Iron-doped carbon dot nano-enzyme assisted ratio type electrochemical aptamer sensor, preparation method and application

Abstract

The invention discloses an iron-doped carbon dot nano-enzyme assisted ratio type electrochemical aptamer sensor, a preparation method and application, and belongs to the field of sensors. The method comprises the following steps: (1) preparing a molybdenum disulfide-gold nanoparticle-methylene blue composite material modified electrode; (2) preparing a MoS2 / Au / MB / cDNA modified electrode; (3) preparing the iron-doped carbon dot nano enzyme; (4) preparing the iron-doped carbon dots modified by the acetamiprid aptamer; and (5) preparing the MoS2 / Au / MB / cDNA (at) Apt / Fe-CDs modified electrode. The preparation method disclosed by the invention is low in cost, simple in experimental operation and easy in reaction condition control. The iron-doped carbon dot nano enzyme-assisted ratio-type electrochemical aptamer sensor prepared by the invention can be used as a working electrode for quantitative analysis and detection of acetamiprid, and has good stability, high sensitivity, high specificity and high accuracy in acetamiprid detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sensors and pesticide residue detection, and particularly relates to a Fe-doped carbon dot nanoszyme-assisted ratio-type electrochemical aptamer sensor, a preparation method and application thereof. BACKGROUND

[0002] Acetamiprid (ACE) as a typical neonicotinoid insecticide, is widely used in the control of pests in various crops due to its high insecticidal activity (characterized by low dosage, rapid onset and long-lasting efficacy). However, this compound has high polarity and low volatility characteristics, and is prone to accumulate in plants to form pesticide residues. Existing studies have shown that acetamiprid may have potential toxic effects such as mutagenicity, teratogenicity and carcinogenicity, which pose a significant threat to the ecological environment and human health.

[0003] Currently, pesticide residue analysis mainly relies on instrumental analysis methods such as high-performance liquid chromatography (HPLC) or chromatography-mass spectrometry (LC-MS). Although these techniques have excellent analytical accuracy and precision, due to the complexity of the actual sample matrix, there is a serious matrix interference effect, and usually complex sample pretreatment is required to improve the selectivity and sensitivity of the method. In addition, these methods also have limitations such as high detection cost and long cycle time. Therefore, developing efficient and convenient acetamiprid rapid detection technology as a supplement to traditional instrumental analysis methods has important practical value for realizing 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 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, the lack of reproducibility and poor accuracy are still the main bottlenecks that restrict 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 by constructing a dual-signal self-calibration system, significantly improving the detection reliability, and thus have attracted widespread attention from researchers.

[0004] As a typical transition metal dichalcogenide, molybdenum disulfide (MoS2) is an ideal electrode modification material due to its excellent specific surface area and electrical conductivity. Nanozymes, as a kind of nanomaterials with enzymatic activity, have become a powerful substitute for natural enzymes because of their advantages such as scalable preparation, controllable structure, easy functional modification, high cost-effectiveness and good stability. In the field of biosensing, nanozymes can significantly improve the detection sensitivity of sensors and improve the response speed and operational stability through efficient catalytic signal amplification mechanism, providing new opportunities for the development of a new generation of biosensors. In particular, it is worth noting that by utilizing the rich interface regulation characteristics of nanozymes, bioactive molecules can be loaded on nanozymes as nanocarriers to construct multifunctional bioconjugates as signal amplification elements, which provides a new idea for developing high-sensitivity and high-stability sensing and detection systems. SUMMARY

[0005] In view of the shortcomings of the prior art, the purpose of the present application is to provide an iron-doped carbon dot nanozyme-assisted ratio-type electrochemical aptamer sensor, a preparation method and an application. The iron-doped carbon dot nanozyme-assisted ratio-type electrochemical aptamer sensor prepared by the present application can quantitatively analyze and detect acetamiprid, 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 solutions.

[0007] The preparation method of the iron-doped carbon dot nanozyme-assisted ratio-type electrochemical aptamer sensor comprises the following steps:

[0008] (1) Preparation of molybdenum disulfide-gold nanoparticle-methylene blue (MB) (MoS2 / Au / MB) composite material modified electrode;

[0009] (2) Preparation of MoS2 / Au / MB / cDNA modified electrode;

[0010] (3) Preparation of iron-doped carbon dot (Fe-CDs) nanozyme;

[0011] (4) Preparation of acetamiprid aptamer modified iron-doped carbon dot (Apt / Fe-CDs);

[0012] (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode.

[0013] Specifically, the following steps are included:

[0014] (1) Preparation of molybdenum disulfide-gold nanoparticle-methylene blue (MB) (MoS2 / Au / MB) composite material modified electrode:

[0015] a. Dissolve the molybdenum source and the sulfur source in deionized water, mix uniformly, and then perform a hydrothermal reaction. After the reaction is completed, perform washing and drying treatment to obtain a MoS2 material;

[0016] b. Dissolve sodium citrate and tannic acid in deionized water, add MoS2, stir, continue to add chloroauric acid, and react under stirring. After the reaction is completed, perform washing and drying to obtain a MoS2 / Au material;

[0017] c. Mix the MoS2 / Au material with methylene blue, perform washing and drying treatment, and obtain a MoS2 / Au / MB material.

