CN113684256A - Method for detecting multiple targets through multiple positioning based on green solvent and programmable oligonucleotide probe - Google Patents

Method for detecting multiple targets through multiple positioning based on green solvent and programmable oligonucleotide probe Download PDF

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CN113684256A
CN113684256A CN202111008240.2A CN202111008240A CN113684256A CN 113684256 A CN113684256 A CN 113684256A CN 202111008240 A CN202111008240 A CN 202111008240A CN 113684256 A CN113684256 A CN 113684256A
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oligonucleotide
target
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CN113684256B (en
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王琛
邹秉杰
宋沁馨
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China Pharmaceutical University
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

The invention discloses a method for detecting multiple targets (such as proteins or other targets in cells) by multiple positioning based on green solvents and programmable oligonucleotide probes, which directly or indirectly connects target-specific oligonucleotides with the targets, specifically connects oligonucleotides connected with one or more targets with corresponding fluorescent probes, and realizes positioning analysis of the corresponding targets by positioning detection of the fluorescent probes. In order to realize more heavy target positioning analysis, the method uses a green solvent to remove the detected fluorescent probe, then the oligonucleotide on other unanalyzed targets is specifically connected with the corresponding fluorescent probe, and further the positioning analysis of other corresponding targets is realized through the positioning detection of the fluorescent probe. Therefore, the method is not influenced by the signal cross of different specific fluorescent probes, is not hindered by the limited number of detection channels of the fluorescent detection equipment, and can realize the positioning analysis of multiple targets.

Description

Method for detecting multiple targets through 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 multi-target positioning detection based on a green solvent and a programmable oligonucleotide probe.
Background
Cells are the basic unit that constitutes an organism; all physiological functions of an organism are performed through various types of intercellular communication and mutual cooperation thereof. Therefore, it is important to elucidate the molecular mechanism by which each cell in organs and tissues of the body exerts its biological function. And therefore in the context of in situ biological environments, the characterization of the molecular composition of single cell and tissue samples is crucial to the in-depth exploration of life activities and mechanisms of disease development.
Immunofluorescence imaging techniques are ideal tools for the characterization of molecular targets in biological samples (e.g., cells or tissues) that maintain the original position of the molecular target in the sample, thereby obtaining accurate positional information. However, due to the overlapping of the spectra of the fluorophores, the detection channel of the fluorescence microscope is limited, the multiplex detection capability of the immunofluorescence method is limited, and the method can only realize the detection of 3-5 targets generally. In the research at home and abroad, various technical methods for avoiding the overlapping limitation of fluorescence spectra have been reported, including batch-wise immune antibody staining and target multi-round imaging methods. However, the method takes a long time, for example, each round of antibody staining incubation generally needs more than 2 hours at normal temperature, and generally needs 12-16 hours at 4 ℃, so the method is not widely used in practice. The other method is to incubate the antibodies against all targets to be analyzed simultaneously, the antibodies are respectively labeled with oligonucleotide tags in advance to distinguish and detect the targets, and then the fluorescent probes are used for carrying out fluorescence imaging on the antibodies of the targets in batches. The method detects the antibody of one or more targets each time, then realizes the dissociation of the fluorescent probe and the target by using denaturants such as formamide and the like, and then detects the other one or more targets, so that the cyclic process of the hybridization combination/dissociation of the fluorescent probes with different specificities and the target is realized in a cyclic reciprocating manner, the limitation of the overlapping of fluorescence spectra is avoided, and the detection and analysis of more targets are realized. Compared with a batch antibody dyeing method, the method improves the speed of multi-target detection, but needs to repeatedly rely on toxic reagents to perform dissociation of fluorescent probes, and is not beneficial to personnel and environment protection in real practice; in addition, such methods have no or insufficient signal amplification process and are difficult to effectively detect low abundance molecular targets, especially under conditions where fluorescence scattering and fluorescence background effects are common in the sample to be tested. Therefore, there is a need to revolutionize the existing technology and provide an efficient technical method for detecting molecular targets in biological samples with multiplex detection capability and high sensitivity.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide an effective technical method for detecting the molecular target in the biological sample, which has multiple detection capabilities 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, which comprises the steps of respectively directly or indirectly connecting a target to be detected with specific oligonucleotides, specifically connecting the oligonucleotides connected with the target with corresponding fluorescent probes, realizing positioning analysis of the corresponding target by positioning detection of the fluorescent probes, removing the detected fluorescent probes by using the green solvent, specifically connecting the oligonucleotides on other targets which are not analyzed with the corresponding fluorescent probes, and further realizing positioning analysis of the other corresponding targets by positioning detection of the fluorescent probes.
