Construction and application of electrochemical luminescence sensor for high-sensitivity detection of N-nitrosodimethylamine content

Abstract

The invention belongs to the field of electrochemical sensing, and particularly relates to construction and application of an electrochemical luminescence sensor for detecting the content of N-nitrosodimethylamine with high sensitivity. The Cas12a enzyme improves the specificity and realizes signal amplification through the recognition mechanism and trans-cleavage capability of the Cas12a enzyme. The ATT-AuNCs is used as an efficient electrochemical luminescence emitter, so that the transition of a signal is promoted. After a sample is added, the Cas12a-crRNA compound can specifically recognize a DNA activator to trigger cutting of non-specific DNA which is modified on a glassy carbon electrode and is marked by ferrocene, so that the electrochemical luminescence intensity is increased. The NDMA in the sample can be detected through the method, so that the content of nitrosamine in food can be detected. The CRISPR / Cas12a is combined with an electrochemical luminescence technology, so that a sensitive and rapid NDMA detection means is provided, and a new idea is provided for detecting the content of cancerogenic substances in various foods.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical sensing, and specifically relates to the construction and application of an electrochemical luminescence sensor for highly sensitively detecting the content of N-nitrosodimethylamine. Background Art

[0002] N-nitrosamines are extremely toxic and carcinogenic compounds, usually produced by the reaction of nitrites and nitrates with proteins in meat during the process of pickling, curing and smoking. These toxic compounds not only pollute the environment, but also have serious impacts on public health. Studies have shown that N-nitrosodimethylamine (NDMA) is one of the strongest carcinogens and has been listed as an animal carcinogen by the American Conference of Governmental Industrial Hygienists (ACGIH) and identified as a possible human carcinogen by the U.S. Environmental Protection Agency (EPA). Therefore, NDMA needs to be monitored at extremely low concentrations to prevent harm to public health. At present, a variety of methods have been developed to detect low concentrations of NDMA. These methods include traditional detection techniques and emerging spectroscopic techniques. Traditional detection methods mainly include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Although these methods can meet the needs of quantitative analysis, their complex operating procedures, high costs and time-consuming problems still limit the rapid detection of NDMA. To overcome these limitations, researchers have proposed a variety of spectroscopic techniques for NDMA screening. Currently, different spectroscopic techniques have been proposed for NDMA screening, including ultraviolet-visible spectroscopy (UV-Vis), Raman spectroscopy, and fluorescence spectroscopy. However, these methods usually require external light source excitation, which may cause signal interference and reduce detection accuracy. Therefore, there is an urgent need for an NDMA monitoring solution that is both environmentally friendly and reliable.

[0003] Electrochemiluminescence (ECL) technology combines the principles of electrochemistry and chemiluminescence. It generates electroactive substances by applying voltage to electrodes, which further react to produce chemiluminescence. In this process, electrical signals are converted into light signals. Due to the advantages of simple equipment requirements, low background noise, high sensitivity and wide dynamic range, electrochemiluminescence has been widely used in medical diagnosis, food analysis and environmental monitoring in recent years. CRISPR-Cas12a is a class II VA type CRISPR-associated enzyme that can be guided by single-stranded crRNA to cut targeted DNA or RNA fragments, and trigger the cutting of non-specific ssDNA through the complementary binding of double-stranded DNA (dsDNA) or crRNA to target ssDNA. Due to its unique functions, CRISPR-Cas12a has been widely used in biological analysis, such as the detection of nucleic acids and small molecules. It is able to convert biological features into detectable signals and amplify the output signals. Based on these advantages, the combination of CRISPR / Cas system and ECL technology may become an ideal choice in the field of biosensing. Summary of the invention

