Preparation method of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor

Abstract

The present invention belongs to the field of biosensing and relates to a method for preparing a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor. The steps include: preparing the magnetic metal-organic framework, introducing Uio-66-NH2 on the surface of magnetic Fe3O4 by an in situ synthesis method to synthesize Fe3O4@Uio-66; constructing a double-stranded DNA functionalized magnetic metal-organic framework, encapsulating a fluorescent dye inside Fe3O4@Uio-66, and connecting the double-stranded DNA to the surface of Fe3O4@Uio-66 to form a DNA-gated Fe3O4@Uio-66; constructing a ratiometric fluorescence sensor, in which an aptamer specifically binds to ochratoxin A, releasing the fluorescent dye, and simultaneously degrading the complementary chain by nuclease exonuclease I, and detecting the fluorescence signal ratio F 550 / F 425 The sensor was used to quantitatively analyze ochratoxin A. The linear response range of the sensor for ochratoxin A was 0.5-1000 ng / mL, with a detection limit of 0.308 ng / mL, indicating good anti-interference and practical application capabilities.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a preparation method and application of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor. Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by various Aspergillus and Penicillium fungi. OTA is widely found in grains, coffee, wine, beer, and dried fruit. Due to its potent nephrotoxicity, hepatotoxicity, and carcinogenicity, its detection and control in food and feed are particularly important. Among commonly used detection methods, enzyme-linked immunosorbent assay (ELISA) does not require complex equipment and is suitable for testing large sample batches. However, other mycotoxins or impurities in the sample may affect the results. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), while offering advantages of high precision and sensitivity, also have the disadvantages of high detector costs and the requirement for specialized technicians to operate and maintain. Therefore, to effectively control OTA contamination and hazards, the development of a new, rapid, convenient, sensitive, and accurate OTA detection method is crucial.

[0003] Fluorescence sensor is a technology that uses fluorescence signals to detect specific target substances. It has the characteristics of good stability and short detection time. Nucleic acid aptamers are molecules composed of nucleic acids that can specifically bind to target molecules. They are stable and low-cost. Fluorescence aptamer sensors combine fluorescence sensing technology with the high selectivity of nucleic acid aptamers, making them excellent in detection sensitivity and specificity. In addition, to further improve sensitivity, enzyme-assisted signal amplification strategies are often used. These include nucleases (Exo I, Exo III, Recjf Exo, T7 Exo), metal ion-specific deoxyribozymes, DNA polymerases, etc. Among them, Exo I is a nuclease that is highly selective for denatured or single-stranded deoxyribonucleic acid and is widely used in biosensors. Metal-organic frameworks (MOFs) are a class of porous materials composed of metal ions or metal clusters and organic ligands through coordination bonds. Among the many MOFs, Uio-66-NH2 is the most common one. Uio-66-NH2 is composed of zirconium metal ions (Zr 4+ ) and 2-aminoterephthalic acid (NH2-BDC), and has attracted considerable attention due to its high chemical stability, moderate pore size, and ease of functionalization. The magnetic material Fe3O4 is widely used in biomedicine, environmental remediation, and magnetic separation due to its excellent magnetic properties and biocompatibility.

[0004] This invention constructs a novel ratiometric fluorescent aptasensor. By introducing Uio-66-NH2 onto the surface of Fe3O4, a magnetic metal-organic framework (Fe3O4@Uio-66) is synthesized. This composite material combines the porous properties of MOFs with the magnetic characteristics of Fe3O4, enabling the loading of a larger number of signal molecules while achieving rapid separation. A DNA-gating strategy is introduced to develop a magnetic metal-organic framework with intelligent response properties. Leveraging the specific recognition and programmability of DNA molecules, Fe3O4@Uio-66 can release rhodamine (Rho 6G) signal molecules in the presence of OTA, enabling precise control. An Exo I-assisted signal amplification strategy is employed. Exo I specifically recognizes and degrades single-stranded DNA, further releasing signal molecules, achieving signal amplification and further improving the biosensor's detection sensitivity. Furthermore, utilizing the inherent fluorescence properties of Uio-66-NH2 and the Rho 6G fluorescence signal, a dual-signal output establishes a self-calibrating fluorescence detection platform, reducing the interference of false-positive signals. The fluorescence sensing strategy based on the present invention not only overcomes the problems of the above-mentioned existing technologies, but also has excellent selectivity and sensitivity, and can also achieve rapid and accurate OTA detection in complex environments, and has broad application prospects. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of currently used detection methods, such as low sensitivity, proneness to false positive signals, slow response, and complex sensor preparation. A method for preparing a DNA-gated magnetic metal-organic framework (Fe₃O₄@Uio-66) ratiometric fluorescence sensor is provided. This method enables high sensitivity, fast response, and high specificity in detecting OTA in food samples.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor comprises the following steps:

[0008] (1) Synthesis of Fe3O4: Ferric chloride hexahydrate was dissolved in ethylene glycol to form a homogeneous solution under ultrasonication. Polyacrylate A, a deionized water mixture, and urea compound B were then added sequentially. The mixture was ultrasonicated and heated overnight in a Teflon-lined stainless steel autoclave before cooling to room temperature. The solid product was washed several times with deionized water and ethanol to remove organic and inorganic impurities, and then the solid was collected with a magnet. Finally, the Fe3O4 was dried under vacuum conditions.

[0009] (2) Preparation of functionalized Fe₃O₄: Dissolve urea compound B in deionized water, then add a certain amount of F₃O₄. Stir the solution, and collect the solid with a magnet. Wash the solid several times with deionized water and N,N-dimethylformamide. Finally, vacuum dry overnight to obtain functionalized Fe₃O₄.

[0010] (3) Synthesis of Fe3O4@UiO-66: Functionalized Fe3O4 was dispersed in a solution of transition metal chloride C and 2-aminoterephthalic acid in N,N-dimethylformamide, heated and stirred, and the solid was collected with a magnet. The above steps were repeated once. The solid was then redispersed in a new solution containing the two reagents at the above concentrations. The solution was transferred to a Teflon-lined stainless steel autoclave, sealed, and heated for a specified period of time. The sample was collected with a magnet, washed with ethanol, and dried under vacuum. This resulted in a magnetic metal-organic framework.

[0011] (4) Preparation of double-stranded DNA functionalized Fe3O4@Uio-66: Fe3O4@Uio-66 was mixed with the dye Rhodamine 6G and reacted for a period of time, thereby loading the dye Rhodamine 6G onto Fe3O4@Uio-66. In order to obtain double-stranded DNA functionalized Fe3O4@Uio-66, the aptamer complementary chain cDNA was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for reaction. Then, the aptamer APT was added to the reaction mixture and treated at a constant temperature shaker at 37°C for a period of time to obtain double-stranded DNA (dsDNA). In order to encapsulate Rhodamine 6G, the double-stranded DNA dsDNA solution and the above-mentioned Rhodamine 6G and Fe3O4@Uio-66 mixture were treated at a constant temperature shaker at 37°C for a period of time. The prepared product was washed three times with buffer to remove the unloaded Rhodamine 6G and double-stranded DNA, and dispersed in buffer.

[0012] (5) Construction of ratiometric fluorescent aptamer sensor: First, double-stranded DNA functionalized Fe3O4@Uio-66 was prepared. Then, different concentrations of OTA were added to the solution and the solution was treated in a constant temperature oscillator at 37°C for a period of time. Subsequently, a certain volume of nuclease Exo I was added to the reaction solution and the solution was treated in a constant temperature oscillator at 37°C for a period of time. Then, magnetic separation was performed using a magnetic stand, the supernatant was discarded, and the fluorescence spectrum of the precipitated solid was recorded. At this point, a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescent sensor was prepared.

[0013] (6) For actual sample detection: Measure the fluorescence signal of the food sample of unknown concentration according to steps (1) to (5), wherein the different concentrations of OTA added in step (5) are food sample extracts in the actual sample detection, and the detection results are substituted into the standard curve to obtain the OTA concentration in the sample.

