Label-free biosensor based on DNA fluorescent aptamer for detection kit of demethylase fto

Abstract

The application discloses a label-free biosensor based on DNA fluorescent aptamer for a detection kit of demethylase FTO, methyl modification is carried out on a specific base site of a DNA aptamer (Bibb Lettuce), so that the binding activity of the DNA aptamer is obviously inhibited, the DNA aptamer cannot be specifically combined with a fluorescent small molecule (DFHBI-1T), and only a weak fluorescent signal can be generated. Only when the target object FTO exists, can the FTO specifically remove the methyl modification on the DNA aptamer, restore the binding activity of the Bibb Lettuce, make the Bibb Lettuce be highly specifically combined with the DFHBI-1T, and a significantly enhanced fluorescent signal can be generated. The detection time of the kit is only 2 h, and the kit can detect FTO to 43.2 nM; the kit is beneficial to realizing simple, rapid and economical detection of the demethylase FTO, and thus solves the problem that the clinical application of existing detection methods is limited due to the defects of a complex probe synthesis process, high cost, insufficient light stability, serious self-fluorescence interference and poor stability.

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, specifically to a label-free biosensor based on DNA fluorescent aptamers for the detection kit of demethylase FTO, belonging to the field of biopharmaceutical technology. Background Technology

[0002] As the first discovered RNA demethylase, FTO participates extensively in various physiological processes by regulating the dynamic balance of methylation modification. Its abnormal expression is closely related to the clinical characteristics of various malignant tumors. It can serve as a novel tumor marker for early cancer diagnosis and a prognostic indicator. Quantitative analysis of FTO is of great significance for studying its biological functions and its application in disease diagnosis.

[0003] However, due to the small size of the N6-methyl group removed by the demethylase FTO and the chemical inertness of the methyl group, traditional detection methods often require expensive instruments and professional personnel, and suffer from problems such as cumbersome procedures, large sample requirements, high costs, long processing times, and radiation hazards, limiting their widespread application. Nucleic acid aptamers, as a type of functional nucleic acid, are short single-stranded oligonucleotide fragments (DNA or RNA) with specific sequences obtained through in vitro screening techniques. Due to their unique structural characteristics, they can bind to corresponding target molecules with high affinity and specificity, and have advantages such as simple preparation, flexible structure, and ease of modification, showing broad application prospects in clinical diagnosis and treatment. Among them, the synthesis process of DNA aptamers is mature and stable, with lower production costs and better stability, making the detection kit simpler and more economical compared to traditional detection methods. However, currently, there is no label-free biosensor based on DNA fluorescent aptamers for the detection of the demethylase FTO. Summary of the Invention

[0004] The purpose of this invention is to provide a label-free biosensor based on DNA fluorescent aptamers for the detection kit of demethylase FTO.

[0005] The objective of this invention is achieved through the following technical solution: a label-free biosensor based on DNA fluorescent aptamers for the detection kit of the demethylase FTO. The recombinant human protein FTO was purchased from Active Motif (Carlsbad, CA, USA).

[0006] A DNA fluorescent aptamer comprising Bibb Lettuce-A, Bibb Lettuce-mA1, and Bibb Lettuce-mA2;

[0007] The Bibb Lettuce-A nucleic acid sequence is shown in SEQ ID NO: 1.

[0008] The Bibb Lettuce-mA1 nucleic acid sequence is shown in SEQ ID NO: 2.

[0009] The Bibb Lettuce-mA2 nucleic acid molecule sequence is shown in SEQ ID NO: 3.

[0010] The application of the aforementioned DNA fluorescent aptamer in the detection of demethylase FTO.

[0011] The application of the aforementioned DNA fluorescent aptamer in the specific recognition of the demethylase FTO.

[0012] A label-free biosensor for detecting the demethylase FTO includes the aforementioned DNA fluorescent aptamer and fluorescent small molecule.

[0013] The fluorescent small molecule includes difluorohydroxybenzimidazole-1-triazole DFHBI-1T; the molar ratio of DNA fluorescent aptamer to fluorescent small molecule is 1:1-3.