[0004] The purpose of the present invention is to provide a construction and application of an electrochemiluminescence sensor for highly sensitively detecting the content of N-nitrosodimethylamine.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for constructing an electrochemiluminescent sensor for highly sensitively detecting the content of N-nitrosodimethylamine comprises the following steps: S1: Take the streptavidin magnetic bead solution, place it on a magnetic stand for magnetic separation, remove the supernatant, wash the magnetic beads with a PBS buffer solution containing 0.05% Tween 20, and obtain washed magnetic beads; add the aptamer DNA solution to the washed magnetic beads, react at 20-45°C for 0.5-3h, and obtain the aptamer-modified magnetic bead solution; add the activator DNA solution to the aptamer-modified magnetic bead solution, react at 20-45°C for 0.5-3h, and then place it on a magnetic stand for magnetic separation, remove the supernatant, wash the magnetic beads with a PBS buffer solution, and resuspend and disperse in a PBS buffer solution to obtain a hybrid complex-modified magnetic bead solution; S2: Mix the Cas12a protein solution with the crRNA solution, react at 20-45°C for 10-45 minutes to obtain a Cas12a-crRNA complex solution; add the sample solution to be tested to the hybrid complex modified magnetic bead solution, react at 20-45°C for 0.5-3 hours, and then place it on a magnetic stand for magnetic separation, transfer the supernatant to the Cas12a-crRNA complex solution, and react at 20-45°C for 10-45 minutes to obtain an activated Cas12a-crRNA complex solution; S3: dripping 6-aza-2-thiothymine-gold nanocluster solution onto the surface of a bare glassy carbon electrode, drying at 37°C overnight, to obtain modified electrode 1; dripping ssDNA solution onto the surface of modified electrode 1, incubating at 20-45°C for 0.5-4h, and then rinsing the electrode with PBS buffer solution, to obtain modified electrode 2; dripping activated Cas12a-crRNA complex solution onto the surface of modified electrode 2, incubating at 20-45°C for 0.5-4h, and then rinsing the electrode with PBS buffer solution, to obtain modified electrode 3; S4: Using PBS buffer solution containing triethanolamine as the electrochemiluminescence working solution, using modified electrode 3 as the working electrode to construct a three-electrode system, using an electrochemiluminescence detection device to measure and collect electrochemiluminescence signals, and thus completing the construction of the electrochemiluminescence sensor; The sequence of the aptamer DNA is: 5'-CAACAGTTGCGGCAGCCTGTCCTG-3'; The sequence of the activator DNA is: 5′-CCCAGGTAAACACACAAACCTTCCGCAACTGTTG-Biotin-3′; The sequence of ssDNA is: 5′-Fc-AACCGCTTCCCCGACTTCC-SH-3′; The sequence of crRNA is: 5′-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3′; Step S1 is specifically as follows: S1-1: Take 200 μL of a 1 mg / mL streptavidin magnetic bead solution, place it on a magnetic stand for magnetic separation, remove the supernatant, and wash the magnetic beads with a PBS buffer solution containing 0.05% Tween 20 to obtain washed magnetic beads; S1-2: Add 200 μL of 10 μM aptamer DNA to the magnetic beads washed in S1-1, react at 37°C for 2 h, and obtain an aptamer-modified magnetic bead solution; S1-3: Add 200 μL of 10 μM activator DNA to the aptamer-modified magnetic bead solution of S1-2, react at 37°C for 2 h, and then place on a magnetic stand for magnetic separation. Remove the supernatant, wash the magnetic beads with PBS buffer solution, and then resuspend and disperse in 200 μL PBS buffer solution to obtain a hybrid complex-modified magnetic bead solution; Step S2 is specifically as follows: S2-1: Mix 10 μL of 50 nM Cas12a protein solution with 10 μL of 50 nM crRNA solution, and react at 37 °C for 30 min to obtain a Cas12a-crRNA complex solution; S2-2: Add 20 μL of the sample solution to be tested to the hybrid complex modified magnetic bead solution of S1-3, react at 37°C for 2 hours, and then place it on a magnetic stand for magnetic separation. Transfer the supernatant and add it to the Cas12a-crRNA complex solution of S2-1, react at 37°C for 30 minutes to obtain an activated Cas12a-crRNA complex solution; Step S3 is as follows: S3-1: synthesizing ATT-AuNCs, dissolving and dispersing them in deionized water to obtain an ATT-AuNCs solution; S3-2: 8 μL of 3 mg / mL ATT-AuNCs solution was added dropwise to the surface of the bare glassy carbon electrode and dried at 37°C overnight to obtain modified electrode 1; S3-3: Take 8 μL of 5 μM ssDNA solution and drop it onto the surface of modified electrode 1 of S3-2, incubate at 37°C for 2 h, and rinse the electrode surface with PBS buffer solution to obtain modified electrode 2; S3-4: Take 10 μL of the activated Cas12a-crRNA complex solution of S2-2 and add it dropwise to the surface of the modified electrode 2 of S3-3, incubate at 37°C for 2 h, and rinse the electrode surface with PBS buffer solution to obtain a modified electrode 3; Step S4 is specifically as follows: S4-1: adding triethanolamine with a final concentration of 0.2 M to the PBS buffer solution to obtain an electrochemiluminescence working solution; S4-2: A three-electrode system is composed of an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and the modified electrode 3 of S3-4 as a working electrode. The system is connected to an electrochemiluminescence detection device. 1 mL of the electrochemiluminescence working solution of S4-1 is added to the electrochemiluminescence cell for electrochemiluminescence test. The test method used is cyclic voltammetry. The applied voltage is 0 V ~ 1.2 V, the scanning rate is 0.1 V / s, and the electrochemiluminescence signal is collected using an ultra-weak luminescence meter. The high voltage of the photomultiplier tube is -800 V, thereby completing the construction of the electrochemiluminescence sensor.

