A Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe and its preparation method and application

Abstract

The present invention relates to the technical field of pesticide residue detection, and in particular to a Cu‑BTC@Ag NPs@TPE‑(COOH)4@DNA aptamer fluorescent probe, and a preparation method and application thereof. In this study, Cu‑trimethylene tricarboxylate MOF-loaded silver nanoparticles were synthesized, and tetracarboxyl tetraphenylethylene with aggregation-inducing fluorescent groups was grafted to obtain a fluorescent material. Then, the DNA aptamer of acetamiprid was used as a recognition element to construct an aptamer probe. It has excellent fluorescence luminescence, and the probe can capture acetamiprid in the sample through the DNA aptamer, resulting in a significant reduction in the fluorescence signal of the aptamer, thereby establishing a fluorescence method for detecting acetamiprid. Since Cu‑BTC@Ag NPs can effectively amplify the fluorescence intensity of TPE‑(COOH)4, the detection limit is 1.16×10 ‑ 12 mol / L.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, and in particular to a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe and a preparation method and application thereof. Background Art

[0002] Acetamiprid is a chloronicotinoid insecticide with the characteristics of broad insecticidal spectrum, high activity, low dosage and long lasting effect, mainly acting as contact and stomach poison. However, as acetamiprid residues accumulate in agricultural products in the natural environment, it poses a potential risk to human health. Therefore, it is necessary to monitor acetamiprid residues in agricultural products and the environment. At present, the detection methods of acetamiprid include liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, electrochemical method, etc., but these methods have the disadvantages of cumbersome and time-consuming pretreatment, high cost, inaccurate results and poor stability. In comparison, fluorescence analysis has become an important direction of current related research due to its advantages of high sensitivity, low cost and simple operation.

[0003] Fluorescence detection technology achieves qualitative and quantitative analysis of the target by stimulating the fluorescent probe or marker to emit a fluorescent signal after interacting with the target. Detection sensitivity and the ability to select and identify target molecules are the core performance of fluorescence analysis technology. These two performances depend to a large extent on the properties of the fluorescent probe itself, such as luminescence intensity, photochemical stability, and whether there are recognition groups or loaded recognition elements. There are many types of fluorescent probe materials at present. According to their luminescence mechanism and properties, they can be divided into organic fluorescent materials, inorganic fluorescent materials and quantum dot fluorescent materials.

[0004] Organic fluorescent materials are materials composed of organic molecules containing aromatic ring structures. Common organic fluorescent materials include organic dyes and organic light-emitting polymers. Organic fluorescent materials have high luminous efficiency and good solubility, but have low photostability and thermal stability. They are easily affected by light and thermal decomposition, resulting in reduced luminous efficiency and shortened lifespan, which affects their application. Representatives of inorganic fluorescent materials are rare earth ion luminescence and rare earth fluorescent materials. Their advantages are strong absorption capacity, high conversion rate, narrow-band emission of the central ion of the rare earth complex, which is conducive to full-color display, and stable physical and chemical properties. However, their biocompatibility is low and the preparation cost is high. Fluorescent quantum dots are a type of nano-sized luminescent particles (such as CdTe, C-dots). This quantum dot can lock electrons in a very small three-dimensional space. When a beam of light is irradiated, the electrons will be excited and jump to a higher energy level. When these electrons return to their original lower energy level, they will emit a beam of light with a certain wavelength. Compared with traditional organic dye molecules and fluorescent proteins, quantum dots have unique optical and electronic properties, such as adjustable emission wavelength, high brightness, resistance to photobleaching, and simultaneous excitation of multiple quantum dots of different colors of fluorescence. However, their luminescence efficiency is often affected by problems such as surface defects of quantum dots, and there are toxicity issues.

[0005] Therefore, in order to obtain fluorescent probes with better performance and thus improve the sensitivity of fluorescence analysis technology, the preparation of composite fluorescent materials would be an effective way. For example, metal-based nanomaterials or new materials can be introduced to achieve the amplification of fluorescence signals. Among them, metal-organic frameworks (MOFs) have the advantages of high adjustability, high sensitivity, reversibility and reusability in signal amplification, providing new ideas and methods for the development of fluorescence amplification technology. For example, although most MOFs do not have fluorescent groups, the fluorescence properties of the fluorescent reagents loaded on them can be regulated by rationally selecting and designing ligands, metal ions and structural topology; and MOFs have a large number of pores and surface areas, which provide good conditions for the adsorption of fluorescent molecules, thereby improving the sensitivity of fluorescence signals. However, as a single material, the electronic conduction ability and stability of MOFs need to be further improved in practical applications. In order to improve the performance of MOFs, many conductive materials (such as metal nanomaterials) can be combined with MOFs, and the synergistic effect between the two components can be used to strengthen their respective advantages and offset their disadvantages, so as to achieve effective amplification of fluorescent reagents.

