A double ring probe and its preparation method and application

Abstract

The present invention relates to a double-ring probe and a preparation method and application thereof, wherein the double-ring probe comprises a recognition module, a signal module and a template chain recognition module; the signal module is complementary connected to the template chain recognition module, and the template chain recognition module is complementary connected to the recognition module. The double-ring probe is applied to a biomarker detection platform, which is a universal platform, and can be switched to different biomarker detections by simply changing the recognition module and the corresponding signal module; the technology of simultaneously detecting proteins and nucleic acids with low-cost and uncomplicated instruments is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a double-ring probe and a preparation method and application thereof. Background Art

[0002] To ensure accurate diagnosis and treatment of diseases, it is necessary to perform multiplex analysis of biomarkers in a single sample volume by various means. In particular, the mechanisms and pathways associated with disease occurrence are complex, and disease biomarkers (such as proteins / peptides, miRNAs, and DNA) may participate in the regulation of biological processes in a synergistic manner. Therefore, the simultaneous detection of different classes of biomolecules, rather than detecting only proteins or nucleic acids, can provide more comprehensive information and help to more accurately diagnose and monitor the progression of diseases. Spectroscopic methods are usually the preferred method for multi-target detection, because the combination of characterized frequency / wavelength or optical labels can be easily used to encode multiplex signal readout. Among them, microarrays are easily used for multiplex detection of proteins and nucleic acids, identifying different targets by their positions in the microarray, but the analysis time increases with the number of targets and lacks sufficient sensitivity. However, complex mechanisms and designs are essential for the combination of antibody recognition and nucleic acid hybridization. To date, except for the work on the simultaneous detection of small molecules, proteins, and microRNAs using immunoarrays, the main strategy for the simultaneous detection of nucleic acids and proteins in one test tube is to amplify the oligonucleotide sequence corresponding to the target after recognition with aptamers. In the example of combined DNA and protein detection using fiber optic arrays, the immunoassay is converted into a rolling circle amplification (RCA) assay by combining antibodies and padlock DNA for ligation to facilitate RCA. However, even with the conversion to detect the released DNA sequence, most methods can only detect one protein and one nucleic acid target in a multiplexed assay, which provides limited information about the test sample. In addition to the need for multiple optical probes, one of the possible reasons is the difficulty in designing the released DNA sequence to avoid crosstalk.

[0003] Quantum dots (QDs) have attracted extensive attention from researchers in the field of optical imaging due to their unique physicochemical properties. Their tunable size allows QDs to emit visible fluorescence at different wavelengths under excitation from a single light source, and they play an unparalleled role in multi-target, simultaneous, multi-color imaging of tissues and cells compared to other molecular biotechniques and traditional fluorescent materials. This technology provides real-time observation of multiple biomarkers, allowing us to quantify their expression levels and helping us gain a deeper understanding of the interactions between biomolecules and the relationship between biomolecules and disease occurrence, progression, and prognosis.

[0004] Based on the above research background, there is still an urgent need to develop a platform and method that can simultaneously detect proteins and nucleic acids with low cost and uncomplicated instrument settings. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a double-ring probe that can identify specific targets and achieve signal amplification with the assistance of DNA polymerase / nuclease.

[0006] This application is achieved through the following technical solutions:

[0007] The present application provides a double-ring probe, comprising a recognition module, a signal module and a template chain recognition module; one of the rings is the signal module, the other ring is the template chain recognition module, and the two rings are connected together by a signal module neck, and the neck is composed of reverse complementary paired base pairs; the recognition module is formed by hybridization of two chains, one of which is a target aptamer and the other is a complementary chain to the target; the template chain recognition module provides more than two nuclease recognition sites for the template chain to start a subsequent signal cycle; the signal module sequence is complementary to the template chain recognition module chain, and the template chain recognition module sequence is complementary to the recognition module sequence; the signal module is a stem-loop structure to reduce interference signals generated by non-specific hybridization.

[0008] The double-ring probe described in the present application has high stability, and there is no significant difference in the fluorescence signal intensity in various buffer solutions such as PBS, DMEM, DEPC and TM. Therefore, the double-ring probe of the present application can be used in various complex experimental environments.

[0009] When the double-loop probe of the present application is used to detect biomarkers, the recognition module of the double-loop probe can specifically recognize the target and expose the template recognition site to trigger subsequent signal amplification. Finally, a double signal cycle is constructed and a large number of transcription signal modules are generated. At this time, the signal module contains the successfully assembled H probe.

[0010] The two ends of the double-ring probe described in the present application can be labeled with a reporter fluorescent group and a quencher fluorescent group, respectively. The fluorescent group described in the present application is not specifically limited, as long as it can be ensured that the fluorescent group can distinguish the target detection object. Preferably, the 5' end of the base sequence described in the present application is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ quencher group. Whether the assembly is successful is confirmed based on the fluorescent signal reaction.

[0011] The present application also provides an application of a double-ring probe in a biomarker detection platform.

[0012] The biomarker detection platform described in the present application includes a double-ring probe, a template chain, a molecular beacon MB probe, a 96-well plate and quantum dot fluorescence detection technology.

[0013] The molecular beacon MB probe described in the present application is modified with biotin, and preferably the biotin is at the 5' end of the molecular beacon. For example, the sequence of the biotin-modified molecular beacon MB probe is Biotin-AAT CCA CCG AAG AAC TTC TTTTTT CTT CGG TGG AAA AAA AA TGA GTG.

[0014] The two ends of the molecular beacon MB probe described in the present application can be labeled with a reporter fluorescent group and a quencher fluorescent group, respectively. The fluorescent group described in the present application is not specifically limited, as long as it can be ensured that the fluorescent group can distinguish the target detection object. Preferably, the 5' end of the base sequence described in the present application is labeled with a FAM fluorescent molecular signal group, and the 3' end is labeled with a BHQ quencher group. Confirm whether the molecular beacon MB probe is successfully assembled with the 96-well plate based on the fluorescent signal reaction.

[0015] The 96-well plate described in the present application is a 96-well plate with streptavidin fixed thereto.

[0016] The molecular beacon MB probe described in the present application can be fixed in a 96-well plate through the interaction of streptavidin. After the molecular beacon MB probe is successfully assembled with the well plate, the molecular beacon MB probe is a hairpin structure and retains the tail end of the Y structure, providing an identification site for the released H probe.

[0017] The quantum dot fluorescence detection technology described in the present application includes the step of labeling DNA with quantum dots to obtain QDs-ssDNA probes.

[0018] For example, the QDs-ssDNA probe sequence described in this application is AAA GAA GTT CTT.

