CN113151401A

CN113151401A - Fluorescence sensor based on quantum dots and three-dimensional hybridization structure and preparation and application thereof

Info

Publication number: CN113151401A
Application number: CN202010928781.6A
Authority: CN
Inventors: 张春阳; 张艳; 李庆楠
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2021-07-23

Abstract

The invention belongs to the technical field of biological detection and molecular biology, and particularly provides a fluorescent sensor based on quantum dots and a three-dimensional hybridization structure, and preparation and application thereof. The fluorescent sensor based on the quantum dots and the three-dimensional hybridization structure comprises the quantum dots and the three-dimensional hybridization structure assembled on the surfaces of the quantum dots, wherein the three-dimensional hybridization structure comprises a cy 5-labeled probe and biotin-modified DNA. The method solves the problems of low sensitivity and long time consumption of the selection method of the FTO inhibitor in the prior art.

Description

Fluorescence sensor based on quantum dots and three-dimensional hybridization structure and preparation and application thereof

Technical Field

The invention belongs to the technical field of biological detection and molecular biology, and particularly provides a fluorescent sensor based on quantum dots and a three-dimensional hybridization structure, and preparation and application thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

N⁶-methyladenosine (m)⁶A) Is the most ubiquitous internal modification in mRNA and plays a key role in post-transcriptional regulation of mRNA splicing, transcription, translation, metabolism, and nuclear export. FTO dependent m⁶A demethylation is a new RNA processing regulation mechanism, and plays an important role in regulating adipogenesis, cell signal transduction and disease-related biological diseases. FTO has been identified as being involved in a number of diseasesDisease processes such as type II diabetes, obesity, alzheimer's disease, cardiovascular disease and cancer. Therefore, FTO may be a potential molecular target for cancer therapy and drug development. In view of the important role of FTO in the disease process, sensitive detection of FTO activity and development of inhibitors against FTO are of great significance in biological research, clinical diagnosis and drug development. The small molecule FTO activity regulator not only plays an important role in understanding the structure and the function of FTO, but also provides unique opportunities for treating diseases such as cancer, obesity and the like. In recent years, researchers have adopted strategies to identify FTO inhibitors in which small molecules are directed to m in vitro and in vivo⁶FTO demethylation of A showed good inhibitory activity. The natural product rhein is identified as the first inhibitor of FTO by biochemical and biophysical methods (i.e., structure-based virtual screening, High Performance Liquid Chromatography (HPLC), PAGE analysis). The high-throughput fluorescence polarization method (FP) is adopted to also prove that Mefenamic Acid (MA) is a high-selectivity inhibitor of FTO. The inventors have found that while these methods all develop FTO inhibitors that are highly selective, these methods are time consuming and require high concentrations of FTO protein, and furthermore, they do not detect FTO activity sensitively at low concentrations.

In the current FTO activity detection experiment, most of the traditional gel electrophoresis and autoradiography detection methods need complex nucleic acid labeling and gel electrophoresis procedures, and the sensitivity is low and time and labor are wasted. And new technologies for detecting FTO activity, such as virtual screening based on structures, fluorescence polarization methods, high performance liquid chromatography and the like, are developed in the later stage. The inventors have found that although these methods can detect the activity of FTO and screen for inhibitors to some extent, these methods often take a long time and consume a large amount of high concentration of protein and probe, and the sensitivity of detection does not meet the requirements of the present disclosure.

Disclosure of Invention

Aiming at the problems of low sensitivity and long time consumption of the selection method of the FTO inhibitor in the prior art. The present disclosure presents a novel method that enables accurate and sensitive determination of FTO activity and for screening of FTO selective inhibitors. Effective FTO inhibitors can be screened from small molecules. The method is simple to operate, economical, high in sensitivity, low in background and good in specificity, and can be used for realizing ultra-sensitive detection of FTO activity in cells.

In one or some embodiments of the present disclosure, there is provided a fluorescence sensor based on quantum dots and a three-dimensional hybridization structure, comprising quantum dots and a three-dimensional hybridization structure assembled on the surface of the quantum dots, the three-dimensional hybridization structure comprising a cy 5-labeled probe and biotin-modified DNA.

In one or some embodiments of the present disclosure, a method for preparing the fluorescence sensor based on the quantum dot and the three-dimensional hybridization structure is provided, which includes the following steps:

1) FTO-mediated demethylation of m6A in ssDNA substrate;

2) dpn II assists in the cleavage of the demethylated dsDNA substrate to generate biotinylated capture probes;

3) QD-DNA-cy5 nanostructures were constructed and FRET signals were subsequently measured.