[0006] In order to improve the ability of fluorescent probes to select and identify target molecules, introducing molecular recognition elements is an effective way. Among them, aptamers and molecular imprinting membranes have attracted much attention due to their strong specific recognition ability for target molecules. Aptamers are a small oligonucleotide sequence or short polypeptide obtained by in vitro screening, which can bind to the corresponding ligand with high affinity and strong specificity. Molecular imprinting membranes refer to polymers that completely match a certain molecule in terms of spatial structure and binding sites. They can all bind specifically to target molecules. Compared with the lack of rigidity of molecular imprinting membranes and the easy collapse of imprinting holes that affect recognition performance, aptamers have the advantages of higher chemical stability, flexible structure, low cost, easy synthesis and easy labeling, so they are more widely used. However. At present, there are no reports on the introduction of MOFs loaded with metal nanocomposites to amplify the signal of fluorescent reagents and the use of DNA aptamers as fluorescent probes for the detection of neonicotinoid pesticide residues. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe and its preparation method and application. In the present invention, tetracarboxyl tetraphenylethylene with aggregation-inducing fluorescent groups is loaded on the surface of Cu-trimethylene tricarboxylic acid MOF (Cu-BTC) and silver nanoparticles (Ag NPs) to achieve signal amplification of tetracarboxyl tetraphenylethylene fluorescent reagent; at the same time, the DNA aptamer of acetamiprid is introduced as a molecular specific recognition element to construct a new type of highly sensitive and highly selective fluorescent probe to achieve the detection of trace acetamiprid in agricultural products ( Figure 1 ). First, Cu-BTC@AgNPs composite nanomaterials were synthesized by high temperature method, and then tetracarboxyl tetraphenylethylene (TPE-(COOH)4) was grafted, and a cross-linker was used to combine with DNA aptamers to obtain a fluorescent probe with high luminescence efficiency. Through the DNA aptamer, the probe can selectively capture acetamiprid in the sample and quench the fluorescence signal of the probe, thereby establishing a new method for detecting acetamiprid. Cu-BTC@Ag NPs amplifies the fluorescence signal of TPE-(COOH)4, making this method highly sensitive; at the same time, the specific recognition ability of the DNA aptamer makes the probe highly selective.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe, comprising the following steps:

[0010] 1) mixing copper chloride, trimesic acid and a solvent and reacting them, centrifuging, washing and drying the obtained reactant to obtain Cu-BTC;

[0011] 2) mixing the Cu-BTC obtained in step 1) with water, silver nitrate solution, sodium citrate solution and sodium hydroxide solution and stirring the mixture for reaction, filtering and drying the stirred reaction mixture to obtain Cu-BTC@AgNPs;

[0012] 3) The Cu-BTC@AgNPs obtained in step 2) are mixed with a buffer solution, tetraphenylethylene-coupled tetraphenylimidazole, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and a DNA aptamer solution of acetamiprid, and then dissolved to obtain a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe;

[0013] The nucleotide sequence of the acetamiprid DNA aptamer is shown in SEQ ID No.1.

[0014] Preferably, in step 1), the volume ratio of the mass of copper chloride to the mass of trimesic acid to the volume ratio of the solvent is 0.81 g:2 g:200 ml;

[0015] The solvent includes double distilled water and ethylene glycol, and the volume ratio of the double distilled water to the ethylene glycol is 3:1.

[0016] Preferably, the reaction conditions of step 1) include: temperature of 280° C. and reaction time of 3 h;

[0017] The mixing is carried out under stirring, and the stirring time is 1.5h;

[0018] The centrifugal conditions include: centrifugation at 10000 rpm for 5 min;

[0019] The reagent used for the washing is anhydrous ethanol, and the number of washing times is 2 times;

[0020] The drying temperature is 60°C.

[0021] Preferably, in step 2), the volume ratio of the mass of Cu-BTC to the volume of water, the volume of silver nitrate solution, the volume of sodium citrate solution, and the volume of sodium hydroxide solution is 0.1 g: 50 mL: 50 μL: 250 μL: 3 mL;

[0022] The concentration of the silver nitrate solution is 0.1 mol / L;

[0023] The concentration of the sodium citrate solution is 0.1 mol / L;

[0024] The concentration of the sodium hydroxide solution is 25 mmol / L.

[0025] Preferably, the stirring reaction conditions in step 2) include: temperature of 50° C. and time of 30 min;

[0026] The pore size of the filter membrane used in the filtration is 0.22 μm;

[0027] The drying temperature is 60°C.

[0028] Preferably, in step 3), the volume ratio of the mass of Cu-BTC@AgNPs to the volume of the buffer, the mass of tetraphenylethylene-coupled tetraphenylimidazole, the mass of N-hydroxysuccinimide, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the volume ratio of the DNA aptamer solution of acetamiprid is 200 mg: 5 mL: 1 mg: 5 mg: 5 mg: 200 μL;

[0029] The concentration of the acetamiprid DNA aptamer solution is 1 mg / mL.

[0030] Preferably, the buffer in step 3) is PBS buffer with a concentration of 0.01 mol / L and a pH value of 7.4;

[0031] The dissolution reaction is carried out under stirring, and the dissolution reaction time is 30 minutes.