[0014] The detection mechanism of this invention: Fe3O4@Uio-66 is synthesized via a stepwise assembly in situ synthesis method. The dye Rhodamine 6G is encapsulated within multiple mesopores due to the porous nature of the MOF in Fe3O4@Uio-66. Furthermore, Fe3O4@Uio-66 possesses abundant reactive amino groups, making it easy to bind to other functional groups. Therefore, carboxyl-containing cDNA can be easily attached to Fe3O4@Uio-66 via an amidation reaction. The aptamer APT hybridizes with the complementary cDNA to form dsDNA. The dsDNA is attached to the Rhodamine 6G-encapsulated Fe3O4@Uio-66, acting as a gate for the functionalized Fe3O4@Uio-66. In the absence of OTA, the capped double-stranded DNA structure hinders the release of the signal molecule. Theoretically, Exo I can specifically cleave the 3' to 5' ends of single-stranded nucleic acid sequences, effectively preventing Exo I from hydrolyzing the dsDNA. Under excitation at 290 nm, the dyes Rho 6G and Fe3O4@Uio-66 have emission peaks at approximately 550 nm and 425 nm, respectively. In the presence of OTA, the aptamer APT specifically binds to the target molecule, thereby destroying the capped double-stranded DNA structure and releasing the signal molecule. In addition, the free single-stranded cDNA on Fe3O4@Uio-66 is degraded from the 3' end by Exo I, thereby reducing steric hindrance and promoting the release of signal molecules. The emission peak of the dye Rho 6G at 550 nm decreases, while the emission peak of Fe3O4@Uio-66 at 425 nm remains unchanged. Finally, according to F 550 / F 425 The values ​​of OTA were used for quantitative analysis.

[0015] It is further defined that the polyacrylic acid salt A in step (1) is one or more of polyacrylamide, sodium polyacrylate, and polyvinyl alcohol;

[0016] It is further defined that the urea compound B in steps (1) and (2) is one or both of thiourea and urea;

[0017] It is further defined that the transition metal chloride in step (3) is one or more of zirconium tetrachloride, copper chloride, and nickel chloride;

[0018] It is further defined that the complementary DNA cDNA in step (4) is 5'-TGT CCG ATG CTC CCT TTA-3'; wherein the group labeled at the 5' end is one or more of COOH, NH2, and SH, and the aptamer APT is 5'-GAT CGG GTGTGG GTG GCG TAA AGG GAG CAT CGG ACA-3';

[0019] It is further defined that the aptamer complementary chain cDNA in step (4) has an aptamer APT dosage range of 50-400 μL and a concentration range of 1-8 μM;

[0020] It is further defined that the volume of exonuclease Exo I in step (5) is 0.5-1.5 μL and the concentration is 5-40 U / mL;

[0021] It is further defined that the constant temperature oscillator treatment time in steps (4) and (5) is 0.5 to 24 hours;

[0022] It is further defined that in step (4), the buffer solution is one or two of Tris-HCl, PBS, and HEPES.

[0023] Compared with the prior art, the present invention has the following significant advantages:

[0024] 1. By introducing Uio-66-NH2 onto the surface of Fe3O4, the present invention successfully prepared a magnetic metal-organic framework that integrates the porous properties of MOFs and the magnetic properties of Fe3O4, which can load more signal molecules and achieve rapid separation.

[0025] 2. Combining the fluorescence properties of Uio-66-NH2 itself and the fluorescence properties of the dye molecule Rhodamine 6G, dual signal output is constructed to construct a self-calibrating ratiometric fluorescence aptamer detection platform to avoid false positives during the detection process.

[0026] 3. The present invention uses Exo I to degrade single-stranded DNA, thereby reducing steric hindrance and further improving sensor sensitivity.

[0027] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation method of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor.

[0029] Figure 2 Fluorescence intensity of the sensor constructed according to Example 1 of the present invention in the absence of OTA (solid line), the presence of 25 ng / mL OTA (dashed line), and the presence of 500 ng / mL OTA (dash-dotted line).

[0030] Figure 3 This is the standard curve for detecting OTA using the sensor constructed in Example 1 of the present invention.