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 is understood that prior to analyzing the sample, its inclusion of the target is known, suspected, unknown or unsuspected.
Wherein each target interacts directly or indirectly only with oligonucleotides of a specific sequence, wherein indirect interaction means that the target interacts directly with a specific intermediate molecule to which the oligonucleotides of the specific sequence are linked, wherein the intermediate molecule is at least one antibody, antibody fragment, oligonucleotide, aptamer, small molecule, which intermediate molecule has target specificity.
Wherein the fluorescent probe comprises at least a nucleic acid moiety and a fluorescent signal emitting moiety; the fluorescent probe is hybridized on the oligonucleotide by a direct mode or an indirect mode, wherein the direct mode refers to that the fluorescent probe is directly hybridized with the oligonucleotide according to the base complementary pairing principle; the indirect method is to hybridize the oligonucleotide to an intermediate nucleic acid molecule, which is then hybridized to the fluorescent probe, wherein the intermediate nucleic acid molecule is one or more nucleic acid molecules having multiple sequences capable of hybridizing to the fluorescent probe.
The green solvent is glycerol aqueous solution or glycerol buffer solution capable of destroying base complementary pairing hydrogen bonds, and the use of the green solvent does not cause chemical and biological hazards to the environment and operators.
Wherein the oligonucleotide probes are programmable oligonucleotide probes, all oligonucleotide probes can be designed based on different sequences of target species in an orthogonal mode, and the number of connectable fluorescent probes on each target-specific oligonucleotide can be increased through the design of nucleic acid sequences so as to achieve the fluorescent signal enhancement. The oligonucleotides can be extended in situ by at least one nucleic acid amplification technique to form long single-stranded nucleic acid molecules having multiple sequences that hybridize to the fluorescent probes.
Wherein, the target-linked oligonucleotides are specifically linked with the corresponding fluorescent probes in batches, the number of targets analyzed in each batch is not more than the number of detection channels of the fluorescent detection equipment in the batch of fluorescent detection completed by hybridizing the oligonucleotides with the specific fluorescent probes, the number of detection batches to be completed is determined by the number of all targets to be analyzed and the number of detection channels of the fluorescent detection equipment, and the fluorescent labels of different fluorescent probes used in the same batch are different.
The step of completing fluorescence detection in the fluorescence detection by hybridizing the oligonucleotides with the specific fluorescent probes in batches refers to the step of detecting fluorescence emitted by the fluorescent probes by using fluorescence detection equipment, and performing positioning analysis on corresponding targets according to the positions and the number of the fluorescence.
Wherein the fluorescence detection device is an epifluorescence microscope or a confocal microscope.
Specifically, the invention discloses a high-sensitivity and multi-target localization detection method, which comprises the following steps:
1) combining a sample (e.g., a tumor cell sample suspected of containing multiple test targets) that may contain multiple test targets (e.g., biomolecules such as proteins) with a specific binding agent (e.g., an oligonucleotide-labeled antibody) against the targets; wherein the specific binders for each target are linked to one signal amplifying nucleic acid strand, and wherein different specific binders are linked to different signal amplifying nucleic acid strands;
2) combining the signal amplifying nucleic acid strand after step 1) with a fluorescent probe having a complementarity to the signal amplifying nucleic acid strand. Wherein each signal amplifying nucleic acid strand is bound to a fluorescent probe;
3) imaging the sample after step 2) to detect the position and number of signal amplifying nucleic acid strands stably bound with the fluorescent probe. Wherein detection of a signal-amplifying nucleic acid strand stably associated with a fluorescent probe indicates the presence of the corresponding target;
4) removing the fluorescent probe bound to the signal amplifying nucleic acid chain after step 3) to quench its fluorescent signal;
5) repeating steps 2) -4), wherein one or more (no more than the number of detection channels of the microscope) of said signal-amplifying nucleic acid strand-specific fluorescent probes are used each time for the analysis.