[0032] The present invention also provides a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe prepared by the preparation method described in the above technical solution.

[0033] The present invention also provides the use of the Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe described in the above technical solution in the detection of acetamiprid.

[0034] Preferably, the application comprises the following steps:

[0035] A. Mixing the sample with the Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe and PBS buffer to react and obtain a reactant;

[0036] The pH value of the PBS buffer is 7.4, and the reaction time is 8 min;

[0037] B. The fluorescence intensity of the reactant detected is calculated as F1, and the blank control is calculated as F0. Substitute it into the following formula to calculate the concentration of acetamiprid:

[0038] △IF=F0-F1;

[0039] △IF=0.98c+56.94, the unit of c is 10 -11 mol / L.

[0040] The beneficial effects of the present invention are:

[0041] The present invention synthesizes Cu-trimethylene tricarboxylate MOF (Cu-BTC) loaded silver nanoparticles, and grafts tetracarboxyl tetraphenylethylene (TPE-(COOH)4) with aggregation-induced fluorescent groups to obtain Cu-BTC@Ag NPs@TPE-(COOH)4 fluorescent material. Then, a DNA aptamer of acetamiprid is used as a recognition element to construct a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe. Cu-BTC@Ag NPs@TPE-(COOH)4 has excellent fluorescence luminescence, and the probe can capture acetamiprid in the sample through the DNA aptamer, resulting in a significant reduction in the fluorescence signal of the Cu-BTC@AgNPs@TPE-(COOH)4 aptamer, thereby establishing a fluorescence method for detecting acetamiprid. Since Cu-BTC@Ag NPs can effectively amplify the fluorescence intensity of TPE-(COOH)4, this method has high sensitivity; at the same time, the DNA aptamer can specifically recognize acetamiprid, thereby effectively eliminating the interference of other pesticides with similar structures and having high selectivity. The linear range of the method is 5~5200×10 -11 mol / L, the detection limit is 1.16×10 -12 mol / L. This method was applied to the detection of actual samples such as banana and cowpea, and the spiked recovery rate was 92.5-103.9%. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0043] Figure 1 A process diagram of a fluorescent probe for detecting acetamiprid;

[0044] Figure 2 (A) SEM image of Cu-BTC@Ag NPs; (B) SEM image of Ag NPs; (C) XRD spectrum of Cu-BTC@AgNPs; (D) XPS spectrum of Cu-BTC@AgNPs;

[0045] Figure 3 Optical spectra and fluorescence spectra of the probe: a. UV-visible absorption spectrum of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe; b. Excitation spectrum of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe; c. Emission spectrum of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe; d. Emission spectrum of TPE-(COOH)4;

[0046] Figure 4 is the fluorescence quenching response of different concentrations of acetamiprid to the probe: a.50×10 -11 mol / L; b.100×10 - 11 mol / L; c.500×10 -11 mol / L; d.2500×10 -11 mol / L;

[0047] Figure 5 is the effect of pH value on the fluorescence intensity of the probe. The concentration of acetamiprid is 1×10 -8 mol / L, n=5;

[0048] Figure 6 is the effect of reaction time on the fluorescence intensity of the probe. The concentration of acetamiprid is 1×10 -8 mol / L, n=5;

[0049] Figure 7 (A) Fluorescence spectra of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe under different concentrations of acetamiprid: (am): (0, 5, 10, 50, 100, 250, 400, 500, 1000, 2500, 3500, 4500, 5200)×10 -11 mol / L; (B) calibration curve;

[0050] Figure 8 To explain the interference of different pesticides on the probe detection signal, the concentration of acetamiprid was 5×10 -10 mol / L, the concentration of other pesticides is 5×10 -8 mol / L, n=5;

[0051] Fig. 9 The five probes were respectively -10 mol / L fluorescence quenching value after acetamiprid reaction, n = 5;

[0052] Fig.10 is the stability of the fluorescence signal of the probe within 1 h, and the concentration of acetamiprid is 5×10 -10 mol / L, n=5. DETAILED DESCRIPTION

[0053] The present invention provides a method for preparing a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe, comprising the following steps:

[0054] 1) mixing copper chloride, trimesic acid and a solvent and reacting them, centrifuging, washing and drying the obtained reactant to obtain Cu-BTC;

[0055] 2) mixing the Cu-BTC obtained in step 1) with water, silver nitrate solution, sodium citrate solution and sodium hydroxide solution and stirring the mixture for reaction, filtering and drying the stirred reaction mixture to obtain Cu-BTC@AgNPs;

[0056] 3) The Cu-BTC@AgNPs obtained in step 2) are mixed with a buffer solution, tetraphenylethylene-coupled tetraphenylimidazole, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and a DNA aptamer solution of acetamiprid, and then dissolved to obtain a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe;

[0057] The nucleotide sequence of the acetamiprid DNA aptamer is shown in SEQ ID No.1.

[0058] The invention comprises the following steps: mixing cupric chloride, trimesic acid and a solvent, and reacting the mixture; centrifuging, washing and drying the obtained reactant to obtain Cu-BTC.