In one or more embodiments of the present disclosure, there is provided a use of the above fluorescence sensor based on quantum dots and three-dimensional hybridization structure or a product prepared by the above method for preparing a fluorescence sensor based on quantum dots and three-dimensional hybridization structure in FTO inhibitor detection.

In one or some embodiments of the present disclosure, a method for detecting an FTO inhibitor is provided, in which a product obtained by the above fluorescence sensor based on a quantum dot and a three-dimensional hybridization structure or the above fluorescence sensor based on a quantum dot and a three-dimensional hybridization structure is excited by laser, and a quantum dot fluorescence signal and a Cy5 fluorescence signal are observed simultaneously.

One or some of the above technical solutions have the following advantages or beneficial effects:

1) the present disclosure is able to enhance the detection signal without amplification: traditional methods of detecting enzyme activity often require amplification, whereas the experimental design of the present disclosure has double-stranded DNA probes labeled with biotin at each end, and the cleavage products are four capture probes labeled with biotin. Compared with the traditional amplification method, the method greatly reduces the background signal and prevents nonspecific amplification.

2) The method has high detection sensitivity by using a quantum dot-based monomolecular sensing technology: and each quantum dot is provided with about 12-15 streptavidin, and one streptavidin can be connected with 3 biotin, so that 45-48 biotin-labeled probes can be theoretically connected onto one quantum dot, and the fluorescence resonance energy transfer efficiency is greatly enhanced. And the background signal of single molecule detection is polar, the sensitivity is very high, and the detection of the target substance can be realized on the level of single molecule.

3) The two ends of the double-stranded DNA probe designed by the method are respectively marked with biotin, and each specific template has a unique identical sequence which is matched with the report probe, so that the design is simplified, and the experiment cost is reduced.

4) The method does not need additional bioluminescence and the marking of a quenching group, greatly reduces the complexity and the cost of operation, and greatly reduces the background signal of detection by using a quantum dot-based single molecule detection technology. And any external separation and elution steps are not needed, the complexity of the experiment is reduced, the designed specific probe is adopted, the cost and the complexity of the operation are reduced, and the resources are saved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.

FIG. 1: example 1 mechanism diagram.

FIG. 2: example 2 non-lipid-lowering polyacrylamide gel electrophoresis (PAGE) to investigate FTO-triggered Dpn ll cleavage patterns, panel A, FTO-affected product analyzed by native polyacrylamide gel electrophoresis. Lane 1, positive control;

lane

2, 2 micromolar DNA substrate + Dpn ll;

lane

3,2 micromolar DNA substrate +500 nanomolar FTO + Dpn ll; panel B, red line is fluorescence intensity in the presence of FTO and black line is fluorescence intensity in the absence of FTO.

FIG. 3: example 3 a significant plot of the detection signal of cy5 was observed in the presence of FTO, wherein A, D is a quantum dot plot in the presence and absence of FTO, respectively; B. e is a graph of cy5 in the presence or absence of FTO. C. F is the overlay of quantum dots and cy5, respectively.

FIG. 4: plot of the effect of the parameters of example 4, wherein the intensity of the fluorescence spectra of panel a, 605QD and Cy5 is a function of the ratio of Cy5 labeled reporter probe to 605 QD; panel B, fluorescence efficiency and Cy5 fluorescence intensity as a function of Cy5 labeled reporter probe to QD ratio; panel C, Cy5 counts as a function of dpntii reaction temperature; panel D, Cy5 counts as a function of the amount of DpnII. Error bars show the standard deviation of three independent experiments.

FIG. 5: example 5 graphs of the results of FTO at various concentrations, graph a, the intensity of the fluorescence spectrum at different concentrations of FTO, tested under optimal experimental conditions using the proposed method; panel B, fluorescence intensity is linear with the logarithm of FTO concentration; panel C, effect of different concentrations of FTO on Cy5 counts; panel D, log of Cy5 counts to FTO concentration at (1X 10)^-13M～1×10^-6M) is linearly related. Error bars show the standard deviation of three independent experiments.

