with different specificities, is not hindered by limited detection channel number of fluorescent detection equipment, and can realize multi-target positioning analysis.

Description

Method for detecting multiple targets by multiple positioning based on green solvent and programmable oligonucleotide probe

Technical Field

The invention belongs to the field of multi-target positioning detection in a sample to be tested, and particularly relates to a method for detecting multiple targets by multi-positioning based on a green solvent and a programmable oligonucleotide probe.

Background

Cells are the basic units that make up an organism; all physiological functions of the organism are implemented through various cell-cell communication and mutual cooperation. Therefore, it is important to elucidate the molecular mechanisms by which individual cells function biologically in organs and tissues of the body. Also in the context of biological environments, therefore, the characterization of molecular composition of single cells and tissue samples is critical for the deep exploration of vital activity and disease occurrence mechanisms.

Immunofluorescence imaging techniques are ideal tools for the characterization of molecular targets in biological samples (e.g., cells or tissues) that can maintain the original position of the molecular targets in the sample, thereby obtaining accurate positional information. However, due to the overlapping of the fluorophore spectra, the fluorescence microscope has a limited detection channel, and the multiplex detection capability of the immunofluorescence method is limited, and the method can only detect 3-5 targets. In domestic and foreign research, various technical methods for circumventing the overlapping limitation of fluorescence spectra have been reported, including methods of batch-wise immunosorbent assay staining and multiple rounds of target imaging. However, such methods take a long time, such as more than 2 hours per round of antibody staining incubation at normal temperature and 12-16 hours at 4 degrees celsius, and thus are not widely used in practice. Another class of methods is to incubate antibodies against all targets to be analyzed simultaneously, which are labeled with oligonucleotide tags in advance, to perform differential detection of the targets, and then to perform batch-wise fluorescence imaging of the antibodies against the targets using fluorescent probes. The method detects antibodies of one or more targets at a time, then uses denaturing agents such as formamide to realize dissociation of the fluorescent probes from the targets, and then detects other targets or targets, so that the cyclic process of hybridization combination/dissociation of different specific fluorescent probes and targets is realized in a cyclic and reciprocating manner, the limitation of fluorescence spectrum overlapping is avoided, and detection analysis of more targets is realized. Compared with the method of antibody staining in batches, the method improves the speed of multi-target detection, but the method repeatedly depends on toxic reagents to dissociate fluorescent probes, which is not beneficial to the protection of personnel and environment in real practice; in addition, such methods do not have a signal amplification process or insufficient signal amplification, and effective detection of low abundance molecular targets is difficult, especially when the sample to be tested is subjected to common fluorescent scattering and fluorescent background effects. Therefore, there is a need to innovate the prior art to provide an efficient technical method for detecting molecular targets in biological samples with multiplex detection capability and high sensitivity.

Disclosure of Invention

The invention aims to: the invention aims to provide an effective technical method for detecting molecular targets in biological samples, which has multiple detection capability and high sensitivity.

The technical scheme is as follows: in order to solve the technical problems, the invention provides a method for detecting multiple targets by multiple positioning based on a green solvent and a programmable oligonucleotide probe, wherein the method for detecting multiple targets by multiple positioning is characterized in that the target to be detected is directly or indirectly connected with a specific oligonucleotide, the oligonucleotide connected with the target is specifically connected with a corresponding fluorescent probe, positioning analysis of the corresponding target is realized by positioning detection of the fluorescent probe, then the detected fluorescent probe is removed by using the green solvent, then the oligonucleotides on other unanalyzed targets are specifically connected with the corresponding fluorescent probe, and positioning analysis of other corresponding targets is realized again by positioning detection of the fluorescent probe.

Wherein the target refers to an analyte present in a sample to be tested, wherein the sample is one or more of a cell, a tissue, a cell extract, a tissue extract, or a cell secretion; the analyte is one or more of nucleic acid, protein, polypeptide, lipoprotein and glycoprotein. It will be appreciated that prior to analysis of the sample, its inclusion target is known, suspected, unknown or not suspected.

Wherein each target interacts directly or indirectly only with an oligonucleotide of a specific sequence, wherein said indirect interaction refers to a direct interaction of the target with a specific intermediate molecule to which the oligonucleotide of the specific sequence is attached, wherein said intermediate molecule is at least one antibody, antibody fragment, oligonucleotide, aptamer, small molecule, the intermediate molecule having target specificity.