[0059] In the present invention, the mass of the cupric chloride, the mass of trimesic acid, and the volume ratio of the solvent are preferably 0.81g:2g:200ml. In the present invention, the solvent preferably includes deionized water and ethylene glycol, and the volume ratio of deionized water to ethylene glycol is preferably 3:1. In the present invention, the reaction conditions preferably include: temperature of 280°C and time of 3h. In the present invention, the mixing is preferably carried out under stirring, and the stirring time is preferably 1.5h. In the present invention, the centrifugal conditions preferably include: centrifugation at 10000rpm for 5min. In the present invention, the reagent used for washing is preferably anhydrous ethanol, and the number of washings is preferably 2 times; in the present invention, the drying temperature is preferably 60°C and the time is 3h, preferably vacuum drying.

[0060] The method comprises mixing the obtained Cu-BTC with water, a silver nitrate solution, a sodium citrate solution and a sodium hydroxide solution, and then stirring the mixture for reaction. The obtained stirred reaction product is filtered and dried to obtain Cu-BTC@Ag NPs.

[0061] In the present invention, the volume ratio of the mass of Cu-BTC to the volume of water, the volume of silver nitrate solution, the volume of sodium citrate solution, and the volume of sodium hydroxide solution is preferably 0.1g:50mL:50μL:250μl:3mL; the concentration of the silver nitrate solution is preferably 0.1mol / L; the concentration of the sodium citrate solution is preferably 0.1mol / L; the concentration of the sodium hydroxide solution is preferably 25mmol / L. In the present invention, the conditions for the stirring reaction preferably include: a temperature of 50°C and a time of 30min. In the present invention, the pore size of the filter membrane used for filtration is preferably 0.22μm. In the present invention, the drying temperature is preferably 60°C and the time is 3h, preferably vacuum drying.

[0062] The present invention mixes the obtained Cu-BTC@AgNPs with a buffer solution, tetraphenylethylene-coupled tetraphenylimidazole, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and a DNA aptamer solution of acetamiprid, and then performs a dissolution reaction to obtain a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe; the nucleotide sequence of the DNA aptamer of acetamiprid is shown in SEQ ID No.1.

[0063] SEQ ID No. 1: 5'-NH2-(CH2)6-CATAACAGCACTACGTTGTGTCTTAACATTATGAGTCGCACTCAAGG-3'.

[0064] In the present invention, the mass of the Cu-BTC@AgNPs and the volume of the buffer, the mass of tetraphenylethylene coupled tetraphenylimidazole, the mass of N-hydroxysuccinimide, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the volume ratio of the DNA aptamer solution of acetamiprid are preferably 200mg:5mL:1mg:5mg:5mg:200μL. In the present invention, the concentration of the DNA aptamer solution of acetamiprid is preferably 1mg / mL. In the present invention, the buffer is preferably PBS buffer, with a concentration of 0.01mol / L and a pH value of 7.4. In the present invention, the dissolution reaction is carried out under stirring, and the time of the dissolution reaction is preferably 30min.

[0065] The present invention also provides a Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe prepared by the preparation method described in the above technical solution.

[0066] The present invention also provides the use of the Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe described in the above technical solution in the detection of acetamiprid.

[0067] In the present invention, the application preferably comprises the following steps:

[0068] A. Mixing the sample with the Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe and PBS buffer to react and obtain a reactant;

[0069] The pH value of the PBS buffer is 7.4, and the reaction time is 8 min;

[0070] B. The fluorescence intensity of the reactant detected is calculated as F1, and the blank control is calculated as F0. Substitute it into the following formula to calculate the concentration of acetamiprid:

[0071] △IF=F0-F1;

[0072] △IF=0.98c+56.94, the unit of c is 10 -11 mol / L.

[0073] In the present invention, the sample preferably includes banana, cowpea, etc. In the present invention, the sample processing method preferably includes: chopping 10g of sample and adding 20mL of acetonitrile, high-speed homogenization for 5min, filtering with qualitative filter paper to remove most of the solid matter, and then centrifuging at 8000rpm for 10min. Then the solution is placed in a 40°C water bath and evaporated to dryness, and finally fixed to 2mL with PBS (0.01mol / L, pH=7.4). The present invention preferably takes 1mL of sample and adds it to 300μL Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe, fixes to 2mL with 0.01mol / LPBS (0.01mol / L, pH=7.4), mixes well and reacts fully for 8min before performing fluorescence detection. In the present invention, the conditions of the fluorescence detection include: excitation wavelength Ex=350nm, emission spectrum range is 400-600nm, slits are all 5nm, and photomultiplier tube is 700V.

[0074] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] 1. Experimental part

[0077] 1.1 Materials and Instruments

[0078] CuCl2, trimesic acid (H3BTC), anhydrous ethanol, AgNO3, trisodium citrate, boric acid (H3BO3), 5 mg N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), acetamiprid, clothianidin, thiamethoxam, imidacloprid, dinotefuran, nitenpyram, chloramphenicol, atrazine, chlorpyrifos, and methyl parathion were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). DNA aptamer single-chain of acetamiprid was purchased from Shanghai Bioengineering Co., Ltd. (Shanghai, China). Tetracarboxytetraphenylethylene (TPE-(COOH)4) was purchased from Xi'an Qiyue Biotechnology Co., Ltd. All chemical reagents were analytical grade products, and ultrapure water was used throughout the experiment.