[0018] d. Add the MoS2 / Au / MB material to deionized water to obtain a MoS2 / Au / MB dispersion liquid. Drop coat the MoS2 / Au / MB dispersion liquid onto the surface of a glassy carbon electrode, and naturally air dry to obtain a MoS2 / Au / MB composite material modified glassy carbon electrode, which is recorded as a MoS2 / Au / MB / GC electrode.

[0019] (2) Preparation of a MoS2 / Au / MB / cDNA modified electrode: Add the acetamiprid aptamer complementary DNA (cDNA) to the surface of the MoS2 / Au / MB / GC electrode prepared in (1), and incubate at 37°C after the addition is completed. Then continue to add 5-10 μL of 6-mercaptohexan-1-ol (MCH) for 1 h. Wash away the unbound MCH with a pH 7.40.01 mol / L phosphate buffer solution, and air dry at room temperature to obtain a MoS2 / Au / MB / cDNA modified electrode.

[0020] (3) Preparation of an iron-doped carbon dot (Fe-CDs) nanoscale enzyme: Dissolve the iron salt and the carbon source in deionized water, and perform microwave heating reaction. After the reaction is completed, dialyze overnight using a dialysis bag with a molecular weight of 500, and freeze-dry to obtain a Fe-CDs nanoscale enzyme.

[0021] (4) Preparation of an acetamiprid aptamer modified iron-doped carbon dot nanoscale enzyme (Apt / Fe-CDs): Disperse the Fe-CDs nanoscale enzyme in a pH 7.410 mM phosphate buffer solution containing glutaraldehyde, and stir at room temperature. After vacuum filtration, wash with deionized water to remove residual glutaraldehyde, and dry under vacuum. Disperse the obtained solid in a pH 7.410 mM phosphate buffer solution, add the amino-functionalized aptamer, and incubate overnight. Wash the unbound aptamer with a pH 7.410 mM phosphate buffer solution to obtain an Apt / Fe-CDs nanoscale enzyme.

[0022] (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode: Disperse Apt / Fe-CDs nanoscale enzyme into pH = 7.410 mM phosphate buffer solution to obtain Apt / Fe-CDs nanoscale enzyme solution, drop Apt / Fe-CDs nanoscale enzyme solution on the surface of MoS2 / Au / MB / cDNA modified electrode, and dry at room temperature to obtain MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode, which is a ratio type electrochemical aptamer sensor assisted by iron-doped carbon dot nanoscale enzyme.

[0023] In an example of the present application, the molybdenum source in step a is selected from ammonium molybdate, sodium molybdate or molybdenum chloride.

[0024] In an example of the present application, the sulfur source in step a is selected from thioacetamide, thiourea or L-cysteine.

[0025] In an example of the present application, the molar ratio of the molybdenum source to the sulfur source in step a is 1:(10-20).

[0026] In an example of the present application, the molar ratio of the molybdenum source to the sulfur source in step a is 1:(10-20).

[0027] In an example of the present application, the hydrothermal reaction temperature in step a is 150-250 DEG C, and the reaction time is 20-10 h.

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

[0029] In an example of the present application, in step b, the mass ratio of sodium citrate to tannic acid is 1:(0.002-0.005).

[0030] In an example of the present application, in step b, the mass-volume ratio (mg / mL) of sodium citrate to deionized water is 1:(0.03-0.1).

[0031] In an example of the present application, in step b, the stirring time after adding MoS2 is 0.2-2 h.

[0032] In an example of the present application, in step b, the mass ratio of chloroauric acid to sodium citrate is 1:(2-8).

[0033] In an example of the present application, in step b, the reaction temperature is 100-200 DEG C, and the reaction time is 24-48 h. After the reaction is completed, the drying temperature is 50-200 DEG C.

[0034] In an example of the present application, in step c, the mass ratio of MoS2 / Au material to methylene blue is 1:(0.2-1) (mg / mL).

[0035] In an example of the present application, in step c, the stirring time of MoS2 / Au material mixed with methylene blue is 4-12 h.

[0036] In an example of the present application, in step d, the concentration of MoS2 / Au / MB dispersion is 1-10 mg / mL.

[0037] Preferably, the volume of MoS2 / Au / MB dispersion solution dropped on the glassy carbon electrode is 1-10 μL.

[0038] In an example of the present application, the base sequence of the complementary DNA of acetamiprid aptamer is: 5'-SH-TCCTCTATAATATGGTGTAGCGATCAAGAACCCTCTGCAGACAAATTACA-3'.

[0039] The base sequence of the acetamiprid aptamer is: 5'-NH2-TGTAATTTGTCTGCAGCGGTTCTTGATCGCTGACACCATATTATGAAGA-3'.