[0006] An electrochemiluminescence sensor for highly sensitively detecting the content of N-nitrosodimethylamine is constructed by adopting the above-mentioned construction method.

[0007] Application of the above electrochemiluminescence sensor in detecting N-nitrosodimethylamine.

[0008] The significant advantages of the present invention are: (1) The required raw materials are simple and easy to obtain, and no complicated synthesis steps are required. At the same time, the use of magnetic separation technology avoids the complex sample pretreatment in traditional detection methods, greatly shortens the detection time, and is suitable for on-site detection and high-throughput screening.

[0009] (2) The present invention uses ATT-AuNCs as a high-efficiency luminescent material. After combining with ssDNA, the stability and responsiveness of the electrochemiluminescence signal are greatly improved through precise modification of the electrode surface.

[0010] (3) The present invention combines the CRISPR / Cas12a system to achieve signal amplification and improve sensitivity.

[0011] (4) The method of the present invention can be directly used to detect the carcinogen NDMA in food, and shows a good linear response to NDMA in the concentration range from 5pM to 500nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Schematic diagram of the detection principle of the present invention.

[0013] Figure 2 :The feasibility verification of the present invention.

[0014] Figure 3 : Electrochemiluminescence spectra corresponding to different concentrations of NDMA. The NDMA concentrations from a to g are 5pM, 10pM, 100pM, 1nM, 10nM, 100nM, and 500nM, respectively.

[0015] Figure 4 : Working curve drawn based on the linear relationship between the luminescence signal intensity value and the logarithmic value of NDMA concentration.

[0016] Figure 5 : The detection specificity results of the present invention. NDEA: N-nitrosodiethylamine; NDPA: N-nitrosodi-n-propylamine; NDPhA: N-nitrosodiphenylamine; NPYR: N-nitrosopyrrolidine; mixture: NDEA+NDPA+NDPhA+NPYR+NDMA. DETAILED DESCRIPTION

[0017] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0018] The streptavidin-labeled magnetic bead solution used in the following examples was purchased from Promega Corporation, with the product number Z5481, and the magnetic bead particle size was 1 μm.

[0019] The sequence of the aptamer DNA used in the following examples is: 5'-CAACAGTTGCGGCAGCCTGTCCTG-3'.