[0014] A kit for detecting demethylase FTO includes the aforementioned DNA fluorescent aptamer, HEPES buffer, fluorescent small molecule, and reaction substrate; the reaction substrate includes L-ascorbic acid, ferrous sulfate hexahydrate, α-ketoglutarate α-KG, and bovine serum albumin (BSA).

[0015] A method for detecting the demethylase FTO, the method being used for non-disease diagnostic or therapeutic purposes; the method comprising:

[0016] (1) Methylated DNA aptamers and demethylase FTO were dissolved in the reaction substrate, and HEPES and ultrapure water were added and incubated together.

[0017] (2) After adding DFHBI-1T fluorescent small molecule solution and HEPES buffer, and continuing to incubate in the dark, the detection of demethylase FTO is achieved by detecting the fluorescence signal intensity.

[0018] DNA aptamers include any one of the following: Bibb Lettuce-A nucleic acid molecule sequence as SEQ ID NO: 1, Bibb Lettuce-mA1 nucleic acid molecule sequence as SEQ ID NO: 2, or Bibb Lettuce-mA2 nucleic acid molecule sequence as SEQ ID NO: 3.

[0019] Incubate together for 20-60 minutes in step (1); Incubate together for 20-60 minutes in step (1).

[0020] The 20-60 minutes refers to any number of minutes or any time period within the range of 20-60 minutes; preferably, it is 40-60 minutes.

[0021] In step (1), the detection limit of demethylase FTO is 43.2 nM, and it is preferred to perform the detection in the range of FTO concentration of 0.85-0.0265625 μM.

[0022] In step (2), the fluorescence signal is 450-465nm.

[0023] The buffer solutions are HEPES buffers: 10 × 50 mM HEPES buffer (pH=7.0), 2 × FTO buffer (containing 10 × HEPES, 10 × L-ascorbic acid, 10 × (NH4)2Fe(SO4)2·6H2O, 100 × α-KG, 100 × BSA), and 5 × 50 mM HEPES buffer (containing 1000 mM KCl, 40 mM MgCl2, pH=8.0).

[0024] Table 1: Sequences of nucleic acid molecules mentioned in the text

[0025]

[0026] Note that in the Bibb Lettuce-mA1 nucleic acid molecule sequence in the sequence listing, N represents N6-Me-dA.

[0027] Note that in the Bibb Lettuce-mA2 nucleic acid molecule sequence in the sequence listing, N represents N6-Me-dA. Attached Figure Description

[0028] Figure 1 A schematic diagram of the structure for screening methylation sites of the Bibb Lettuce aptamer.

[0029] Figure 2 The images show the effects of methylation modification at different sites. A represents the fluorescence signal intensity before and after methylation at site A1 of the Bibb Lettuce aptamer and after demethylation by FTO. B represents the fluorescence signal intensity before and after methylation modification at site A2 of the Bibb Lettuce aptamer and after demethylation by FTO.

[0030] Figure 3 This is a schematic diagram illustrating the principle of the present invention for detecting target objects.

[0031] Figure 4 This is a diagram for mechanism verification.

[0032] Figure 5To illustrate the effect of aptamer concentration on its binding with the fluorescent small molecule DFHBI-1T, A shows the fluorescence spectra of different concentrations of aptamer reacting with the fluorescent small molecule, and B shows the fluorescence signal intensity of different concentrations of aptamer reacting with the fluorescent small molecule.

[0033] Figure 6 The reaction concentration ratio of the aptamer and the fluorescent small molecule DFHBI-1T was optimized. A shows the fluorescence spectra of the aptamer and the fluorescent small molecule after the reaction at different reaction concentration ratios, and B shows the fluorescence signal intensity of the aptamer and the fluorescent small molecule after the reaction at different reaction concentration ratios.

[0034] Figure 7 Optimize the reaction time for demethylation.

[0035] Figure 8 Optimize the reaction time between the aptamer and the fluorescent small molecule DFHBI-1T.

[0036] Figure 9 This is a specific detection diagram for FTO.

