Ratio aptamer sensor based on metal organic framework nano-enzyme assistance, preparation method and application

Abstract

The invention discloses a ratio aptamer sensor based on metal organic framework nano-enzyme assistance, a preparation method and application, and relates to sensors and pesticide residue detection. The preparation method disclosed by the invention comprises the following steps: (1) preparing a UiO-66-NH2 / THI modified electrode; (2) preparation of a UiO-66-NH2 / THI / cDNA modified electrode; (3) preparing an electrode modified by the UiO-66-NH2 / THI / cDNA and the biotin functionalized aptamer; (4) preparing a cobalt metal organic framework nano enzyme modified by streptavidin; and (5) preparing the ratio aptamer sensor based on metal organic framework nano enzyme assistance. The preparation method disclosed by the invention is low in cost, simple in experimental operation and easy in reaction condition control. The ratio aptamer sensor prepared by the invention can be used as a working electrode for quantitative analysis and detection of thiamethoxam, and is good in stability, high in sensitivity and high in accuracy in thiamethoxam detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sensors and pesticide residue detection, and particularly relates to a ratio aptamer sensor based on metal-organic framework nanozyme assistance, a preparation method and application. BACKGROUND

[0002] Thiamethoxam (Thi) is a neonicotinoid insecticide with high efficiency and broad-spectrum activity, which exerts insecticidal effect by targeting the nicotinic acetylcholine receptor in the insect nervous system, and has been widely used in the planting of various crops. However, it has poor persistence in the environment, high dosage requirement, and large residual amount, resulting in large amounts of residues in the environment and crops, and potential risks to aquatic ecosystems.

[0003] Currently, pesticide residue analysis mainly relies on instrumental analysis methods such as high-performance liquid chromatography (HPLC) or high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Although these techniques have high detection accuracy, they require expensive equipment, professional operators, and tedious sample pretreatment steps, making it difficult to meet the real-time detection needs in agricultural and food products. Therefore, developing efficient and convenient thiamethoxam rapid detection technology as a complementary means to traditional instrumental analysis methods has important value for food safety monitoring and health risk assessment. Electrochemical aptamer sensing technology has shown good application prospects in the field of point-of-care testing (POCT) due to its advantages such as rapid detection, high sensitivity, low cost, simple operation, and easy miniaturization. It is worth noting that the performance of electrochemical aptamer sensors mainly depends on three key indicators: accuracy, specificity, and sensitivity. However, issues such as lack of reproducibility and poor accuracy are still major bottlenecks that hinder its development as a standard method replacement technology. Compared with traditional single-signal output electrochemical detection methods, ratio-type electrochemical aptamer sensors can effectively eliminate environmental interference and significantly improve detection reliability by constructing a dual-signal self-calibration system, thus attracting widespread attention from researchers.

[0004] Metal-organic framework materials are a class of compounds with high porosity, high specific surface area, and adjustable structure, which can improve the adsorption capacity by combining with biomolecules and internal reference signal molecules. Nanozymes, as nano materials with enzyme-like activity, have advantages such as scalable preparation, adjustable structure, easy functional modification, high cost-effectiveness, and good stability. In the field of biosensing, nanozymes can significantly improve the detection sensitivity of sensors through efficient catalytic signal amplification mechanism, and can also improve the response speed and operational stability. In addition, the rich interface regulation characteristics of nanozymes can be used as nano carriers to load bioactive molecules, providing a new idea for developing high-sensitivity and high-stability sensing and detection systems. SUMMARY

[0005] In view of the above prior art defects, the purpose of the present application is to provide a metal organic framework nanoscale enzyme assisted ratio aptamer sensor, a preparation method and application. The prepared ratio aptamer sensor can be used for quantitative analysis and detection of thiamethoxam with high accuracy and high selectivity, effectively solving the technical barriers existing in the prior art.

[0006] In order to achieve the above-mentioned purpose or other purposes, the present application is realized by the following technical scheme.

[0007] A preparation method of a metal organic framework nanoscale enzyme assisted ratio aptamer sensor, comprising the following steps:

[0008] (1) Preparation of UiO-66-NH2 / THI modified electrode; (2) Preparation of UiO-66-NH2 / THI / cDNA modified electrode; (3) Preparation of UiO-66-NH2 / THI / cDNA and biotin functionalized aptamer modified electrode; (4) Preparation of streptavidin modified cobalt metal organic framework nanoscale enzyme; (5) Preparation of metal organic framework nanoscale enzyme assisted ratio aptamer sensor (UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode).

[0009] Specifically, the following steps are included:

[0010] (1) Preparation of UiO-66-NH2 / THI modified electrode:

[0011] a. Dissolve zirconium source and 2-amino terephthalic acid in DMF, add a regulator, ultrasonically treat the mixture until uniform, then perform hydrothermal reaction, after the reaction is completed, perform washing and drying treatment to obtain UiO-66-NH2 material;

[0012] b. Mix the UiO-66-NH2 material with thionine (THI), perform washing and drying treatment to obtain UiO-66-NH2 / THI material;

[0013] c. Disperse the UiO-66-NH2 / THI material in water to obtain UiO-66-NH2 / THI dispersion liquid, drop coat the UiO-66-NH2 / THI dispersion liquid onto the surface of a glassy carbon electrode, and naturally air dry to obtain a UiO-66-NH2 / THI modified electrode, denoted as UiO-66-NH2 / THI / GCE;