[0020] The sequence of the activator DNA used in the following examples is: 5'-CCCAGGTAAACACACAAACCTTCCGCAACTGTTG-Biotin-3'.

[0021] The sequence of the ssDNA used in the following examples is: 5'-Fc-AACCGCTTCCCCGACTTCC-SH-3'.

[0022] The sequence of the crRNA used in the following examples is: 5'-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3'.

[0023] The Cas12a protein used in the following examples was purchased from Nearshore Protein Company, catalog number E373-YH01.

[0024] Embodiment 1: Construct an electrochemiluminescent sensor for highly sensitive detection of N-nitrosodimethylamine (NDMA) content based on CRISPR / Cas12a and use electrochemiluminescence method to detect NDMA. The steps are as follows: S1: Preparation of hybrid complex modified magnetic beads: S1-1: Take 200 μL of 1 mg / mL streptavidin-labeled magnetic bead solution, place it on a magnetic stand for magnetic separation, remove the supernatant, and wash the magnetic beads three times with PBS buffer solution (0.01 M, pH 7.4) containing 0.05% Tween 20, each time for 3 min, to obtain washed magnetic beads; S1-2: Add 200 μL of 10 μM aptamer DNA to the magnetic beads washed in S1-1, and react at 37°C for 2 h to modify the aptamer DNA on the surface of the magnetic beads, thereby obtaining an aptamer-modified magnetic bead solution; S1-3: Add 200 μL of 10 μM activator DNA to the aptamer-modified magnetic bead solution of S1-2, react at 37°C for 2 h to allow the aptamer DNA modified on the surface of the magnetic beads to hybridize with the activator DNA, then place on a magnetic stand for magnetic separation, remove the supernatant, wash the magnetic beads with PBS buffer solution (0.01 M, pH 7.4) 3 times, 3 min each time, then resuspend in 200 μL PBS buffer solution (0.01 M, pH 7.4) to obtain a hybrid complex-modified magnetic bead solution, which is stored at 4°C for later use.

[0025] S2: Activation of non-specific cleavage activity of CRISPR / Cas12a: S2-1: Mix 10 μL of 50 nM Cas12a protein solution with 10 μL of 50 nM crRNA solution, and react at 37 °C for 30 min to obtain a Cas12a-crRNA complex solution; S2-2: Add 20 μL of the sample solution containing NDMA to the hybrid complex modified magnetic bead solution of S1-3, react at 37 ° C for 2 hours, and then place it on a magnetic stand for magnetic separation. Transfer the supernatant and add it to the Cas12a-crRNA complex solution of S2-1, and react at 37 ° C for 30 minutes to activate the cleavage activity of Cas12a protein to obtain an activated Cas12a-crRNA complex solution.

[0026] S3: Modification process of working electrode S3-1: Add NaOH with a final concentration of 0.2 M to the ATT solution with a concentration of 0.08 M to obtain a NaOH-ATT solution; take 15 mL of the NaOH-ATT solution and add it to 15 mL of the HAuCl4 solution with a concentration of 10 mg / mL, stir at room temperature in the dark for 1 hour to obtain a golden solution; dialyze with a 50 kDa dialysis bag for 24 hours and freeze-dry overnight to obtain a golden solid powder, namely ATT-AuNCs, which is dissolved and dispersed in deionized water to obtain an ATT-AuNCs solution; S3-2: 8 μL of 3 mg / mL ATT-AuNCs solution was added dropwise to the surface of the bare glassy carbon electrode and dried at 37°C overnight to obtain modified electrode 1; S3-3: Take 8 μL of 5 μM ssDNA solution and drop it onto the surface of modified electrode 1 of S3-2. Incubate at 37 °C for 2 h to fix ssDNA to the ATT-AuNCs modified electrode through Au-S bonds. Rinse the electrode surface with PBS buffer solution (0.01 M, pH 7.4) to obtain modified electrode 2. S3-4: Take 10 μL of the activated Cas12a-crRNA complex solution of S2-2 and add it dropwise to the surface of the modified electrode 2 of S3-3, incubate at 37°C for 2 h, rinse the electrode surface with PBS buffer solution (0.01 M, pH 7.4) to obtain the modified electrode 3, which is the working electrode.