[0019] The quantum dots QDs described in the present application are a kind of semiconductor nanomaterial with a particle size of 1-10nm, which can be selected from 1nm, 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm, 9nm, and 10nm.

[0020] The quantum dots described in the present application can be selected from cadmium sulfide quantum dots, cadmium selenide quantum dots or cadmium telluride quantum dots, preferably cadmium selenide quantum dots.

[0021] The excitation wavelength of the quantum dots described in the present application is 300-400 nm, preferably 350 nm, and the emission wavelength is 500-650 nm, preferably 550-605 nm.

[0022] The template chain sequence described in the present application is TTG CTG AGG TGA GTG TAA TGA GGG AAT GAT GT.

[0023] The bioassay platform provided by the present application, the target is hybridized with the recognition module, the double-stranded structure of the recognition module is opened, and the template chain recognition site is exposed. The template chain contains two endonuclease recognition sites. When the template chain is hybridized with the endonuclease recognition site to form a double-stranded structure, the endonuclease recognizes and cuts to produce a gap. Under the action of DNA polymerase, the sequence fragments at both ends of the gap are extended, during which the target is released to hybridize with the next double-loop probe to form a signal cycle. In addition, a large number of fragment sequences consistent with the target sequence are generated, and they are continuously hybridized with the recognition module to trigger the signal cycle. The ssDNA chain transcribed with the signal module as a template is assembled into a hairpin structure (H probe mixture). The biotin-modified molecular beacon MB probe is fixed on a 96-well plate by streptavidin interaction to form MB@plate. When the H probe mixture is added to a 96-well plate, the H probe can collide with the 3' end of MB to form a blunt end when the H probe mixture is mixed with MB@plate. Exonuclease Exo-III is added to the above 96-well plate. Exonuclease Exo-III recognizes the 3' blunt end and cuts the fragment of MB, leaving a fragment@plate complex. Then the quantum dot-labeled QDs-ssDNA probe is hybridized with the fragment@plate complex to form a QDS-SSDNA-fragment@plate complex. The fluorescence signal in the 96-well plate is analyzed by a microplate reader.

[0024] The biomarker detection platform described in the present application only requires replacing the recognition module and the corresponding signal module in the double-ring probe, and the technology can be extended to multi-target detection.

[0025] The present application also provides a method for detecting biomarkers using a biomarker detection platform, comprising the following steps:

[0026] After the assembled double-circle probe solution, the sample solution to be tested, DNA polymerase, the template chain sequence, the endonuclease, and the buffer are mixed and incubated to obtain a mixture containing the H probe;

[0027] The mixture containing the H probe and the molecular beacon MB probe solution were added dropwise to a 96-well plate and incubated;

[0028] Add exonuclease to the 96-well plate and incubate;

[0029] Then, the QDs-ssDNA probe solution labeled with quantum dots is added and incubated to obtain a product solution;

[0030] Analyze the fluorescence signal of the product solution on the 96-well plate to determine whether the sample to be tested contains the biomarker to be detected.

[0031] The mixed incubation described in the present application further includes heating to degrade the enzymes used in the step, such as DNA polymerase and endonuclease, after the mixed incubation. The heating temperature described in the present application is 60-80° C. and the heating time is 5-20 minutes.

[0032] The method for detecting biomarkers using a biomarker detection platform described in the present application also includes a double-ring probe assembly method, including the steps of centrifuging the synthesized double-ring probe, dissolving it in DEPC-treated water, and incubating to obtain the assembled double-ring probe.

[0033] Preferably, the double-ring probe assembly method described in the present application comprises the steps of: centrifuging the synthesized double-ring probe, dissolving it in DEPC-treated water, heating it to 70-100° C., incubating it for 5-30 min, and then slowly cooling it to room temperature.

[0034] The preparation method of the QDs-ssDNA probe solution described in the present application is as follows: quantum dots are added to a borate buffer solution to prepare a quantum dot solution; ssDNA is added to DEPC water to prepare an ssDNA solution; an appropriate amount of EDC solution is added to the quantum dot solution for activation, and then an ssDNA solution is added for coupling, and incubation is performed to obtain a QD-ssDNA probe conjugate.

[0035] The biomarker described in the present application can be selected from miRNA or CRP protein. The miRNA described in the present application can be miRNA-21, miRNA-211, miRNA-155 or let-7a.

[0036] In some embodiments, when the biomarker described in the present application is miRNA-21, its sequence is UAG CUU AUC AGACUG AUG UUG A. The sequence of the double-loop probe used to measure miRNA-21 is TAG

[0037]

[0038] The bold characters are recognition modules, and the underlined characters are signal modules.

[0039] In some embodiments, when the target described in the present application is CRP protein, the sequence of the double-ring probe used to measure CRP is

[0040] The bold ones are recognition modules, and the underlined ones are signal modules.

[0041] The template chain sequence described in the present application is TTG CTG AGG TGA GTG TAA TGA GGG AAT GAT GT.

[0042] The molecular beacon MB probe sequence described in the present application is AAT CCA CCG AAG AAC TTC TTT TTT CTT CGGTGG AAA AAA AA TGA GTG.

[0043] The QDs-ssDNA probe sequence described in this application is AAA GAA GTT CTT.

[0044] The nuclease described in the present application is Nb.BbvCI and / or Nb.Bpu101, preferably Nb.BbvCI.

[0045] The exonuclease described in the present application is selected from any one or more of Exo-I, Exo-II, and Exo-III; preferably, the exonuclease is selected from Exo-III.

[0046] The buffer described in the present application is selected from PBS, DMEM or TM, and the preferred buffer is PBS.

[0047] In some embodiments, a method for detecting biomarkers using a biomarker detection platform described in the present application comprises the steps of: mixing a double-ring probe solution, a DNA polymerase with a concentration of 10-60U / L, a sample solution to be tested, a template chain sequence and a nuclease with a concentration of 10-60U / L in a tube containing a PBS buffer, mixing and incubating at room temperature for 10-60 minutes, and then heating to 60-80°C for 5-20 minutes to degrade the enzyme used to obtain a mixture containing an H probe; adding the mixture containing the H probe and a molecular beacon MB probe solution to a 96-well plate and incubating at room temperature for 10-60 minutes; adding a nuclease with a concentration of 2.5-12.5U / L and incubating for 15-75 minutes; adding a QDs-ssDNA probe solution and mixing, and incubating at room temperature for 10-40 minutes; after washing the supernatant with PBS buffer, analyzing the fluorescence signal in the 96-well plate by a microplate reader.