[0031] Figure 4This is a comparison of the selectivity of the sensor constructed in Example 1 of the present invention to OTA in the presence of other interfering toxins. DETAILED DESCRIPTION

[0032] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Example 1

[0033] A preparation method and application of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor, the implementation method of which is as follows Figure 1 shown.

[0034] The preparation method of the DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor comprises the following steps:

[0035] (1) Synthesis of Fe3O4: 0.54 g of ferric chloride hexahydrate was dissolved in 20 mL of ethylene glycol under ultrasonication to form a homogeneous solution. 0.15 mL of sodium polyacrylate (PASS), 1.5 mL of deionized water, and 1.2 g of urea were then added sequentially. The mixture was ultrasonicated for 10 min and then placed in a Teflon-lined stainless steel autoclave (100 mL). The autoclave was heated at 200 °C for 12 h and then cooled to room temperature. The solid product was washed several times with deionized water and ethanol to remove organic and inorganic impurities, and then the solid was collected with a magnet. Finally, the Fe3O4 was dried under vacuum at 60 °C.

[0036] (2) Preparation of functionalized Fe3O4: 0.64 mL of 30% PASS was dissolved in 11.36 mL of deionized water, and then 0.2 g of F3O4 was added. The solution was stirred at 37°C for 24 h, and the solid was collected with a magnet. The solid was washed several times with deionized water and N,N-dimethylformamide. Finally, the functionalized Fe3O4 was obtained after vacuum drying at 60°C overnight.

[0037] (3) Synthesis of Fe3O4@UiO-66: Functionalized Fe3O4 (0.2 g) was dispersed in zirconium tetrachloride (11.46 mmol) and diaminoterephthalic acid (11.46 mmol) dissolved in 46 mL of N,N-dimethylformamide solution. The mixture was stirred at 100 °C for 4 h, and the solid was collected with a magnet. The above steps were repeated once. The mixture was then redispersed in a new solution containing the two reagents at the above concentrations. The solution was transferred to a 100 mL Teflon-lined stainless steel autoclave, sealed, heated to 120 °C, and reacted for 12 h. The sample was collected by a magnet, washed with ethanol, and dried in vacuum at 60 °C. A magnetic metal-organic framework was obtained.

[0038] (4) Preparation of double-stranded DNA functionalized Fe3O4@UiO-66: The signal molecule Rhodamine 6G was loaded onto Fe3O4@UiO-66. 400 μL of Fe3O4@UiO-66 (1 mg / mL) was mixed with 400 μL of Rhodamine 6G (0.6 mg / mL) and reacted at room temperature for 3 h. To obtain double-stranded DNA functionalized Fe3O4@Uio-66, 100 μL of cDNA (4 μM) was mixed with 50 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1 mg / mL) and 50 μL of N-hydroxysuccinimide (1 mg / mL) for 30 min. Then, 200 μM APT (4 μM) was added to the reaction mixture and incubated at 37°C for 30 min to obtain double-stranded DNA (dsDNA). To encapsulate rhodamine 6G, the dsDNA solution and the aforementioned mixture of rhodamine 6G and Fe3O4@Uio-66 were stirred at 37°C for 12 h. The prepared product was washed three times with tris to remove unloaded rhodamine 6G and DNA, and then dispersed in 400 μL of tris.

[0039] (5) Construction of ratiometric fluorescence aptasensor: First, 30 μL of double-stranded DNA-functionalized Fe3O4@UiO-66 was taken, and then 10 μL of OTA at different concentrations was added to the solution and incubated at 37°C for 30 min. Subsequently, 0.75 μL of Exo I (20 U / mL) was added to the reaction solution and incubated at 37°C for 60 min. Then, magnetic separation was performed using a magnetic stand, the supernatant was discarded, and the fluorescence spectrum of the precipitated solid was recorded. Fluorescence was excited at 290 nm and recorded in the range of 400-650 nm.