[0037] Figure 10 The images show the detection of different concentrations of FTO using a label-free biosensor based on DNA fluorescent aptamers for the detection kit of the demethylase FTO. A is the fluorescence spectrum of different concentrations of FTO after demethylation, and B is the scatter plot and linear correlation curve of the fluorescence intensity of different concentrations of FTO after demethylation. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following description.

[0039] The reagents used in the embodiments of the present invention are as follows:

[0040] HEPES: Purchased from Sigma, 99.5%.

[0041] HEPES sodium salt: purchased from Sigma, 96%.

[0042] Potassium chloride: purchased from Sinopharm Chemical Reagent Co., Ltd., with a content of ≥99.5%.

[0043] Magnesium chloride hexahydrate: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0044] Dimethyl sulfoxide: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] TE buffer: purchased from Solarbio Ltd., pH=8.0.

[0046] L-Ascorbic acid: purchased from Sangon Biotech.

[0047] Ferrous sulfate hexahydrate: purchased from Sangon Biotech.

[0048] Bovine serum albumin (BSA): purchased from Sangon Biotech.

[0049] α-Ketoglutarate (α-KG): Purchased from Sangon Biotech.

[0050] Recombinant human protein FTO: purchased from Active Motif.

[0051] Ultrapure water: supplied by Millipore Milliq water purification system (18.2 MΩ·cm, Millipore, USA);

[0052] Preparation method of HEPES buffer (10 × 50 mM, pH=7.0): Weigh 9.6 g HEPES and 2.5 g HEPES sodium salt into a beaker, add ultrapure water to dissolve and make up to 100 mL, and then adjust the pH to 7.0 with 1 M sodium hydroxide solution at 25℃.

[0053] Preparation method of 100 × α-KG (300 μM): Weigh 0.0219 g α-KG into a beaker, add ultrapure water to dissolve and make up to 5 mL, dispense into portions and store in a refrigerator at -20℃.

[0054] Preparation method of 10 × L-ascorbic acid (2 mM) + 10 × (NH4)2Fe(SO4)2·6H2O (300 μM): Weigh 0.0352 g of L-ascorbic acid and 0.0118 g of (NH4)2Fe(SO4)2·6H2O into beakers, add ultrapure water to dissolve and make up to 10 mL, dispense into portions and store in a refrigerator at -20℃.

[0055] Preparation method of 100 × BSA (50 μg / mL): Weigh 0.025 g BSA into a beaker, add ultrapure water to dissolve and make up to 5 mL, dispense into aliquots and store at -4℃.

[0056] Preparation method of 2× FTO buffer: Take 20 μL HEPES buffer (10 × 50 mM, pH=7.0), 20 μL 10 × L-ascorbic acid + 10 × (NH4)2Fe(SO4)2·6H2O, 2 μL 100 × α-KG, and 2 μL 100 × BSA into an EP tube, add 56 μL of ultrapure water and mix well to obtain 100 μL 2× FTO buffer.

[0057] Preparation method of HEPES buffer (5 × 50 mM, containing 1000 mM KCl, 40 mM MgCl2, pH=8.0): Weigh 1.43 g HEPES, 4.92 g HEPES sodium salt, 6.043 g potassium chloride, and 0.813 g magnesium chloride into a beaker, add ultrapure water to dissolve and make up to 100 mL, and then adjust the pH to 8.0 with 1 M sodium hydroxide solution at 25℃.

[0058] Preparation of DNA stock solution (100 μM): Dissolve DNA powder in TE buffer (pH=8.0) according to the amount indicated on the DNA tube label, and store at -20℃.

[0059] Preparation method of DFHBI-1T stock solution (1 mM): Weigh 1 mg of DFHBI-1T powder and dissolve it in 3.123 mL of dimethyl sulfoxide to obtain a 1 mM DFHBI-1T stock solution. After dispensing, store it in a refrigerator at -20℃ protected from light.