[0079] The morphology and microstructure of Cu-BTC@Ag NPs were observed by scanning electron microscopy (SEM) (ZEISS∑IGMA), and the crystal structure of Cu-BTC@Ag NPs was characterized by X-ray powder diffraction (XRD) (ultimaIV). X-ray photoelectron spectroscopy (XPS) (EscaLab 250Xi) was used to characterize and analyze the elements on the surface of Cu-BTC@AgNPs. Fourier transform infrared (FT-IR) (IRTracer-100) was used to characterize the functional groups of Cu-BTC@AgNPs. The spectrum of Cu-BTC@AgNPs was detected by ultraviolet spectrophotometer (UV5200) and fluorescence spectrometer (Hitachi F-7000), respectively.

[0080] 1.2 Preparation of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe

[0081] Cu-BTC was synthesized by the following steps. 0.81 g CuCl2 and 2.00 g H3BTC were slowly added to a mixed solvent containing 150 mL of double distilled water and 50 mL of ethylene glycol, and the mixed solution was continuously stirred for 1.5 h and reacted at 280 ° C for 3 h. After the reaction was completed, it was cooled to room temperature, centrifuged at 10000 rpm / min for 5 min, then washed with 150 mL of anhydrous ethanol and centrifuged again. After repeating the same operation three times, the solid after centrifugation was vacuum dried at 60 ° C to obtain Cu-BTC.

[0082] Take 0.1g Cu-BTC powder and disperse it in 50mL deionized water. Under stirring, add 50μL 0.1mol / LAgNO3 and 250μL 0.1mol / L trisodium citrate. Then, add 3mL 25mmol / LNaOH and stir the reaction in a 50℃ water bath for 30min. Finally, filter the reaction solution through a 0.22μm water filter membrane to obtain Cu-BTC@Ag NPs. The solid after centrifugation is vacuum dried at 60℃ to obtain Cu-BTC@Ag NPs. 20 mg Cu-BTC@Ag NPs were dispersed in 5 mL PBS (0.01 mol / L, pH = 7.4), and then 1 mg tetraphenylethylene-coupled tetraphenylimidazole, 5 mg N-hydroxysuccinimide (NHS) and 5 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 200 μL single-stranded DNA aptamer 5'-NH2-(CH2)6-CATAACAGCACTACGTTGTGTCTT AACAT TATGAGTCGCACTCAAGG-3' of acetamiprid were added. The mixture was fully dissolved and reacted for 30 min under stirring to finally obtain the Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe (4 mg / mL) that can specifically capture acetamiprid.

[0083] 1.3 Fluorescence detection method

[0084] 0, 5, 10, 50, 100, 250, 400, 500, 1000, 2500, 3500, 4500, 5200)×10 -11 mol / L acetamiprid was added to 300μL Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe of 4mg / mL, and the volume was fixed to 2mL with 0.01mol / LPBS (0.01mol / L, pH=7.4), mixed well and reacted for 8min, and the fluorescence intensity before and after the addition of acetamiprid was detected. The excitation wavelength Ex=350nm, the emission spectrum range was 400-600nm, the slit was 5nm, and the photomultiplier tube was 700V.

[0085] 1.4 Sample pretreatment

[0086] Take 10g of each sample of banana, cowpea, etc., chop it, add 20mL of acetonitrile, homogenize it at high speed for 5min, filter it with qualitative filter paper to remove most of the solid matter, and then centrifuge it at 8000rpm for 10min. Then place the solution in a 40℃ water bath to evaporate to dryness, and finally dilute it to 2mL with PBS (0.01mol / L, pH=7.4). Take 1mL of sample and add it to 300μL Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe, dilute it to 2mL with 0.01mol / L PBS (0.01mol / L, pH=7.4), mix it evenly and react it for 8min, and detect the fluorescence intensity before and after the addition of acetamiprid.