[0048] In some embodiments, a method for detecting a biomarker using a biomarker detection platform described in the present application comprises the steps of: mixing 5-30 μL of a double-ring probe solution, 1-3 μL of a DNA polymerase having a concentration of 10-60 U / L, 1-3 μL of a sample solution to be tested, 1-3 μL of a template sequence, and 1-3 μL of a nuclease having a concentration of 10-60 U / L in a tube containing 5-15 μL of a PBS buffer, incubating at room temperature for 10-60 minutes, then heating to 60-80° C., and incubating for 5-20 minutes with The enzyme used for degradation is used to obtain a mixture containing H probes; the mixture containing H probes and 8-12 μl of molecular beacon MB probe solution are added to a 96-well plate, and incubated at room temperature for 10-60 min; 1-3 μL of nuclease with a concentration of 2.5-12.5 U / L is added, and incubated for 15-75 min; 1-3 μL of QDs-ssDNA probe solution is added and mixed, and incubated at room temperature for 10-40 min; after the supernatant is washed with PBS buffer, the fluorescence signal in the 96-well plate is analyzed by a microplate reader.

[0049] The concentration of the double ring probe described in the present application is 5-30 μM / L. The concentration of the double ring probe can be selected from 5 μM / L, 10 μM / L, 15 μM / L, 16 μM / L, 17 μM / L, 18 μM / L, 19 μM / L, 20 μM / L, 21 μM / L, 22 μM / L, 23 μM / L, 24 μM / L, 25 μM / L, 26 μM / L, 27 μM / L, 28 μM / L, 29 μM / L or 30 μM / L. Preferably, the concentration of the double ring probe is 20-25 μM / L.

[0050] The concentration of the template strand described in the present application is 5-30 μM / L. The concentration of the template can be selected from 5 μM / L, 10 μM / L, 15 μM / L, 16 μM / L, 17 μM / L, 18 μM / L, 19 μM / L, 20 μM / L, 21 μM / L, 22 μM / L, 23 μM / L, 24 μM / L, 25 μM / L, 26 μM / L, 27 μM / L, 28 μM / L, 29 μM / L or 30 μM / L. Preferably, the concentration of the template strand is 20-25 μM / L.

[0051] The concentration and reaction time of DNA polymerase described in the present application are key parameters, because the DNA template generated by DNA polymerase serves as the trigger of the next enzymatic cycle reaction, and the insufficient amount and reaction time of DNA polymerase will make the fluorescent signal not significant enough. Therefore, the concentration of DNA polymerase described in the present application is 20-60U / L. The enzyme concentration of DNA polymerase described in the present application can be selected from 20U / L, 25U / L, 30U / L, 35U / L, 40U / L, 45U / L, 55U / L or 60U / L. The enzyme concentration of DNA polymerase is preferably 40U / L.

[0052] The concentration of the nuclease described in the present application is 10-60U / L, and the concentration of the DNA polymerase described in the present application can be selected from 10U / L, 15U / L, 20U / L, 25U / L, 30U / L, 35U / L, 40U / L, 45U / L, 50U / L, 55U / L, 60U / L; preferably 40U / L.

[0053] The enzyme concentration of the exonuclease described in the present application is 2.5-12.5 U / L. The enzyme concentration of the exonuclease described in the present application can be selected from 2.5 U / L, 5.0 U / L, 7.5 U / L, 10.0 U / L or 12.5 U / L. More preferably, 10 U / L.

[0054] The incubation temperature of step (1) described in the present application is room temperature, and the incubation time is 10-60 min, which can be selected from 10 min, 20 min, 30 min, 40 min, 50 min or 60 min, preferably 40-60 min.

[0055] The incubation temperature of step (2) described in the present application is room temperature, and the incubation time is 10-60 min, which can be selected from 10 min, 20 min, 30 min, 40 min, 50 min or 60 min, preferably 30 min.

[0056] The incubation temperature of step (3) described in the present application is room temperature, and the incubation time is 15-75 min, which can be selected from 15 min, 30 min, 45 min, 60 min or 75 min, preferably 45-75 min.

[0057] The incubation temperature of step (4) described in the present application is room temperature, and the incubation time is 10-40 minutes.

[0058] The borate buffer described in the present application has a concentration of 10-30 mmol / L and a pH of 6-9.

[0059] The concentration of the quantum dot solution in the present application is 0.5-5.0 mg / dL, which can be selected from 0.5 mg / dL, 1.0 mg / dL, 1.5 mg / dL, 2.0 mg / dL, 2.5 mg / dL, 3.0 mg / dL, 3.5 mg / dL, 4.0 mg / dL, 4.5 mg / dL or 5.0 mg / dL. Preferably, the concentration of the quantum dot solution is 1.0 mg / dL.

[0060] The concentration of the ssDNA solution in the present application is 10-30 mM / L, which can be selected from 10 mM / L, 15 mM / L, 20 mM / L, 25 mM / L or 30 mM / L. Preferably, the concentration of the ssDNA solution is 20 mM / L.

[0061] The volume ratio of the quantum dot solution to the ssDNA solution described in the present application is 1-3:3-1, which can be selected from 1:2, 1:3, 1:1, 2:1 or 3:1; preferably 1:1.

[0062] Beneficial Effects

[0063] 1. Universal biomarker detection platform of the double-ring probe of the present application. The double-ring probe is designed with three different functional areas, integrating specific target recognition (recognition module), signal cycle based on template chain (template chain recognition module), and connection sequence (signal module) mediating QDs labeling on 96-well plates. The platform constructed in the present application can be switched to different biomarker detections by changing the recognition module; the DNA polymerase / endonuclease-assisted chain displacement cycle and exonuclease-mediated signal cycle used in the present application give the method high detection sensitivity; the unique optical properties of QDs enable the method to be used in the simultaneous detection of multiple targets, realizing the technology of simultaneous detection of proteins and nucleic acids with low cost and uncomplicated instruments.

[0064] 2. The double-ring probe and detection platform of the present application exhibit excellent accuracy, high sensitivity, high selectivity, stability and reproducibility, and the analysis and detection are rapid and convenient.

[0065] 3. The detection platform of the present application has been successfully applied to the detection of miRNA and CRP. The detection results of this method for detecting miRNA and CRP are consistent with those of the classical or traditional miRNA and CRP detection methods, and have good accuracy.

[0066] 4. The detection limit of the detection platform of the present application is lower than that of traditional methods, and it can detect low-concentration clinical samples, and has great application potential in biomedical analysis and early clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Working principle diagram of the biomarker detection platform of this application.

[0068] Figure 2 Feasibility analysis diagram of the biomarker detection platform of this application.