Wherein, in step 1) said sample, which may contain a plurality of targets to be analyzed, is understood to mean that the targets thereof are unknown, known, suspected, or unsuspected prior to analyzing the sample to be analyzed; whether a specific binder is capable of binding to the 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 binder is capable of binding to the sample). By "bound to the sample" is meant that the specific binding agent binds to its corresponding target.
Wherein, in step 1), the sample is a cell, a cell group, a tissue, or a secretion or an extract thereof, and the target can be a nucleic acid, a protein, a polypeptide, a lipoprotein, or a glycoprotein.
Wherein, in step 1), said signal amplifying nucleic acid strand is at least one single-stranded nucleic acid molecule individually synthesized or engineered with a plurality of repeating nucleotide sequence units, each "unit" being capable of hybridization binding with at least one fluorescent probe. Therefore, the method is a signal amplification detection method, and the detection sensitivity of the low-abundance target is improved.
Wherein, the signal amplifying nucleic acid chain in the step 1) is at least one functionalized material containing a plurality of nucleotide sequences, and the material can be a metal ion, a non-metal particle and a quantum dot.
Wherein in step 1) the specific binders comprise a target specific binding molecule and an oligonucleotide fragment, wherein binders with different specificities comprise different oligonucleotide fragments; wherein the oligonucleotide fragments function as labels for different targets, the different oligonucleotide fragments being used to distinguish between the different targets; in addition, the oligonucleotide fragment functions as a docking chain capable of linking to a specific signal amplification nucleic acid chain; wherein the specific binding molecule is an antibody, an antibody fragment, an aptamer, an oligonucleotide, or a small molecule.
Wherein in step 1) each specific binder binds at most only one target to be analyzed, and wherein the oligonucleotide fragments in said specific binders bind only one signal amplifying nucleic acid strand, whereby each signal amplifying nucleic acid strand corresponds to one target to be analyzed; wherein the signal amplifying nucleic acid strand is bound only to the specific fluorescent probe.
Thus, the detection of a signal amplifying nucleic acid strand stably bound with said fluorescent probe in step 3) indicates the presence of the corresponding target, and therefore, the position and number of the signal amplifying nucleic acid strand stably bound with said fluorescent probe detected in step 3) indicates the position and number of the corresponding target.
Wherein, the fluorescent probe bound to the signal amplifying nucleic acid chain is removed in step 4) using a green reagent, an aqueous glycerol solution or a glycerol buffer, to quench the signal thereof; wherein the green reagent is that the reagent has no harm 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 of metal particles, non-metal particles, nucleic acids, quantum dots, the material being a sub-microscale or nanoscale structure.
Wherein the fluorescent probe can emit specific fluorescence under specific excitation light and can be detected and analyzed by a fluorescence microscope or a fluorescence scanning instrument; therefore, when a plurality of targets (not more than the number of detection channels of the fluorescence detection device) are detected simultaneously, different fluorescence labels contained in the fluorescence probes aiming at different targets can emit different specific fluorescence under different specific excitation light respectively, and therefore, the targets can be distinguished and identified through the specific fluorescence emitted by the labeled imaging material.
Wherein the different fluorescent probes may be identically labeled, so that in step 5) only one fluorescent probe specific for the signal amplifying nucleic acid 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 (no more than the number of microscopic detection channels) of the signal-amplifying nucleic acid chain-specific fluorescent probes are used at a time, i.e. a plurality (no more than the number of microscopic detection channels) of the targets to be analyzed are detected at a time. This method can be used with fluorescence microscopes or fluorescence scanners using multiple excitation wavelengths and multi-channel detectors.
Wherein, in step 3), the sample imaging refers to the imaging detection of the fluorescence signal by using a fluorescence detection device, wherein the fluorescence detection device is an epi-fluorescence microscope or a laser confocal microscope.