[0040] (6) Establishment of standard curve: 10 μL of OTA standard solution of different concentrations was added to step (5), and after incubation in step (5), the fluorescence intensity signal was detected. A linear fit was performed with the logarithm of OTA concentration as the horizontal axis and the fluorescence intensity signal as the vertical axis to establish the standard curve of the sensor for OTA.

[0041] The specific steps for detecting the fluorescence intensity of the sample are as follows: use a micro-cuvette to hold the sample to be tested, set the excitation wavelength of the fluorescence spectrophotometer to 290 nm, the emission wavelength measurement range to 400-650 nm, the slit to 10 nm, the measurement voltage to 300 V, and record the fluorescence intensity at emission wavelengths of 425 nm and 550 nm.

[0042] like Figure 2Shown are the fluorescence intensities of the sensor constructed in Example 1 of the present invention in the absence of OTA (solid line), the presence of 25 ng / mL OTA (dashed line), and the presence of 500 ng / mL OTA (dotted line).

[0043] like Figure 3 As shown in Figure 2, the signal value of the dye at 550 nm decreases with the increase of OTA concentration in the solution, and the signal of Fe3O4@UiO-66 at 425 nm remains unchanged with the increase of OTA concentration in the solution. The linear regression equation is Y=-0.54 lgC OTA +2.96 (R 2 =0.993), the linear range was 0.5-1000 ng / mL, and the limit of detection (LOD) was 0.308 ng / mL, where Y represents the fluorescence signal value, C OTA It represents the concentration of OTA in the solution (ng / mL). Example 2

[0044] A preparation method and application of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor, the practical application of which includes the following steps:

[0045] To verify that the prepared DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor has specific recognition for OTA, an OTA standard was added to 50 mM Tris-HCl to a concentration of 25 ng / mL in the sample. Other interfering toxins (AFB1, ZEN, OTB, and T2) were also prepared in 50 mM Tris-HCl at a concentration of 250 ng / mL. The detection system constructed in Example 1 was used to detect the above-mentioned interfering toxin standards. The detection results are shown in Figure 1. Figure 4 As shown, it is shown that the method of the present invention has high selectivity for OTA. Example 3

[0046] A preparation method and application of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor, the practical application of which includes the following steps:

[0047] (1) Take 1 g of OTA-spiked corn flour and soak it in 3 mL of methanol / water (7:3) solution for 30 min. Then centrifuge it at 12,000 rpm. Filter the supernatant 2-3 times with a 0.45 μm organic filter membrane to obtain the supernatant. Dilute the filtrate to the required concentration with a methanol / Tris-HCl solution (2:8). Take 1 mL of wine sample. Add 1 mL of OTA standard solution of different concentrations and filter it with a 0.45 μm organic filter membrane. The resulting supernatant containing OTA is diluted with a methanol / Tris-HCl solution (2:8).

[0048] (2) The sample extract was processed according to steps (1) to (5) of Example 1 to obtain the fluorescence intensity signal, which was then inserted into the standard curve to obtain the concentration of OTA in the sample.

[0049] (3) When corn flour was used as the actual sample for determination, 10 times and 100 times the standard amount of OTA standard was added to the corn flour, respectively, with a reference amount of 5 ng / mL. 10 μL of sample solution was taken, and the fluorescence intensity at the emission wavelengths of 425 nm and 550 nm was measured according to steps (1) to (5) of Example 1. The OTA concentration in the sample was obtained by substituting the standard curve detected in Example 1 into the standard curve. Each sample was measured three times and the average value was taken. The average recovery rate of the ratiometric fluorescence sensor was calculated to be 94.8%-98.3%. When wine was used as the actual sample for determination, 10 times and 100 times the standard amount of OTA standard was added to the wine, respectively, with a reference amount of 5 ng / mL. 10 μL of sample solution was taken, and the fluorescence intensity at the emission wavelengths of 425 nm and 550 nm was measured according to steps (1) to (5) of Example 1. The standard curve of Example 1 was used to determine the OTA concentration in the sample. Each sample was measured three times and the average value was taken. The average recovery rate of the ratiometric fluorescence sensor was calculated to be 92.4%-116.0%.