[0060] The design of aptamer methylation site modification is as follows: First, the three-dimensional structure of Bibb Lettuce was predicted and optimized using a protein structure prediction model (AlphaFold3); simultaneously, the three-dimensional structure of DFHBI-1T was obtained from the PubChem database. Then, using DFHBI-1T as a ligand, molecular docking (MD) simulations were performed on the unmodified and methylated aptamer structures. The potential binding region between DFHBI-1T and the Bibb Lettuce aptamer was predicted using the AutoDock tool, and docking simulation analysis was performed using AutoDock Vina software to evaluate the impact of different methylation site modifications on the binding affinity and interaction forces between the aptamer and the DFHBI-1T molecule. Figure 1 This is a schematic diagram of the structure for screening DNA aptamer methylation sites. By selecting the key base sites A1 and A2 on the Bibb Lettuce aptamer for methylation modification, the methylated aptamers Bibb Lettuce-mA1 and Bibb Lettuce-mA2 can be obtained, and subsequent experiments are carried out based on this.

[0061] Example 1

[0062] To screen for the optimal methylation modification sites, two key A bases on the DNA aptamer Bibb Lettuce were methylated. The feasibility of using DNA aptamers methylated at two different sites for FTO detection was evaluated. The specific nucleic acid sequences are: Bibb Lettuce-A, Bibb Lettuce-mA1, and Bibb Lettuce-mA2.

[0063] First, 1.56 μM single nucleic acid sequences (Bibb Lettuce-A, Bibb Lettuce-mA1, and Bibb Lettuce-mA2) were used as control groups and mixed with 13 μL of 2× FTO buffer and ultrapure water to obtain a total DNA mixture of 26 μL. These mixtures were then incubated together at 37°C for 1 h. Next, each mixture was mixed with 3.12 μM DFHBI-1T, 10 μL of 5× 50 mM HEPES buffer, and ultrapure water to obtain a total DNA mixture of 50 μL. This mixture was then incubated at 37°C in the dark for another 1 h. The fluorescence intensity at an excitation wavelength of 465 nm was measured using an FS5 fluorescence spectrometer. The samples were divided into three groups: Bibb Lettuce-A, Bibb Lettuce-mA1, and Bibb Lettuce-mA2.

[0064] Next, 1.56 μM of single methylated nucleic acid sequences (Bibb Lettuce-mA1, Bibb Lettuce-mA2) and 3.12 μM of demethylase FTO were used as experimental groups. These were then mixed with 13 μL of 2×FTO buffer and ultrapure water to obtain a total DNA mixture of 26 μL, and incubated together at 37°C for 1 h. Afterward, these were mixed with 3.12 μM of DFHBI-1T, 10 μL of 5 × 50 mM HEPES buffer, and ultrapure water to obtain a total DNA mixture of 50 μL. This mixture was then incubated at 37°C in the dark for another 1 h. The fluorescence intensity at an excitation wavelength of 465 nm was detected using an FS5 fluorescence spectrometer. The groups were designated as the Bibb Lettuce-mA1+FTO group and the Bibb Lettuce-mA2+FTO group.

[0065] The experimental results are attached. Figure 2As shown in Figure A, compared with the unmethylated Bibb Lettuce-A, the binding activity of Bibb Lettuce-mA1 after methylation at site A1 was effectively inhibited, and the fluorescence intensity after binding with the fluorescent small molecule DFHBI-1T decreased. However, when it reacted with the demethylase FTO (Bibb Lettuce-mA1+FTO), the fluorescence intensity after the aptamer bound to the fluorescent small molecule did not rebound, indicating that methylation at site A1 of the Bibb Lettuce aptamer cannot be used as an effective site for FTO demethylation. As shown in Figure B, compared with the unmethylated Bibb Lettuce-A, the binding activity of Bibb Lettuce-mA2 after methylation at site A2 was significantly inhibited, and the fluorescence intensity after binding with the fluorescent small molecule DFHBI-1T decreased significantly. Furthermore, after reacting with the demethylase FTO (Bibb Lettuce-mA2 + FTO), the fluorescence intensity of the aptamer bound to the fluorescent small molecule was significantly enhanced. This indicates that methylation modification at site A2 of the Bibb Lettuce aptamer can significantly inhibit the binding activity of the aptamer, and FTO can effectively remove the methylation modification at this site, restoring the high specificity of the aptamer's binding ability to the fluorescent small molecule. Therefore, site A2 (Bibb Lettuce-mA2) of the Bibb Lettuce aptamer was ultimately selected as the optimal methylation modification site.

