Preparation method of pesticide fluorescent aptamer sensor based on ZIF-8 skeleton material

Abstract

A method for preparing a pesticide fluorescent aptamer sensor based on a ZIF-8 skeleton material and its application in the specific and sensitive detection of acetamiprid belong to the field of biosensor technology. The pesticide fluorescent aptamer sensor based on a ZIF-8 skeleton material constructed by the present invention has the advantages of fast response speed, simple operation, low cost, etc., and realizes the selective and highly sensitive detection of acetamiprid. It is mainly based on the electrostatic interaction, hydrogen bond and Zn 2+ The coordination effect adsorbs cDNA-FAM and regulates its fluorescence intensity, improving the stability of the sensor by enhancing the resistance to the influence of biological ligand displacement on cDNA-FAM. By utilizing the highly specific binding of acetamiprid and its aptamer ABA, as well as the complementary effect of ABA and cDNA-FAM chains, the adsorption process of cDNA-FAM on the ZIF-8 surface is regulated, thereby regulating the fluorescence response of the system and improving the detection sensitivity. The present invention provides scientific insights into the biointerface adsorption effect between DNA and ZIF-8, and provides a rapid detection method for the sensitive and specific detection of acetamiprid in food.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a method for preparing a pesticide detection fluorescent aptamer sensor based on a ZIF-8 skeleton material and an application of the sensor in the specific and sensitive detection of acetamiprid. Background Art

[0002] Aptasome-based biosensors have shown great potential for detecting biomarkers and environmental pollutants. Aptasomes are a class of DNA or RNA sequences generated by selective exponential enrichment (SELEX). They are highly specific, easy to synthesize, and modify, selectively binding to their targets with high affinity, making them suitable for a variety of analytical techniques and biosensors. In recent years, a variety of signal transduction aptamer sensors have been developed. Among them, fluorescent aptamer sensors offer advantages such as fast response, small reaction volume, and simple readout, making them ideal biosensors for pesticide detection. Currently, most fluorescent aptamer sensors use absorptive nanomaterials as fluorescence quenchers, including metal nanoparticles, carbon materials, and metal oxide nanosheets. However, nonspecific DNA displacement by other biomolecules makes accurate detection in biological fluids difficult, leading to false-positive results. Therefore, developing interfaces for specific DNA adsorption remains challenging.

[0003] Metal organic frameworks (MOFs) are organic-inorganic hybrid materials with intramolecular pores formed by self-assembly of organic ligands and metal ions or clusters through coordination bonds. They have adsorption properties, optical properties, electromagnetic properties, etc., and have the advantages of stable structure, large specific surface area, and strong fluorescence quenching ability. They can effectively adsorb DNA through covalent or non-covalent interactions, thereby developing biosensors combining aptamers and MOFs. Non-covalent binding does not have strict requirements on the pore size of MOFs, and the fixation process is usually faster and simpler than covalent binding. The present invention uses ZIF-8 as a model framework structure to develop a fluorescent aptamer sensor based on dsDNA / ZIF-8 to detect acetamiprid. By specifically binding to acetamiprid to change the fluorescence, highly sensitive and selective detection of acetamiprid is achieved, which has great application potential in food safety. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a pesticide fluorescent aptamer sensor based on a ZIF-8 skeleton material and its application in the rapid and specific detection of acetamiprid.

[0005] This paper constructs a fluorescent aptasensor for the specific detection of acetamiprid, which exhibits tunable fluorescence behavior and acetamiprid concentration-dependent response. The dsDNA / ZIF-8 aptasensor combines the efficient fluorescence quenching properties of ZIF-8 with DNA-specific adsorption, thereby improving detection stability and optical response. This allows for highly sensitive and selective detection of acetamiprid, providing a promising monitoring method for food safety testing.