[0069] (A) Fluorescence spectra of the FAM-labeled double-ring probe before and after assembly;

[0070] (B) Fluorescence statistics of SYBR Green signal and incubation time of the double-ring probe;

[0071] (C) Fluorescence intensity of the FAM-labeled dual-ring probe in different experimental solutions;

[0072] (D) SYBR Green signal during the Exo-III auxiliary signal cycle;

[0073] (E) Electron micrograph of quantum dots;

[0074] (F) Emission spectrum of quantum dots;

[0075] (G) Fluorescence spectra of the biobeacon MB before and after immobilization on the plate surface.

[0076] Figure 3 Optimization profile of parameters in the biomarker detection platform of this application

[0077] (A) Fluorescence intensity of target molecules under different incubation times and different concentrations of DNA polymerase;

[0078] (B) Fluorescence intensity of target molecules under different concentrations of endonuclease;

[0079] (C) Fluorescence intensity of target molecules of Exo-III at different incubation times and different concentrations.

[0080] Figure 4 Performance analysis of the biomarker detection platform of the present application for detecting miRNA-21

[0081] (A) Fluorescence spectra of different concentrations of miRNA-21;

[0082] (B) Correlation equation between fluorescence intensity F / F0 and miRNA-21 concentration;

[0083] (C) Comparison of fluorescence intensity F / F0 of the detected target miRNA-21 and other targets;

[0084] (D) The fluorescence intensity F / F0 of 10 samples was detected.

[0085] Figure 5 Clinical application of the biomarker detection platform of this application

[0086] (A) Fluorescence spectra of different concentrations when miRNA-21 and CRP were detected simultaneously;

[0087] (B) Correlation diagram between fluorescence intensity F / F0 and miRNA-21 concentration;

[0088] (C) Correlation diagram between fluorescence intensity F / F0 and CRP concentration;

[0089] (D) Correlation diagram between the miRNA-21 concentration measured by the biomarker detection platform of the present application and the miRNA-21 concentration measured by the PCR detection method;

[0090] (E) Correlation diagram between the CRP concentration measured by the biomarker detection platform of the present application and the CRP concentration measured by the ELISA method. DETAILED DESCRIPTION

[0091] The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.

[0092] Carboxyl water-soluble quantum dots were obtained from Wuhan Jiayuan Quantum Dot Co., Ltd., China.

[0093] The DNA sequences listed in Tables 1 and 2 were synthesized by Shanghai Sangong Biotechnology Co., Ltd., China and purified by HPLC. The 5' end of the molecular beacon was labeled with biotin.

[0094] 1-Ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride (EDC·HCl, 250 g, 98%) was purchased from Shanghai Biotech Biotechnology Co., Ltd., China.

[0095] DMEM was purchased from Biotechnology Co., Ltd.

[0096] The streptavidin-immobilized 96-well plate was purchased from NEB (Beijing) Co., Ltd.

[0097] The water used in the experiments was treated with diethyl pyrocarbonate (DEPC).

[0098] PBS buffer: NaCI 8.0g, KCI 0.2g, KH2PO4 0.24g, Na2HPO4.12H2O

[0099] 3.63g, add distilled water to 800ml, adjust the pH to 7.4 with HCl, make up to 1000ml with distilled water, sterilize with high pressure steam for 20min, and store for 40 seconds until use.

[0100] TM buffer: composed of 0.5M Tris-HCl (pH 7.5), 80mM MgSO4.

[0101] DNA polymerase, endonuclease Nb.BbvCI, and exonuclease Exo-III were provided by Sangon Biotech Co., Ltd (Shanghai, China).

[0102] All reagents were of analytical grade.

[0103] Embodiment 1:

[0104] A biomarker detection platform includes a double ring probe, a biotin-modified molecular beacon MB probe, a 96-well plate and quantum dot fluorescence detection technology. Figure 1 As shown, the double-loop probe comprises a recognition module, a signal module and a template chain recognition module. One of the loops is a signal module, and the other loop is a template chain recognition module. The two loops are connected together by the neck of the signal module, and the neck is composed of reverse complementary paired base pairs. The template chain recognition module provides two endonuclease recognition sites for the template chain to start the subsequent signal cycle; the recognition module is formed by hybridization of two chains, one of which is an adapter sequence and the other is a complementary chain to the target; the signal module sequence is complementary to the template chain recognition module sequence, and the template chain recognition module sequence is complementary to the recognition module sequence; the stem-loop structure of the signal module can reduce the interference signal generated by nonspecific hybridization.

[0105] The biotin-modified molecular beacon MB probe is fixed in a 96-well plate through the interaction of streptavidin. After the molecular beacon MB probe is successfully assembled with the well plate, the molecular beacon MB probe is a hairpin structure and retains the tail end of the Y structure, providing an identification site for the released H probe.

[0106] The quantum dot fluorescence detection technology includes the step of labeling DNA with quantum dots.

[0107] The double-ring probe and quantum dot-labeled DNA are added to a 96-well plate fixed with a molecular beacon MB probe, and the quantum dot fluorescence detection technology is used to determine the presence and content of the biomarker based on the fluorescence signal.

[0108] The working mechanism of the biomarker detection platform is as follows: Taking the target miRNA-21 as an example, Figure 1The target shown hybridizes with the "1" fragment in the recognition module, opens the double-stranded structure of the recognition module, and exposes the template chain recognition site. The template chain contains two endonuclease recognition sites ("2" fragments). When the template chain hybridizes with the endonuclease recognition site to form a double-stranded structure, the endonuclease recognizes and cuts the "2" fragment to produce a gap. Under the action of DNA polymerase, the "7" and "6" fragments are extended, during which miRNA-21 is released to hybridize with the next double-loop probe to form a signal cycle. In addition, a large number of "8" fragments consistent with the miRNA-21 sequence are generated and continuously hybridize with the recognition module to trigger the signal cycle. The ssDNA chain transcribed with the signal module as a template is assembled into a hairpin structure (H probe mixture). The H probe mixture is incubated at 70°C for 10 minutes to degrade the enzyme. The molecular beacon MB probe is fixed on a 96-well plate through streptavidin interaction to form MB@plate. When the H probe mixture is added to the 96-well plate, the H probe can collide with the 3' end of MB to form a blunt end when the H probe mixture is mixed with MB@plate. Exonuclease Exo-III was added to the above 96-well plate. Exonuclease Exo-III recognized the 3' blunt end and cut the "3" and "10" fragments of MB, leaving a "11" fragment@plate complex. Then the quantum dot-labeled QDs-ssDNA probe was hybridized with the "11" fragment@plate complex to form a QDS-SSDNA-"11"@plate complex. The fluorescence signal in the 96-well plate was analyzed by a microplate reader.