[0066] The concept for DNA fluorescent aptamers is as follows: The optimal methylation modification sites for the aptamers will be determined through methylation site screening experiments, and the relevant nucleic acid sequences will be synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Attached) Figure 3 This diagram illustrates the principle of using DNA fluorescent aptamers for target analyte detection. The methylated aptamer Bibb Lettuce-mA2 exhibits significantly inhibited binding activity. In the absence of the target analyte FTO, Bibb Lettuce-mA2 binds weakly to DFHBI-1T, producing only a low background signal. Only in the presence of FTO can FTO specifically remove the methylation on Bibb Lettuce-mA2, restoring it to the highly binding Bibb Lettuce-A aptamer, which can then bind highly specifically to the fluorescent small molecule DFHBI-1T, resulting in a significantly enhanced fluorescence signal. By detecting changes in fluorescence signal intensity, the quantitative detection of the target analyte demethylase FTO can be achieved.

[0067] Example 2

[0068] To verify the feasibility of using a DNA aptamer-based fluorescent biosensor in the detection of the demethylase FTO, after screening for the optimal methylation modification site, the fluorescence intensity of the Bibb Lettuce aptamer before methylation (Bibb Lettuce-A), after methylation (Bibb Lettuce-mA2), before methylation reacting with FTO (Bibb Lettuce-A+FTO), after methylation reacting with FTO (Bibb Lettuce-mA2+FTO), and after inactivating FTO protein by high-temperature heating, was compared and analyzed.

[0069] First, in the control groups (Bibb Lettuce-A and Bibb Lettuce-mA2), a single 0.78 μM nucleic acid sequence was mixed with 13 μL of 2 × FTO buffer and ultrapure water to obtain a total DNA mixture of 26 μL. Then, in the experimental groups (Bibb Lettuce-A+FTO and Bibb Lettuce-mA2+FTO), a single 0.78 μM methylated nucleic acid sequence was mixed with 1.56 μM of demethylase FTO, 13 μL of 2 × FTO buffer and ultrapure water to obtain a total DNA mixture of 26 μL. They were co-incubated at 37°C for 1 h; then, they were mixed thoroughly with 1.56 μM DFHBI-1T, 10 μL of 5 × 50 mM HEPES buffer, and ultrapure water to obtain a total DNA mixture of 50 μL. This mixture was then incubated at 37°C in the dark for another 1 h, and the fluorescence intensity at an excitation wavelength of 465 nm was detected using an FS5 fluorescence spectrometer. The samples were grouped into Bibb Lettuce-A, Bibb Lettuce-mA2, Bibb Lettuce-A+FTO, and Bibb Lettuce-mA2+FTO groups.

[0070] To further verify the effect of FTO activity on the experiment, the inactivated FTO required for the experiment was obtained by heating at 95℃ for 5 min. 0.78 μM of a single methylated modified nucleic acid sequence (Bibb Lettuce-A and Bibb Lettuce-mA2) was mixed with 1.56 μM of inactivated FTO (Heated FTO), 13 μL of 2× FTO buffer, and ultrapure water to obtain a total DNA mixture of 26 μL. All groups were incubated together at 37℃ for 1 h. Then, the mixture was thoroughly mixed with 1.56 μM DFHBI-1T, 10 μL of 5 × 50 mM HEPES buffer, and ultrapure water to obtain a total DNA mixture of 50 μL. This mixture was then incubated at 37℃ in the dark for another 1 h. The fluorescence intensity at an excitation wavelength of 465 nm was detected using an FS5 fluorescence spectrometer. The groups were designated as Bibb Lettuce-A + Heated FTO and Bibb Lettuce-mA2 + Heated FTO.