[0006] In the present invention, ZIF-8 interacts with Zn through electrostatic interactions, hydrogen bonds and 2+ The complex readily adsorbs 6-carboxyfluorescein-labeled complementary DNA (cDNA-FAM), and through photoaptamer electron transfer (PET), FAM fluorescence intensity decreases. Metal ions enhance cDNA-FAM adsorption by ZIF-8, a phenomenon difficult to achieve with other nanomaterials. This adsorption enhances resistance to displacement of cDNA-FAM by different sequences and biological ligands. The aptamer (ABA), specifically binding to acetamiprid, binds to cDNA-FAM on the ZIF-8 surface, forming weakly adsorbed double-stranded DNA (dsDNA), resulting in a poor PET effect and increased fluorescence intensity. In the presence of acetamiprid, cDNA-FAM is released from the dsDNA and adsorbed by ZIF-8, causing fluorescence quenching again. By observing changes in fluorescence intensity and the strength of the fluorescence, acetamiprid residues can be detected. The dsDNA / ZIF-8-based aptamer sensor exhibits excellent sensitivity and stability for the determination of acetamiprid, with a detection limit of 0.05 ng / mL.

[0007] The preparation of a pesticide fluorescent aptamer sensor based on a ZIF-8 framework material according to the present invention comprises the following steps:

[0008] A. Preparation of ZIF-8

[0009] A methanolic solution of Zn(NO₃)₂·6H₂O and a methanolic solution of 2-methylimidazole (2-MIM) were mixed in a 1:1 volume ratio. The reaction mixture was then allowed to stand at room temperature for 10–15 hours and centrifuged at 8,000–10,000 rpm for 8–15 minutes to collect the precipitate. The precipitate was then washed 3–5 times with methanol to remove unreacted precursors and freeze-dried to obtain ZIF-8 powder. The ZIF-8 solution was then dissolved in methanol and stored at 4°C until ready for use.

[0010] B. Preparation of dsDNA / ZIF-8-based aptamer sensors

[0011] First, PBS buffer (pH 7.4), ABA (10 nM), and cDNA-FAM (10 nM) were mixed in a 2:1:1 volume ratio in a 1.5 mL test tube, mixed thoroughly to obtain Solution A, and maintained at room temperature for 1–1.5 hours. ZIF-8 was then added to Solution A and incubated at room temperature for 15–30 minutes to obtain the test solution; the volume ratio of ZIF-8 to mixed Solution A was 1:4. The test solution was then diluted to 450–500 μL with ultrapure water, and fluorescence was measured on a fluorescence spectrophotometer with an excitation wavelength of 480 nm and emission intensity at 520 nm. The excitation and emission slits were 10.0 nm, respectively. This resulted in an aptamer sensor for detecting acetamiprid.

[0012] C. Create a standard curve of "fluorescence intensity-acetamiprid concentration"

[0013] Acetamiprid standard solution was diluted with ultrapure water to obtain solutions of varying concentrations (0.05 ng / mL to 100 ng / mL). These solutions were then mixed with PBS buffer (pH 7.4) and ABA (10 nM) in a volume ratio of 1:2:1 and incubated at room temperature for 1 to 1.5 hours to obtain Solution B. Next, cDNA-FAM (10 nM) was added, mixed, and incubated for 1 to 1.5 hours. Finally, ZIF-8 was added and incubated at room temperature for 15 to 30 minutes to obtain the test solution. The volume ratio of cDNA-FAM, ZIF-8, and Solution B was 1:1:4. After incubation, the solution was further diluted with ultrapure water to 450 to 500 μL. Fluorescence was measured on a fluorescence spectrophotometer with an excitation wavelength of 480 nm and emission intensity at 520 nm. Fluorescence spectra were recorded with excitation and emission slits of 10.0 nm, respectively. A standard relationship curve was generated.

[0014] D. Detection of acetamiprid in actual spiked samples

[0015] Actual samples such as tap water, Songhua River water, and apples were processed as research targets. 5-15 mL of water sample was filtered through a 0.22 mm membrane, and apples were ground and filtered. Then, 0.5-1 g of sample was mixed evenly with a standard solution of acetamiprid (0.5, 5, 25, and 50 ng / mL). Acetonitrile (mL) and NaCl (g) were added in a ratio of 1 g: 10 mL: 3 g, and the mixture was mixed evenly again and incubated for 20-40 min to obtain the sample to be tested. Finally, these products were collected and repeated in step C. The obtained data was substituted into the standard relationship curve obtained in step C to calculate the concentration of acetamiprid in the actual sample solution, which was then compared with the spiked amount to verify the feasibility of the device of the present invention.