[0014] (2) Preparation of the UiO-66-NH2 / THI / cDNA modified electrode: 2.5% glutaraldehyde was added to the surface of the UiO-66-NH2 / THI modified electrode prepared in (1), and after drying at room temperature, the complementary DNA (cDNA) of the thiamethoxam aptamer was added dropwise, and after the addition was completed, incubation was carried out at 4°C, followed by the continuous addition of 10 μL of 1% bovine serum albumin (BSA) for 1 h, and then the unbound cDNA and BSA were washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA modified electrode;

[0015] (3) Preparation of the UiO-66-NH2 / THI / cDNA and biotin-functionalized aptamer modified electrode: biotin-functionalized thiamethoxam aptamer was added dropwise to the surface of the UiO-66-NH2 / THI / cDNA modified electrode prepared in step (2), and incubation was carried out at 25°C, and then the unbound biotin-functionalized thiamethoxam aptamer was washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA and biotin-functionalized aptamer modified electrode, which is denoted as the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode;

[0016] (4) Preparation of the streptavidin modified cobalt metal organic framework nanoscale enzyme:

[0017] ① Cobalt salt and 2-methyl imidazole were dissolved in methanol respectively, mixed thoroughly, and then transferred to a reaction kettle for further reaction, and after the reaction was completed, the Co-MOF material was obtained by washing and drying;

[0018] ② The Co-MOF material was dispersed in DMF, excess succinic anhydride was added, and the reaction was carried out under stirring at room temperature, and after the reaction was completed, the carboxylated Co-MOF material was obtained by washing and drying;

[0019] ③ The carboxylated Co-MOF material was dispersed in water to obtain a carboxylated Co-MOF dispersion liquid, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbonyl diimide (EDC) were added to the carboxylated Co-MOF dispersion liquid, and after incubation, the unbound cDNA and BSA were washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and then streptavidin (SA) was added, and after overnight reaction, the SA / Co-MOF nanoscale enzyme was obtained by washing with a pH 7.4 0.01 mol / L phosphate buffer solution and drying;

[0020] (5) Preparation of the ratio aptamer sensor based on metal organic framework nanoszyme assisted (UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode): The SA / Co-MOF nanoszyme is dispersed into water to obtain a SA / Co-MOF nanoszyme dispersion liquid, and the SA / Co-MOF nanoszyme dispersion liquid is added dropwise to the surface of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode, and the mixture is dried at room temperature to obtain a UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode, that is, the ratio aptamer sensor based on metal organic framework nanoszyme assisted.

[0021] UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode surface dropwise SA / Co-MOF nanoszyme dispersion liquid, room temperature, dry, get UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode, that is, the ratio aptamer sensor based on metal organic framework nanoszyme assisted.

[0022] In an example of the present application, the zirconium source in step a is selected from zirconium tetrachloride or zirconium oxychloride.

[0023] In an example of the present application, the molar ratio of 2-amino terephthalic acid to zirconium source in step a is 1:(1-1.2).

[0024] In an example of the present application, the molar volume (mmol / mL) ratio of 2-amino terephthalic acid to DMF in step a is 1:(20-60).

[0025] In an example of the present application, the regulator in step a is selected from acetic acid or benzoic acid.

[0026] In an example of the present application, the molar volume (mmol / mL) ratio of 2-amino terephthalic acid to the regulator in step a is 1:(1.8-6).

[0027] In an example of the present application, the hydrothermal reaction temperature in step a is 80-150°C, and the reaction time is 24-30h.

[0028] In an example of the present application, the drying treatment in step a is a conventional technical operation. Preferably, the drying temperature in step a is 50-200°C, and the time is 1-24h. Preferably, DMF and ethanol are used for washing in turn, and the washing is performed 3 times.

[0029] In an example of the present application, in step b, the mass ratio of UiO-66-NH2 material to THI (thionine) is 1:(0.1-0.5).

[0030] In an example of the present application, in step b, the mixing time of UiO-66-NH2 material and THI is 12h-48h.

[0031] In an example of the present application, in step c, the concentration of the UiO-66-NH2 / THI dispersion liquid is 1-5mg / mL.

[0032] The glassy carbon electrode used in the present application is a standard glassy carbon electrode commercially available. The glassy carbon electrode surface drop-coating solution mentioned in the present application refers to the amount of drop-coating solution on a glassy carbon electrode.

[0033] The volume of the UiO-66-NH2 / THI dispersion solution drop-coated on the glassy carbon electrode is 1-10 μL.

[0034] In an example of the present application, the volume of glutaraldehyde drop-coated on the UiO-66-NH2 / THI modified electrode is 5-15 μL.

[0035] In an example of the present application, the base sequence of the complementary DNA (cDNA) of the thiamethoxam aptamer is 5'-SH-ATCC GAATTA TCT AGT GAT CGG CTCACAGGACGACCAGTTAAGAACATA-3'. The base sequence of the thiamethoxam aptamer is 5'-streptavidin-TAT GTT CTTAAC TGG TCG TCC TGT GAG CCGATCACT AGATAATTAGGAT-3'.

[0036] Preferably, the concentration of the complementary DNA of the thiamethoxam aptamer is 0.1-0.5 μM. The volume of cDNA drop-coated on the UiO-66-NH2 / THI modified electrode is 2-10 μL.

[0037] In an example of the present application, the incubation time in step (2) is 12-16 h.

[0038] The biotin-functionalized thiamethoxam aptamer used in the present application is commercially available.

[0039] In an example of the present application, the concentration of the biotin-functionalized thiamethoxam aptamer is 0.1-0.5 μM.

[0040] Preferably, the volume of biotin-functionalized thiamethoxam aptamer drop-coated on the UiO-66-NH2 / THI / cDNA modified electrode is 2-10 μL.

[0041] In an example of the present application, the incubation time in step (3) is 2-16 h.