Among them, it should be understood that: in step 1) different signal amplifying nucleic acid strands are directed against different targets, and in step 5) one or more (no more than the number of microscopic detection channels) of said signal amplifying nucleic acid strand-specific fluorescent probes are used at a time, i.e. one or more (no more than the number of microscopic detection channels) targets to be analyzed are detected at a time; if the number of targets to be analyzed exceeds the detection channel of the fluorescence detector, the fluorescent probe bound to the signal amplification nucleic acid chain of the current target can be removed by using a green reagent, namely glycerol aqueous solution or 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 breaking the limit of the detection channel of the fluorescence detector; wherein the signal quenching is the actual dissociation of the labeled imaging material from the bound signal amplifying nucleic acid strands, the signal amplifying nucleic acid strands with the labeled imaging material removed can still be bound to the specific labeled imaging material. Therefore, the method of the invention is a high-sensitivity multi-target detection method based on the target batch detection of green reagents and programmable oligonucleotide probes.
Has the advantages that: compared with the prior art, the invention has the following advantages: the invention can detect multiple targets exceeding the detection channel number through the fluorescence microscope, and avoids the spectrum overlapping of fluorophores and the obstruction of limited detection channels of the fluorescence microscope. Compared with a multiple target detection method based on batch immune antibody dyeing, the method has shorter detection time, can realize target signal amplification detection, and is particularly suitable for high-sensitivity detection of low-abundance targets. Compared with a multiple target detection method based on a nucleic acid label and a formamide buffer solution, the method uses a green and environment-friendly glycerol aqueous solution or glycerol buffer solution, is a personnel and environment-friendly method, and has a faster speed for destroying nucleic acid hybridization hydrogen bonds by the glycerol solution. The sensitivity of the invention can reach the expression of the low-abundance protein Epithelial Cell adhesion molecule (EpCAM) protein in 1 Cell of the breast Cancer Cell Line MDA-MB-231 (the general expression quantity of mRNA is 9.899 units, and the data is derived from the Cancer Cell Line Encyclopedia (CCLE) database); the batch detection round of the invention 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 provided by the present invention;
FIG. 2 is a schematic diagram of the principle of protein target detection in a cell sample based on glycerol aqueous solution, signal amplification chain and fluorescent probe imaging provided by the present invention;
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 present invention;
FIG. 4 is the result of one embodiment of target detection in a sample to be tested provided in the present invention;
FIG. 5 is a graph of the results of one embodiment of the imaging probe dissociation removal based on glycerol in water to achieve signal quenching and target detection in a sample to be tested provided in the present invention;
FIG. 6 is a result of one embodiment of ten repeated imaging tests based on the same target in a sample to be tested using an aqueous glycerol solution as provided in the present invention;
FIG. 7 shows the results of a ten-pass reiterated imaging test of the same target in the sample to be tested of FIG. 6, according to an embodiment of the present invention.
Detailed Description
The invention provides a target height multi-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) via an intermediate substance (e.g., streptavidin and/or biotin) to form a target-specific conjugate; these target-specific binders with different "tags" are then brought into contact with the sample to be tested and bound to the corresponding targets; the tags are then ligated to long single-stranded nucleic acid molecules (e.g., signal amplification strands) (see fig. 2) or the "tags" to form long single-stranded nucleic acid molecules in situ (see fig. 3) that are capable of hybridizing to multiple fluorescent probes, and the long single-stranded nucleic acid molecules stably bound with fluorescent probes are then detected under a signal detection system (e.g., a fluorescence microscope), i.e., the position and number of specific targets in the sample to be tested can be reflected by their position and number. In addition, the fluorescence stably bound on the long single-stranded nucleic acid molecule can be removed by the green reagent (such as glycerol solution) in the invention, so that the detection of all the targets to be detected in batches can be realized, namely, because the number of detection channels of the fluorescence microscope is limited, the detection based on the fluorescence probe is firstly carried out on one or more targets (not more than the number of detection channels of the fluorescence microscope), then the hybridized fluorescence probe is removed by the green reagent (such as glycerol solution), and the detection based on the fluorescence probe is carried out on the other one or more targets (not more than the number of detection channels of the fluorescence microscope), and the steps are repeated until all the targets are completely detected. Therefore, the invention can realize the multiple target positioning detection analysis exceeding the number of detection channels of the fluorescence microscope.