[0071] The experimental results are attached. Figure 4 As shown, the unmethylated Bibb Lettuce-A aptamer specifically binds to the DFHBI-1T fluorescent small molecule, generating a strong fluorescence signal. After the addition of the demethylase FTO, the fluorescence intensity shows a slight upward trend, but it remains essentially the same as the fluorescence signal after the addition of inactivated FTO, indicating that the enhanced fluorescence signal is not due to the key role played by the demethylase FTO. In contrast, the binding activity of the methylated Bibb Lettuce-mA2 aptamer is significantly inhibited, and its fluorescence intensity decreases significantly after binding to the DFHBI-1T fluorescent small molecule. However, the fluorescence intensity significantly increases after the addition of the demethylase FTO, and the fluorescence signal intensity does not increase after the addition of inactivated FTO. This indicates that methylation significantly inhibits the binding activity of the aptamer to the fluorescent small molecule, and only the catalytically active demethylase FTO can effectively remove the methylation modification on the aptamer, thereby restoring the binding activity and generating a significantly enhanced fluorescence signal. Therefore, this invention can achieve a simple detection of the target demethylase FTO by detecting changes in fluorescence signal.

[0072] Example 3

[0073] To investigate the effect of substrate concentration on the fluorescence intensity of aptamers (Bibb Lettuce-A and Bibb Lettuce-mA2) after binding with the fluorescent small molecule DFHBI-1T, we designed reactions of aptamers with different concentrations and fluorescent small molecules with a fixed concentration, as well as reactions of aptamers with different concentration ratios and fluorescent small molecules, to determine the optimal reaction concentration ratio of aptamers and fluorescent small molecules.

[0074] First, 100 μM Bibb Lettuce-A and Bibb Lettuce-mA2 were serially diluted to 3.12 μM, 1.56 μM, 0.8 μM, and 0.4 μM. Then, 2 μL of Bibb Lettuce-A and Bibb Lettuce-mA2 were mixed with 13 μL of 2 × FTO buffer and 11 μL of ultrapure water, respectively, and incubated together at 37 °C for 1 h. Then, they were mixed with 1.56 μM DFHBI-1T and 10 μL of 5 × 50 mM HEPES buffer, and incubated at 37 °C in the dark for another 1 h. The fluorescence intensity at an excitation wavelength of 465 nm was then detected using an FS5 fluorescence spectrometer. The samples were divided into four groups: Bibb Lettuce-A 0.4 μM, Bibb Lettuce-A 0.8 μM, Bibb Lettuce-A 1.56 μM, Bibb Lettuce-A 3.12 μM, Bibb Lettuce-mA2 0.4 μM, Bibb Lettuce-mA2 0.8 μM, Bibb Lettuce-mA2 1.56 μM, and Bibb Lettuce-mA2 3.12 μM. The detection results are attached. Figure 5 As shown in Figure A, the fluorescence signal gradually increases with the increase of aptamer concentration, and the fluorescence signal intensity is shown in Figure B. This indicates that when the concentration of fluorescent small molecules remains constant, the fluorescence signal intensity after the aptamer binds to the fluorescent small molecules gradually increases with the increase of aptamer concentration, showing a good concentration dependence.

[0075] To further improve the binding efficiency between the aptamers (Bibb Lettuce-A and Bibb Lettuce-mA2) and the fluorescent small molecule DFBHI-1T, enhance signal sensitivity, and reduce experimental costs while ensuring binding efficiency, the reaction concentration ratio of the aptamer and the fluorescent small molecule in the reaction system was optimized. Specifically, the reaction concentration ratio of the aptamer to the DFHBI-1T small molecule was 1:1, 1:2, and 1:4.