[0109] Example 2

[0110] A method for detecting miRNA-21 using a biomarker detection platform comprises the following steps:

[0111] (1) Assembly of the double-ring probe: The synthesized double-ring probe was centrifuged at 5000 rpm for 5 min, and 12.5 μL of DEPC water was added to prepare a 20 μM double-ring probe solution. Then 10 μL of the double-ring probe solution was heated to 90°C, incubated for 20 min, and then slowly cooled to room temperature. The double-ring probe sequence is shown in SEQ ID NO: 1 in Table 1.

[0112] (2) Preparation of QDs-DNA probe: Cadmium selenide quantum dots were added to borate buffer solution (20mmol / L, pH7.4) to prepare carboxyl water-soluble quantum dot solution with a solubility of 1.0mg / dL; ssDNA probe was added to DEPC water to prepare ssDNA probe solution with a concentration of 20mM / L. An appropriate amount of EDC solution was added to the quantum dot solution for activation, and then the ssDNA probe solution was added for coupling. The volume ratio of the quantum dot solution to the ssDNA probe solution was 1:1. The solution was incubated at room temperature for 2h in a constant temperature oscillator, centrifuged at 6000rpm for 5min in an ultrafiltration tube (molecular weight cutoff of 50KD), and washed 3 times with PBS buffer to obtain a QD-ssDNA conjugate. The QD-ssDNA conjugate was dissolved in PBS buffer and stored in the dark at 4°C. The sequence of the QDs-ssDNA probe is shown in SEQ ID NO: 2 in Table 1.

[0113] (3) Solution preparation: The template chain sequence is prepared into a 20 μM / L template chain solution using DEPC water;

[0114] (4) The miRNA-21 sample was added with DEPC water to prepare a sample solution with a concentration of 5 μM / L; the biotin-modified molecular beacon MB probe was added with DEPC water to prepare a molecular beacon MB probe solution with a concentration of 1 μM / L.

[0115] (5) Detection of miRNA-21: 10 μL of double-ring probe solution (concentration 5 μM / L), 2 μL of DNA polymerase (concentration 40 U / L), 2 μL of target miRNA-21 sample to be tested (concentration 5 μM / L), 2 μL of template chain solution (concentration 5 μM / L) and 2 μL of nuclease (concentration 40 U / L) were mixed in a tube containing 12 μL of PBS buffer, incubated at room temperature for 40 minutes, and then heated to 70°C for 10 minutes to degrade the enzyme used to obtain a mixture. The mixture and 10 μL of molecular beacon MB probe solution (concentration 1 mM / L) were added to a streptavidin-fixed 96-well plate and incubated at room temperature for 20 minutes. Add 2 μL Exo-III (concentration 10 U / L) and incubate for 45 min; add 2 μL QDs-ssDNA probe solution, mix and incubate; after washing the supernatant with PBS buffer, analyze the fluorescence signal in the 96-well plate by a microplate reader. The quantum dot excitation wavelength is 350 nm and the maximum fluorescence emission wavelength is 525±5 nm.

[0116] The sequence of the biotin-modified molecular beacon MB probe is shown in SEQ ID NO: 3 in Table 1; the sequence of the template strand is shown in SEQ ID NO: 4 in Table 1.

[0117] Table 1 Sequence Listing

[0118]

[0119] The bold font is the recognition module, the underline font is the signal module, and the normal font font is the template chain recognition module. Molecular beacon MB probe, the biotin is at the 5' end of the molecular beacon.

[0120] Example 3 Biomarker Detection Platform Detects CRP Protein

[0121] A method for detecting CRP protein using a biomarker detection platform comprises the following steps:

[0122] (1) Assembly of the double-loop probe: The synthesized double-loop probe was centrifuged at 5000 rpm for 5 min, and 12.5 μL of DEPC water was added to dilute the double-loop probe to 20 μM. Then 10 μL of the double-loop probe was heated to 90°C, incubated for 20 min, and then slowly cooled to room temperature. The nucleotide sequence of the double-loop probe described in the present application is shown in SEQ ID NO: 5 in Table 2.

[0123] (2) Preparation of QDs-DNA probe: Cadmium selenide quantum dots were added to borate buffer solution (20mmol / L, pH7.4) to prepare carboxyl water-soluble quantum dot solution with a solubility of 1.0mg / dL; ssDNA probe was added to DEPC water to prepare ssDNA probe solution with a concentration of 20mM / L. An appropriate amount of EDC solution was added to the quantum dot solution for activation, and then the ssDNA probe solution was added for coupling. The volume ratio of the quantum dot solution to the ssDNA probe solution was 1:1. The solution was incubated at room temperature for 2h in a constant temperature oscillator, centrifuged at 6000rpm for 5min in an ultrafiltration tube (molecular weight cutoff of 50KD), and washed 3 times with PBS buffer to obtain a QD-ssDNA conjugate. The QD-ssDNA conjugate was dissolved in PBS buffer and stored in the dark at 4°C. The nucleotide sequence of the QDs-ssDNA is shown in SEQ ID NO: 2 in Table 2.

[0124] (3) Solution preparation: The template sequence is prepared into a 5 μM / L template solution using DEPC water;

[0125] The sample to be tested is added with DEPC water to prepare a sample solution with a concentration of 5 μM / L; the molecular beacon MB probe is added with DEPC water to prepare a molecular beacon MB probe solution with a concentration of 1 μM / L

[0126] (4) CRP protein detection: 10 μL of double ring probe solution (concentration 5 μM / L), 2 μL of DNA polymerase (concentration 40 U / L), 2 μL of target miRNA-21 sample to be tested (concentration 5 μM / L), 2 μL of template sequence (concentration 5 μM / L) and 2 μL of nuclease (concentration 40 U / L) were mixed in a tube containing 12 μL of PBS buffer, incubated at room temperature for 40 minutes, and then heated to 70°C for 10 minutes to degrade the enzyme used to obtain a mixture. The mixture and 10 μL of molecular beacon MB probe solution (concentration 1 mM / L) were added to a 96-well plate and incubated at room temperature for 20 minutes. 2 μL of Exo-III (concentration 10 U / L) was added, incubated for 45 minutes, and then mixed with 2 μL of QDs-ssDNA probe solution. After the supernatant was washed with PBS buffer, the fluorescence signal in the 96-well plate was analyzed by a microplate reader. The excitation wavelength of the quantum dots was 350 nm, and the maximum fluorescence emission wavelength was 605 ± 5 nm.