[0040] Preferably, the volume of cDNA added to the surface of MoS2 / Au / MB modified electrode in step (2) is 30-200 μL.

[0041] In an example of the present application, the incubation time is 0.5-3 h.

[0042] In an example of the present application, in step (3), the iron salt is selected from ferric chloride, ferric nitrate or ferric sulfate. The carbon source is selected from o-phenylenediamine or p-phenylenediamine.

[0043] In an example of the present application, in step (3), the mass ratio of iron salt to carbon source is 1:(1-15).

[0044] In an example of the present application, in step (3), the mass-volume ratio (g / mL) of iron salt to deionized water is 1:(10-100).

[0045] In an example of the present application, in step (3), the reaction temperature is 150-250℃ and the reaction time is 2-16 h.

[0046] In an example of the present application, in step (4), the mass-volume ratio (g / mL) of Fe-CDs nanoscale enzyme to glutaraldehyde is 1:(3-8).

[0047] In one example of the present invention, the mass molar ratio (g / μmol) of Fe-CDs nanozyme to amino-functionalized aptamer (Apt) in step (4) is 1:(50-150).

[0048] In one example of the present invention, the incubation temperature in step (4) is 37°C.

[0049] In one example of the present invention, the concentration of the Apt / Fe-CDs nanozyme solution in step (5) is 1 to 10 mg / mL.

[0050] Preferably, in step (5), the volume of Apt / Fe-CDs nanozyme solution added to the MoS2 / Au / MB / cDNA modified electrode is 1 to 10 μL.

[0051] This invention also protects an iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor prepared by the above-described preparation method.

[0052] This invention also protects the application of the above-mentioned iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor in the detection of acetamiprid.

[0053] A method for detecting imidacloprid uses an iron-doped carbon nanoparticle-assisted ratiometric electrochemical aptamer sensor as the working electrode.

[0054] Includes the following steps:

[0055] 1) Preparation of standard solutions: Weigh different masses of acetamiprid and add them to ethanol to prepare standard solutions, so that the concentration of acetamiprid solution is between 0.1 nmol / L and 100 nmol / L;

[0056] 2) Construction of the standard curve: Using the iron-doped carbon nanoparticle-assisted ratiometric electrochemical aptamer sensor prepared above—MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode—as the working electrode, Ag-AgCl as the reference electrode, and platinum as the counter electrode, differential pulse voltammetry was used to detect the acetamiprid standard solutions of different concentrations prepared in step 1). The electrochemical signal detected at -0.3V was denoted as I. (-0.3V) The electrochemical signal detected at -0.5V is denoted as I. (-0.5V) The ratio of the two is I (-0.5V) / I (-0.3V) Construct a standard curve with the corresponding acetamiprid standard solution;

[0057] 3) Actual sample testing: Following the testing method in step 2, replace the standard solution with the acetamiprid sample solution to be tested and perform the test. The result is I. (-0.3V) and I (-0.5V) , will I(-0.5V) / I (-0.3V) Substituted into the standard curve, the detection of acetamiprid in the actual sample can be realized.

[0058] Specifically, the detection method in step 2) is as follows: the MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode is placed in an acetamiprid standard solution for adsorption for 10 min, after adsorption, the MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode is washed with double distilled water for 3 times, then, a three-electrode system composed of a working electrode, an Ag-AgCl reference electrode and a platinum counter electrode is placed in a 0.01 mol / L phosphate buffer solution with pH=7.4 containing 0.1 mM H2O2 and 0.1 mM o-phenylenediamine (OPD), after reaction for 10 min, differential pulse voltammetry is adopted for detection with a scanning voltage of-0.1 V to (-0.7) V.

[0059] wherein the detected electrochemical signal I (-0.3V) is the methylene blue electrochemical signal, the detected electrochemical signal I (-0.5V) is the o-phenylenediamine oxidation product 2,3-diaminophenazine electrochemical signal.

[0060] In the application, the molybdenum disulfide-gold (MoS2 / Au) composite modified material is innovatively used as a sensing interface, methylene blue (MB) is used as an internal reference electrochemical signal probe, the electrochemical signal generated by nanoscale enzyme catalysis is used as a detection signal, complementary DNA (cDNA) and specific aptamer are introduced as molecular recognition elements, a ratio type electrochemical sensing platform is constructed, and high accuracy and high selectivity quantitative analysis of acetamiprid is realized.

[0061] In summary, the application discloses a preparation method of a ratio type electrochemical aptamer sensor assisted by iron-doped carbon dot nanoscale enzyme, the preparation method of the application first prepares a molybdenum disulfide-gold composite nanomaterial with good electrical conductivity, and modifies the molybdenum disulfide-gold composite nanomaterial on an electrode, so that the molybdenum disulfide-gold composite nanomaterial has good chemical stability and high efficient electron transfer efficiency; then, the iron-doped carbon dot nanoscale enzyme is prepared, and the Apt / Fe-CDs nanoscale enzyme is modified on the electrode, and the activity of the nanoscale enzyme is enhanced by iron doping. The preparation method of the application has low cost, simple experimental operation and easy control of reaction conditions. The ratio type electrochemical aptamer sensor assisted by the iron-doped carbon dot nanoscale enzyme prepared by the application can be used as a working electrode for quantitative analysis and detection of acetamiprid, and has good stability, high sensitivity, high specificity and high accuracy in acetamiprid detection. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 It is an SEM graph of MoS2 / Au prepared in Example 1.