The methods of the invention allow for localized detection of multiple targets (e.g., proteins, nucleic acids) in a sample (e.g., a biological sample) to be tested. In some cases, it is unknown, suspect whether the target is present in the sample to be tested, and a 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 signal amplification detection of the target by increasing the number of the fluorescent probes hybridized with the oligonucleotide specific to the target, and is particularly suitable for high-sensitivity detection of low-abundance targets.
The method breaks through the limitation of a fluorescence microscope detection channel, and realizes the multiple target detection flux exceeding the number of the fluorescence microscope detection channel by using a green reagent (such as glycerol solution).
Furthermore, the method of the invention can be distinguished by different fluorescently labeled fluorescent probes regardless of the location of the targets in the sample to be tested and the proximity of the relative distances.
These methods have applicability, for example, in 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 for which a localized or quantitative analysis is desired and to which specific binding molecules capable of binding are present. 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 DNA products of RNA that undergoes reverse transcription (in general).
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: a fibrous protein; globular proteins, and acute phase proteins; a heme protein; a cell adhesion protein; transporting the protein across a membrane; co/reverse transport of proteins; 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 beo olsen, beijing); human breast cancer cell line MDA-MB 231 (purchased from Shanghai ATCC cell Bank); citrate buffer, phosphate buffered saline (1 × PBS solution, pH 7.4) (purchased from Gibco); DMEM medium (containing penicillin-streptomycin diabody) (purchased from triumyl organisms); sterile Fetal Bovine Serum (FBS) (available from Natocor-Industria Biol Logi); bovine Serum Albumin (BSA) was purchased from Amresco; cell culture dishes (purchased from tin-free naisi); biotin, formamide, polyethylene glycol tert-octylphenyl ether (Triton X-100) (purchased from Sigma-Aldrich); 4', 6-diamidino-2-phenylindole (DAPI) (available from bosd, wuhan); all probes, oligonucleotides, template molecules were prepared by Shanghai Biometrics, Inc.; dNTPs (available from Baisheng, Beijing Sai); rnase a, Salmon Sperm DNA Solution (Salmon Sperm DNA Solution) (purchased from seimer feishel); biotinylated antibody Anti-EpCAM (Biotin) (purchased from Abcam); t4 DNA ligase, phi29 DNA polymerase (from New England Biolabs); ammonium chloride, sodium hydroxide (purchased from the national pharmaceutical group); experimental deionized Water (18.2M Ω effluent measurement) from a Water purification instrument Explorer series waters purification system (available from Blue Water); the cell grade experimental water was from autoclaved analytical grade experimental water; other water for molecular biology experiments (ex drochen); other reagents are 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 molecule 1: PO (PO)4-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 procedures, contents and conditions:
oligonucleotide tag specific antibody modification: 25 μ L of 2.5 μ M oligonucleotide 1 was mixed well with 25 μ L of 2.5 μ M streptavidin and incubated at 37 ℃ for 30 minutes. Then, 50. mu.L of 1.25. mu.M biotinylated antibody Anti-EpCAM (Biotin) was added to the reaction mixture, mixed well, incubated at 25 ℃ for 30 minutes, followed by addition of 1mM biotin and incubation at 25 ℃ for 20 minutes to obtain an oligonucleotide 1-labeled Anti-EpCAM antibody solution (the component of the dilution solution was 8mM Na)2HPO4，2mM NaH2PO4150mM NaCl, 0.1% BSA, 0.025% Tween 20, pH 7.4, 0.5mg/mL salmon sperm DNA, where reagents not indicated in step 1 were purchased from Bos Takara Shuzo).
The specific steps for detecting the Epithelial cell adhesion molecule (EpCAM) protein expressed in the human breast cancer cell line MDA-MB 231 are as follows: culturing the human breast cancer cell line MDA-MB 231 in a glass-bottom culture dish, culturing by using a DMEM medium containing 10% sterile fetal bovine serum, culturing in a sterile cell culture box with the relative humidity of 95%, the carbon dioxide gas of 5% and the temperature of 37 ℃ until the cell fusion degree is 30%, and obtaining the cell sample of the embodiment.