[0127] The sequence of the molecular beacon MB probe described in the present application is shown in SEQ ID NO: 3 in Table 2; the sequence of the template strand is shown in SEQ ID NO: 4 in Table 2;

[0128] Table 2 Sequence Listing

[0129]

[0130] The bold font is the recognition module, the underlined font is the signal module, and the normal font is the template chain recognition module. The molecular beacon MB probe is labeled with biotin, and the biotin is at the 5' end of the molecular beacon. Example 4 Feasibility analysis of the biomarker detection platform of this application Experimental purpose: Taking miRNA-21 as an example, the feasibility of the constructed universal biomarker detection platform was investigated

[0131] 1. Verify the assembly of the double ring probe

[0132] 1.1 Sample preparation:

[0133] Assembly of the double-ring probe: The synthesized double-ring probe was centrifuged at 5000 rpm for 5 min, and 12.5 μL of DEPC water was added to dilute the double-ring probe to 20 μM. Then 10 μL of the double-ring probe was heated to 90°C, incubated for 20 min, and then slowly cooled to room temperature.

[0134] Double-ring probe assembly verification: The fluorescently labeled double-ring probe was assembled as described above. The fluorescent signal of the double-ring probe before and after assembly was detected.

[0135] 1.2 Experimental method: Both ends of the bicyclic probe were labeled with SYBR Green I fluorescent dye, the 5' end was labeled with FAM fluorophore, and the 3' end was labeled with BHQ quencher group. Considering that the assembly of the bicyclic probe mainly depends on the two-segment double-stranded structure of the recognition module, SYBR Green I dye was used to characterize the "stem" part of the bicyclic probe. The fluorescence signal intensity in the sample was detected at a wavelength of 500-540nm using a fluorescence spectrophotometer to evaluate the fluorescence changes of the denatured bicyclic probe to examine whether the recognition module of the bicyclic probe was successfully assembled.

[0136] 1.3 Experimental results: The fluorescent moieties FAM and BHQ were labeled at both ends of the linear double-ring probe, such as Figure 2 As shown in A, after the double-ring probe was assembled, the FAM fluorescence signal was significantly reduced, indicating that the recognition module of the double-ring probe was successfully assembled.

[0137] 2. Screening experiment of incubation time for double-ring probe assembly

[0138] 2.1 Sample group to be tested: Take 10 μL of the double-ring probe solution and heat it to 90°C, incubate for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively, and then slowly cool to room temperature to obtain each detection solution.

[0139] 2.2 Experimental results: Figure 2 B shows the relationship between the SYBR Green signal and incubation time of the double-ring probe. As the incubation time increases, the SYBR Green signal increases, and the SYBR Green signal of the assembled double-ring probe is significantly higher than the linear state, indicating that two double-stranded structures have been formed. However, when the incubation time reaches 20 minutes, the SYBR Green signal enhancement at 25 minutes and 30 minutes is not significantly enhanced, so the preferred incubation time is 20 minutes.

[0140] 3. Stability study of the double ring probe

[0141] 3.1 Experimental purpose: To investigate the stability of the designed double-ring probe by detecting the fluorescence intensity of the double-ring probe in different buffer solutions.

[0142] 3.2 Experimental method: The PBS solution in Example 2 was replaced with DMEM, DEPC and TM buffer respectively, and other conditions were the same.

[0143] 3.3 Experimental results: Figure 2 As shown in C, there is no significant difference in the SYBR signal intensity of the double-ring probe in PBS, DMEM, DEPC and TM buffers, which indicates that the stem structure of the probe remains relatively stable even in a complex experimental environment, thus endowing the double-ring probe with high structural stability.

[0144] 4. Verify that the molecular probe MB@plate is successfully assembled

[0145] 4.1 Experimental purpose: Use SYBR Green I dye to label the double-stranded structure formed by MB and MB-H probe complex, and use a fluorescence spectrophotometer to detect the fluorescence signal intensity in the sample at a wavelength of 500-540nm to verify MB assembly and MB recognition of H probe.

[0146] 4.2 Detection method: Same as Example 2

[0147] 4.3 Experimental results: Figure 2 As shown in D, when MB is assembled into a hairpin structure, the fluorescence signal of the MB group is significantly enhanced compared to the SYBR Green I signal of the linear group MB (L), indicating that the MB assembly is successful. When MB is hybridized with the H probe, the SYBR signal only increases slightly, which can be explained by the fact that the H probe itself has a double-stranded structure in the stem. When Exo-III is added, the SYBR Green I signal is significantly weakened, indicating that when the H probe mixture is mixed with the MB@plate, the H probe can collide with the 3' end of the MB to form a blunt end. Exonuclease Exo-III is added to the above 96-well plate. The exonuclease Exo-III recognizes the 3' blunt end, cuts the MB part of the fragment and degrades it, leaving only the single-stranded @plate. MB labeled with FAM is fixed on the plate, and the unfixed MB is washed away with PBS to verify the assembly of MB@plate. The results are shown in Figure 2. Figure 2 As shown in G, the FAM fluorescence signal of @plate is significantly lower than that of MB@plate, indicating that the FAM labeled with MB is successfully immobilized on the surface of the 96-well plate.

[0148] 5. Verification of Quantum Dots Labeled on Single-stranded ss-DNA

[0149] 5.1 Experimental Methods

[0150] The particle size of cadmium selenide quantum dots is about 1nm, and the transmission electron microscope image is as follows Figure 2 As shown in E.

[0151] Cadmium selenide quantum dot solution: Preparation of cadmium selenide quantum dot solution according to Example 2

[0152] QDs-DNA probe solution: prepared according to the QDs-DNA probe solution in Example 2

[0153] Detection method: Same as Example 2

[0154] 5.2 Experimental results: Figure 2As shown in F, the fluorescence signal can be observed by fluorescence microscope. The optimal excitation wavelength of the quantum dots of the present application is 350nm, and 350nm is the excitation light that can obtain the emission wavelength in the range of 550-600nm. The overlap of the absorption spectrum and the emission spectrum through spectral analysis proves that the single-stranded ss-DNA has been successfully labeled by quantum dots.

[0155] Example 5 Optimization of detection parameters

[0156] 1. DNA polymerase concentration screening and reaction time

[0157] 1.1 Experimental Group 1: In step (4) of the miRNA-21 detection method of Example 2, the DNA polymerase concentration was set to 10 U / L, 20 U / L, 30 U / L, 40 U / L, 50 U / L, and 60 U / L. Except for the different DNA polymerase concentrations, the other processes were the same as in Example 2.

[0158] 1.2 Experimental Group 2: Except for the DNA polymerase incubation time in step (4), which is different from that in Example 2, other steps and parameters are the same as those in Example 2.