Wherein the fluorescent probe comprises at least a nucleic acid portion and a fluorescent signal emitting portion; the fluorescent probe hybridizes to the oligonucleotide by direct or indirect means, wherein the direct means that the fluorescent probe hybridizes directly to the oligonucleotide according to the base complementary pairing principle; by indirect means, the oligonucleotide hybridizes to an intermediate nucleic acid molecule which hybridizes to the fluorescent probe, wherein the intermediate nucleic acid molecule is one or more nucleic acid molecules having a plurality of sequences capable of hybridizing to the fluorescent probe.

Wherein the green solvent is a glycerol aqueous solution or a glycerol buffer solution capable of breaking base complementary pairing hydrogen bonds, and the use of the green solvent does not generate chemical and biological harm to the environment and operators.

Wherein the oligonucleotide probes are programmable oligonucleotide probes, all the oligonucleotide probes can be designed orthogonally based on different sequences of target species, and the number of fluorescent probes which can be connected on each target specific oligonucleotide can be increased through the design of nucleic acid sequences so as to achieve fluorescence signal enhancement. The oligonucleotides may be extended in situ by at least one nucleic acid amplification technique to form long single stranded nucleic acid molecules having multiple stretches of sequence capable of hybridizing to the fluorescent probes.

Wherein the specific ligation of the target-ligated oligonucleotides to the corresponding fluorescent probes is performed in batches, the number of targets analyzed per batch is not greater than the number of detection channels of the fluorescent detection device in the batch of fluorescent detection performed by hybridizing the specific fluorescent probes to the oligonucleotides, wherein the number of detection batches to be completed is determined jointly by the number of targets to be analyzed and the number of detection channels of the fluorescent detection device, and wherein the fluorescent labels of the different fluorescent probes used in the same batch are different.

Wherein, the batch hybridization of the specific fluorescent probe to the oligonucleotide completes the fluorescent detection in the fluorescent detection, namely, the fluorescent detection equipment is utilized to detect the fluorescent light emitted by the fluorescent probe, and the positioning analysis is carried out on the corresponding target according to the position and the number of the fluorescent light.

Wherein the fluorescence detection device is an epifluorescence microscope or a confocal microscope.

Specifically, the invention discloses a high-sensitivity multi-target positioning detection method, which comprises the following steps:

1) Binding a sample (e.g., a tumor cell sample suspected of containing a plurality of targets to be tested) that may contain a plurality of targets to be tested (e.g., biomolecules such as proteins) to a specific binding agent (e.g., an oligonucleotide-labeled antibody) directed against the targets; wherein the specific binders for each target are linked to one signal amplifier chain, and wherein different specific binders are linked to different signal amplifier chains;

2) Binding the signal amplifier strand to a fluorescent probe having a complementary strand to the signal amplifier strand after step 1). Wherein each signal amplifier strand is bound to a fluorescent probe;

3) Imaging the sample after step 2) to detect the position and number of signal amplifier strands to which the fluorescent probe is stably bound. Wherein the presence of the corresponding target is detected in the signal amplification nucleic acid chain to which the fluorescent probe is stably bound;

4) Removing the fluorescent probe bound to the signal amplifier strand after step 3) to quench its fluorescent signal;

5) Repeating steps 2) -4) wherein each time an analysis is performed using one or more (no more than the number of microscopic detection channels) fluorescent probes specific for the signal amplifier strand.

Wherein, in step 1) the sample, which may contain a plurality of targets to be analyzed, is understood to be that the targets are unknown, known, suspected, or not suspected prior to analysis of the sample to be analyzed; whether a specific binding partner can bind to a sample depends on whether a given target is present in the sample (e.g., when a given target is present on the sample, the specific binding partner can bind to the sample). "bind to a sample" means that the specific binding member binds to its corresponding target.

Wherein in step 1) the sample is a cell, cell population, tissue, or secretion, extract thereof, and the target may be a nucleic acid, protein, polypeptide, lipoprotein, glycoprotein.

Wherein in step 1) the signal amplifier strand is at least one single-stranded nucleic acid molecule having a plurality of repeating nucleotide sequence units, individually synthesized or engineered, each "unit" being capable of hybridization binding to at least one fluorescent probe. Therefore, the method provided by the invention is a signal amplification detection method, and the detection sensitivity of the low-abundance targets is improved.