The cell samples were rinsed 3 times with 1 XPBS, incubated for 45 minutes at room temperature using 4% paraformaldehyde solution, and then incubated at 100mM NH4Reacting in 1 XPBS solution of Cl for 20 minutes, and washing with 1 XPBS for 5 minutes; followed by reaction in 1 XPBS with 0.1% Triton X-100 for 2 minutes, rinsing with 1 XPBS solution, then incubation in 5% BSA solution for 2 hours at room temperature, and reaction in 0.05mg/mL RNase A at 37 ℃ for 30 minutes, rinsing with 1 XPBS three times; 0.1mg/mL of streptavidin (containing 0.5mg/mL of Salmonon Sperm DNA) was added and incubated at 37 ℃ for 30 minutes, followed by addition of 1mM biotin and reaction at 37 ℃ for 30 minutes, and rinsing with 1 XPBS 3 times. After all the above treatments, the cell samples were incubated with the oligonucleotide 1-labeled antibody Anti-EpCAM overnight (14 hours) at 4 ℃, washed 3 times the next day with 1X PBS containing 0.1% Triton X-100 and 2% BSA for 10 minutes each, then washed twice with 1X PBS for 5 minutes each, and rinsed once with distilled water, to obtain cell samples bound with the oligonucleotide 1-labeled antibody Anti-EpCAM. (reagents involved in this section are all noted in step 1).
A sample of cells bound to the Anti-EpCAM oligonucleotide 1-labeled antibody described above was contacted with 100nM template molecule 1 (diluted in a solution containing 2 × citrate buffer, 20% formamide, 0.5mg/mL Salmonon Sperm DNA), incubated at 37 deg.C for 30 minutes, rinsed three times with 1 × PBS, rinsed once with distilled water, and immediately after aspirating all the liquid, a T4 ligase system (50mM Tris-HCl, 10mM MgCl. RTM. DNA) was added210mM DTT, 1mM ATP, 0.1U/. mu. L T4 DNA ligase, pH 7.5), incubated at 37 ℃ for 30 minutes, rinsed three times with 1 XPBS, rinsed once with distilled water, aspirated all the liquid and immediately added to the phi29 polymerase system (0.5mM dNTPs, 0.25U)/μL phi29 DNA polymerase，0.2mg/mL BSA，50mM Tris-HCl，10mM MgCl2，10mM(NH4)2SO44mM DTT, pH 7.5), incubated at 37 ℃ for 60 minutes, rinsed three times with 1 XPBS and once with distilled water. mu.M target-specific fluorescent probe 1 (diluted in a solution containing 2 × citrate buffer, 20% formamide, 0.5mg/mL Salmonon Sperm DNA) was added and incubated at 37 ℃ for 30 minutes. Wash with 1 XPBS containing 0.1% Triton X-100 for 10 minutes, then wash twice with 1 XPBS for 5 minutes each, followed by incubation in 1 ug/. mu.L DAPI solution for 5 minutes, then wash with 1 XPBS for 5 minutes. And finally, imaging through a fluorescence microscope. After the imaging was completed, a 95% glycerol aqueous solution was added, left to stand for 1 minute, rinsed three times with 1 × PBS, and imaged again by a fluorescence microscope. See fig. 4, 5 and 6 for experimental results. (reagents involved in this section are noted in step 1, where the T4 ligase system and phi29 polymerase system were purchased from New England Biolabs).