[0042] In an example of the present application, the cobalt salt in ① of step (4) is selected from cobalt nitrate or cobalt chloride.

[0043] In an example of the present application, the molar ratio of the cobalt salt to 2-methylimidazole in ① of step (4) is 1:(0.2-1.3).

[0044] In an example of the present application, the amount of methanol in ① of step (4) is sufficient to dissolve the cobalt salt and 2-methylimidazole.

[0045] In an example of the present application, the reaction time in ① of step (4) is 1-5 h, and the reaction temperature is 50-150℃.

[0046] In an example of the present application, the drying temperature in ① of step (4) is 50-200℃, and the drying time is 1-24 h. Preferably, the washing is performed by using methanol and water in sequence, and the washing is performed for 3 times.

[0047] In an example of the present application, the mass-volume ratio (mg / mL) of Co-MOF material and DMF in ② of step (4) is 1:(1-3).

[0048] In an example of the present application, the mass ratio of Co-MOF material and succinic anhydride in ② of step (4) is not more than 1:1.

[0049] In an example of the present application, the reaction time of Co-MOF material and succinic anhydride in ② of step (4) is 12-24 h.

[0050] In an example of the present application, the mass ratio of carboxylated Co-MOF material and NHS in ③ of step (4) is 1:(0.05-0.12).

[0051] In an example of the present application, the molar ratio of EDC and NHS in ③ of step (4) is 1:2.

[0052] In an example of the present application, the incubation time in ③ of step (4) is 0.5-2 h, and the incubation temperature is 37℃.

[0053] In an example of the present application, the mass ratio of carboxylated Co-MOF and streptavidin in ③ of step (4) is 1:(0.1-0.5).

[0054] In an example of the present application, the concentration of SA / Co-MOF nanosensor dispersion solution in step (5) is 1-10 mg / mL.

[0055] Preferably, the volume of SA / Co-MOF nanosensor dispersion solution added on the surface of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode is 1-10 μL.

[0056] The second aspect of the present application protects a ratio aptamer sensor based on metal organic framework nanosensor assisted prepared by the above preparation method.

[0057] The third aspect of the present application protects the application of a metal-organic framework nanoscale enzyme assisted ratio aptamer sensor in thiamethoxam detection.

[0058] A method for thiamethoxam detection, using a metal-organic framework nanoscale enzyme assisted ratio aptamer sensor as a working electrode. Specifically, the method comprises the following steps:

[0059] 1) Preparation of standard solution: different amounts of thiamethoxam are weighed and added to ethanol to prepare a standard solution, so that the concentration of the thiamethoxam solution is 0.1 nmol / L to 100 nmol / L;

[0060] 2) Preparation of standard curve: using the ratio aptamer sensor-UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode prepared in the above step as a working electrode, Ag-AgCl as a reference electrode, platinum as a counter electrode, and using differential pulse voltammetry, different concentrations of thiamethoxam standard solution prepared in step 1) are detected; wherein the detected electrochemical signal at-0.25V is denoted as I (-0.25V) , and the detected electrochemical signal at 0.25V is denoted as I (0.25V) , and the ratio of the two is I (0.25V) / I (-0.25V) , and a standard curve is constructed with the corresponding thiamethoxam standard solution;

[0061] 3) The thiamethoxam sample solution to be detected is replaced by the standard solution for detection, and the current ratio I (0.25V) / I (-0.25V) between the measured 0.25V position signal I (0.25V) and the-0.25V position signal I (-0.25V) is used to obtain the concentration of the thiamethoxam sample solution through the standard curve.

[0062] Specifically, the detection method in step 2) is as follows: the working electrode is placed in different concentrations of thiamethoxam standard solution prepared in step 1) for adsorption for 10 minutes, and then washed with distilled water for 3 times, and the three-electrode system composed of the washed working electrode, Ag-AgCl reference electrode, and platinum counter electrode is placed in 0.2mM tetramethyl benzidine (TMB) in 0.01mol / L phosphate buffer solution with pH=7.4 for reaction for 10 minutes, and then tested by differential pulse voltammetry with a potential scanning range of 0.5 to (-0.4)V. Two electrochemical signals are detected, which are the thionine signal at-0.25V position I (-0.25V) and the signal at 0.25V position I (0.25V) , which is the reduction signal of TMB oxidation product oxTMB.

[0063] The application innovatively uses metal organic framework (UiO-66-NH2) composite modified material as a sensing interface, the surface of which is loaded with sulfur speck internal reference electrochemical signal probes, and the electrochemical signal generated by nanometer enzyme catalysis is used as a detection signal, and meanwhile, complementary DNA (cDNA) and specific aptamer are introduced as molecular recognition elements, so that high-accuracy and high-selectivity quantitative analysis of thiamethoxam is realized through construction of a ratio type electrochemical sensing platform.

[0064] In summary, the application discloses a preparation method of a ratio aptamer sensor assisted by metal organic framework nanometer enzyme, the metal organic framework (UiO-66-NH2) material is prepared, has good chemical stability and easy modification, and is combined with biomolecules and internal reference signal molecules to improve the adsorption capacity by taking advantage of the properties; the metal organic framework nanometer enzyme (Co-MOF) prepared by the application has peroxidase-like activity, does not need to add hydrogen peroxide, and can only use oxygen to carry out a reaction, thereby enhancing the stability of the sensor. The preparation method is low in cost, simple in experimental operation, and easy to control the reaction conditions. The ratio aptamer sensor assisted by the metal organic framework nanometer enzyme prepared by the application can be used as a working electrode for quantitative analysis and detection of thiamethoxam, has good stability in thiamethoxam detection, and is high in sensitivity, specificity and accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 SEM image of UiO-66-NH2 prepared for example 1.