[0063] Figure 2 Apt / Fe-CDs nanoszyme peroxidase activity graph prepared for Example 1.

[0064] Figure 3 Ratio-type electrochemical aptamer sensor with iron-doped carbon dot nanoszyme as working electrode for detecting 1 nM acetamiprid solution in order of five times of repeated experiments for Example 1.

[0065] Figure 4 Ratio current obtained by ratio-type electrochemical aptamer sensor as working electrode for detecting 1 nM acetamiprid solution for Example 1 to Example 4.

[0066] Figure 5 Standard linear curve of I (-0.5V) / I (-0.3V) for different concentrations of acetamiprid standard solution and the detected I DETAILED DESCRIPTION

[0067] The present application will be described in detail below with specific reference being made to certain embodiments. The advantages and features of the present application will become apparent to those skilled in the art upon examination of the following details. The detailed description of the application is preceded by a description of the background of the application, which is not a admission of prior art. The following detailed description, given by way of example, is not intended to limit the present application to the specific embodiments disclosed, but rather the application can be practiced with the stated elements and steps in various embodiments of the application. The described embodiments are to be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each example apply equally to other examples unless otherwise indicated. Moreover, although the application is susceptible to embodiments in both hardware and software, certain embodiments of the application are described in terms of procedures performed by or on a computer. It is to be understood that the various embodiments can be implemented in software, hardware, or a combination thereof. It is to be further understood that the software can be initially stored on a computer readable medium before it is loaded into memory or other storage devices of the computer.

[0068] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", 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 which can be implemented. The change or adjustment of the relative relationship without substantial change of the technical content is also regarded as the scope of the application which can be implemented.

[0069] 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 prepared by conventional methods in the art. Unless otherwise specified, the detection methods used in the examples of the present application are conventional detection methods in the industry. Unless otherwise specified, the raw materials used in the present application are commercially available. The complementary DNA of acetamiprid aptamer, acetamiprid aptamer, amino-functionalized aptamer, and glassy carbon electrode used in the examples of the present application are commercially available. 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 on a glassy carbon electrode.

[0070] Example 1

[0071] Preparation of a ratio electrochemical aptamer sensor assisted by iron-doped carbon dot nanometer enzyme based on MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode:

[0072] (1) Preparation of MoS2 / Au / MB modified electrode:

[0073] a. Dissolve 1 mmol of ammonium molybdate and 14 mmol of thioacetamide in 40 mL of deionized water, mix thoroughly, and place in a 50 mL inner liner of polytetrafluoroethylene reaction kettle, react at 200°C for 15 h. After the reaction is completed, wash with ethanol and deionized water for 3 times, and dry at 60°C for 2 h to obtain MoS2 nanoflower;

[0074] b. Dissolve 0.32 g of sodium citrate and 1.4 mg of tannic acid in 15 mL of deionized water, add MoS2 and stir for 30 min, add 0.06 g of chloroauric acid and react at 200°C for 24 h. After the reaction is completed, wash with ethanol and deionized water, and dry at 60°C to obtain MoS2 / Au material;

[0075] c. Add 100 μL of MB to 200 μL of 1 mg / mL MoS2 / Au and mix and stir for 8 h. Wash with ethanol and deionized water, and dry at 60°C to obtain MoS2 / Au / MB composite material;

[0076] d. Take MoS2 / Au / MB material and add to deionized water to obtain a MoS2 / Au / MB dispersion solution with a concentration of 2 mg·mL -1 MoS2 / Au / MB dispersion solution, add 5 μL of MoS2 / Au / MB dispersion solution on the surface of a glassy carbon electrode, and dry at room temperature to obtain a MoS2 / Au / MB modified electrode.

[0077] (2) Preparation of MoS2 / Au / MB / cDNA modified electrode: 100 μL of complementary DNA (cDNA) of acetamiprid aptamer was added to the surface of MoS2 / Au / MB modified electrode, incubated at 37 °C for 1 h, and then 10 μL of 6-mercaptohexan-1-ol (MCH) was continuously added for 1 h. The unbound MCH was washed away with pH 7.40.01 mol / L phosphate buffer solution, and the MoS2 / Au / MB / cDNA modified electrode was obtained after air drying at room temperature.

[0078] (3) Preparation of iron-doped carbon dot (Fe-CDs) nanoscale enzyme: 0.45 g of iron chloride and 0.7 g of o-phenylenediamine were dissolved in 20 mL of deionized water, and then microwave heating was performed at 200 °C for 4 h. The product was dialyzed overnight using a dialysis bag with a molecular weight of 500, and then freeze-dried to obtain Fe-CDs.