[0159] Step (4) 10 μL of double-ring probe solution (concentration 5 μM / L), 2 μL of DNA polymerase (concentration 40 U / L), 2 μL of target miRNA-21 test sample (concentration 5 μM / L), 2 μL of template sequence (concentration 5 μM / L) and 2 μL of nuclease (concentration 40 U / L) are mixed in a tube containing 12 μL of PBS buffer, and incubated at room temperature for 10, 20, 30, 40, 50, and 60 minutes, respectively, and then heated to 70° C. for 10 minutes to degrade the enzyme used to obtain a mixture.

[0160] 1.3 Experimental results: Figure 3 A. As shown in Table 3, the fluorescence intensity increases with the increase of DNA polymerase concentration and reaches the highest intensity at 40-60U / L. The fluorescence intensity at 50U / L and 60U / L is no different from that at 40U / L. Based on the principle of energy saving and consumption reduction, 40U / L is the preferred concentration of DNA polymerase. Figure 3 A. As shown in Table 3, as the incubation time increases, the fluorescence intensity increases and then reaches a maximum within 40 min. When the reaction time is longer than 40 min, the fluorescence signal hardly changes. Therefore, a reaction time of 40 min is preferred.

[0161] Table 3 Effects of DNA polymerase concentration and incubation time on fluorescence intensity

[0162]

[0163] 2. Endonuclease concentration screening

[0164] 2.1 Experimental group: In the miRNA-21 detection method of Example 2, the nuclease concentration was set to 10U / L, 20U / L, 30U / L, 40U / L, 50U / L, and 60U / L. Except for the different nuclease concentrations, the other processes were the same as Example 2.

[0165] 2.2 Experimental results: Figure 3 As shown in Figure B, the fluorescence intensity increases with the increase of nuclease concentration and reaches the highest intensity at a concentration of 40-60U / L. The fluorescence intensity at concentrations of 50U / L and 60U / L is no different from that at 40U / L. Based on the principle of energy saving and consumption reduction, 40U / L is preferred as the nuclease concentration.

[0166] 3. Exonuclease concentration and incubation time screening

[0167] 3.1 Experimental Group 1: In the miRNA-21 detection method of Example 2, the exonuclease concentration was set to 2.5 U / L, 5.0 U / L, 7.5 U / L, 10.0 U / L, and 12.5 U / L. Except for the different exonuclease concentrations, the other processes were the same as Example 2.

[0168] 3.2 Experimental Group 2: Except for the different incubation time of Exo-III in step (4) of Example 2, other steps were the same as those of Example 2.

[0169] Step (4) Add 2 μL Exo-iii (concentration 10 U / L) and incubate for 15 min, 30 min, 45 min, 60 min, and 75 min, respectively.

[0170] 3.3 Experimental results: Figure 3 C. As shown in Table 4, the fluorescence intensity increases with the increase of Exo-iii concentration, and the fluorescence intensity tends to a constant value when the amount of Exo-iii is 10U / L. As the incubation time increases, the fluorescence intensity increases rapidly and then reaches equilibrium at 45 minutes. Therefore, it is preferred that the Exo-iii concentration is 10U / L and the incubation time is 45-60 minutes.

[0171] Table 4 Effect of Exo-iii concentration and incubation time on fluorescence intensity

[0172]

[0173] Example 6 Sensitivity of the detection platform

[0174] Experimental group: The miRNA-21 solution was diluted with DEPC water to prepare concentrations of 1fM, 10fM, 100fM, 1pM, 10pM, and 100pM, respectively.

[0175] Other conditions are the same as in Example 2.

[0176] Experimental results: Figure 4 As shown in A, as the concentration of miRNA-21 increases, the fluorescence intensity (F / F0) is observed to gradually increase, where F is the fluorescence after adding miRNA-21 to the fluorescent substance, and F0 is the fluorescence without adding miRNA-21 to the fluorescent substance. Figure 4 As shown in B, the logarithm of the fluorescence intensity (F / F0) and the miRNA-21 concentration showed a good linear relationship, the linear equation was Y=1.217*lgC+1.094, and the minimum detection limit (under 3 times the mean square error of the background) was estimated to be 586aM. Therefore, the detection platform of the present application has a high sensitivity.

[0177] Example 7 Specificity experiment of the detection method of the present application

[0178] Experimental method: The target miRNA-21, miRNA-211, miRNA-155 and let-7a were respectively detected according to the method of Example 2. Except for the different targets, other parameters were the same as Example 2, and the concentration of the target solution was 5uM / lL.

[0179] Experimental results: Four miRNA families with similar and identical sequences were tested, including miRNA-21, miRNA-211, miRNA-155 and let-7a. Figure 4 As shown in C, the fluorescence intensity of miRNA-21 is much higher than that of other miRNAs, indicating that the double-ring probe and detection platform method of the present application have strong sensitivity and selectivity to miRNA-21.

[0180] Example 8 Repeatability experiment of the detection platform of this application

[0181] Experimental method: Sample size: 10, detection method is the same as in Example 2,

[0182] Experimental results: Figure 4 As shown in D, the miRNA-21 fluorescence intensity of the miRNA-21 in 10 samples measured by the miRNA-21 detection method of the present application is between 4.2-4.5, and it can be seen that the coefficient of variation is low, indicating that the method has good repeatability.

[0183] Example 9: The sensitivity of the detection platform of the present application for simultaneous detection of miRNA-21 and CRP protein

[0184] Experimental group: 10 μL miRNA-21 double-loop probe solution (concentration 5 μM / L), 10 μL CRP double-loop probe solution (concentration 5 μM / L), 2 μL DNA polymerase (concentration 40 U / L), 2 μL target, 2 μL template strand solution (concentration 5 μM / L) and 2 μL endonuclease (concentration 40 U / L) were mixed in a tube containing 12 μL PBS buffer, and the mixture was incubated at room temperature for 40 minutes, and then the mixture was heated to 70 ° C for 10 minutes to degrade the enzyme used in the method. Then the mixture and 10 μl molecular beacon MB probe solution (concentration 1 mM / L) were added to a 96-well plate and incubated at room temperature for 20 minutes. 2 μL Exo-iii (concentration 10 U / L) was added, incubated for 30 minutes, and 2 μL QDs-ssDNA-1 was added and mixed. After the supernatant was washed with PBS buffer, the fluorescence signal was recorded.

[0185] The target is miRNA-21, and the concentrations are 10fM, 100fM, 1pM, 10pM, and 100pM. The concentrations of CRP are 0.3mg / L, 0.5mg / L, 1.0mg / L, 2.5mg / L, 5.0mg / L, 10.0mg / L, 15.0mg / L, and 20.0mg / L.

[0186] Comparison group 1: Traditional miRNA-21 immunofluorescence method

[0187] Comparative group 2: Detected by enzyme-linked immunosorbent assay (ELISA), kit KHA0031; Thermo Fisher; Invitrogen CRP Human ELISA Kit.