[0066] Figure 2 SA / Co-MOF nanometer enzyme peroxidase activity graph prepared for example 1.

[0067] Figure 3 The ratio of photocurrent obtained by using the ratio aptamer sensor assisted by the metal organic framework nanometer enzyme prepared by repeating the experiment of example 1 for five times in turn as a working electrode to detect 10nM thiamethoxam solution is shown in the following figure.

[0068] Figure 4 The ratio current obtained by using the ratio aptamer sensors obtained in example 1 to example 4 as a working electrode to detect 10nM thiamethoxam solution is shown in the following figure.

[0069] Figure 5 The standard linear curve of the ratio of different concentrations of thiamethoxam standard solution and I (0.25V) / I (-0.25V) . DETAILED DESCRIPTION

[0070] The present application is described in greater detail by the following specific examples, and other advantages and permutations of the present application will become apparent to those skilled in the art upon completion of this description. The present application also can be implemented in different embodiments and of its aspects without departing from the spirit or essential characteristics thereof. Any reference signs in the examples of the drawings and the specification shall not be construed as limiting of the scope of the application. There are many alternative ways of implementing both the process and apparatus of the present application. It is understood that the application is not limited to the specific examples described in this specification and that no component of the application is essential to the practice of the application unless the context clearly indicates otherwise. The following examples further illustrate the application. Unless otherwise indicated, the reagents and starting materials used in the following examples are commercially available or can be readily prepared by conventional methods known in the art. Unless otherwise indicated, the detection methods used in the following examples are conventional detection methods in the art. Unless otherwise indicated, the starting materials used in the following examples are commercially available.

[0071] It should be noted that the terms such as "upper", "lower", "left", "right", "intermediate", and "one" and the like used in the specification are only for the convenience of clear description, and are not intended to limit the scope of the application. Changes or adjustments of the relative relationship without substantial changes in the technical content are also considered as the scope of the application.

[0072] The technical solutions of the present application are described in detail below through several specific examples and comparative examples. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be readily prepared by conventional methods known in the art. Unless otherwise specified, the detection methods used in the following examples are conventional detection methods in the art. Unless otherwise specified, the starting materials used in the following examples are commercially available.

[0073] Example 1

[0074] Preparation of a ratio aptamer sensor assisted by a metal-organic framework nanoscale enzyme: preparation of a UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode:

[0075] (1) Preparation of a UiO-66-NH2 / THI modified electrode:

[0076] a. 40 mg of zirconium tetrachloride (0.17 mmol) and 31 mg of 2-amino terephthalic acid (0.17 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF), 1 mL of acetic acid was added, and after ultrasonic reaction for 5 min, the solution was transferred to a 50 mL inner liner of polytetrafluoroethylene reaction kettle, and reacted at 120°C for 24 h. The product was washed with DMF and ethanol for 3 times, and dried at 60°C for 5 h to obtain the UiO-66-NH2 material;

[0077] b. 20 μL of 1 mg / mL THI was added into 100 μL of 1 mg / mL UiO-66-NH2 and mixed for 12 h, washed with ethanol and deionized water, and dried at 60°C to obtain the UiO-66-NH2 / THI composite material;

[0078] c. 5 μL of 2 mg / mL UiO-66-NH2 / THI dispersion liquid was added dropwise on the surface of the glassy carbon electrode, and dried at room temperature to obtain the UiO-66-NH2 / THI modified electrode.

[0079] (2) Preparation of the UiO-66-NH2 / THI / cDNA modified electrode: 10 μL of 2.5% glutaraldehyde was added dropwise on the surface of the prepared UiO-66-NH2 / THI modified electrode, dried at room temperature, and then 10 μL of 0.1 μM complementary DNA (cDNA) of the thiamethoxam aptamer was added dropwise, and after the addition was completed, incubation was performed at 4°C for 12 h, and then 10 μL of 1% bovine serum albumin (BSA) was continuously added dropwise for 1 h, and the unbound cDNA and BSA were washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA modified electrode.

[0080] (3) Preparation of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode: 10 μL of 0.1 μM biotin-functionalized thiamethoxam aptamer (biotion-aptamer) was added dropwise on the surface of the UiO-66-NH2 / THI / cDNA modified electrode prepared in (2), and incubation was performed at 25°C for 12 h, and the unbound biotion-aptamer was washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode.

[0081] (4) Preparation of the SA / Co-MOF nanozyme:

[0082] ① 15 mmol of cobalt nitrate was dissolved in 30 mL of methanol, 3 mmol of 2-methylimidazole was dissolved in 10 mL of methanol, and after the two were fully mixed, they were transferred into a 100 mL inner liner of a polytetrafluoroethylene reaction kettle, and reaction was performed at 120°C for 2 h, and then the reaction product was sequentially washed with methanol and deionized water for 3 times, and dried at 60°C for 5 h to obtain the Co-MOF material;

[0083] ② 1 mg of the Co-MOF material was dispersed in 1 mL of DMF, 1 mg of succinic anhydride was added, and reaction was performed at room temperature for 24 h, and after the reaction was completed, the product was washed with DMF, and dried at 60°C for 5 h to obtain the carboxylated Co-MOF material;

[0084] (3) 1 mg of carboxylated Co-MOF material was dispersed in 1 mL of pH 7.40.01 mol / L phosphate buffer solution, 0.06 mg of NHS (5.21 x 10 -7 mol) and 0.04 mg of EDC (2.6 x 10 -7 mol) were added, and after incubation for 2 h, washing was performed with pH 7.40.01 mol / L phosphate buffer solution, 0.2 mg of streptavidin (SA) was added, and the reaction was allowed to proceed overnight. After washing with pH 7.40.01 mol / L phosphate buffer solution and drying, SA / Co-MOF nanoscale enzyme was obtained.