Wherein in step 1) the signal amplifier strand is at least one functionalized material comprising a plurality of nucleotide sequences, which material may be a metal ion, a non-metal particle, a quantum dot.

Wherein in step 1) the specific binding member comprises a target specific binding molecule and an oligonucleotide fragment, wherein the binding members having different specificities comprise different oligonucleotide fragments; wherein the oligonucleotide fragments function as tags for different targets, different oligonucleotide fragments being used to distinguish between different targets; furthermore, the oligonucleotide fragment functions as a docking strand capable of ligating with a specific signal amplifier strand; wherein the specific binding molecule is an antibody, antibody fragment, aptamer, oligonucleotide or small molecule.

Wherein in step 1) each specific binding partner binds at most only to one target to be analyzed, and wherein the oligonucleotide fragments in the specific binding partner bind only to one signal amplifier strand, whereby each signal amplifier strand corresponds to one target to be analyzed; wherein the signal amplifier strand binds only to specific fluorescent probes.

Thus, the detection of the presence of the signal amplifier chain to which the fluorescent probe is stably bound in step 3) indicates the presence of the corresponding target, and thus, the position and number of the signal amplifier chain to which the fluorescent probe is stably bound detected in step 3) indicates the position and number of the corresponding target.

Wherein, in step 4), the fluorescent probe bound to the signal amplifier chain is removed using a green reagent, glycerol aqueous solution or glycerol buffer, to quench the signal; wherein the green reagent is that the reagent is harmless to operators and environment in the process of using the reagent.

Wherein the fluorescent probe is a material comprising at least a fluorescent signal emitting moiety and an oligonucleotide moiety; wherein the material is at least one metal particle, non-metal particle, nucleic acid, quantum dot, and the material is a submicron or nanoscale structure.

Wherein the fluorescent probe can emit specific fluorescence under specific excitation light and can be detected and analyzed by a fluorescent microscope or a fluorescent scanning instrument; therefore, when a plurality of targets (not more than the number of detection channels of the fluorescence detection device) are detected simultaneously, the fluorescence probes for the different targets contain different fluorescent labels, and can emit different specific fluorescence under different specific excitation lights respectively, so that the targets can be distinguished and identified through the specific fluorescence emitted by the labeled imaging materials.

Wherein the different fluorescent probes may be labeled identically, so that in step 5) only one fluorescent probe specific for the signal amplifier strand is used at a time, i.e. only one target to be analyzed is detected at a time. This method requires only a single excitation wavelength and a single detector fluorescence microscope or fluorescence scanning instrument.

Wherein the different fluorescent probes may be differently labeled, so that in step 5) a plurality (not more than the number of microscopic detection channels) of fluorescent probes specific for the signal amplifier strand are used at a time, i.e. a plurality (not more than the number of microscopic detection channels) of targets to be analyzed are detected at a time. This method may utilize fluorescence microscopy or fluorescence scanning instruments for a variety of excitation wavelengths and multi-channel detectors.

Wherein, imaging the sample in step 3) refers to imaging detection of fluorescent signals using a fluorescent detection device, wherein the fluorescent detection device is an epifluorescent microscope or a confocal laser microscope.

Among other things, it should be understood that: in step 1) different signal amplifier strands are directed against different targets, and in step 5) one or more (no more than the number of microscopy detection channels) fluorescent probes specific for said signal amplifier strands are used each time, i.e. one or more (no more than the number of microscopy detection channels) targets to be analyzed are detected each time; if the number of targets currently analyzed exceeds the fluorescence detector detection channel, the fluorescent probe bound to the signal amplifier chain for the current target can be removed by using a green reagent, i.e., an aqueous glycerol solution or a glycerol buffer solution, so as to quench the signal, and then the steps 2-4 are repeated. Here, the purpose of the signal quenching is to prevent the signal of the current target from affecting the detection of other targets in the next round, and thus break through the limitation of the fluorescence detector detection channel; wherein the signal quenching is in effect the dissociation of the labeled imaging material from the bound signal amplifier strands, the signal amplifier strands from which the labeled imaging material is removed can still be bound again to the specific labeled imaging material. Therefore, the method of the invention is a high-sensitivity multi-target detection method based on the batch detection of targets by green reagents and programmable oligonucleotide probes.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: the invention can detect multiple targets exceeding the number of detection channels by the fluorescence microscope, and avoids the spectral overlap of fluorophores and the limited obstruction of the detection channels of the fluorescence microscope. Compared with a multi-target detection method based on batch immune antibody staining, the method has the advantages that the detection time is shorter, the amplification detection of target signals can be realized, and the method is particularly suitable for high-sensitivity detection of low-abundance targets. Compared with a multi-target detection method based on nucleic acid labels and formamide buffer, the method uses environment-friendly glycerol aqueous solution or glycerol buffer, is a personnel and environment-friendly method, and has higher speed of destroying nucleic acid hybridization hydrogen bonds. The sensitivity of the invention can reach the expression of low-abundance protein epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) protein in 1 cell of breast cancer cell line MDA-MB-231 (the mRNA general expression quantity is 9.899 units, and the data is derived from CANCER CELL LINE Encyclopedia (CCLE) database); the batch detection round can reach at least 10 rounds.