In fig. 4, it is a graph of the result of detecting the protein of Epithelial cell adhesion molecule (EpCAM) expressed in the cell sample (human breast cancer cell line MDA-MB 231) by 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 result shows that the signal of the protein target is distributed in a dotted manner, and the signal can be clearly distinguished from the background, and the size is in the range of several hundred nanometers, so the method can detect the target signal without relying on a high-resolution microscope (the result is taken by a Nikon ECLIPSE Ni microscope). Referring to the principle diagram of FIG. 3, the dotted signal in FIG. 4 is a single-stranded nucleic acid molecule stably bound with the specific imaging probe 1 detected by fluorescence microscopy. It is to be understood that the single-stranded nucleic acid molecule is linked to the oligonucleotide 1-labeled antibody Anti-EpCAM via oligonucleotide 1, and the target EpCAM binds to the antibody Anti-EpCAM, such that the detection of a spot signal from the "single-stranded nucleic acid molecule bound to the imaging probe 1" indicates the presence of the target Epithelial cell adhesion molecule (EpCAM) protein in the cell sample (human breast cancer cell line MDA-MB 231), and such that the position and amount of the spot signal indicates the position and number of the target Epithelial cell adhesion molecule (EpCAM) in the cell sample (human breast cancer cell line MDA-MB 231), i.e. the position of the spot is the position of the target Epithelial cell adhesion molecule (EpCAM) in the cell sample (human breast cancer cell line MDA-MB 231), the number of target Epithelial cell adhesion molecules (EpCAM) was 370 ± 24 (in cell 1) and 330 ± 30 (in cell 2), respectively. Among them, according to the single cell sequencing data of the tumor cell line disclosed in international "encyclopedia of tumor cell line (CCLE)", EPCAM (ENSG00000119888.6) gene is low expressed in human breast cancer cell line MDA-MB 231 cells, i.e., it is indicated that Epithelial cell adhesion molecule (EPCAM) measured in this example is a low abundance protein in human breast cancer cell line MDA-MB 231 cells. The results in fig. 4 show the characteristic of the method of the present invention for highly sensitive detection of the low-abundance protein, the method of the present invention can convert the signal of the protein into a fluorescent bright spot which is easy to identify through signal amplification, and the protein quantification results are respectively 370 ± 24 (in cell 1) and 330 ± 30 (in cell 2) through fluorescent bright spot counting.
In FIG. 5, the top left is an imaging diagram of cell nucleus showing the specific location of the cell in the sample and the approximate subcellular relative location distribution of the cell, and the bottom left is a result diagram of detecting the Epithelial cell adhesion molecule (EpCAM) protein expressed in the cell sample (human breast cancer cell line MDA-MB 231) by the method of the present invention (see FIG. 3 for the principle), which shows that the signal of the protein target shows a dotted distribution and can be clearly distinguished from the background, the signal is from the single-stranded nucleic acid molecule combined with the imaging probe 1, and the single-stranded nucleic acid molecule is formed by oligonucleotide 1 through in situ amplification and extension of nucleic acid. As can be seen from the principle of the method shown in FIG. 3, in order to detect the oligonucleotides in the second batch, it is critical that the fluorescent probe stably bound to the single-stranded nucleic acid molecule can be dissociated and removed by the glycerol aqueous solution. Therefore, in this example, after the cell sample is detected by imaging, 95% glycerol aqueous solution is added, and the cell sample is left to stand for 1 minute and rinsed with 1 × PBS three times, and the result is shown in fig. 5 (lower right), from which it can be seen that after the glycerol aqueous solution with the content of 95% is used, the spot-like signal (see lower left of fig. 5) originally detected in the sample is quenched to disappear (see lower right of fig. 5). The principle of the method according to fig. 3 can be explained as follows: when 95% glycerol aqueous solution is added to a sample distributed with a dot-shaped signal, the hydrogen bonding force of the fluorescent probe stably bound on the single-stranded nucleic acid molecule is weakened and dissociated, and after 1 × PBS rinsing, the fluorescent probe is removed, so that signal quenching disappears.
Based on the results in fig. 5, this example performed ten times of repeated imaging on the same cell (human breast cancer cell line MDA-MB 231) in the cell sample (human breast cancer cell line MDA-MB 231) based on the results in fig. 5, and the results are shown in fig. 6, that is, the method in this example performed imaging analysis on the target Epithelial cell adhesion molecule (EpCAM) in the sample to obtain the first round of imaging results in fig. 6, then the fluorescent probe was removed with 95% glycerol aqueous solution to quench the signal, and the method in this example performed repeated imaging analysis on the target Epithelial cell adhesion molecule (EpCAM) in the sample to obtain the second round of imaging results in fig. 6. The procedure is repeated again, then the fluorescent probe is removed by using 95% glycerol aqueous solution to quench the signal, the method of the embodiment is used again to perform imaging analysis on the target Epithelial cell adhesion molecule (EpCAM) in the sample, and finally 10 imaging rounds are performed to obtain 10 imaging results in total as shown in fig. 6. From the results, it can be seen that the use of the glycerol aqueous solution does not significantly affect the signal presentation, i.e. the position of the fluorescent bright spot appearing in each imaging run does not change. In addition, the present embodiment also quantitatively analyzes the EpCAM signal in the imaging results of 10 rounds of fig. 6, and as a result, as shown in fig. 7, the result shows that the number of EpCAM signals (fluorescent bright spots) in 10 imaging rounds does not significantly change, and the use of the glycerol aqueous solution does not significantly affect the significant change of the number of signals (fluorescent bright spots). In conclusion, the strategy principle of the method of the invention for multi-batch multi-target (broadly) imaging using aqueous glycerol solutions is enabled.