[0087] 2. Results and Discussion

[0088] 2.1 Characterization of Cu-BTC@Ag NPs

[0089] SEM, XRD and XPS were used to characterize the morphology and composition of Cu-BTC@Ag NPs. Figure 2 As shown in A. Cu-BTC has a regular octahedral morphology ( Figure 2 In the figure (A), Ag NPs are spherical in shape, without aggregation, and are evenly distributed on the surface of Cu-BTC, with a diameter of about 5 nm ( Figure 2 (B) The XRD pattern of the prepared Cu-BTC@Ag NPs is shown in Figure 2 As shown in Figure C, the prepared sample has characteristic diffraction peaks at 2θ of 11.6°, 13.4°, 17.5°, 19.1°, 21.3° and 29.3°, which correspond to the (020), (021), (022), (031), (112) and (023) crystal planes of Cu-BTC, respectively, which are basically the same as those reported in the literature. Therefore, it can be determined that the prepared sample is CuBTC. Figure 2 It can also be seen from Figure C that the diffraction peak intensity is moderate and the peak shape is good, indicating that the experimentally prepared Cu-BTC has a high degree of crystallinity and a well-developed crystal structure. The sharp peaks at 2θ=37.9°, 44.1°, 64.6°, and 77.1° correspond to the (111), (200), (220), and (311) crystal planes in Ag NPs. The diffraction peak intensity is moderate and the peak shape is good, indicating that the experimentally prepared Ag NPs have a high degree of crystallinity and a well-developed crystal structure. XPS spectra are shown in Figure 5. Figure 2 The peak at 285 eV corresponds to C (1s), the peak at 404 eV corresponds to N (1s), the peak at 533 eV corresponds to O (1s), and the peaks at 368 eV and 374 eV correspond to Ag (3d 5 / 2 ) and Ag(3d 3 / 2), the peak at 938 eV corresponds to Cu(2p). The above results show that Cu-BTC@AgNPs contains only five elements, C, O, N, Ag and Cu, and does not contain other impurity elements. The above results show that Cu-BTC@AgNPs were successfully synthesized.

[0090] 2.2. Optical properties of fluorescent probes

[0091] The optical properties of the fluorescent probe were investigated using UV-visible absorption and fluorescence spectra. Figure 3 As shown in the figure, the Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer probe has strong absorption at 350nm (curve a). The maximum excitation wavelength of the probe is around 357nm (curve b), which is consistent with the absorption spectrum results. The probe is excited at a wavelength of 350nm, and the maximum emission wavelength of the probe is around 471nm (curve c). The larger Stokes shift effectively avoids the overlap between the excitation and emission spectra. In addition, the emission spectrum of the probe is high in intensity, and the peak shape is symmetrical and narrow, which indicates that the probe has good fluorescence properties. The amplification effect of Cu-BTC@AgNPs on TPE-(COOH)4 fluorescent molecules was further studied. As shown in the figure, TPE-(COOH)4 has a strong fluorescence emission spectrum (curve d). When bound to Cu-BTC@AgNPs, the fluorescence intensity of TPE-(COOH)4 is effectively amplified (curve c, about 2.1 times). The reason is speculated to be that the scattering of light by Cu-BTC@AgNPs is enhanced and surface plasmon resonance occurs, which leads to an enhancement of the local electric field, thereby increasing the excited state lifetime and fluorescence intensity of the fluorescent molecules.

[0092] The quantum yield of fluorescent substances is an important parameter of fluorescent reagents. By comparing the integrated fluorescence intensity (i.e., the area included in the corrected fluorescence spectrum) of two dilute solutions of the fluorescent sample to be tested and the reference fluorescent standard substance with known quantum yield obtained under the same excitation conditions, as well as the absorbance of the incident light (ultraviolet-visible light) of the same excitation wavelength, and then substituting these values ​​into a specific formula (1), the quantum yield of the fluorescent sample to be tested can be obtained after calculation.

[0093]

[0094] Where Φ u , Φ s is the fluorescence quantum yield of the substance to be tested and the reference standard substance; F u 、F s is the integrated fluorescence intensity of the test substance and the reference substance; A u , A sis the absorbance of the incident light of the test substance and the reference substance at the excitation wavelength (A=εbc). The present invention uses rhodamine B (RhB) solution as a reference fluorescent standard substance. According to the literature report, the quantum yield of RhB is 0.89. The quantum yield of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe measured by this method is 0.86, which indicates that the Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe in this study has a high quantum yield and can be used as an excellent fluorescent reagent. The relevant parameters are shown in Table 1:

[0095] Table 1 Fluorescence quantum yield parameters of Rhodamine B and Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamers

[0096]

[0097] 2.3. Mechanism of acetamiprid quenching probe fluorescence intensity

[0098] With the addition of different concentrations of acetamiprid, the probe captured more and more acetamiprid through the aptamer, and the fluorescence intensity of the Zn-MOF@AuNPs / DNA aptamer continued to decrease, such as Figure 4 As shown, this indicates that acetamiprid can effectively quench the fluorescence intensity of the probe. Therefore, the mechanism of acetamiprid quenching the fluorescence of the probe was studied. The target (quencher) generally affects the fluorescence intensity of the fluorescent probe by interacting with the quantum dots at the binding site through electrostatic force, hydrogen bonding, van der Waals interaction or stereo conformation change. First, the fluorescence lifetime of the probe was measured in the presence of different concentrations of acetamiprid. The test results show that the intrinsic lifetime τ0 of the material is 12.36ns. When 50, 100, 500, and 2500×10 -11 When acetamiprid was added into the system, the fluorescence lifetimes of C-dots were 12.33, 12.30, 12.28, and 12.22 ns, respectively. The concentration of acetamiprid increased from 0 mol / L to 2.5×10 -9 When the concentration of acetamiprid was 0.14 ns, the probe fluorescence lifetime only decreased by 0.14 ns, and the probe lifetime did not change much. Therefore, the fluorescence quenching of the Zn-MOF@AuNPs / DNA aptamer probe by acetamiprid was mainly a static process. In addition, when acetamiprid was captured by the DNA aptamer, it formed hydrogen bonds with the bases of the aptamer. This interaction changed the conformation of the aptamer, causing the distance and angle between the two to change, and also affected the charge transfer between the two, shortening the distance between acetamiprid and the probe, making it easier to bind to the probe.