[0016] The mechanism of the present invention is as follows:

[0017] Zn was added by coprecipitation 2+ and 2-MIM in methanol to obtain ZIF-8 with a hexagonal shape. ZIF-8 is formed by electrostatic interactions, hydrogen bonds and Zn 2+ The coordination effect adsorbs the fluorescent indicator cDNA-FAM and quenches its fluorescence. 2+ The four 2-MIM ligands form a framework, and the metal ions on its surface are not yet fully coordinated. Therefore, the adsorption capacity of ZIF-8 is also related to the unsaturated Zn 2+ In addition, the free nucleoside of DNA failed to trigger DNA desorption, while phosphate could moderately desorb cDNA-FAM. The phosphate group of cDNA-FAM desorbed the incompletely coordinated Zn in ZIF-8 through the Zn-OP bond. 2+ It has high affinity and enhances the adsorption of ZIF-8 to cDNA-FAM. The fluorescence quenching mechanism of MOFs for cDNA-FAM is mainly based on fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) effects. Moreover, as the temperature increases, the quenching effect is significantly enhanced, that is, there is a dynamic quenching mechanism. And the Zn in the MOFs framework structure 2+ It can be inserted into the base pair of cDNA-FAM, electrostatically bind to the phosphate group, and trigger the PET process from FAM to ZIF-8.

[0018] The aptamer sensor based on dsDNA / ZIF-8 used in the present invention exhibits better detection performance than the ABA-FAM / ZIF-8 sensor. Due to the high specificity of DNA and the adsorption and fluorescence quenching ability of ZIF-8, the fluorescence emission intensity of the aptamer sensor decreases with the increase of acetamiprid concentration, and has better biostability, showing a good linear relationship in the range of 0.05 ng / mL to 100 ng / mL (such as Figure 4 The limit of detection (LOD) was 0.05 ng / mL, which is much lower than the maximum residue limit (500 ng / mL) set by the Chinese National Food Safety Standard (GBT 2763-2021). This indicates that the proposed aptasensor can meet the detection requirements and is expected to be practically applied in the detection of acetamiprid.

[0019] Compared with existing methods, the dsDNA / ZIF-8 fluorescent aptamer sensor proposed in the present invention has the following characteristics:

[0020] (1) ZIF-8, as a quencher of fluorescent aptamer sensor, has the advantages of fast response, high quenching efficiency, and simple operation, and can be used for sensitive detection of acetamiprid.

[0021] (2) DNA adsorption on ZIF-8 is achieved through electrostatic interactions, hydrogen bonds and Zn2+ Coordination occurs, which helps to resist the displacement of cDNA-FAM by biological ligands, has high stability and specificity in detecting acetamiprid, and has a certain anti-interference ability in complex environments.

[0022] (3) ZIF-8 acts as an “ion pump” and contains a large amount of Zn 2+ , promoting cDNA-FAM adsorption and triggering the PET effect, improving detection efficiency and sensitivity, with a detection limit of 0.05 ng / mL, showing broad application prospects in the field of pesticide biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 (A) shows the synthesis process of ZIF-8 described in Example 1; (B) shows a photograph of ZIF-8 under natural light; (C) shows a scanning electron microscopy image of ZIF-8 prepared in Example 1; (D) shows a transmission electron microscopy image of ZIF-8 prepared in Example 1; (E) shows a Fourier transform infrared spectrum of ZIF-8 prepared in Example 1. (F) shows the fluorescence quenching spectrum of cDNA-FAM by ZIF-8 described in Example 2; (G) shows the fluorescence intensity of cDNA-FAM after centrifugation of cDNA-FAM / ZIF-8 in Example 2.

[0024] Figure 2 (A) is the Zeta potential of ZIF-8, cDNA-FAM and cDNA-FAM / ZIF-8 described in Example 2; (B) is the fluorescence absorption of Example 2 after adding urea (a) and DMSO (b) to the system; (C) is the desorption degree after adding EDTA to the system in Example 2; (D) is the fluorescence absorption of Example 2 after adding free nucleosides (a) and phosphates (b) to the system; (E) is a schematic diagram of the adsorption behavior of cDNA-FAM on the ZIF-8 surface described in Example 2.