[0050] The prepared ratiometric fluorescent aptasensor has been validated for OTA detection with high accuracy, a wide linear range, and a fast response speed. Furthermore, the results of tests on real samples (such as corn flour and wine) demonstrate the excellent practical application value of the prepared sensor.

[0051] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0052] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims

1. A method for preparing a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor, characterized in that: The following steps are involved: (1) Preparation of magnetic metal organic framework: dissolve ferric chloride hexahydrate in ethylene glycol, then add polyacrylate A, deionized water and urea compound B to obtain magnetic metal Fe3O4; Fe3O4 is treated with an aqueous solution of urea compound B to obtain functionalized Fe3O4; disperse it in an N, N-dimethylformamide solution containing transition metal chloride C and 2-aminoterephthalic acid, heat and stir, and collect the solid with a magnet; repeat the above steps once; then redisperse the collected solid in an N, N-dimethylformamide solution containing transition metal chloride C and 2-aminoterephthalic acid, the solution concentration is consistent with the previous two treatment concentrations, the solution is placed in an autoclave, sealed and heated for a certain time, the sample is collected by a magnet, washed with ethanol, and vacuum dried to obtain a magnetic metal organic framework Fe3O4@Uio-66; (2) Double-stranded DNA functionalized magnetic metal organic framework: Fe3O4@Uio-66 and rhodamine 6G were mixed and reacted for a period of time, thereby loading the dye rhodamine 6G onto Fe3O4@Uio-66; in order to obtain double-stranded DNA functionalized Fe3O4@Uio-66, the aptamer complementary chain cDNA was mixed and reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and the cDNA was 5'-TGT CCG ATG CTC CCTTTA-3', wherein the group labeled at the 5' end was COOH; then, the aptamer APT was added to the reaction mixture, and the mixture was incubated at 37°C in a constant temperature oscillator for a period of time to obtain double-stranded DNA dsDNA; in order to encapsulate rhodamine 6G, the dsDNA solution and the above-mentioned mixed solution of rhodamine 6G and Fe3O4@Uio-66 were incubated at 37°C in a constant temperature oscillator for a period of time according to a certain volume ratio; The prepared product was washed three times with buffer to remove unloaded Rho 6G and double-stranded DNA (dsDNA) and dispersed in the buffer; (3) Construction of ratiometric fluorescent aptamer sensor: First, double-stranded DNA functionalized Fe3O4@Uio-66 was prepared, and then different concentrations of OTA were added to the solution and incubated at 37 °C in a constant temperature oscillator for a period of time; Then, a certain volume of exonuclease Exo I was added to the reaction solution and incubated in a constant temperature shaker at 37°C for a period of time; magnetic separation was performed using a magnetic stand, and the fluorescence spectrum of the precipitated solid was recorded; At this point, a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor was prepared.

2. The preparation method of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor according to claim 1, characterized in that: In step (1), the polyacrylate A is one or more of potassium polyacrylate, sodium polyacrylate, and lithium polyacrylate, the urea compound B is one or both of thiourea and urea, and the transition metal chloride is zirconium tetrachloride.

3. The preparation method of a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor according to claim 1, characterized in that: In step (2), the aptamer APT is 5'-GAT CGG GTG TGG GTG GCGTAA AGG GAG CAT CGG ACA-3'.

4. The method for preparing a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor according to claim 1, characterized in that: In step (2), the amount of the aptamer complementary chain cDNA and the aptamer APT is in the range of 50-400 μL, the concentration range is 1-8 μM, the constant temperature shaking incubation time is 0.5-24 h, and the volume ratio of the double-stranded DNA dsDNA solution and the mixed solution of rhodamine 6G and Fe3O4@Uio-66 is one of 1:1, 1:2, and 1:

3.

5. The method for preparing a DNA-gated magnetic metal-organic framework Fe3O4@Uio-66 ratiometric fluorescence sensor according to claim 1, characterized in that: In step (3), the volume of the exonuclease Exo I is 0.5-1.5 μL, the concentration is 5-40 U / mL, and the constant temperature shaking incubation time is 0.1-1.5 h.

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