[0099] Optimization of detection conditions

[0100] The effects of buffer pH and reaction time on the detection of acetamiprid by Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probe were investigated. Figure 5 As shown, when the pH value of the buffer solution is in the range of 6.8 to 8.0, the quenching value (△IF) of the probe fluorescence before and after the addition of acetamiprid is detected. As the pH value continues to increase from 6.8, △IF continues to increase until pH = 7.4, when △IF reaches its maximum value. After that, as the pH continues to increase, △IF decreases instead. Therefore, PBS with a pH of 7.4 is selected as the optimal system buffer. At the same time, the effect of the reaction time between acetamiprid and the fluorescent probe on the quenching effect is studied. Figure 6 As shown in the figure, as the reaction proceeds, more and more acetamiprid binds to the probe, and the fluorescence intensity of the probe continues to decrease. When the reaction time reaches 8 minutes, △IF reaches the maximum and remains basically unchanged. Therefore, the reaction time is selected as 8 minutes.

[0101] 2.5 Calibration curve

[0102] According to the experimental method, the probe was used to adsorb different concentrations of acetamiprid, and the fluorescence intensity of the Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe was detected before and after the addition of acetamiprid, △IF=F0-F1 was calculated, and a calibration curve was drawn. Figure 7 As shown in Figure 2, with the increase of acetamiprid concentration, the probe fluorescence was quenched and the intensity decreased continuously. The acetamiprid concentration (c) was between 5 and 5200×10 -11 mol / L has a good linear relationship with the fluorescence quenching intensity (△IF) of the probe, and its linear regression equation is: △IF=0.98c(10 -11 mol / L)+56.94, r=0.994, the detection limit is 1.16×10 -12 mol / L (LOD = 3σ / m, where σ is the standard deviation of the blank and m is the slope of the calibration graph). In addition, the experimental results were compared with the existing acetamiprid residue detection method, as shown in the table. The present method has a higher sensitivity in detecting acetamiprid than the existing reported methods (Table 2).

[0103] Table 2 Comparison of different methods for detecting acetamiprid

[0104]

[0105]

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[0118]

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[0120]

[12] Su,C.,Dong,C.,Jiang,D.,Shan,X.,Chen,Z.(2023).Construction ofelectrochemiluminescence aptasensor for acetamiprid detection using flower-liked SnO2 nanocrystals encapsulated Ag3PO4 composite asluminophore.Microchemical Journal,187,108374.

[0121]

[13] Shi,X.,Sun,J.,Yao,Y.,Liu,H.,Huang,J.,Guo,Y.,Sun,X.(2020).Novelelectrochemical aptasensor with dual signal amplification strategy fordetection of acetamiprid.Science of The Total Environment,705,135905.

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[14] Yi,J.,Liu,Z.,Liu,J.,Liu,H.,Xia,F.,Tian,D.,Zhou,C.(2020).A label-free electrochemical aptasensor based on 3D porous CS / rGO / GCE for acetamipridresidue detection.Biosensors and Bioelectronics,148,111827.

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[0127] 2.6 Fluorescent probe selectivity, reproducibility and stability

[0128] The selective recognition ability of the probe to acetamiprid is the key to the method of the present invention. Therefore, the addition of other pesticide molecules to verify the selective recognition performance of the probe to acetamiprid was studied. According to the experimental method, the probe was used to adsorb 5×10 -10 mol / L acetamiprid fluorescence signal intensity (I0) and 5×10 -10 mol / L acetamiprid and 5×10 -8 The fluorescence signal intensity (I1) of the mixed solution of thiothiazide, imidacloprid, clothianidin, dinotefuran, nitenpyram, carbofuran, parathion-methyl, atrazine, chlorpyrifos and the mixture of the above 9 pesticides was calculated. The relative deviation of the fluorescence intensity before and after the mixture of acetamiprid and the interfering substance was calculated as follows: RD = (I0-I) / I0×100%. The results are shown in Figure 8As shown, the RD is less than 3%. The above results show that the probe has excellent selective recognition performance for acetamiprid.

[0129] Reproducibility and stability are important indicators that determine the actual application effect of fluorescent probes. -10 mol / L acetamiprid was reacted with five Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer probes, and the fluorescence intensity before and after the addition of acetamiprid was detected, and the △IF was calculated. The relative standard deviation (RSD) of the five experiments was 1.19%, indicating that the method has good reproducibility ( Fig. 9 ). In addition, the prepared Cu-BTC@Ag Ps@TPE-(COOH)4@DNA aptamer fluorescent probe was combined with 5×10 -10 After 8 min of reaction with 1 mol / L acetamiprid, the fluorescence signal was detected every 5 min, and the fluorescence intensity after 1 h was 98.39% of the initial value ( Fig.10 ), indicating that the fluorescent probe has good stability.