[0076] First, 0.8 μM Bibb Lettuce-A and Bibb Lettuce-mA2 were mixed with 13 μL of 2 × FTO buffer and 11 μL of ultrapure water and incubated together at 37 °C for 1 h. Then, Bibb Lettuce-A and Bibb Lettuce-mA2 were incubated with 0.8 μM, 1.56 μM, and 3.12 μM DFHBI-1T and 10 μL of 5 × 50 mM HEPES buffer, respectively, at 37 °C in the dark for 1 h before fluorescence testing and S / N calculation. The samples were divided into four groups: Bibb Lettuce-A+DFHBI-1T 0.8 μM, Bibb Lettuce-A+DFHBI-1T 1.56 μM, Bibb Lettuce-A+DFHBI-1T 3.12 μM, Bibb Lettuce-mA2+DFHBI-1T 0.8 μM, Bibb Lettuce-mA2+DFHBI-1T 1.56 μM, and Bibb Lettuce-mA2+DFHBI-1T 3.12 μM. The detection results of this kit are shown in the attached figure. Figure 6 As shown in Figure A, the fluorescence signal curves of the aptamer and the fluorescent small molecule DFHBI-1T at different concentration ratios are obtained. By comparing the fluorescence signal intensity and the reaction signal-to-background ratio (S / N) at different reaction concentration ratios (Figure B), it was found that the reaction signal-to-background ratio of the system is the highest when the reaction concentration ratio of the aptamer and the fluorescent small molecule DFHBI-1T is 1:2. Therefore, the optimal reaction concentration ratio of the aptamer and the fluorescent small molecule DFHBI-1T is finally determined to be 1:2.

[0077] Example 4

[0078] To achieve rapid detection of the target analyte, the reaction time required for the demethylation reaction was optimized through kinetic experiments. The specific steps were as follows: 0.4 μM Bibb Lettuce-mA2 and 13 μL 2 × FTO buffer were mixed thoroughly with FTO and incubated at 37°C for 0 min, 20 min, 40 min, 60 min, and 80 min, respectively. Then, the mixture was mixed thoroughly with 1.56 μM DFHBI-1T and 10 μL 5 × 50 mM HEPES buffer, and incubated at 37°C in the dark for 1 h before detecting the fluorescence intensity. The detection results of this kit are shown in the attached figure. Figure 7As shown, by changing the reaction time of FTO demethylation, it was found that the fluorescence signal intensity after FTO demethylation gradually increased with the extension of reaction time and gradually stabilized at 60 min, indicating that the removal of methyl groups by FTO was almost complete at this time. Therefore, 60 min was finally selected as the optimal incubation time for FTO demethylation.

[0079] Example 5

[0080] This embodiment optimized the reaction time between the aptamer and the fluorescent small molecule. The specific steps were as follows: 0.78 μM Bibb Lettuce-mA2, 13 μL 2 × FTO buffer, 1.56 μM FTO, and 11 μL ultrapure water were mixed thoroughly and incubated at 37°C for 1 h. Then, the mixture was mixed with 0.78 μM DFHBI-1T and 10 μL 5 × 50 mM HEPES buffer and incubated at 37°C in the dark for 0 min, 20 min, 40 min, 60 min, and 80 min before detecting the fluorescence intensity. The detection results of this kit are shown in the attached figure. Figure 8 As shown, it was found that when the incubation time between the aptamer and the small molecule reached 60 min, the fluorescence signal gradually stabilized and the response signal-to-background ratio reached its maximum value. Therefore, the optimal incubation time between the aptamer and the fluorescent small molecule was finally determined to be 60 min.

[0081] Example 6

[0082] This embodiment designed a system-specific experiment to detect the fluorescence signal intensity of the system for different target substances (FTO, BSA, T4 PNK, GSH, ATP, H2O2). The specific steps were as follows: 0.78 μM Bibb Lettuce-mA2 was incubated with 13 μL of 2 × FTO buffer and 13 μL of 2 × FTO buffer in the experimental group, 1.56 μM FTO, 50 μg / mL BSA, 20 U / mL T4 PNK, 5 mM GSH, 5 mM ATP, and 100 μM H2O2 (30%), as well as the blank control group (Blank) with only ultrapure water, for 1 h at 37°C. Subsequently, 1.56 μM DFHBI-1T and 10 μL of 5 × 50 mM HEPES buffer were added to each experimental group and the blank control group, mixed thoroughly, and incubated at 37°C in the dark for 1 h before detecting the fluorescence intensity. The subjects were divided into four groups: FTO, BSA, T4 PNK, GSH, ATP, H2O2, and Blank. Experimental results are attached. Figure 9As shown, compared to the experimental group with significant fluorescence intensity after the addition of FTO, the fluorescence signal intensity of other experimental groups was almost the same as that of the blank control group. This indicates that only the demethylase FTO can specifically remove the methylation modification on the Bibb Lettuce aptamer, thereby restoring the aptamer's binding activity and enabling it to specifically bind to the DFHBI-1T fluorescent small molecule, producing a significant fluorescence signal. Therefore, this detection kit can specifically identify and detect FTO in complex samples, and has good application potential.