Drawings

FIG. 1 is a schematic diagram of the principle of protein target detection in a cell sample based on glycerol aqueous solution and fluorescent probe imaging;

FIG. 2 is a schematic diagram of the principle of protein target detection in a cell sample based on glycerol aqueous solution, a signal amplification chain and fluorescent probe imaging;

FIG. 3 is a schematic diagram of the principle of protein target detection in a cell sample based on glycerol aqueous solution, nucleic acid in situ amplification technology and fluorescent probe imaging provided by the invention;

FIG. 4 is a graph showing the results of one embodiment of target detection in a sample to be tested provided in the present invention;

FIG. 5 is a graph showing the results of one embodiment of the invention based on the use of aqueous glycerol for the dissociation of imaging probes to achieve signal quenching and target detection in a sample to be tested;

FIG. 6 is a graph showing the results of one embodiment of the present invention based on ten-pass repeated imaging detection of the same target in a sample to be tested using an aqueous glycerol solution;

FIG. 7 is a graph showing the results of one embodiment of the present invention for quantitative comparison analysis of the results of ten repeated imaging tests of the same target in the sample to be tested of FIG. 6.

Detailed Description

The invention provides a target height multiple positioning detection method based on a green solvent and a programmable oligonucleotide probe. The method employs an orthogonally designed oligonucleotide tag that can be stably attached to a target-specific binding molecule (e.g., an antibody) by an intermediate substance (e.g., streptavidin and/or biotin) to form a target-specific conjugate; then contacting the target specific binders with different 'labels' with a sample to be tested and binding the target specific binders on the corresponding targets; the labels are then attached to long single-stranded nucleic acid molecules (e.g., signal amplification strands) (see FIG. 2) or the "labels" are formed in situ to long single-stranded nucleic acid molecules (see FIG. 3) that are capable of hybridization binding to a plurality of fluorescent probes, and the long single-stranded nucleic acid molecules to which the fluorescent probes are stably bound are then detected under a signal detection system (e.g., fluorescent microscope), i.e., the location and number of specific targets in the sample to be tested can be reflected by their location and number. In addition, the fluorescence stably bound to the long single-stranded nucleic acid molecules can be removed by the green reagent (such as glycerol solution) in the invention, so that batch detection of all targets to be detected can be realized, namely, due to the limited number of detection channels of a fluorescence microscope, detection based on the fluorescence probes is firstly carried out on one or more targets (not exceeding the number of detection channels of the fluorescence microscope), then hybridized fluorescence probes are removed by the green reagent (such as glycerol solution), and detection based on the fluorescence probes is carried out on the other one or more targets (not exceeding the number of detection channels of the fluorescence microscope), and the cycle is repeated until all targets are detected completely. Therefore, the invention can realize the multi-target positioning detection analysis exceeding the number of detection channels of the fluorescence microscope.

The method of the invention can be used for positioning and detecting a plurality of targets (such as proteins and nucleic acids) in a sample to be tested (such as a biological sample). In some cases, whether the target is unknown, suspicious, in the sample to be tested, one sample to be tested may contain one or more 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. The method can realize the signal amplification detection of the target by increasing the number of fluorescent probes hybridized with the target-specific oligonucleotides, and is particularly suitable for high-sensitivity detection of low-abundance targets.

The method breaks through the limitation of the detection channels of the fluorescence microscope, and realizes the detection flux of multiple targets exceeding the number of the detection channels of the fluorescence microscope by using a green reagent (such as glycerol solution).