FIG. 6: example 6 comparison of the enzyme inhibition by rhein (A), diacerein (B) or plumbagin (C) on FTO (0.5. mu.M) demethylation and ALKBH5 (0.5. mu.M) demethylation by PAGE analysis. The inhibitors rhein (D), diacerein (E) or plumbagin (F) IC50 values analyzed using single molecule imaging for demethylation of FTO (0.05 μ M) and ALKBH5(0.05 μ M).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

Fat and obesity associated RNA demethylase (FTO) can catalyze N in mRNA⁶-methyladenosine (m)⁶A) Demethylation of the residue, converting m⁶A becomes A. FTO plays a key role in human obesityAnd may affect the development and prognosis of cancer. Accurate detection of FTO activity and screening for inhibitors of FTO are critical to biological research, clinical diagnosis and therapy. Here, the present disclosure develops a single Quantum Dot (QDs) -based Fluorescence Resonance Energy Transfer (FRET) sensing platform for detecting FTO activity and screening for effective FTO inhibitors. FTO mediated m⁶After A demethylation, the demethylated DNA sequence can be cleaved by the Dpn II endonuclease. All DNA fragments resulting from cleavage were modified with biotin (biotin) and used as capture probes to trigger the assembly of Cy 5-labeled reporter probes on the QD surface, resulting in FRET between the QD and Cy5, resulting in emission of Cy5, which can be quantified by single molecule detection techniques. The method can sensitively detect the FTO activity, and the detection limit is 7.9 multiplied by 10^-14And M. The disclosure also screens several small molecule inhibitors of FTO and identifies Diacerein as a highly selective inhibitor of FTO. In the mechanism of inhibiting FTO demethylation, Diacenein is neither a structural mimic of 2-oxoglutarate (2OG) nor a chelator of metal ions. In addition, Diacherein also inhibits the demethylation activity of endogenous FTO extracted from HeLa cells, providing a new approach for the treatment of FTO-related diseases and the development of drugs.

Preferably, the biotin is FTO-specific biotin.

Preferably, the biotin is modified at the end of the DNA.

1) FTO-mediated demethylation of m6A in ssDNA substrate;

Preferably, in step 1), m is bound site-specifically⁶A(5’-G-m⁶A-T-C-3') ssDNA as substrate, which has biotin labels at both 3' and 5 'ends, in the presence of FTO, which will make 5' -G-m⁶The sequence of A-T-C-3' is demethylated to produce a demethylated sequence of 5' -G-A-T-C-3', and the complementary strand, modified with biotin at both the 3' and 5' ends, can hybridize to the demethylated ssDNA portion to form dsDNA having a Dpn II endonuclease recognition site (5' -G-A-T-C-3 ').

Preferably, in step 2), the Dpn II endonuclease can specifically recognize and cleave demethylated dsDNA at the recognition site (5 '-G-A-T-C-3').

Preferably, in step 3), the three-dimensional hybridization structure is formed by hybridization of 4 different sequences and 1 identical sequence with 4 different biotin-modified cleavage products and the same cy 5-labeled reporter gene probe.

Preferably, the laser is a 488nm argon laser and FTO activity is quantitatively monitored by simply counting the number of Cy5 signals based on single molecule imaging of TIRF.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

The oligonucleotide sequences (5 '-3') used in the examples are as follows:

DNA substrate 1: biotin-AAG CTC CCA TGT TAG GAT CAG TGT CTCG A G biotin (SEQ ID NO.1)

Complement 1 sequence: biotin-ACG ATA CAT CCC ACT GAT CCT AACATGC T C-biotin (SEQ ID NO.2)

DNA substrate 2: CAT GTT AGG ATC AGT G (SEQ ID NO.3)

Complement 2 sequence: CAC TGA TCC TAA CAT G (SEQ ID NO.4)

Demethylated DNA: CAT GTT AGG ATC AGT G (SEQ ID NO.5)

Template 1: AAT GAG GAC TAG GAG CTA ACA TGG GAG CTT (SEQ ID NO.6)

Template 2: CTC GAG ACA CTG ATC AAT GAG GAC TAG GAG (SEQ ID NO.7)

Template 3: AAT GAG GAC TAG GAG AGT GGG ATG TAT CGT (SEQ ID NO.8)

And (4) template: GAG CAT GTT AGG ATC AAT GAG GAC TAG GAG (SEQ ID NO.9)

And (3) reporting the probe: P-CTC CTA GTC CTC A T (SEQ ID NO.10)

siRNA：AAA UAG CCG CUG CUU GUG AGA(SEQ ID NO.11)

anti-siRNA：UCU CAC AAG CAG CGG CUA UUU(SEQ ID NO.12)

Example 1

This example provides a method for preparing a fluorescence sensor based on quantum dots and three-dimensional hybridization structure, which includes three consecutive steps:

(1) FTO mediated m⁶Demethylation of a in ssDNA substrate;

(2) dpn II assists in the cleavage of the demethylated dsDNA substrate to generate biotinylated capture probes;

(3) QD-DNA-cy5 nanostructures were constructed and FRET signals were subsequently measured. FTO has high oxidative demethylation activity.