[0025] Figure 3 (A) is the fluorescence intensity of the system in Example 3 by adding different DNA chain sequences into the system; (B) is the circular dichroism spectrum of dsDNA before and after the aptamer sensor based on dsDNA / ZIF-8 recognizes acetamiprid in Example 3; (C) is the feasibility of the aptamer sensor based on dsDNA / ZIF-8 in Example 3; (D) is the fluorescence intensity of the system in Example 3 using the aptamer sensor based on dsDNA / ZIF-8 at different concentrations of ABA (0-100nM).

[0026] Figure 4(A) is a schematic diagram of the use of the ABA-FAM / ZIF-8 aptamer sensor in Example 3 to detect the fluorescence of acetamiprid; (B) is the response of the ABA-FAM / ZIF-8 aptamer sensor described in Example 3 to different concentrations of acetamiprid; (C) is a schematic diagram of the use of the dsDNA / ZIF-8-based aptamer sensor in Example 3 to detect the fluorescence of acetamiprid; (D) is the response of the dsDNA / ZIF-8-based aptamer sensor in Example 3 to different concentrations of acetamiprid; (E) is the fluorescence intensity of the dsDNA / ZIF-8-based aptamer sensor system in Example 4 to different concentrations of acetamiprid (0, 0.05, 0.5, 5, 25, 50, 100 ng / mL); (F) is a linear regression relationship diagram of the detection of different concentrations of acetamiprid by the dsDNA / ZIF-8-based aptamer sensor in Example 4.

[0027] Figure 5 (A) is the selective anti-interference ability of the dsDNA / ZIF-8 aptamer sensor based on Example 5 for common pesticides and interfering substances (excluding acetamiprid); (B) is the selective anti-interference ability of the dsDNA / ZIF-8 aptamer sensor based on Example 5 for common pesticides and interfering substances (containing acetamiprid). DETAILED DESCRIPTION

[0028] Example 1: Synthesis and Characterization of ZIF-8

[0029] ZIF-8 was synthesized by coprecipitation method (such as Figure 1 A). Zn(NO3)2·6H2O methanol solution (25mM) and 2-methylimidazole methanol solution (2-MIM) (25mM) were mixed in a volume ratio of 1:1. The reaction mixture was allowed to stand at room temperature for 15h to obtain a milky white suspension (such as Figure 1 B). The mixture was then centrifuged at 8000 rpm for 10 min to collect the precipitate. The supernatant was removed, and the precipitate was washed three times with methanol to remove unreacted precursor. Finally, the precipitate was freeze-dried to obtain ZIF-8.

[0030] The freeze-dried precipitate was characterized and analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 As shown in Figures C and D, the synthesized ZIF-8 has a hexagonal shape, is uniformly distributed, and has a size of approximately 900 nm.

[0031] Example 2: Study on the adsorption mechanism of ZIF-8 and cDNA-FAM

[0032] ZIF-8 was synthesized by the operation of Example 1 to explore the adsorption effect between ZIF-8 and cDNA-FAM. First, PBS buffer (pH = 7.4), ABA (10nM) and cDNA-FAM (10nM) were mixed in a volume ratio of 2:1:1 and added to a 1.5mL test tube to obtain a mixed solution A, which was kept at room temperature for 1h. Then, the mixed solution A was mixed with the ZIF-8 solution in a volume ratio of 4:1, incubated at room temperature for 20min, and then diluted to 500μL with ultrapure water to measure fluorescence. Equal amounts of DNA denaturant and surfactant (urea, dimethyl sulfoxide, etc.) were added to the above system to further explore the adsorption force. Finally, different concentrations of ethylenediaminetetraacetic acid (EDTA) were added to the system to explore the effect of ions on adsorption.