[0079] (4) Preparation of acetamiprid aptamer modified iron-doped carbon dot (Apt / Fe-CDs): 1.0 g of Fe-CDs nanoscale enzyme was dispersed in 50 mL of pH 7.410 mM phosphate buffer solution containing 10% glutaraldehyde, and then stirred at room temperature for 24 h. After vacuum filtration, the residual glutaraldehyde was removed by washing with deionized water, and then the product was dried under vacuum. The obtained solid was dispersed in pH 7.410 mM phosphate buffer solution, and then 100 μmol of amino-functionalized aptamer was added and incubated overnight. The unbound aptamer was washed with pH 7.410 mM phosphate buffer solution to obtain Apt / Fe-CDs nanoscale enzyme.

[0080] (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode: 10 μL of 1 mg / mL Apt / Fe-CDs nanoscale enzyme was added to the surface of MoS2 / Au / MB / cDNA modified electrode, and then air dried at room temperature to obtain MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode.

[0081] Example 2

[0082] Preparation of a ratio electrochemical aptamer sensor based on iron-doped carbon dot nanoscale enzyme assistance--MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode:

[0083] (1) Preparation of MoS2 / Au / MB modified electrode:

[0084] a. 1 mmol of ammonium molybdate and 10 mmol of thioacetamide were dissolved in 40 mL of deionized water, mixed thoroughly, placed in a 50 mL inner liner of polytetrafluoroethylene reaction kettle, and then reacted at 200 °C for 18 h. After the reaction, the product was washed with ethanol and double-distilled water for 3 times, and then dried at 70 °C for 2 h to obtain MoS2 nanoflower.

[0085] b. 0.32 g of sodium citrate, 1.2 mg of tannic acid were dissolved in 20 mL of deionized water, 0.1 g of chloroauric acid was added and stirred for 50 min, and then reacted at 150°C for 30 h. After the reaction, ethanol and deionized water were used for washing, and drying was performed at 60°C to obtain a MoS2 / Au material;

[0086] c. 100 μL of MB was added to 300 μL of 1 mg / mL MoS2 / Au and mixed and stirred for 5 h. Ethanol and deionized water were used for washing, and drying was performed at 60°C to obtain a MoS2 / Au / MB composite material.

[0087] d. 10 μL of 1 mg·mL-1 MoS2 / Au / MB dispersion solution was added dropwise to the surface of a glassy carbon electrode, and dried at room temperature to obtain a MoS2 / Au / MB modified electrode. -1 MoS2 / Au / MB dispersion solution was added dropwise to the surface of a glassy carbon electrode, and dried at room temperature to obtain a MoS2 / Au / MB modified electrode.

[0088] (2) Preparation of a MoS2 / Au / MB / cDNA modified electrode: 150 μL of complementary DNA (cDNA) of acetamiprid aptamer was added dropwise to the surface of a MoS2 / Au / MB modified electrode, and incubated at 37°C for 40 min. 10 μL of 6-mercaptohexan-1-ol (MCH) was further added dropwise and blocked for 1 h. Unbound MCH was washed away with a pH 7.4 0.01 mol / L phosphate buffer solution, and dried at room temperature to obtain a MoS2 / Au / MB / cDNA modified electrode.

[0089] (3) Preparation of iron-doped carbon dot (Fe-CDs) nanoszyme: 0.25 g of iron chloride and 0.7 g of o-phenylenediamine were dissolved in 10 mL of deionized water, and microwave heating was performed at 150°C for 16 h. Dialysis was performed overnight using a dialysis bag with a molecular weight of 500, and freeze-drying was performed to obtain Fe-CDs.

[0090] (4) Preparation of acetamiprid aptamer-modified iron-doped carbon dots (Apt / Fe-CDs): 1.2 g of Fe-CDs nanoszyme was dispersed in 50 mL of pH 7.4 10 mM phosphate buffer solution containing 10% glutaraldehyde, and stirred at room temperature for 24 h. After vacuum filtration, deionized water was used for washing to remove residual glutaraldehyde, and drying was performed under vacuum. The obtained solid was dispersed in pH 7.4 10 mM phosphate buffer solution, and 150 μmol of amino-functionalized aptamer was added and incubated overnight. Unbound aptamer was washed away using pH 7.4 10 mM phosphate buffer solution to obtain Apt / Fe-CDs nanoszyme.

[0091] (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode: 5 μL of 2 mg / mL Apt / Fe-CDs nanoscale enzyme was added on the surface of MoS2 / Au / MB / cDNA modified electrode, and dried at room temperature to obtain MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode.