[0027] S4: Electrochemiluminescence test S4-1: Add triethanolamine (TEA) with a final concentration of 0.2 M to a PBS buffer solution (0.1 M, pH 11.5) to obtain an electrochemiluminescence working solution; S4-2: The Ag / AgCl electrode is used as the reference electrode, the platinum wire electrode is used as the counter electrode, and they form a three-electrode system with the working electrode of S3-4, which is connected to the electrochemiluminescence detection equipment. 1mL of the electrochemiluminescence working solution of S4-1 is added to the electrochemiluminescence cell for electrochemiluminescence test. The test method used is cyclic voltammetry. The applied voltage is 0V~1.2V, the scanning rate is 0.1V / s, and the electrochemiluminescence signal is collected using an ultra-weak luminescence meter. The high voltage of the photomultiplier tube is -800V. The peak value of the electrochemiluminescence signal is taken as the signal readout method to obtain the luminescence signal intensity value.

[0028] The detection principle of the present invention is shown in Figure 1 The design idea mainly consists of two parts: one part is electrode modification: the surface of the bare glassy carbon electrode is modified with the luminescent reagent ATT-AuNCs. When the ssDNA modified with thiol at one end and ferrocene at the other end is connected to the electrode surface through Au-S bond, ferrocene quenches the luminescence of ATT-AuNCs; the other part is the activation of CRISPR-Cas12a and its shearing of ssDNA: the surface of the magnetic beads is connected with aptamer DNA and activation chain DNA with complementary base pairing. NDMA specifically binds to the aptamer DNA to compete with the activation chain DNA and collect it by magnetic separation, which is used to further activate the nonspecific shearing activity of CRISPR-Cas12a. After the Cas12a protein shears the ssDNA on the electrode surface, ferrocene is far away from the electrode surface, and the luminescence signal of ATT-AuNCs is restored.

[0029] Embodiment 2: a: The construction of the electrochemiluminescence sensor is basically the same as that of Example 1, except that the modified electrode 1 is used as the working electrode to detect the NDMA standard solution with a concentration of 50 ng / mL.

[0030] b: The construction of the electrochemiluminescence sensor is basically the same as that of Example 1, except that the modified electrode 2 is used as the working electrode to detect the NDMA standard solution with a concentration of 50 ng / mL.

[0031] c: The construction of the electrochemiluminescence sensor is basically the same as that of Example 1, except that: the NDMA standard solution with a concentration of 50 ng / mL is detected.

[0032] d: The construction of the electrochemiluminescence sensor is basically the same as that in Example 1, except that: S2-2: 20 μL of ultrapure water was added to the hybrid complex modified magnetic bead solution of S1-3, reacted at 37 ° C for 2 h, and then placed on a magnetic stand for magnetic separation, and the supernatant was transferred and added to the Cas12a-crRNA complex solution of S2-1, and reacted at 37 ° C for 30 min to obtain an unactivated Cas12a-crRNA complex solution; S3-4: 10 μL of the unactivated Cas12a-crRNA complex solution of S2-2 was added dropwise to the surface of the modified electrode 2 of S3-3, incubated at 37 ° C for 2 h, and the electrode surface was rinsed with PBS buffer solution (0.01 M, pH 7.4) to obtain a working electrode.

[0033] The feasibility verification results are as follows Figure 2 As shown in the figure, the electrochemiluminescence signal of group a is strong; the working electrode of group b is connected to ssDNA modified with ferrocene on the basis of group a, and the electrochemiluminescence signal is quenched; the working electrode of group c is modified on the basis of group b, the Cas12a protein whose shearing activity is activated due to the presence of NDMA, the ssDNA is sheared so that the ferrocene is away from the electrode surface, and the electrochemiluminescence signal is restored; the working electrode of group d is modified on the basis of group b, the Cas12a protein without NDMA and the shearing activity is not activated, the ssDNA is not sheared, and the electrochemiluminescence signal is not restored.