In addition, the method of the invention can distinguish between different fluorescent probes labeled with fluorescent labels, regardless of the proximity of the target position and relative distance in the sample to be tested.

These methods have applicability in, for example, medical diagnostics (e.g., detection and characterization of circulating tumor cells, detection of multiple targets of molecular biological signaling pathways).

In the present invention, the term "target" may be any biological component to which localization or quantitative analysis is desired and to which a specific binding molecule is capable of binding is present. In some embodiments, the target may be an engineered or non-naturally occurring biomolecule. The term "biomolecule" is any molecule produced by a living organism, including macromolecules such as proteins, proteoglycans, lipids and nucleic acids, as well as small molecules such as metabolites and natural products. Examples of biomolecules include, but are not limited to: DNA, RNA, cDNA, or a DNA product of RNA subjected to reverse transcription (in general).

In some embodiments, a target may be a protein target, for example, a protein of the 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; globular proteins, and acute phase proteins; heme protein; cell adhesion proteins; transporting proteins across a membrane; protein is transported in the same direction or in opposite directions; hormones and growth factors; a receptor; a DNA binding protein; a transcriptional regulator; immune system proteins; nutrient storage/transport proteins; an enzyme.

Example 1

(1) Experimental materials and reagents:

Streptavidin (purchased from beijing boaosen); human breast cancer cell line MDA-MB 231 (purchased from Shanghai ATCC cell bank); citrate buffer, phosphate buffered saline (1 XPBS solution, pH 7.4) (available from Gibco); DMEM medium (containing penicillin-streptomycin diabodies) (purchased from keyl organisms); sterile Fetal Bovine Serum (FBS) (purchased from Natocor-Industrial Biol Twill gica); bovine serum albumin (bovine serum albumin, BSA) was purchased from (amerco); cell culture dishes (from tin-free resistant); biotin, formamide, polyethylene glycol tert-octylphenyl ether (Triton X-100) (from Sigma-Aldrich); 4', 6-diamidino-2-phenylindole (DAPI) (available from armed bosrad); all probes, oligonucleotides, and template molecules were prepared by Shanghai Biotechnology Co., ltd; dNTPs (purchased from beijing centenasia); RNaseA, salmon sperm DNA solution (Salmon Sperm DNA Solution) (available from Simer's Feier); biotinylated antibody Anti-EpCAM (Biotin) (purchased from Abcam); t4 DNA LIGASE ligase, phi29 DNA polymerase (from NEW ENGLAND Biolabs); ammonium chloride, sodium hydroxide (purchased from the national drug group); deionized Water for experiments (effluent measurement 18.2mΩ) was from Water purifier Explorer series WaterPurification system (available from Blue Water); the cell grade test water is from an autoclaved analytical grade test water; other molecular biology experimental waters (from drohens); other reagents were all analytically pure; the fluorescence microscope was a Nikon ECLIPSE NI microscope (available from Nikon, japan).

Oligonucleotide 1: biotin-AAAAA AAAAA AAAAA GAGAG CGACA CTATG AGACA GGTGA TCCCA TCCTG AGC

Template molecules 1：PO₄-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

(2) Experimental steps, content and conditions:

Oligonucleotide tag-specific antibody modification: mu.L of 2.5. Mu.M oligonucleotide 1 was taken and mixed well with 25. Mu.L of 2.5. Mu.M streptavidin and incubated for 30 minutes at 37 ℃. Then, 50. Mu.L of 1.25. Mu.M biotinylated antibody Anti-EpCAM (Biotin) was added to the above reaction mixture, and the mixture was thoroughly mixed, incubated at 25℃for 30 minutes, followed by 1mM Biotin, and incubated at 25℃for 20 minutes, to obtain an oligonucleotide 1-labeled Anti-EpCAM antibody solution (the diluted solution had a composition of ：8mM Na₂HPO₄,2mM NaH₂PO₄,150mM NaCl,0.1％BSA,0.025％Tween 20,pH 7.4,0.5mg/mL salmon sperm DNA, and the reagents not shown in step 1 were all purchased from the national drug group).