[0092] Example 3

[0093] The preparation method and conditions of this example are the same as those of Example 1, except that:

[0094] In step (1), the molybdenum source is sodium molybdate, the sulfur source is L-cysteine, the molar ratio of the molybdenum source to the sulfur source is 1:15, the hydrothermal reaction temperature is 150°C, and the reaction time is 20 h; in b, the mass ratio of sodium citrate to tannic acid is 1:0.005, the mass ratio of chloroauric acid to sodium citrate is 1:2, the reaction temperature is 100°C, and the reaction time is 48 h. In step (2), 30 μL of cDNA is added on the surface of the MoS2 / Au / MB modified electrode. In step (3), the iron salt is ferric nitrate, the mass ratio of the iron salt to the carbon source is 1:10, the reaction temperature is 230°C, and the reaction time is 7 h. In step (4), the mass-volume ratio of Fe-CDs nanoscale enzyme to glutaraldehyde satisfies 1:3

[0095] Example 4

[0096] The preparation method and conditions of this example are the same as those of Example 1, except that:

[0097] In step (1), the molybdenum source is molybdenum chloride, the sulfur source is thiourea, the molar ratio of the molybdenum source to the sulfur source is 1:20, the hydrothermal reaction temperature is 200°C, and the reaction time is 15 h; in b, the mass ratio of sodium citrate to tannic acid is 1:0.003, and the mass ratio of chloroauric acid to sodium citrate is 1:8. In step (2), 100 μL of cDNA is added on the surface of the MoS2 / Au / MB modified electrode; in step (3), the iron salt is ferric sulfate, the carbon source is p-phenylenediamine, the mass ratio of the iron salt to the carbon source is 1:15, the reaction temperature is 250°C, and the reaction time is 2 h. In step (4), the mass-volume ratio of Fe-CDs nanoscale enzyme to glutaraldehyde satisfies 1:5

[0098] Example 5

[0099] The preparation method and conditions of this example are the same as those of Example 1, except that:

[0100] The molar ratio of the molybdenum source to the sulfur source in step (1) a is 1:17, the hydrothermal reaction temperature is 250°C, and the reaction time is 10 h; the mass ratio of sodium citrate to tannic acid in step (1) b is 1:0.002, and the mass ratio of chloroauric acid to sodium citrate is 1:5. The volume of the cDNA added to the MoS2 / Au / MB modified electrode surface in step (2) is 200 μL. The carbon source in step (3) is p-phenylenediamine, the mass ratio of the iron salt to the carbon source is 1:1, the reaction temperature is 150°C, and the reaction time is 10 h. The mass-to-volume ratio of the Fe-CDs nanoscale enzyme to glutaraldehyde in step (4) is 1:8.

[0101] Performance characterization

[0102] 1. The MoS2 / Au material prepared in step (1) of Example 1 was subjected to SEM testing, and the results are shown in FIG. 1. As can be clearly seen from the figure, gold nanoparticles are dispersed on the MoS2 nanosheets. Figure 1

[0103] 2. The Apt / Fe-CDs nanoscale enzyme prepared in step (4) of Example 1 was dispersed in water to obtain an Apt / Fe-CDs 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 μL of the 1 mg / mL Apt / Fe-CDs nanoscale enzyme solution, 50 μL of 40 mM 3,3',5,5'-tetramethylbenzidine (TMB), and 50 μL of 50 mM hydrogen peroxide were added, and the ultraviolet-visible absorption spectrum was measured after 3 minutes of reaction. Meanwhile, in 1 mL of an acetic acid buffer with a pH of 3.6, 50 μL of 40 mM 3,3',5,5'-tetramethylbenzidine (TMB) and 50 μL of 50 mM hydrogen peroxide were added, and the ultraviolet-visible absorption spectrum was measured after 3 minutes of reaction as a control. The results are shown in FIG. 2. As can be seen from the figure, after the addition of the Apt / Fe-CDs 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 Apt / Fe-CDs nanoscale enzyme has strong peroxidase-like activity. Figure 2

[0104] ​​3、To verify the stability of the experiment, the preparation method of Example 1 was repeated 5 times, and 5 iron-doped carbon dot nanoscale enzyme assisted ratio type electrochemical aptamer sensors MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrodes prepared under the same experimental conditions were obtained. Four iron-doped carbon dot nanoscale enzyme assisted ratio type electrochemical aptamer sensors 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 1nM acetamiprid solution for 10 minutes, then the working electrode was washed twice with distilled water, and then the working electrode, reference electrode and counter electrode were placed in a 0.01 mol / L phosphate buffer solution containing 0.1mM H2O2 and 0.1mM o-phenylenediamine (OPD) at pH=7.4 for 10 minutes. After reaction, differential pulse voltammetry was used for detection, and the scan voltage was-0.1V~(-0.7)V. The ratio of I (-0.5V) and I (-0.3V ) was I (-0.5V) / I (-0.3V) The results are shown in Figure 3 , and it can be seen from the figure that the ratio signals obtained by the modified electrodes as working electrodes when testing 1nM acetamiprid solution are basically consistent, indicating that the preparation method of the application has good reproducibility.