Therefore, to study FTO vs m⁶A demethylation Activity, the present disclosure contemplates site-specific binding of m⁶A(5’-G-m⁶A-T-C-3') ssDNA as substrate, which has biotin labels at both 3' and 5 'ends, in the presence of FTO, which will make 5' -G-m⁶Demethylation of the A-T-C-3' sequence to produce 5-Demethylation sequence of G-A-T-C-3'. Complementary strands modified with biotin at both the 3 'and 5' ends can hybridize to the demethylated ssDNA portion to form dsDNA having a Dpn II endonuclease recognition site (5 '-G-A-T-C-3'). In step 2), the Dpn II endonuclease can specifically recognize and cleave the demethylated dsDNA at the recognition site (5 '-G-A-T-C-3'). This partially complementary structure facilitates cleavage after cleavage, resulting in four different ssDNA cleavage products. The present disclosure also contemplates 4 different templates (templates 1-4) comprising 4 different sequences and 1 identical sequence that can hybridize to the newly generated 4 different biotin-modified cleavage products and the same cy 5-labeled reporter probe to form a three-dimensional hybridization structure. Subsequently, all biotin-modified cleavage products can be used as capture probes to drive the three-dimensional hybridization structure to the surface of 605QDs, forming 605QD-DNA-cy5 complex through specific biotin-streptavidin interaction. The principle of FTO activity assay is described in figure 1: when the 605QD-DNA-Cy5 complex is excited by 488nm argon laser, the fluorescence signal of 605QD and the fluorescence signal of Cy5 can be observed simultaneously, because the donor of 605QD and the acceptor of Cy5 generate fluorescence resonance transfer, therefore, the FTO activity can be quantitatively monitored by simply counting the number of Cy5 signals through single molecule imaging based on TIRF. In contrast, in the absence of FTO, the ssDNA substrate remains methylated (5' -G-m)⁶A-T-C-3') and is not cleaved by Dpn II. Therefore, neither the capture probe nor the 605QD-DNA-Cy5 complex could be generated, and therefore, FRET between QD and Cy5 would not occur.

Example 2

This example provides a method for detecting an FTO inhibitor, comprising the following steps:

detecting the activity of FTO: first, 0.3 micromolar methylated ssDNA was reacted with different concentrations of FTO protein in 10 microliters of reaction buffer (containing 50 millimolar HEPES, pH 7.5, 100 micromolar 2-OG,100 micromolar L-ascorbic acid and 150 micromolar (NH4)2Fe (SO4)2) at 30 ℃ for 2 hours, then inactivated at 95 ℃ for 5 minutes, and then the ssDNA was annealed to the complementary strand in a system of 1 Xannealing solution (containing 50 millimolar sodium chloride and 10 millimolar Tris-HCl (pH 8.0) in a total volume of 20 milliliters). 8 microliters of the resulting dsDNAs,5 units of DpnII, and 1 XDpnII buffer solution were reacted at 37 ℃ for 60 minutes in a mixed solution of 10 microliters, and then inactivated at 80 ℃ for 20 minutes. 10 μ L biotinylated digest, 1 μmol Cy5 labeled reporter probe, 0.5

μmol template

1,2,3,4 were added to a reaction containing 100 mmol Tris-HCl, 10 mmol (NH4)2SO4 and 3 mmol MgCl2 in a final volume of 20 μ L, pH 8.0. The mixture is heated for 5 minutes at 95 ℃, then is incubated for 30 minutes at room temperature to obtain the sandwich hybrid, and after the hybridization reaction, the sandwich hybrid product is assembled on the surface of 605QDs through the specific biotin-streptavidin interaction to form a 605QD-DNA-cy5 nano composite structure. And then detecting the mixed product.

Gel electrophoresis experiment: the amplification products were analyzed by 14% native polyacrylamide gel electrophoresis (PAGE), which was followed by 14% native polyacrylamide gel electrophoresis (PAGE) at 110V constant voltage for 50 min in 1 XTBE buffer (89 mmol per liter Tris-HCl, 89 mmol per liter boric acid, 2 mmol per liter EDTA, pH8.3) with 1 XSSYBR Gold as the fluorescence indicator, and finally the gel electrophoresis images were imaged by a ChemiDoc MP imaging system.