[0130] 2.7. Actual sample testing

[0131] The probe was used to detect acetamiprid residues in actual samples such as bananas and cowpeas, and the test results were compared with the results of HPLC-MS analysis, and a spike recovery test was performed. As shown in Table 3, the spike recovery of the method was 92.5-103.9%, RSD < 3%, and the test results were consistent with the HPLC-MS results. These results show that this fluorescent probe method can be used for actual sample detection with satisfactory results.

[0132] Table 3 Actual sample test results and spike recovery experiment

[0133]

[0134] It can be concluded from the above embodiments that the present invention grafts TPE-(COOH)4 molecules with aggregation-induced fluorescent groups onto Cu-BTC@Ag NPs materials to obtain Cu-BTC@Ag NPs@TPE-(COOH)4 fluorescent materials with excellent fluorescence properties, and combines DNA aptamers to prepare Cu-BTC@Ag NPs@TPE-(COOH)4 probes that can specifically identify acetamiprid. Although Cu-BTC@Ag NPs itself does not fluoresce, it can effectively amplify the fluorescence intensity of TPE-(COOH)4, and the composite fluorescent material exhibits stable and good fluorescence properties; at the same time, the probe introduces DNA aptamers, so that the probe exhibits good selective recognition ability for the target molecule acetamiprid. Therefore, the probe has the characteristics of high sensitivity, high selectivity, and easy operation in the application of acetamiprid residue detection in actual samples.

[0135] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of Cu-BTC@Ag NPs@TPE-(COOH)4@DNA aptamer fluorescent probe in the detection of acetamiprid; The preparation method of Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe includes the following steps: 1) mixing copper chloride, trimesic acid and a solvent and reacting them, centrifuging, washing and drying the obtained reactant to obtain Cu-BTC; 2) mixing the Cu-BTC obtained in step 1) with water, silver nitrate solution, sodium citrate solution and sodium hydroxide solution and stirring the mixture for reaction, filtering and drying the stirred reaction mixture to obtain Cu-BTC@AgNPs; 3) The Cu-BTC@AgNPs obtained in step 2) are mixed with a buffer solution, tetraphenylethylene-coupled tetraphenylimidazole, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and a DNA aptamer solution of acetamiprid, and then dissolved to obtain a Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe; The nucleotide sequence of the DNA aptamer of acetamiprid is shown in SEQ ID No. 1; In the step 1), the volume ratio of the mass of copper chloride to the mass of trimesic acid to the volume of the solvent is 0.81 g:2 g:200 mL; The solvent includes double distilled water and ethylene glycol, and the volume ratio of the double distilled water to the ethylene glycol is 3:1; The reaction conditions of step 1) include: temperature of 280° C. and reaction time of 3 h; The mixing is carried out under stirring, and the stirring time is 1.5h; The centrifugal conditions include: centrifugation at 10000 rpm for 5 min; The reagent used for the washing is anhydrous ethanol, and the number of washing times is 2 times; The drying temperature is 60°C; In the step 2), the volume ratio of the mass of Cu-BTC to the volume of water, the volume of silver nitrate solution, the volume of sodium citrate solution, and the volume of sodium hydroxide solution is 0.1 g: 50 mL: 50 μL: 250 μL: 3 mL; The concentration of the silver nitrate solution is 0.1 mol / L; The concentration of the sodium citrate solution is 0.1 mol / L; The concentration of the sodium hydroxide solution is 25mmol / L; The stirring reaction conditions in step 2) include: temperature of 50° C. and time of 30 min; The pore size of the filter membrane used in the filtration is 0.22 μm; The drying temperature is 60°C; In the step 3), the volume ratio of the mass of Cu-BTC@AgNPs to the volume of the buffer solution, the mass of tetraphenylethylene-coupled tetraphenylimidazole, the mass of N-hydroxysuccinimide, the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the DNA aptamer solution of acetamiprid is 200 mg: 5 mL: 1 mg: 5 mg: 5 mg: 200 μL; The concentration of the acetamiprid DNA aptamer solution is 1 mg / mL.

2. The use according to claim 1, characterized in that: The application comprises the following steps: A. Mixing the sample with the Cu-BTC@AgNPs@TPE-(COOH)4@DNA aptamer fluorescent probe and PBS buffer to react and obtain a reactant; The pH value of the PBS buffer is 7.4, and the reaction time is 8 min; B. The fluorescence intensity of the reactant detected is calculated as F1, and the blank control is calculated as F0. Substitute it into the following formula to calculate the concentration of acetamiprid: △IF=F0-F1; △IF=0.98c+56.94, the unit of c is 10 -11 mol / L.

3. The use according to claim 1, characterized in that: The buffer in step 3) is PBS buffer with a concentration of 0.01 mol / L and a pH value of 7.4; The dissolution reaction is carried out under stirring, and the dissolution reaction time is 30 minutes.

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