[0034] Embodiment 3: The electrochemiluminescent sensor was constructed by referring to the steps of Example 1, and NDMA standard solutions of different concentrations (5pM, 10pM, 100pM, 1nM, 10nM, 100nM, 500nM) were tested to obtain the change of electrochemiluminescent signal with the increase of NDMA concentration. Figure 3 Further, according to the linear relationship between the obtained luminescent signal intensity value and the logarithmic value of the NDMA standard solution concentration, a working curve is drawn, as shown in FIG. Figure 4 As shown, the corresponding linear regression equation is: ECL=1554.9lg C+ 1384.8 (R 2 =0.9971), the detection range of the linear regression equation was 5pg / mL~500ng / mL, and the minimum detection limit was 0.37pg / mL.

[0035] Embodiment 4: An electrochemiluminescent sensor was constructed by referring to the steps of Example 1, and NDEA standard solution (1 μM), NDPA standard solution (1 μM), NDPhA standard solution (1 μM), NPYR standard solution (1 μM), NDMA standard solution (100 nM) and mixed standard solution (1 μM NDEA + 1 μM NDPA + 1 μM NDPhA + 1 μM NPYR + 100 nM NDMA) were tested. The results are shown in Figure 1. Figure 5 As shown in the figure, it can be seen that the electrochemiluminescent sensor of the present invention has a high specificity for NDMA.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for constructing an electrochemiluminescent sensor for highly sensitive detection of N-nitrosodimethylamine content, characterized in that: The following steps are involved: S1: Take the streptavidin magnetic bead solution, place it on a magnetic stand for magnetic separation, remove the supernatant, wash the magnetic beads with a PBS buffer solution containing 0.05% Tween 20, and obtain washed magnetic beads; add the aptamer DNA solution to the washed magnetic beads, react at 20-45°C for 0.5-3h, and obtain the aptamer-modified magnetic bead solution; add the activator DNA solution to the aptamer-modified magnetic bead solution, react at 20-45°C for 0.5-3h, and then place it on a magnetic stand for magnetic separation, remove the supernatant, wash the magnetic beads with a PBS buffer solution, and resuspend and disperse in a PBS buffer solution to obtain a hybrid complex-modified magnetic bead solution; S2: Mix the Cas12a protein solution with the crRNA solution, react at 20-45°C for 10-45 minutes to obtain a Cas12a-crRNA complex solution; add the sample solution to be tested to the hybrid complex modified magnetic bead solution, react at 20-45°C for 0.5-3 hours, and then place it on a magnetic stand for magnetic separation, transfer the supernatant to the Cas12a-crRNA complex solution, and react at 20-45°C for 10-45 minutes to obtain an activated Cas12a-crRNA complex solution; S3: dripping 6-aza-2-thiothymine-gold nanocluster solution onto the surface of a bare glassy carbon electrode, drying at 37°C overnight, to obtain modified electrode 1; dripping ssDNA solution onto the surface of modified electrode 1, incubating at 20-45°C for 0.5-4h, and then rinsing the electrode with PBS buffer solution, to obtain modified electrode 2; dripping activated Cas12a-crRNA complex solution onto the surface of modified electrode 2, incubating at 20-45°C for 0.5-4h, and then rinsing the electrode with PBS buffer solution, to obtain modified electrode 3; S4: Using PBS buffer solution containing triethanolamine as the electrochemiluminescence working solution, using modified electrode 3 as the working electrode to construct a three-electrode system, using an electrochemiluminescence detection device to measure and collect electrochemiluminescence signals, and thus completing the construction of the electrochemiluminescence sensor; The sequence of the aptamer DNA is: 5'-CAACAGTTGCGGCAGCCTGTCCTG-3'; The sequence of the activator DNA is: 5′-CCCAGGTAAACACACAAACCTTCCGCAACTGTTG-Biotin-3′; The sequence of ssDNA is: 5′-Fc-AACCGCTTCCCCGACTTCC-SH-3′; The sequence of crRNA is: 5′-UAAUUUCUACUAAGUGUAGAUAAGGUUUGUGUGUUUACCUG-3′.