Inhibitor experiments: to investigate the effect of FTO inhibitors on FTO activity, various concentrations of inhibitors, 0.3 micromoles per liter of DNA sequence, were incubated at 30 ℃ for 15 minutes, then 50 nanomoles of FTO were added to the solution and incubated at 30 ℃ for 2 hours, followed by inactivation at 95 ℃ for 5 minutes, after which additional protocols were performed as described previously. Finally, assays are performed in an assay system to determine FTO protein activity. The Relative Activity (RA) of FTO is calculated by the following formula:

RA＝(N_i-N₀)/(N_t-N₀)_*100％

wherein N is₀Is the cy5 single molecule count, N, in the absence of FTO_tIs the cy5 count at 50 nanomoles present, N_iIs the single molecule count in the presence of 50 nanomolar FTO and FTO inhibitor.

Cell culture and preparation of protein extract: hela cells (human cervical cancer cell line) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin streptomycin and placed in an incubator containing 5% carbon dioxide at 37 ℃. The extraction of the cell extract was performed using a protein extraction kit according to the instructions and the cell extract was finally frozen in a freezer at minus 80 ℃ for use.

FTO activity was turned off by transient transfection: the target sequence for FTO was 5'-AAA UAG CCG CUG CUU GUG AGA-3', and expression of FTO was turned off in HeLa cells by transient transfection of siRNA for 48 h. Using Lipofectamine ^TM3000 transfection reagents transfection was performed according to the manufacturer's instructions. 48 hours after transfection, nuclear extracts were prepared using a nuclear extraction kit (ActiveMotif, Carlsbad, Calif., U.S.A.) according to the manufacturer's instructions.

Example 3

As shown in FIG. 2, non-lipid-lowering polyacrylamide gel electrophoresis (PAGE) was performed to investigate FTO-triggered Dpn ll cleavage reactions. A distinct band of cleavage product at 60 min reaction time was observed in the presence of FTO and Dpn ll (FIG. 2A, lane 3), indicating that FTO can initiate the Dpn ll-mediated cleavage reaction. However, no cleavage product band was observed in the absence of FTO (FIG. 2A, lane 2). The present disclosure further demonstrates the feasibility of using fluorimetry and single molecule assays to demonstrate this experiment, a significant cy5 detection signal was observed in the presence of FTO (fig. 2B, fig. 3C, panel B), but no significant cy5 signal was observed in the absence of FTO (fig. 2B, fig. 3F, panel E). This result indicates that cy5 signal was detected only in the presence of FTO, and it is important that the method be sensitive to FTO activity.

Example 4

To obtain the highest assay sensitivity, this example investigated the effect of several parameters, including cy5 to QD ratio, Dpn ll dose, and reaction temperature. As shown in fig. 4A, as the ratio of cy5 to QD was increased from 1:1 to 48:1, the fluorescence intensity of QD was decreased as the ratio was increased before the ratio of cy5 to QD was 24:1, the fluorescence intensity of cy5 was increased, and there was no significant change over 24:1, so that the ratio was determined to be 24:1 as the optimum reaction condition. As can be seen from FIG. 4B, the Cy5 count at 37 ℃ was much higher than the Cy5 counts at 25 ℃ and 45 ℃ and therefore a reaction temperature of 37 ℃ was used in subsequent experiments. As seen in fig. 4C, the Cy5 count increased with the increase of dpnti from 1U to 5U, and leveled off at 5U, so 5U dpnti was used in subsequent experiments.

Example 5

To quantitatively detect FTO, this example tested various concentrations of FTO using the proposed method under optimal experimental conditions. As shown in fig. 5A and 5C, fluorescence and single molecule signals increased in both a time-dependent and a concentration-dependent manner. The higher the concentration of FTO, the more sequences are demethylated by FTO, and therefore more biotinylated probes are cleaved, resulting in a stronger detection signal. As shown in FIG. 5D, the intensity of the detection signal varied from 1X 10 with the concentration of FTO^-13M to 1X 10^-6The concentration of M increases. The detection signal is linearly related to the logarithm of the FTO concentration over a dynamic range of 7 orders of magnitude. The linear relationship can be described as N-58.63 log₁₀C +789.80, correlation coefficient 0.9806, where N is the number of cy5 molecules and C is the FTO concentration (in moles), with a limit of detection of 7.9X 10 by calculating the standard deviation of the mean of the controls plus 3 times^-14. Specific fluorescence spectroscopy (3.3X 10)^-12M) increased by a factor of 100 (5B). The improvement in sensitivity can be attributed to the following: (1) FTO-mediated specific demethylation and subsequent efficient Dpn ll-catalyzed cleavage of demethylated DNA. (2) All cleavage products modified with biotin as capture probes can hybridize with template and Cy5 labeled reporter probes to form multiple biotin/template/Cy 5 complexes. (3) The assembly of multiple biotin/template/Cy 5 complexes on a single QD surface improves fluorescence energy conversion efficiency. (4) Near zero background signal for single molecule detection.