Specific procedure for the detection of epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) protein expressed in the human breast cancer cell line MDA-MB 231: the human breast cancer cell line MDA-MB 231 was cultured in a glass bottom petri dish, cultured in a DMEM medium containing 10% sterile fetal bovine serum, and cultured in a sterile cell incubator at 37℃with 95% relative humidity and 5% carbon dioxide gas until the cell fusion degree was 30%, to obtain the cell sample of this example.

Taking the cell sample, rinsing 3 times with 1 XPBS, then incubating for 45 minutes at normal temperature by using 4% paraformaldehyde solution, then reacting for 20 minutes in 1 XPBS solution of 100mM NH ₄ Cl, and washing for 5 minutes with 1 XPBS; next, the mixture was reacted in 1 XPBS of 0.1% Triton X-100 for 2 minutes, rinsed with 1 XPBS solution, then incubated in 5% BSA solution for 2 hours at room temperature, and reacted in 0.05mg/mL RNase A for 30 minutes at 37℃and rinsed three times with 1 XPBS; 0.1mg/mL streptavidin (containing 0.5mg/mL Salmon Sperm DNA) was added and incubated at 37℃for 30min followed by 1mM biotin at 37℃for 30min and rinsed 3 times with 1 XPBS. After all the above treatments were completed, the cell samples were incubated with the oligonucleotide 1-labeled antibody Anti-EpCAM overnight (14 hours) at 4 ℃ and washed 3 times with 1×pbs containing 0.1% triton X-100 and 2% bsa every day for 10 minutes, then washed twice with 1×pbs every 5 minutes and rinsed once with distilled water, to obtain the oligonucleotide 1-labeled antibody Anti-EpCAM-conjugated cell samples. (the reagents involved in this section are all noted in step 1).

The above cell sample bound with oligonucleotide 1 labeled antibody Anti-EpCAM was contacted with 100nM template molecule 1 (diluted in a solution containing 2×citrate buffer, 20% formamide, 0.5mg/mL Salmon Sperm DNA), incubated at 37 ℃ for 30 minutes, 1×pbs rinsed three times, distilled water rinsed once, T4 ligase system (50 mM Tris-HCl,10mM MgCl ₂, 10mM DTT,1mM ATP,0.1U/μ L T4 DNA LIGASE, pH 7.5) was added immediately after all liquid was absorbed, incubated at 37 ℃ for 30 minutes, 1×pbs rinsed three times, distilled water rinsed once, phi29 polymerase system (0.5mM dNTPs,0.25U/μL phi29 DNA polymerase,0.2mg/mL BSA,50mM Tris-HCl,10mM MgCl₂,10mM(NH₄)₂SO₄,4mM DTT,pH 7.5), was added immediately after all liquid was absorbed for 60 minutes at 37 ℃,1×pbs rinsed three times, distilled water rinsed once. 0.5. Mu.M target-specific fluorescent probe 1 (diluted in a solution containing 2 Xcitrate buffer, 20% formamide, 0.5mg/mL Salmon Sperm DNA) was added and incubated at 37℃for 30 minutes. Washing with 1 XPBS containing 0.1% Triton X-100 for 10 minutes followed by 1 XPBS washing twice for 5 minutes each followed by incubation in 1. Mu.g/. Mu.L DAPI solution for 5 minutes followed by 1 XPBS washing for 5 minutes. Finally, imaging was performed by fluorescence microscopy. After the end of imaging, 95% aqueous glycerol solution was added, left to stand for 1 minute, rinsed three times with 1×pbs, and imaged again by fluorescence microscopy. The experimental results are shown in fig. 4, 5 and 6. (the reagents involved in this section are all indicated in step 1, wherein the reagents involved in the T4 ligase system and the phi29 polymerase system are purchased from NEW ENGLAND Biolabs).