[0085] (5) Preparation of a UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOFF modified electrode: 10 μL of a 1 mg / mL SA / Co-MOF nanoscale enzyme dispersion was added to the surface of a UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode, and the electrode was allowed to dry at room temperature to obtain a UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode.

[0086] Example 2

[0087] Preparation of a ratio aptamer sensor assisted by a metal-organic framework nanoscale enzyme--UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode:

[0088] (1) Preparation of a UiO-66-NH2 / THI modified electrode:

[0089] a. 31 mg of zirconium oxychloride and 31 mg of 2-amino terephthalic acid (0.17 mmol) were dissolved in 5 mL of N,N-dimethylformamide (DMF), 0.6 mL of benzoic acid was added, and after ultrasonic reaction for 5 min, the solution was transferred to a 50 mL inner liner of polytetrafluoroethylene, and the reaction was allowed to proceed at 150°C for 26 h. The product was washed with DMF and ethanol three times in sequence, and dried at 60°C for 5 h to obtain a UiO-66-NH2 material;

[0090] b. 50 μL of 1 mg / mL THI was added to 100 μL of 1 mg / mL UiO-66-NH2, and the mixture was stirred for 30 h. After washing with ethanol and deionized water, the product was dried at 60°C to obtain a UiO-66-NH2 / THI composite material;

[0091] c. 10 μL of a 1 mg / mL UiO-66-NH2 / THI dispersion was added to the surface of a glassy carbon electrode, and the electrode was allowed to dry at room temperature to obtain a UiO-66-NH2 / THI modified electrode.

[0092] (2) Preparation of UiO-66-NH2 / THI / cDNA modified electrode: 15 μL of 2.5% glutaraldehyde was added to the surface of the prepared UiO-66-NH2 / THI modified electrode, and after drying at room temperature, 2 μL of 0.5 μM thiamethoxam aptamer complementary DNA (cDNA) was added, and after the end of the addition, it was incubated at 4°C for 14 h, and then 10 μL of 1% bovine serum albumin (BSA) was continuously added for 1 h, and the unbound MCH was washed away with a pH 7.40.01 mol / L phosphate buffer solution, and after drying at room temperature, the UiO-66-NH2 / THI / cDNA modified electrode was obtained.

[0093] (3) Preparation of UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode: 5 μL of 0.2 μM biotin-functionalized thiamethoxam aptamer (biotion-aptamer) was added to the surface of the UiO-66-NH2 / THI / cDNA modified electrode prepared in (2), and incubated at 25°C for 2 h, and the unbound biotion-aptamer was washed away with a pH 7.40.01 mol / L phosphate buffer solution, and after drying at room temperature, the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode was obtained.

[0094] (4) Preparation of SA / Co-MOF nanozyme:

[0095] ① 15 mmol of cobalt nitrate was dissolved in 30 mL of methanol, 5 mmol of 2-methylimidazole was dissolved in 10 mL of methanol, and after the two were mixed thoroughly, they were transferred to a 100 mL inner liner of a polytetrafluoroethylene reaction kettle, and reacted at 20°C for 24 h, and then washed with methanol and deionized water for 3 times, and dried at 100°C for 2 h to obtain the Co-MOF material;

[0096] ② 1 mg of Co-MOF material was dispersed in 2 mL of DMF, 1 mg of succinic anhydride was added, and stirred at room temperature for 20 h, and after the reaction was completed, it was washed with DMF, and dried at 60°C for 5 h to obtain the carboxylated Co-MOF material;

[0097] ③ 1 mg of the carboxylated Co-MOF dispersion was added to 1 mL of a pH 7.40.01 mol / L phosphate buffer solution, 0.08 mg of NHS and 0.05 mg of EDC were added, and after incubation for 2 h, it was washed with a pH 7.40.01 mol / L phosphate buffer solution, 0.1 mg of streptavidin (SA) was added, and after overnight reaction, it was washed with a pH 7.40.01 mol / L phosphate buffer solution and dried to obtain the SA / Co-MOF material.

[0098] (5) Preparation of the UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode: 5 μL of 5 mg / mL SA / Co-MOF nanoscale enzyme was added dropwise to the surface of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode.

[0099] Example 3

[0100] Preparation of the UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode:

[0101] (1) Preparation of the UiO-66-NH2 / THI modified electrode:

[0102] a. 31 mg of zirconium tetrachloride and 31 mg of 2-amino terephthalic acid (0.17 mmol) were dissolved in 3.4 mL of N,N-dimethylformamide (DMF), and after ultrasonic reaction for 5 min, the solution was transferred into a 50 mL inner liner of a polytetrafluoroethylene reaction kettle, and the reaction was carried out at 80°C for 30 h. The product was washed with DMF and ethanol for 3 times, respectively, and dried at 150°C for 10 h to obtain the UiO-66-NH2 material;

[0103] b. 10 μL of 1 mg / mL THI was added to 100 μL of 1 mg / mL UiO-66-NH2, and mixed and stirred for 48 h. The product was washed with ethanol and deionized water, and dried at 60°C to obtain the UiO-66-NH2 / THI composite material;

[0104] c. 1 L of 3 mg / mL UiO-66-NH2 / THI dispersion liquid was added dropwise to the surface of a glassy carbon electrode, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI modified electrode.