Example 6

This example further investigates the effect of Diacenin as a model inhibitor on FTO-induced demethylation reactions. FTO can form a very specific and stable complex with the inhibitor. As shown in fig. 6, the relative activity of FTO decreased with increasing inhibitor concentration. This result demonstrates that the proposed method can be further developed into a low-cost high-throughput platform for screening FTO inhibitors, providing great potential for further biological applications.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.

SEQUENCE LISTING

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<120> fluorescent sensor based on quantum dots and three-dimensional hybridization structure, preparation and application thereof

<130> 202025775

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<170> PatentIn version 3.3

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Claims

1. a fluorescence sensor based on quantum dots and three-dimensional hybrid structure, is characterized in that, comprises quantum dot and the three-dimensional hybrid structure that is assembled on quantum dot surface, and described three-dimensional hybrid structure comprises the probe of cy5 label and the DNA of biotin modification .

2 . The fluorescence sensor based on quantum dots and three-dimensional hybrid structure according to claim 1 , wherein the biotin is FTO-specific biotin. 3 .

3 . The fluorescence sensor based on quantum dots and three-dimensional hybrid structure according to claim 1 , wherein the biotin is modified at the end of DNA. 4 .

4. The preparation method of the fluorescence sensor based on quantum dots and three-dimensional hybrid structure according to any one of claims 1-3, characterized in that, comprising the steps of:

1) FTO-mediated demethylation of m6A in ssDNA substrates;

2) Dpn II assists in the cleavage of demethylated dsDNA substrates to generate biotinylated capture probes;

3) Construction of QD-DNA-cy5 nanostructures and subsequent measurement of FRET signal.

5. The method for preparing a fluorescence sensor based on quantum dots and three-dimensional hybrid structure as claimed in claim 4, wherein in step 1), m ⁶ A (5'-Gm ⁶ ATC-3 is combined with site specificity) ') ssDNA as a substrate, the ssDNA substrate is biotin-labeled at the 3' and 5' ends, in the presence of FTO, FTO will demethylate the sequence of 5'-Gm ⁶ ATC-3' to A demethylated sequence of 5'-GATC-3' is generated, and a complementary strand modified with biotin at both the 3' and 5' ends can hybridize to the demethylated ssDNA moiety to form a recognition site with Dpn II endonuclease (5'-GATC-3') dsDNA.

6. The preparation method of the fluorescence sensor based on quantum dots and three-dimensional hybrid structure as claimed in claim 5, wherein in step 2), Dpn II endonuclease can be at the recognition site (5'-G-A-T-C-3' ) specifically recognizes and cleaves demethylated dsDNA.

7. The preparation method of the fluorescence sensor based on quantum dots and three-dimensional hybrid structure as claimed in claim 4, characterized in that, in step 3), 4 different sequences and 1 identical sequence are included, and 4 different sequences are included. The biotin-modified cleavage product hybridizes with the same cy5-labeled reporter gene probe to form a three-dimensional hybrid structure.

8. The fluorescence sensor based on quantum dots and three-dimensional hybrid structure according to any one of claims 1-3 or the preparation method of the fluorescence sensor based on quantum dots and three-dimensional hybrid structure according to any one of claims 4-7 is obtained The application of the products in the detection of FTO inhibitors.

9. A method for detecting an FTO inhibitor, characterized in that the fluorescent sensor based on quantum dots and three-dimensional hybrid structure described in any one of claims 1-3 or the fluorescence sensor described in any one of claims 4-7 is excited by laser light The product obtained by the preparation method of the fluorescence sensor based on quantum dots and three-dimensional hybrid structure simultaneously observed the fluorescence signal of quantum dots and the fluorescence signal of Cy5.

10 . The method for detecting FTO inhibitors according to claim 9 , wherein the laser is a 488 nm argon laser, and the FTO activity is quantitatively monitored by simply counting the number of Cy5 signals by TIRF-based single-molecule imaging. 11 .