[0033] The results are as follows Figure 1 As shown in F and G, after ZIF-8 was added to the cDNA-FAM system, obvious fluorescence quenching was observed under the excitation of 490 nm (dark area of the pattern) ( Figure 1 F), and the fluorescence intensity in the supernatant was weak ( Figure 1 G line d), which reduces the initial strength by almost 100% ( Figure 1 G line a), the fluorescence of cDNA-FAM / ZIF-8 (precipitate) also strongly decayed ( Figure 1 G line e), indicating that ZIF-8 can adsorb cDNA-FAM. In order to further study the adsorption mode of ZIF-8 on cDNA-FAM, the Zeta potential of the system was tested, such as Figure 2 As shown in A, when pH is 7.4, ZIF-8 is +48.76mV, while cDNA-FAM / ZIF-8 is +20.93mV, indicating that ZIF-8 and cDNA-FAM have electrostatic interactions. Urea can destroy hydrogen bonds, and DMSO can dissolve hydrophobic molecules. When urea is introduced, the fluorescence intensity of the system increases by 33.42% ( Figure 2 B, a), no fluorescence intensity enhancement was observed after the introduction of DMSO (fluorescence change was less than 10%) ( Figure 2 B, b), the results show that hydrogen bonding is more conducive to the adsorption of cDNA-FAM by ZIF-8 than hydrophobic interaction. Ethylenediaminetetraacetic acid (EDTA) was further used to replace cDNA-FAM in a concentration-dependent manner. The results showed that in the presence of 3.0 mmol / L EDTA, the desorption rate exceeded 40.9% ( Figure 2 C), confirmed that Zn 2+ The coordination effect also facilitates the adsorption of cDNA-FAM on the ZIF-8 surface. Finally, the binding affinity of cDNA-FAM to ZIF-8 was studied using free nucleosides and phosphates. Figure 2As shown in D, free nucleosides (adenosine, cytidine, guanosine, and thymidine) failed to trigger DNA desorption ( Figure 2 D, a), while 0.1 mmol / L phosphate began to moderately desorb cDNA-FAM ( Figure 2 D, b), indicating that the phosphate group of cDNA-FAM binds to the incompletely coordinated Zn in ZIF-8 through the Zn-OP bond. 2+ It has high affinity, thus enhancing the adsorption of ZIF-8 to cDNA-FAM. In summary, ZIF-8 can enhance the adsorption of cDNA-FAM through electrostatic interactions, hydrogen bonds and Zn 2+ Coordinates to effectively adsorb the DNA phosphate backbone ( Figure 2 E).

[0034] Example 3: Preparation and performance evaluation of dsDNA / ZIF-8 based aptamer sensor

[0035] ZIF-8 can adsorb cDNA-FAM, allowing the synthesis of aptamer sensors through simple mixing and incubation. The procedure was the same as in Example 2. First, PBS buffer (pH = 7.4), ABA (10 nM), and cDNA-FAM (10 nM) were mixed in a 2:1:1 volume ratio in a 1.5 mL test tube to obtain mixed solution A. The mixture was then maintained at room temperature for 1 hour. Next, mixed solution A was mixed with ZIF-8 solution in a 4:1 volume ratio, incubated at room temperature for 20 minutes, and diluted to 500 μL with ultrapure water. Fluorescence was then measured.

[0036] The results are as follows Figure 3 As shown: In order to verify the specificity of acetamiprid detection, four DNA chains with different sequences were selected as complementary chains to interact with cDNA-FAM. Except for ABA, other chains could not fully restore the cDNA-FAM fluorescence, indicating that the aptamer sensor based on dsDNA / ZIF-8 has high selectivity for the complementary chain ABA ( Figure 3 A). Circular dichroism (CD) spectroscopy shows that dsDNA has two unique peaks, a positive peak at about 275 nm and a negative peak at about 246 nm ( Figure 3 B), when acetamiprid was added, both peaks decreased, indicating that dsDNA was desorbed from the ZIF-8 surface. In the absence of acetamiprid, the double-stranded dsDNA formed by ABA and cDNA-FAM was desorbed from the ZIF-8 surface, thereby blocking the PET effect between FAM and ZIF-8, resulting in enhanced fluorescence; when acetamiprid was present, it specifically bound to ABA to form an acetamiprid / ABA complex, bringing cDNA-FAM closer to the ZIF-8 surface, causing fluorescence recovery ( Figure 3 C), and the fluorescence intensity increased with the increase of ABA concentration (0-100 nmol / L) ( Figure 3D), indicating that ABA can be developed in a dsDNA / ZIF-8-based aptasensor for acetamiprid detection.