[0105] (2) Preparation of the UiO-66-NH2 / THI / cDNA modified electrode: 5 L of 2.5% glutaraldehyde was added dropwise to the surface of the prepared UiO-66-NH2 / THI modified electrode, and the electrode was dried at room temperature. Then, 5 L of 0.2 M thiamethoxam aptamer complementary DNA (cDNA) was added dropwise, and the electrode was incubated at 4°C for 16 h after the addition was completed. Subsequently, 10 L of 1% bovine serum albumin (BSA) was added dropwise for blocking for 1 h. The unbound MCH was washed away with a pH 7.40 0.01 mol / L phosphate buffer solution, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA modified electrode.

[0106] (3) Preparation of UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode: 2L of 0.5M biotion-aptamer functionalized with thiamethoxam was added dropwise to the surface of the UiO-66-NH2 / THI / cDNA modified electrode prepared in (2), and incubated at 25°C for 16h. The unbound biotion-aptamer was washed away with pH 7.4 0.01mol / L phosphate buffer solution, and the electrode was dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode.

[0107] (4) Preparation of SA / Co-MOF nanoscale enzyme:

[0108] ① 15mmol of cobalt chloride was dissolved in 30mL of methanol, and 19.5mmol of 2-methylimidazole was dissolved in 10mL of methanol. After mixing, they were transferred to a 100mL inner liner of polytetrafluoroethylene reaction kettle, and reacted at 200°C for 1h. Then they were washed with methanol and deionized water for 3 times, and dried at 100°C for 2h to obtain the Co-MOF material;

[0109] ② 1mg of Co-MOF was dispersed in 3mL of DMF, and 1mg of succinic anhydride was added and stirred at room temperature for 20h. After the reaction, it was washed with DMF and dried at 60°C for 5h to obtain the carboxylated Co-MOF material;

[0110] ③ 1mg of the carboxylated Co-MOF dispersion was added to 1mL of pH 7.4 0.01mol / L phosphate buffer solution, and 0.08mg of NHS and 0.05mg of EDC were added. After incubation for 2h, it was washed with pH 7.4 0.01mol / L phosphate buffer solution, and 0.5mg of streptavidin (SA) was added and reacted overnight. After washing with pH 7.4 0.01mol / L phosphate buffer solution, the SA / Co-MOF material was dried to obtain the SA / Co-MOF material.

[0111] (5) Preparation of UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode: 1L of 10mg / mL SA / Co-MOF nanoscale enzyme was added dropwise to the surface of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode, and dried at room temperature to obtain the UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode.

[0112] Example 4

[0113] The reaction conditions in this example are the same as in Example 1, except that in step a, the molar ratio of 2-amino terephthalic acid to zirconium source is 1:1.1; in step b, the mass ratio of UiO-66-NH2 material to THI (thionine) is 1:0.4.

[0114] The mass ratio of the carboxylated Co-MOF material in ③ of step (4) to NHS is 1:0.12.

[0115] Performance characterization

[0116] 1. The UiO-66-NH2 prepared in step (1) of Example 1 was subjected to SEM testing, and the results are shown in Figure 1 From the figure, it can be seen that the UiO-66-NH2 is in the shape of uniform particles.

[0117] 2. The SA / Co-MOF nanoscale enzyme prepared in step (4) of Example 1 was dispersed in water to obtain a SA / Co-MOF nanoscale enzyme solution with a concentration of 1 mg / mL. In 1 mL of an acetic acid buffer with a pH of 3.6, 50 microliters of the 1 mg / mL SA / Co-MOF nanoscale enzyme solution and 50 microliters of 40 mM 3,3',5,5'-tetramethylbenzidine (TMB) were added, and the ultraviolet-visible absorption spectrum was measured after 2 minutes of reaction. At the same time, in 1 mL of an acetic acid buffer with a pH of 3.6, 50 microliters of 40 mM 3,3',5,5'-tetramethylbenzidine (TMB) were added, and the ultraviolet-visible absorption spectrum was measured after 2 minutes of reaction as a control. The results are shown in Figure 2 From the figure, it can be seen that after the addition of the SA / Co-MOF nanoscale enzyme, the oxidation product of TMB has a characteristic absorption peak at 652 nm, and the absorption peak at 652 nm is strong, indicating that the SA / Co-MOF nanoscale enzyme has strong peroxidase-like activity.

[0118] 3、To verify the stability of the experiment, the preparation method of Example 1 was repeated 5 times to obtain 5 metal-organic framework nanoscale enzyme assisted ratio aptamer sensors UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrodes prepared under the same experimental conditions. The 5 UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrodes were used as working electrodes, Ag-AgCl as reference electrodes, and platinum as counter electrodes to obtain a three-electrode system. The working electrode was placed in a 10 nM thiamethoxam solution for adsorption for 10 min, then the working electrode was washed with double distilled water twice, and then the working electrode, the reference electrode and the counter electrode were placed in a 0.01 mol / L phosphate buffer solution containing 0.2 mM tetramethyl benzidine (TMB) at pH = 7.4 for reaction for 10 min, and then differential pulse voltammetry was used for detection, the scanning voltage was 0.5 to (-0.4) V, and the ratio of I (0.25V) and I (-0.25V) was detected (0.25V) / I (-0.25V) , and the results are shown in Figure 3 From the figure, it can be seen that the ratio signal obtained by the modified electrode as the working electrode when testing the 10 nM thiamethoxam solution is basically consistent, which indicates that the preparation method of the application has good reproducibility.