Sequence listing
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Claims (9)

1. The method for detecting multiple targets in a multiple positioning mode based on green solvents and programmable oligonucleotide probes is characterized in that the method for detecting multiple targets in a multiple positioning mode is characterized in that targets to be detected are respectively directly or indirectly connected with specific oligonucleotides, the oligonucleotides connected with the targets are specifically connected with corresponding fluorescent probes, positioning analysis of the corresponding targets is achieved through positioning detection of the fluorescent probes, then the detected fluorescent probes are removed through the green solvents, then the oligonucleotides on other targets which are not analyzed are specifically connected with the corresponding fluorescent probes, and then positioning analysis of the other corresponding targets is achieved through positioning detection of the fluorescent probes.
2. The method for detecting multiple targets based on multiple localization of green solvent and programmable oligonucleotide probes according to claim 1, wherein the target refers to an analyte existing in a test sample, wherein the test 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.
3. The method of claim 1, wherein each target interacts directly or indirectly only with oligonucleotides of a specific sequence, wherein the indirect interaction means that the target interacts directly with a specific intermediate molecule to which the oligonucleotides of a specific sequence are linked, wherein the intermediate molecule is at least one antibody, antibody fragment, oligonucleotide, aptamer or small molecule.
4. The method for multiple target detection based on green solvent and programmable oligonucleotide probes according to claim 1, wherein the fluorescent probe comprises at least a nucleic acid moiety and a fluorescent signal emitting moiety; the fluorescent probe is hybridized on the oligonucleotide by a direct mode or an indirect mode, wherein the direct mode refers to that the fluorescent probe is directly hybridized with the oligonucleotide according to the base complementary pairing principle; the indirect method is to hybridize the oligonucleotide to an intermediate nucleic acid molecule, which is then hybridized to the fluorescent probe, wherein the intermediate nucleic acid molecule is one or more nucleic acid molecules having multiple sequences capable of hybridizing to the fluorescent probe.
5. The method for multiple target detection based on green solvent and programmable oligonucleotide probe in multi-localization manner according to claim 1, wherein the green solvent is glycerol aqueous solution or glycerol buffer solution capable of breaking the hydrogen bond of base complementary pairing.
6. The method for multiple target detection based on green solvent and programmable oligonucleotide probes according to claim 1, wherein the oligonucleotide probes are programmable oligonucleotide probes, all oligonucleotide probes can be designed orthogonally based on different sequences of target species, and the number of fluorescent probes that can be ligated to each target-specific oligonucleotide can be increased by designing nucleic acid sequences to achieve fluorescence signal enhancement.
7. The method for multiple target detection based on green solvent and programmable oligonucleotide probes according to claim 1, wherein the target-linked oligonucleotides are specifically linked to the corresponding fluorescent probes in batches, and the number of targets analyzed in each batch is not greater than the number of detection channels of the fluorescent detection equipment in the batch of fluorescent detection by hybridizing the oligonucleotides with the specific fluorescent probes, wherein the number of detection batches to be completed is determined by the number of targets to be analyzed and the number of detection channels of the fluorescent detection equipment, and wherein the fluorescent labels of different fluorescent probes used in the same batch are different.
8. The method for multi-target detection based on green solvent and programmable oligonucleotide probe as claimed in claim 7, wherein the fluorescence detection in fluorescence detection by specific hybridization of fluorescent probe in batches is performed by detecting the fluorescence emitted from fluorescent probe with fluorescence detection equipment and performing location analysis of corresponding targets according to the position and number of fluorescence.
9. The method for multi-localized detection of multiple targets based on green solvent and programmable oligonucleotide probes according to claim 8, wherein the fluorescence detection device is an epifluorescence microscope or a confocal microscope.
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