[0037] Example 4: Pesticide fluorescent aptamer sensor based on ZIF-8 framework material for acetamiprid detection

[0038] Acetamiprid standard solution was diluted with ultrapure water to obtain solutions of varying concentrations (0.05 ng / mL to 100 ng / mL). The solution was then mixed with PBS buffer (pH 7.4) and ABA (10 nM) in a volume ratio of 1:2:1 and incubated at room temperature for 1 hour to obtain Solution B. Next, cDNA-FAM (10 nM) was added and incubated for 1 hour. Finally, ZIF-8 was added and incubated at room temperature for 20 minutes to obtain the test solution. The volume ratio of cDNA-FAM, ZIF-8, and Solution B was 1:1:4. The solution was further diluted to 500 μL with ultrapure water, and fluorescence was measured and the fluorescence spectrum recorded.

[0039] The results are as follows Figure 4 As shown in Figures E and 4F, the fluorescence intensity of the aptamer sensor decreased dynamically with the increase of acetamiprid concentration in the range of 0.05 to 100 ng / mL ( Figure 4 E). The linear regression equation is F0-F / F0=0.054log[acetamiprid]+0.107, and the correlation coefficient (R 2 ) is 0.992( Figure 4 The detection limit (LOD) of acetamiprid was 0.05 ng / mL, which is much lower than the maximum residue limit (500 ng / mL) set by the Chinese National Food Safety Standard (GBT 2763-2021), indicating that the proposed aptasensor can meet the detection requirements.

[0040] To further evaluate the specificity and safety of the aptasensor, interfering pesticides such as tebuconazole, thiamethoxam, cyhalothrin, fipronil, imidacloprid, permethrin, methyl paraoxon, carbaryl, and cypermethrin, as well as tryptophan, threonine, glutamic acid, arginine, cysteine, vitamin C, ovalbumin (OVA), K + , Na + , Ca 2+ and Fe 3+ The results are as follows Figure 5 As shown in Figures A and 5B, when the concentration of other pesticides and interfering substances (500 ng / mL) is five times higher than that of acetamiprid (100 ng / mL), the fluorescence intensity of the system remains almost unchanged, indicating that the system has good selectivity and anti-interference ability for acetamiprid. Therefore, the dsDNA / ZIF-8 fluorescent aptasensor with excellent selectivity can be used for practical acetamiprid detection applications.

[0041] Example 5: dsDNA / ZIF-8 fluorescent aptamer sensor for the detection of acetamiprid in actual samples

[0042] To evaluate the practical usability and reliability of the dsDNA / ZIF-8 aptasensor, recovery tests were conducted by spiking tap water, Songhua River water, and apple samples with different concentrations of acetamiprid standard solutions. 10 mL of water sample was filtered through a 0.22 mm membrane, and apples were ground and filtered. Afterward, 0.5 g of sample was mixed with acetamiprid standard solutions (0.5, 5, 25, and 50 ng / mL). This mixture was then added to acetonitrile (mL) and NaCl (g) in a ratio of 1 g:10 mL:3 g and incubated for 30 minutes to obtain the test samples. These products were then collected for further experiments.

[0043] The results are shown in Table 1. The recoveries ranged from 98.94% to 105.81%, and the relative standard deviation (RSD) was less than 6.18%, indicating that the method has good reliability for the detection of acetamiprid in environmental and food samples.

[0044] Table 1 Determination of acetamiprid in spiked samples using dsDNA / ZIF-8 aptamer sensor (n=3).

[0045]

Claims

1. A method for preparing a pesticide fluorescent aptamer sensor based on a ZIF-8 framework material, comprising the following steps: A. Preparation of ZIF-8: Mix Zn(NO3)2‧6H2O methanol solution and 2-methylimidazole 2-MIM methanol solution in a volume ratio of 1:1, then let the reaction mixture stand at room temperature for 10-15 hours, centrifuge at 8000-10000 rpm for 8-15 minutes, collect the precipitate, and then wash the precipitate 3-5 times with methanol to remove unreacted precursors. Freeze-dry to obtain ZIF-8 powder, dissolve it in methanol to prepare ZIF-8 solution, and store it at 4°C for future use. B. Preparation of dsDNA / ZIF-8-based aptamer sensor: First, PBS buffer (pH = 7.4), 10 nM ABA, and 10 nM cDNA-FAM were mixed in a volume ratio of 2:1:1 and added to a 1.5 mL test tube. The mixture was uniformly mixed to obtain solution A, and the mixture was maintained at room temperature for 1-1.5 h. Then, ZIF-8 was added to solution A, and the mixture was incubated at room temperature for 15-30 min to obtain the test solution. The volume ratio of ZIF-8 to mixed solution A was 1:

4. The test solution was then diluted with ultrapure water to 450-500 μL. The fluorescence was measured on a fluorescence spectrophotometer with an excitation wavelength of 480 nm and the emission intensity at 520 nm. The excitation and emission slits were 10.0 nm, respectively. This resulted in an aptamer sensor for detecting acetamiprid.

2. The method for preparing a pesticide fluorescent aptamer sensor based on a ZIF-8 framework material according to claim 1, wherein: In step B, ZIF-8 prepared in step A is used to synthesize Zn 2+ The fluorescently labeled complementary DNA, i.e. cDNA-FAM, is coordinated and adsorbed, and the FAM fluorescence intensity is reduced through electron transfer PET by the photoaptamer. The highly specific binding of acetamiprid to its specific binding aptamer ABA, as well as the complementary effect of ABA and cDNA-FAM chains, are used to regulate the adsorption process of cDNA-FAM on the ZIF-8 surface in the system, thereby regulating the fluorescence intensity of the system. The sensor stability is improved by enhancing the resistance to the influence of biological ligand displacement on cDNA-FAM. Based on the adjustable fluorescence behavior and acetamiprid concentration response dependence, a fluorescent aptamer biosensor with high specificity and detection stability is constructed to detect the pesticide acetamiprid.

3. A pesticide fluorescent aptamer sensor based on a ZIF-8 framework material, characterized by: The method according to claim 1 or 2 is used to prepare the present invention.

4. The pesticide fluorescent aptamer sensor based on the ZIF-8 framework material according to claim 3, characterized in that: The acetamiprid standard solution was diluted with ultrapure water to obtain acetamiprid solutions of different concentrations ranging from 0.05 ng / mL to 100 ng / mL. The solutions were then mixed with PBS buffer (pH = 7.4) and 10 nM ABA at a volume ratio of 1:2:1 to allow for sufficient interaction between acetamiprid and the aptamer. The solution was then incubated at room temperature for 1–1.5 h to obtain a mixed solution. 10 nM cDNA-FAM was then added to the mixed solution, mixed evenly, and incubated for 1–1.5 h. Finally, ZIF-8 was added, mixed evenly, and incubated at room temperature for 15–30 min to obtain a reaction solution. The volume ratio of cDNA-FAM, ZIF-8, and the mixed solution was 1:1:

4. The reaction solution was further diluted to 500 μL with ultrapure water. Fluorescence was measured on a fluorescence spectrophotometer at an excitation wavelength of 480 nm and a recording time of 520 nm. The emission intensity at nm was measured to obtain fluorescence intensity curves corresponding to different concentrations of acetamiprid. The acetamiprid concentration standard relationship curve was obtained by software analysis. Then, three actual samples, tap water, Songhua River water, and apple, were processed. 5-15 mL of water sample was filtered through a 0.22 mm membrane, and the apple was ground and filtered. After filtration, 0.5-1 g of sample was mixed evenly with acetamiprid standard solutions of different concentrations of 0.5, 5, 25, and 50 ng / mL. The mixed solution was then mixed with acetonitrile (mL) and NaCl (g) in a ratio of 1 g:10 mL:3 g and incubated for 20-40 min to obtain the reaction product. Finally, the treated product was collected and the fluorescence image of the solution with unknown acetamiprid concentration was acquired using the above experimental operation to obtain the corresponding data. The corresponding data was substituted into the acetamiprid concentration standard relationship curve to calculate the acetamiprid concentration in the actual sample solution.

5. The pesticide fluorescent aptamer sensor based on the ZIF-8 framework material according to claim 4, characterized in that: Used for rapid and sensitive detection of acetamiprid pesticide residue levels in real food samples with high specificity and stability.

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