[0119] 4、The metal-organic framework nanoscale enzyme assisted ratio aptamer sensors UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrodes prepared in Examples 1 to 4 were used as working electrodes, Ag-AgCl as reference electrodes, and platinum as counter electrodes to obtain a three-electrode system. The working electrode was placed in a 10 nM thiamethoxam solution for adsorption for 10 min, then the working electrode was washed with double distilled water twice, and then the working electrode, the reference electrode and the counter electrode were placed in a 0.01 mol / L phosphate buffer solution containing 0.2 mM tetramethyl benzidine (TMB) at pH = 7.4 for reaction for 10 min, and then differential pulse voltammetry was used for detection, the scanning voltage was 0.5 to (-0.4) V, and the ratio of I (0.25V) and I (-0.25V) was detected (0.25V) / I (-0.25V) , and the results are shown in Figure 4 From the figure, it can be seen that the signals measured by the ratio aptamer sensors prepared in the examples as the working electrode in the 10 nM thiamethoxam solution are all strong and stable, and the thiamethoxam can be detected better.

[0120] Preparation of standard curve

[0121] 1. Preparation of standard solutions: Weigh different masses of thiamethoxam and add them to ethanol to prepare standard solutions with concentrations of 0.1 nmol / L, 1.0 nmol / L, 10 nmol / L, and 100 nmol / L, respectively, with a concentration range of 0.1 nmol / L to 100 nmol / L;

[0122] 2. Construction of the standard curve: The UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode prepared in Example 1 was sequentially placed in thiamethoxam solutions of different concentrations prepared in Step 1 for adsorption for 10 min. The electrode was then washed three times with double-distilled water. A three-electrode system consisting of the washed UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode, Ag-AgCl reference electrode, and platinum-paired electrode was placed in a 0.01 mol / L phosphate buffer solution containing 0.2 mM tetramethylbenzidine (TMB) at pH 7.4 and reacted for 10 min. Differential pulse voltammetry was used for detection, with a scan voltage of 0.5–(-0.4) V. The electrical signal I at the 0.25 V position was measured. (0.25V) Signal I at -0.25V position (-0.25V) The current ratio between I (0.25V) / I (-0.25V) A linear relationship was established between the concentration of the thiamethoxam solution and the corresponding standard thiamethoxam solution concentration, and a working curve was plotted. For example... Figure 5 As shown, the linear equation is: Y = 3.62 - 0.84X, the correlation coefficient is 0.995, and the detection concentration range is 0.1 nmol / L to 100 nmol / L.

[0123] Application examples

[0124] Actual water sample processing: Collect actual river and lake water samples, filter them through a 0.22 μm pore size filter membrane, take 5.0 mL of filtrate, add 5.0 mL of 0.01 mol / L phosphate buffer solution with pH 7.4 to obtain the sample solution to be tested.

[0125] The UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode prepared in Example 1 was placed in the sample solution to be detected for adsorption for 10 min, and then the electrode was taken out and washed with double-distilled water for 3 times. Subsequently, the three-electrode system composed of the UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode, an Ag-AgCl reference electrode and a platinum counter electrode was placed in 0.2 mM TMB in a pH 7.4 0.01 mol / L phosphate buffer solution for reaction for 10 min, and then the differential pulse voltammetry was used for detection with a scanning voltage of 0.5 to (-0.4) V. The detection results showed that I (0.25V) = 48 μA, I (-0.25V) = 17.7 μA, I (0.25V) / I (-0.25V) = 2.71, and the concentration of thiamethoxam in the sample solution to be detected was 12 nmol / L according to the standard curve.

[0126] In summary, the ratio aptamer sensor based on the metal organic framework nanoscale enzyme prepared in the application can be used for quantitative analysis and detection of thiamethoxam, and has high detection sensitivity, specificity and accuracy. The method is simple, rapid, low-cost and does not require professional personnel for detection of thiamethoxam.

[0127] The above examples only illustrate the principles and effects of the application, and are not used to limit the application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought of the application should be covered by the claims of the application.

Claims

1. A method for the preparation of a ratio aptamer sensor assisted by metal-organic framework nanoszyme, characterized in that, The method comprises the following steps: (1) preparation of a UiO-66-NH2 / THI modified electrode; (2) preparation of a UiO-66-NH2 / THI / cDNA modified electrode; (3) preparation of a UiO-66-NH2 / THI / cDNA / biotin-functionalized aptamer modified electrode; (4) preparation of a streptavidin-modified cobalt metal organic framework nanoscale enzyme; (5) preparation of a metal organic framework nanoscale enzyme-assisted ratio aptamer sensor.