[0083] Example 7

[0084] A label-free biosensor detection kit based on DNA fluorescent aptamers is used for the detection of demethylase FTO in solution.

[0085] The kit consists of 26 μL of nucleic acid incubation solution and 24 μL of reaction substrate solution; the nucleic acid incubation solution includes 2 μL Bibb Lettuce-mA2, 1.26 μL FTO, 13 μL 2 × FTO buffer and 9.74 μL ultrapure water; the 24 μL reaction substrate solution includes 3.9 μL LDFHBI-1T, 10 μL 5 × 50 mM HEPES buffer and 10.1 μL ultrapure water. FTO at concentrations of 0.85 μM, 0.425 μM, 0.2125 μM, 0.10625 μM, 0.053125 μM, and 0.0265625 μM was mixed with Bibb Lettuce-mA2 aptamer according to the volume and ratio of the nucleic acid incubation solution described above. After incubation at 37°C for 1 h, 24 μL of reaction substrate solution was added, mixed thoroughly, and incubated in the dark for another 1 h. The fluorescence signal was then detected immediately using an FS5 fluorescence spectrometer. The detection results of this kit are shown in the attached figure. Figure 10 As shown in Figure A, the fluorescence signal intensity exhibits a good linear relationship with FTO concentration (Figure B). Within the FTO concentration range of 0.85–0.0265625 μM, the obtained linear curve is y = 27.094x + 10.667 (R²). 2 =0.9507), the detection limit was calculated to be 43.2 nM, which can be used to calculate the concentration of FTO in the sample by fluorescence signal intensity, thus achieving sensitive detection of FTO.

Claims

1. A DNA fluorescent aptamer, characterized in that, The DNA aptamer is Bibb Lettuce-mA2; The Bibb Lettuce-mA2 nucleic acid molecule sequence is shown in SEQ ID NO:

3.

2. The use of the DNA fluorescent aptamer according to claim 1 in the preparation of a formulation for the detection of demethylase FTO.

3. The use of the DNA fluorescent aptamer according to claim 1 in the preparation of formulations that specifically recognize the demethylase FTO.

4. A label-free biosensor for detecting the demethylase FTO, characterized in that, Includes the DNA fluorescent aptamer and fluorescent small molecule as described in claim 1, wherein the fluorescent small molecule includes difluorohydroxybenzimidazole-1-triazole DFHBI-1T.

5. The label-free biosensor for detecting demethylase FTO according to claim 4, characterized in that, The molar ratio of DNA fluorescent aptamer to fluorescent small molecule is 1:1-3.

6. A kit for detecting the demethylase FTO, characterized in that, The invention comprises the DNA fluorescent aptamer, HEPES buffer, fluorescent small molecule, and reaction substrate as described in claim 1; the reaction substrate comprises L-ascorbic acid, ferrous sulfate hexahydrate, α-ketoglutarate α-KG, and bovine serum albumin BSA; the fluorescent small molecule comprises difluorohydroxybenzimidazole-1-triazole DFHBI-1T.

7. A method for detecting the demethylase FTO, characterized in that, The method is used for non-disease diagnosis or treatment purposes; the method includes: (1) Dissolve the DNA fluorescent aptamer and demethylase FTO as described in claim 1 in the reaction substrate, and incubate them together with HEPES and ultrapure water; (2) After adding DFHBI-1T fluorescent small molecule solution and HEPES buffer, and continuing to incubate in the dark, the detection of demethylase FTO is achieved by detecting the fluorescence signal intensity.

8. The method for detecting demethylase FTO according to claim 7, characterized in that, Incubate together for 20-60 minutes in step (1).

9. The method for detecting demethylase FTO according to claim 7, characterized in that, The limit of detection for the demethylase FTO is 43.2 nM. In step (2), the fluorescence signal is 450-465nm.

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