[0105] 4、The iron-doped carbon dot nanoscale enzyme assisted ratio type electrochemical aptamer sensors 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 1nM acetamiprid solution for 10 minutes, then the working electrode was washed twice with distilled water, and then the working electrode, reference electrode and counter electrode were placed in a 0.01 mol / L phosphate buffer solution containing 0.1mM H2O2 and 0.1mM o-phenylenediamine (OPD) at pH=7.4 for 10 minutes. After reaction, differential pulse voltammetry was used for detection, and the scan voltage was-0.1V~(-0.7)V. The ratio of I (-0.5V) and I (-0.3V ) was I (-0.5V) / I (-0.3V) , and the results are shown in Figure 4 , and it can be seen from the figure that the signals measured by the ratio type electrochemical aptamer sensors prepared in the examples as working electrodes in 1nM acetamiprid solution are strong and stable, and can better detect acetamiprid.

[0106] Preparation of standard curve

[0107] 1. Preparation of standard solution: different amounts of acetamiprid were weighed and added 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, in the range of 0.1 nmol / L to 100 nmol / L.

[0108] 2. Preparation of standard curve: the MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode prepared in Example 1 was placed in the above acetamiprid standard solution with different concentrations for 10 min, and then washed with double distilled water for 3 times. Subsequently, the three-electrode system composed of the working electrode, Ag-AgCl reference electrode and platinum counter electrode was placed in a 0.01 mol / L phosphate buffer solution containing 0.1 mM H2O2 and 0.1 mM o-phenylenediamine (OPD) at pH 7.4 for 10 min, and then detected by differential pulse voltammetry with a scanning voltage of -0.1 to -0.7 V. The ratio of the electrochemical signal at -0.5 V to that at -0.3 V was calculated, and the standard curve was plotted as shown in FIG. 2, with the linear equation Y = 1.95-0.85X, the correlation coefficient of 0.994 and the detection concentration range of 0.1 nmol / L to 100 nmol / L. (-0.5V) / I (-0.3V) , and the linear relationship with imidacloprid solution was obtained. Figure 5

[0109] Application Example

[0110] Actual water sample treatment: river water and lake water samples were collected and filtered through a filter membrane with a pore size of 0.22 μm. 5.0 mL of the filtrate was taken and added to 5.0 mL of 0.01 mol / L phosphate buffer solution at pH 7.4 to obtain a sample solution to be detected.

[0111] The MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode prepared in Example 1 was placed in the sample solution to be detected for 10 min, and then taken out and washed with double distilled water for 3 times. The electrode was placed in a 0.01 mol / L phosphate buffer solution containing 0.1 mM H2O2 and 0.1 mM o-phenylenediamine (OPD) at pH 7.4 for 10 min, and then detected by differential pulse voltammetry with a scanning voltage of -0.1 to -0.7 V. The detection results were I (-0.3V) = 20.4 μA, I (-0.5V) = 42 μA, and I (-0.5V) / I (-0.3V) = 2.06. Substituting into the standard curve, the concentration of acetamiprid in the sample solution to be detected was 1.35 nmol / L.

[0112] ​In summary, the iron-doped carbon dot nanoszyme assisted ratio type electrochemical aptamer sensor prepared by the application realizes high accuracy and high selectivity quantitative analysis of acetamiprid, has good stability in acetamiprid detection, and has high sensitivity, specificity and accuracy. Moreover, the method realizes simple operation, rapidness, low cost and can detect acetamiprid without professional personnel.