2. The preparation method according to claim 1, characterized in that, The method comprises the following steps: (1) preparation of a UiO-66-NH2 / THI modified electrode: a. dissolving a zirconium source and 2-amino terephthalic acid in DMF, adding a regulator, uniformly treating by ultrasonic, and then performing a hydrothermal reaction, after the reaction is completed, performing washing and drying treatment to obtain a UiO-66-NH2 material; b. mixing the UiO-66-NH2 material with thionine, performing washing and drying treatment to obtain a UiO-66-NH2 / THI material; c. dispersing the UiO-66-NH2 / THI material in water to obtain a UiO-66-NH2 / THI dispersion, and dropping the UiO-66-NH2 / THI dispersion on the surface of a glassy carbon electrode to naturally dry to obtain a UiO-66-NH2 / THI modified electrode, which is denoted as UiO-66-NH2 / THI / GCE; (2) preparation of a UiO-66-NH2 / THI / cDNA modified electrode: adding 2.5% glutaraldehyde on the surface of the UiO-66-NH2 / THI modified electrode prepared in (1), drying at room temperature, then adding complementary DNA of the thiamethoxam aptamer, incubating at 4 ℃ after the adding is completed, then continuously adding 10 mL of 1% bovine serum albumin for 1 h, washing away the unbound cDNA and BSA with a pH 7.4 0.01 mol / L phosphate buffer solution, and drying at room temperature to obtain a UiO-66-NH2 / THI / cDNA modified electrode; (3) preparation of a UiO-66-NH2 / THI / cDNA / biotin-functionalized aptamer modified electrode: adding biotin-functionalized thiamethoxam aptamer on the surface of the UiO-66-NH2 / THI / cDNA modified electrode prepared in step (2), incubating at 25 ℃, washing away the unbound biotin-functionalized thiamethoxam aptamer with a pH 7.4 0.01 mol / L phosphate buffer solution, and drying at room temperature to obtain a UiO-66-NH2 / THI / cDNA / biotin-functionalized aptamer modified electrode, which is denoted as a UiO-66-NH2 / THI / cDNA / biotin-aptamer modified electrode; (4) preparation of a streptavidin-modified cobalt metal organic framework nanoscale enzyme: ① dissolving a cobalt salt and 2-methyl imidazole in methanol respectively, transferring the mixture to a reaction kettle for continuous reaction, and performing washing and drying after the reaction is completed to obtain a Co-MOF material; ② dispersing the Co-MOF material in DMF, adding an excess amount of succinic anhydride, and stirring and reacting at room temperature, and performing washing and drying after the reaction is completed to obtain a carboxylated Co-MOF material; ③dispersing the carboxylated Co-MOF material into water to obtain a carboxylated Co-MOF dispersion, adding N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide into the carboxylated Co-MOF dispersion, incubating, washing with a pH 7.4 0.01 mol / L phosphate buffer solution, adding streptavidin, reacting overnight, washing with a pH 7.4 0.01 mol / L phosphate buffer solution, and drying to obtain a streptavidin-modified cobalt metal organic framework nanoscale enzyme, denoted as SA / Co-MOF nanoscale enzyme; (5) Preparation of a ratio aptamer sensor assisted by a metal organic framework nanoscale enzyme: dispersing the SA / Co-MOF nanoscale enzyme into water to obtain a SA / Co-MOF nanoscale enzyme dispersion, dropping the SA / Co-MOF nanoscale enzyme dispersion on the surface of the UiO-66-NH2 / THI / cDNA@biotion-aptamer modified electrode, and drying at room temperature to obtain a UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode, which is a ratio aptamer sensor assisted by a metal organic framework nanoscale enzyme.

3. The preparation method according to claim 2, characterized in that, One or more of the following technical features are included: The zirconium source in step a is selected from zirconium tetrachloride or zirconium oxychloride; The molar ratio of 2-amino terephthalic acid to the zirconium source in step a is 1:(1-1.2); The adjusting agent in step a is selected from acetic acid or benzoic acid; The hydrothermal reaction temperature in step a is 80-150°C, and the reaction time is 24-30 h; In step b, the mass ratio of the UiO-66-NH2 material to THI is 1:(0.1-0.5).

4. The preparation method according to claim 2, characterized in that, One or more of the following technical features are included: The cobalt salt in ① of step (4) is selected from cobalt nitrate or cobalt chloride; The molar ratio of the cobalt salt to 2-methylimidazole in ① of step (4) is 1:(0.2-1.3); The reaction time in ① of step (4) is 1-5 h, and the reaction temperature is 50-150°C.

5. The preparation method according to claim 2, characterized in that, One or more of the following technical features are included: The reaction time of the Co-MOF material and succinic anhydride in ② of step (4) is 12-24 h; The mass ratio of the carboxylated Co-MOF material to NHS in ③ of step (4) is 1:(0.05-0.12); The mass ratio of the carboxylated Co-MOF to streptavidin in ③ of step (4) is 1:(0.1-0.5).

6. The preparation method according to claim 2, characterized in that, The concentration of the SA / Co-MOF nanoscale enzyme dispersion in step (5) is 1-10 mg / mL.

7. The ratio aptamer sensor assisted by a metal organic framework nanoscale enzyme prepared by the preparation method of any one of claims 1-6.

8. The application of the ratio aptamer sensor assisted by a metal organic framework nanoscale enzyme prepared by the preparation method of any one of claims 1-6 in thiamethoxam detection.

9. A method for thiamethoxam detection, which uses the ratio aptamer sensor assisted by a metal organic framework nanoscale enzyme prepared by the preparation method of any one of claims 1-6 as a working electrode.

10. The method of claim 9, wherein, The following steps are included: 1) Preparation of standard solution: different mass of thiamethoxam was added into ethanol to prepare standard solution, and the concentration of thiamethoxam solution was 0.1 nmol / L~100 nmol / L; 2) Standard curve drawing: The ratio aptamer sensor prepared above - UiO-66-NH2 / THI / cDNA@biotion-aptamer@SA / Co-MOF modified electrode was used as the working electrode, Ag-AgCl as the reference electrode, platinum as the counter electrode, and differential pulse voltammetry was used to detect the thiamethoxam standard solution with different concentrations prepared in step 1); wherein the detected electrochemical signal at-0.25 V is recorded as I (-0.25V) , and the detected electrochemical signal at 0.25 V is recorded as I (0.25V) . The ratio of the two I (0.25V) / I (-0.25V) and the corresponding thiamethoxam standard solution to construct a standard curve; 3) The sample solution of thiamethoxam to be tested is used instead of the standard solution, and the concentration of the sample solution of thiamethoxam is obtained by the current ratio I (0.25V) between the signals I (-0.25V) at -0.25 V position (0.25V) / I (-0.25V) .

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