[0188] Experimental results: Figure 5 As shown in A, the fluorescence signal of miRNA-21 quantum dots QDs at 525 nm increases as the concentration of miRNA-21 increases from 10 fM to 100 pM. Figure 5 As shown in B, the logarithm of the fluorescence intensity and miRNA-21 concentration showed a good linear relationship from 10 fM to 100 pM. The detection limit (3 times the background standard deviation) was estimated to be 4.2 fM.

[0189] like Figure 5 As shown in A, the fluorescence signal of CRP quantum dots QDs at 605 nm increases as the CRP concentration increases from 0.3 mg / L to 20 mg / L. Figure 5 As shown in Figure C, the fluorescence intensity from 0.3 mg / L to 20 mg / L showed a good linear relationship with the logarithm of the CRP concentration, with an R2 of 0.9942 and a detection limit of 1.23 μg / mL.

[0190] The results of miRNA-21 and CRP were compared with those of control group 1 and control group 2. Figure 5 As shown in D, the miRNA-21 detected by the detection platform of the present application maintained a high consistency with the PCR detection result of comparison 1; Figure 5 As shown in E, the CRP test results determined by the detection platform of the present application maintained a high consistency with the ELISA results of comparison 2, indicating that the detection platform of the present application has a high clinical application value in the detection of multiple biomarkers.

Claims

1. A double-ring probe for detecting biomarkers, comprising a recognition module, a signal module and a template chain recognition module; one of the rings is a signal module, the other ring is a template chain recognition module, and the two rings are connected together by a signal module neck, the neck consisting of reverse complementary paired base pairs; the recognition module is formed by hybridization of two chains, one of which is a target aptamer and the other is a chain complementary to the target; the template chain recognition module provides more than two nuclease recognition sites for the template chain to start a subsequent signal cycle; the signal module is complementary to the template chain recognition module, and the template chain recognition module is complementary to the recognition module; the signal module is a stem-loop structure to reduce interference signals generated by non-specific hybridization; The biomarker includes miRNA-21, the sequence of the double-loop probe is shown in SEQ ID NO: 1; the sequence of the QDs-ssDNA probe is shown in SEQ ID NO: 2; the sequence of the molecular beacon MB probe is shown in SEQ ID NO: 3; the sequence of the template strand is shown in SEQ ID NO: 4; or The biomarker includes CRP protein, the sequence of the double-ring probe is shown in SEQ ID NO: 5; the sequence of the QDs-ssDNA probe is shown in SEQ ID NO: 2; the sequence of the molecular beacon MB probe is shown in SEQ ID NO: 3; and the sequence of the template chain is shown in SEQ ID NO:

4.

2. Use of the double-ring probe according to claim 1 in preparing a biomarker detection kit.

3. The use of claim 2 in preparing a biomarker detection kit, characterized in that: The detection kit also includes a template chain, a molecular beacon MB probe, a 96-well plate and a quantum dot fluorescence detection technology; the quantum dot fluorescence detection technology includes the step of labeling DNA with quantum dots to obtain a QDs-ssDNA probe.

4. The use of claim 3 in preparing a biomarker detection kit, characterized in that: The application includes a method for detecting a biomarker, which includes one or more of the following steps: The assembled double-ring probe solution, the sample solution to be tested, DNA polymerase, template strand, endonuclease, and buffer are mixed and incubated to obtain a mixture containing the H probe; The mixture containing the H probe and the molecular beacon MB probe solution were added dropwise to a 96-well plate and incubated; Add exonuclease to the 96-well plate and incubate; Then, the QDs-ssDNA probe solution labeled with quantum dots is added and incubated to obtain a product solution; Analyze the fluorescence signal of the product solution on the 96-well plate to determine whether the sample to be tested contains the biomarker to be detected.

5. The use of claim 3 in preparing a biomarker detection kit, characterized in that: The molecular beacon MB probe is a biotin-modified molecular beacon, or the 96-well plate is a streptavidin-fixed 96-well plate, and the molecular beacon is fixed in the 96-well plate through the interaction of streptavidin.

6. The use of claim 4 in preparing a biomarker detection kit, characterized in that: The steps include: The assembled double-ring probe solution, DNA polymerase with a concentration of 10-60 U / L, the sample solution to be tested, the template chain and the nuclease with a concentration of 10-60 U / L are mixed in a tube containing PBS buffer, mixed and incubated at room temperature for 10-60 minutes, and then heated to 60-80°C for 5-20 minutes to degrade the enzyme used to obtain a mixture containing H probes; the mixture containing H probes and the molecular beacon MB probe solution are added to a 96-well plate and incubated at room temperature for 10-60 minutes; a nuclease with a concentration of 2.5-12.5 U / L is added and incubated for 15-75 minutes; the QDs-ssDNA probe solution is added and mixed, and incubated at room temperature for 10-40 minutes; after the supernatant is washed with PBS buffer, the fluorescence signal in the 96-well plate is analyzed by a microplate reader.

7. The use of claim 4 in preparing a biomarker detection kit, characterized in that: The method for assembling the double-ring probe comprises the steps of: centrifuging the synthesized double-ring probe, dissolving it in DEPC-treated water, and incubating it to obtain the assembled double-ring probe; or The preparation method of the QDs-ssDNA probe solution comprises: adding quantum dots to borate buffer to prepare a quantum dot solution; adding ssDNA to DEPC water to prepare an ssDNA solution; adding an appropriate amount of EDC solution to the quantum dot solution for activation, then adding ssDNA solution for coupling, and incubating to obtain a QD-ssDNA probe conjugate.

8. The use of claim 7 in preparing a biomarker detection kit, characterized in that: The assembly method of the double-ring probe comprises the steps of: centrifuging the synthesized double-ring probe, dissolving it in DEPC-treated water, heating it to 70-100° C., incubating it for 5-30 min, and then slowly cooling it to room temperature to obtain an assembled double-ring probe having a concentration of 5-30 μM / L; or The preparation method of the QDs-ssDNA probe solution comprises the following steps: adding quantum dots to a borate buffer to prepare a quantum dot solution, wherein the borate buffer has a concentration of 10-30 mmol / L and a pH of 6-9; adding ssDNA to DEPC water to prepare an ssDNA solution, wherein the concentration of the quantum dot solution is 0.5-5.0 mg / dL; adding an appropriate amount of EDC solution to the quantum dot solution for activation, then adding an ssDNA solution for coupling, and incubating to obtain a QD-ssDNA probe conjugate, wherein the volume ratio of the quantum dot solution to the ssDNA solution is 1-3:3-1.

Images