[0113] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for preparing an iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor, characterized in that, Includes the following steps: (1) Preparation of a molybdenum disulfide-gold nanoparticle-methylene blue composite material modified electrode; (2) Preparation of MoS2 / Au / MB / cDNA modified electrode; (3) Preparation of iron-doped carbon dot nanozymes; (4) Preparation of iron-doped carbon dots modified with acetamiprid aptamer; (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: (1) Preparation of molybdenum disulfide-gold nanoparticle-methylene blue composite material modified electrode: a. Dissolve molybdenum source and sulfur source in deionized water, mix them evenly, and carry out a hydrothermal reaction. After the reaction is completed, wash and dry the material to obtain MoS2 material. b. Dissolve sodium citrate and tannic acid in deionized water, add MoS2, stir, continue to add chloroauric acid, and react with stirring. After the reaction is complete, wash and dry to obtain MoS2 / Au material. c. Mix MoS2 / Au material with methylene blue, and then wash and dry to obtain MoS2 / Au / MB material; d. Add MoS2 / Au / MB material to deionized water to obtain MoS2 / Au / MB dispersion. Drop the MoS2 / Au / MB dispersion onto the surface of a glassy carbon electrode and let it air dry to obtain a glassy carbon electrode modified with MoS2 / Au / MB composite material, denoted as MoS2 / Au / MB / GC electrode. (2) Preparation of MoS2 / Au / MB / cDNA modified electrode: Acetamiprid aptamer complementary DNA was added dropwise to the surface of the MoS2 / Au / MB / GC electrode prepared in (1). After the addition was completed, the electrode was incubated at 37 °C. Then, 5~10 mL of 6-mercaptohex-1-ol was added dropwise for 1 h to block the electrode. The unbound MCH was washed away with pH 7.4 0.01 mol / L phosphate buffer solution and the electrode was dried at room temperature to obtain the MoS2 / Au / MB / cDNA modified electrode. (3) Preparation of iron-doped carbon nanozymes: iron salt and carbon source were dissolved in deionized water and reacted by microwave heating. After the reaction was completed, the mixture was dialyzed overnight using a dialysis bag with a molecular weight of 500 and then freeze-dried to obtain Fe-CDs nanozymes. (4) Preparation of iron-doped carbon nanozymes modified with acetamiprid aptamers: Fe-CDs nanozymes were dispersed in a pH 7.4 10 mM phosphate buffer solution containing glutaraldehyde, stirred at room temperature, filtered under vacuum, washed with deionized water to remove residual glutaraldehyde, and dried under vacuum; the obtained solid was dispersed in a pH 7.4 10 mM phosphate buffer solution, amino-functionalized aptamers were added and incubated overnight, and unbound aptamers were washed with a pH 7.4 10 mM phosphate buffer solution to obtain Apt / Fe-CDs nanozymes; (5) Preparation of MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode: Apt / Fe-CDs nanozyme was dispersed in pH=7.410 mM phosphate buffer solution to obtain Apt / Fe-CDs nanozyme solution. Apt / Fe-CDs nanozyme solution was dropped onto the surface of MoS2 / Au / MB / cDNA modified electrode and dried at room temperature to obtain MoS2 / Au / MB / cDNA@Apt / Fe-CDs modified electrode, which is a ratiometric electrochemical aptamer sensor assisted by iron-doped carbon dot nanozyme.

3. The preparation method according to claim 2, characterized in that, Includes one or more of the following technical features: In step (1), the molybdenum source in step a is selected from ammonium molybdate, sodium molybdate, or molybdenum chloride, and the sulfur source is selected from thioacetamide, thiourea, or L-cysteine. In step (1), the molar ratio of molybdenum source to sulfur source in step a is 1:(10~20). In step (1), the hydrothermal reaction temperature in step a is 150℃~250℃, and the reaction time is 20 h~10 h. In step (1), step b, the mass ratio of sodium citrate to tannic acid is 1:(0.002~0.005). In step (1), step b, the mass ratio of chloroauric acid to sodium citrate is 1:(2~8). In step (1), the reaction temperature in step b is 100℃~200℃ and the reaction time is 24 h~48 h.

4. The preparation method according to claim 2, characterized in that, In step (2), the volume of cDNA added to the surface of the MoS2 / Au / MB modified electrode is 30-200 mL.

5. The preparation method according to claim 2, characterized in that, Includes one or more of the following technical features: In step (3), the iron salt is selected from ferric chloride, ferric nitrate, or ferric sulfate, and the carbon source is selected from o-phenylenediamine or p-phenylenediamine; In step (3), the mass ratio of iron salt to carbon source is 1: (1~15). In step (3), the reaction temperature is 150~250℃ and the reaction time is 2 h~16 h.

6. The preparation method according to claim 2, characterized in that, Includes one or more of the following technical features: In step (4), the mass-to-volume ratio (g / mL) of Fe-CDs nanozyme to glutaraldehyde is 1:(3~8). In step (5), the concentration of the Apt / Fe-CDs nanozyme solution is 1~10 mg / mL.

7. The iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor prepared by any one of claims 1 to 6, or the iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor of claim 7, in the detection of acetamiprid.

9. A method for detecting imidacloprid, characterized in that, The iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor prepared by any one of the preparation methods of claims 1 to 6, or the iron-doped carbon dot nanozyme-assisted ratiometric electrochemical aptamer sensor of claim 7, is used as the working electrode for detection.

10. The method as described in claim 9, characterized in that, Includes the following steps: 1) Preparation of standard solutions: Weigh different masses of acetamiprid and add them to ethanol to prepare standard solutions, so that the concentration of acetamiprid solution is between 0.1 nmol / L and 100 nmol / L; 2) Construction of the standard curve: Using an iron-doped carbon nanoparticle-assisted ratiometric electrochemical aptamer sensor as the working electrode, Ag-AgCl as the reference electrode, and platinum as the counter electrode, differential pulse voltammetry was used to detect the acetamiprid standard solutions of different concentrations prepared in step 1). The electrochemical signal detected at -0.3 V was denoted as I. (-0.3V) The electrochemical signal detected at -0.5V is denoted as I. (-0.5V) The ratio of the two is I (-0.5V) / I (-0.3V) Construct a standard curve with the corresponding acetamiprid standard solution; 3) Actual sample testing: Following the testing method in step 2, replace the standard solution with the acetamiprid sample solution to be tested and perform the test. The result is I. (-0.3V) and I (-0.5V) , will I (-0.5V) / I (-0.3V) By substituting the values ​​into the standard curve, acetamiprid in actual samples can be detected.

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