In fig. 4, there is a graph of the results of detecting the epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) protein expressed in the cell sample (human breast cancer cell line MDA-MB 231) using the method of the present invention, wherein cell 1 and cell 2 are any two adjacent human breast cancer cell lines MDA-MB 231 in the cell sample, and the results indicate that the signal of the protein target exhibits a punctiform distribution, and that such signal can be clearly distinguished from the background, and the size is in the range of several hundred nanometers, so that the detection of the target signal by the method can be performed without relying on a high resolution microscope (the results are photographed by the Nikon ECLIPSE NI microscope of japan). Referring to the schematic diagram of FIG. 3, the dot signal is a single-stranded nucleic acid molecule stably bound to the specific imaging probe 1 detected by a fluorescence microscope in FIG. 4. It will be appreciated that the single stranded nucleic acid molecule is linked to the oligonucleotide 1 labelled antibody Anti-EpCAM by oligonucleotide 1, whereas the target EpCAM and antibody Anti-EpCAM bind to each other, thus detection of a punctual signal from the "single stranded nucleic acid molecule bound to imaging probe 1" indicates the presence of the target epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) protein in the cell sample (human breast cancer cell line MDA-MB 231), and thus the location and number of punctual signals indicates the location and number of target epithelial cell adhesion molecules (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) in the cell sample (human breast cancer cell line MDA-MB 231), i.e. the location of the bright spot is the location of the target epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) in the cell sample (human breast cancer cell line MDA-MB 231), and the number of target epithelial cell adhesion molecules (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) is 370±24 (in cell 1) and 330±30 (cell 2), respectively. Among them, according to the single cell sequencing data of the tumor cell line disclosed in International encyclopedia of tumor cell lines (CCLE) ", it was shown that the EPCAM (ENSG 00000119888.6) gene was low-expressed in the human breast cancer cell line MDA-MB 231 cells, that is, it was shown that the epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) measured in this example was a low-abundance protein in the human breast cancer cell line MDA-MB 231 cells. The results in FIG. 4 show the high sensitivity detection characteristics of the low abundance protein by the method of the invention, the signal of the protein can be amplified and converted into fluorescent bright spots which are easy to identify by the method of the invention, and the quantitative results of the protein are 370+/-24 (in cell 1) and 330+/-30 (in cell 2) respectively through the counting of the fluorescent bright spots.

In fig. 5, the upper left is an image of the nucleus, showing the specific location of the cells in the sample and the approximate distribution of subcellular relative locations of the cells, and the lower left is a graph of the results of detection of the epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) protein expressed in the sample of cells (human breast cancer cell line MDA-MB 231) using the method of the invention (principle see fig. 3) prior to the use of glycerol aqueous solution, showing that the signal of the protein target exhibits a punctiform distribution and that such signal is clearly distinguishable from the background, the signal being derived from a single-stranded nucleic acid molecule bound to imaging probe 1, formed by an in situ amplification extension of the nucleic acid from oligonucleotide 1. Referring to the method principle of FIG. 3, if the detection of the oligonucleotides in the second batch is to be achieved, it is critical that the fluorescent probes stably bound to the single-stranded nucleic acid molecules can be dissociated by an aqueous glycerol solution. Thus, in this example, after the cell sample was subjected to imaging detection, a 95% aqueous glycerol solution was added, left standing for 1 minute, and rinsed three times with 1×pbs, and as a result, see fig. 5 (lower right panel), it was found that the originally detected punctiform signal in the sample (see lower left panel of fig. 5) quenched and disappeared after the use of the 95% aqueous glycerol solution (see lower right panel of fig. 5). The principle of the method of fig. 3 can be explained as: after 95% glycerol aqueous solution is added to the sample distributed with the dot-shaped signals, the hydrogen bonding force of the fluorescent probe stably combined on the single-stranded nucleic acid molecules is weakened and dissociated, and after 1×PBS is rinsed, the fluorescent probe is removed, so that signal quenching disappears.

Based on the results of the method shown in fig. 3, the present embodiment repeatedly images the same cell (human breast cancer cell line MDA-MB 231) in the cell sample (human breast cancer cell line MDA-MB 231) ten times, and the results refer to fig. 6, namely, the imaging analysis is performed on the target epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) in the sample by using the method of the present embodiment, so as to obtain the first imaging result in fig. 6, then the fluorescent probe is removed by using a 95% glycerol aqueous solution, so that the signal is quenched, and the repeated imaging analysis is performed on the target epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) in the sample again by using the method of the present embodiment, so as to obtain the second imaging result in fig. 6. The procedure was repeated, "the fluorescent probe was then removed using 95% glycerol in water, the signal quenched, and the target epithelial cell adhesion molecule (EPITHELIAL CELLULAR ADHESION MOLECULE, epCAM) in the sample was again subjected to imaging analysis using the method of this example," and finally imaging was performed through 10 rounds to obtain imaging results for a total of 10 rounds as shown in fig. 6. From this result, it can be seen that the use of the glycerol aqueous solution does not significantly affect the signal presentation, i.e., the position at which the fluorescent bright spot appears in each round of imaging does not change. In addition, the signal of EpCAM in the imaging results of 10 rounds in total in fig. 6 was quantitatively analyzed, and the results are shown in fig. 7, which show that the number of EpCAM signals (fluorescent bright points) in 10 imaging is not significantly changed, and the use of the glycerol aqueous solution does not significantly affect the significant change of the number of signals (fluorescent bright points). Taken together, the strategy principle of the method of the present invention for multi-batch multi-target (broadly) imaging using aqueous glycerol solutions is capable of implementation.