2. The construction method according to claim 1, characterized in that: Step S1 is specifically as follows: S1-1: Take 200 μL of a 1 mg / mL streptavidin magnetic bead solution, place it on a magnetic stand for magnetic separation, remove the supernatant, and wash the magnetic beads with a PBS buffer solution containing 0.05% Tween 20 to obtain washed magnetic beads; S1-2: Add 200 μL of 10 μM aptamer DNA to the magnetic beads washed in S1-1, react at 37°C for 2 h, and obtain an aptamer-modified magnetic bead solution; S1-3: Add 200 μL of 10 μM activator DNA to the aptamer-modified magnetic bead solution of S1-2, react at 37°C for 2 h, and then place on a magnetic stand for magnetic separation. Remove the supernatant, wash the magnetic beads with PBS buffer solution, and then resuspend them in 200 μL PBS buffer solution to obtain a hybrid complex-modified magnetic bead solution.

3. The construction method according to claim 2, characterized in that: Step S2 is specifically as follows: S2-1: Mix 10 μL of 50 nM Cas12a protein solution with 10 μL of 50 nM crRNA solution, and react at 37 °C for 30 min to obtain a Cas12a-crRNA complex solution; S2-2: Add 20 μL of the sample solution to be tested to the hybrid complex modified magnetic bead solution of S1-3, react at 37 ° C for 2 hours, and then place it on a magnetic stand for magnetic separation. Transfer the supernatant and add it to the Cas12a-crRNA complex solution of S2-1, react at 37 ° C for 30 minutes to obtain an activated Cas12a-crRNA complex solution.

4. The construction method according to claim 3, characterized in that: Step S3 is as follows: S3-1: synthesizing ATT-AuNCs, dissolving and dispersing them in deionized water to obtain an ATT-AuNCs solution; S3-2: 8 μL of 3 mg / mL ATT-AuNCs solution was added dropwise to the surface of the bare glassy carbon electrode and dried at 37°C overnight to obtain modified electrode 1; S3-3: Take 8 μL of 5 μM ssDNA solution and drop it onto the surface of modified electrode 1 of S3-2, incubate at 37°C for 2 h, and rinse the electrode surface with PBS buffer solution to obtain modified electrode 2; S3-4: Take 10 μL of the activated Cas12a-crRNA complex solution of S2-2 and add it dropwise to the surface of the modified electrode 2 of S3-3, incubate at 37°C for 2 h, rinse the electrode surface with PBS buffer solution to obtain the modified electrode 3.

5. The construction method according to claim 4, characterized in that: Step S4 is specifically as follows: S4-1: adding triethanolamine with a final concentration of 0.2 M to the PBS buffer solution to obtain an electrochemiluminescence working solution; S4-2: A three-electrode system is composed of an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and the modified electrode 3 of S3-4 as a working electrode. The system is connected to an electrochemiluminescence detection device. 1 mL of the electrochemiluminescence working solution of S4-1 is added to the electrochemiluminescence cell for electrochemiluminescence test. The test method used is cyclic voltammetry. The applied voltage is 0 V ~ 1.2 V, the scanning rate is 0.1 V / s, and the electrochemiluminescence signal is collected using an ultra-weak luminescence meter. The high voltage of the photomultiplier tube is -800 V, thereby completing the construction of the electrochemiluminescence sensor.

6. An electrochemiluminescent sensor for highly sensitive detection of N-nitrosodimethylamine content, characterized in that: The method is constructed by any one of claims 1 to 5.

7. Use of the electrochemiluminescence sensor according to claim 6 in detecting N-nitrosodimethylamine.

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