Sequence listing

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<211> 11

<212> DNA

<213> Fluorescent Probe 1 (ARTIFICIAL SEQUENCE)

<400> 3

cgcgccgagg t 11

Claims

1. A method for detecting multiple targets by multiple positioning based on green solvent and programmable oligonucleotide probe is characterized in that the method comprises the steps of respectively directly or indirectly connecting the target to be detected with specific oligonucleotide, specifically connecting the oligonucleotide connected with the target with corresponding fluorescent probe, realizing positioning analysis of the corresponding target by positioning detection of the fluorescent probe, removing the detected fluorescent probe by using green solvent, specifically connecting the oligonucleotide on other unanalyzed targets with corresponding fluorescent probe, realizing positioning analysis of other corresponding targets by positioning detection of the fluorescent probe,

The fluorescent probe comprises at least a nucleic acid portion and a fluorescent signal emitting portion; the fluorescent probe hybridizes to the oligonucleotide by direct or indirect means, wherein the direct means that the fluorescent probe hybridizes directly to the oligonucleotide according to the base complementary pairing principle; the indirect mode refers to that the oligonucleotide hybridizes to an intermediate nucleic acid molecule which hybridizes to the fluorescent probe, wherein the intermediate nucleic acid molecule is one or more nucleic acid molecules with multiple sequences capable of hybridizing to the fluorescent probe;

The green solvent refers to a glycerol aqueous solution or a glycerol buffer solution capable of breaking base complementary pairing hydrogen bonds; the concentration of glycerol was 95%;

An oligonucleotide: SEQ ID NO.1: biotin-AAAAA AAAAA AAAAA GAGAG CGACA CTATG AGACA GGTGA TCCCA TCCTG AGC

Intermediate nucleic acid molecules ：SEQ ID NO.2：PO₄-GTCTC ATAGT GTCGC TCTCT GA TTC GCGCC GAGGT TGTCT CAGCT TTAGT TTAAT ACGCG CCGAG GTAGG GCTCA GGATG GGATC ACCT

Fluorescent probe: SEQ ID NO.3: alexa Fluor 488-CGCGC CGAGG T.

2. The method of claim 1, wherein the target is an analyte present in a sample to be tested, wherein the sample to be tested is one or more of a cell, a tissue, a cell extract, a tissue extract, or a cell secretion; the analyte is one or more of nucleic acid, protein, polypeptide, lipoprotein and glycoprotein.

3. The method of claim 1, wherein each target interacts directly or indirectly with only oligonucleotides of a specific sequence, wherein the indirect interaction refers to the target interacting directly with a specific intermediate molecule to which the oligonucleotides of the specific sequence are attached, wherein the intermediate molecule is at least one antibody, antibody fragment, oligonucleotide, aptamer, or small molecule.

4. The method of claim 1, wherein the specific ligation of the target-linked oligonucleotides to the corresponding fluorescent probes is performed in batches, wherein the number of targets analyzed per batch is not greater than the number of detection channels of the fluorescent detection device, wherein the number of detection batches required to be completed is determined by the total number of targets to be analyzed and the number of detection channels of the fluorescent detection device, and wherein the fluorescent labels of the different fluorescent probes used in the same batch are different.

5. The method for detecting multiple targets by multiple localization based on green solvents and programmable oligonucleotide probes according to claim 4, wherein the batch fluorescent detection in fluorescent detection by hybridization of specific fluorescent probes with oligonucleotides is to detect fluorescence emitted by fluorescent probes by using a fluorescent detection device and to perform localization analysis on the corresponding targets according to the positions and the number of the fluorescence.

6. The method of multiple localization detection of multiple targets based on green solvents and programmable oligonucleotide probes according to claim 4, wherein the fluorescence detection device is an epifluorescence microscope or a confocal microscope.