Electrochemiluminescence sensor based on defect engineering strategy enhancement and preparation method and application thereof

Abstract

The invention belongs to the technical field of electrochemical luminescence analysis and detection, and relates to an electrochemical luminescence sensor based on defect engineering strategy enhancement and a preparation method and application thereof. Comprising the following steps: mixing g-C3N4, thiourea and Nb2C Mxene, calcining to obtain a g-C3N4-NV / S-Nb2C compound, dispensing a g-C3N4-NV / S-Nb2C dispersion liquid on a GCE electrode, drying, dropwise adding a split aptamer 1 solution, incubating at room temperature, dropwise adding a split aptamer 2 and enrofloxacin mixed solution, finally obtaining an aptamer-modified ENR / apt / g-C3N4-NV / S-Nb2C electrode as a working electrode, constructing a three-electrode system, and detecting the temperature of the three-electrode system to obtain a g-C3N4 / NV / S-Nb2C composite electrode. And introducing into a PBS (Phosphate Buffer Solution) containing potassium persulfate, and detecting the enrofloxacin. The electrochemical luminescence sensor has the characteristics of simplicity, convenience and flexibility in operation, wide detection range, low detection limit, low detection cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemiluminescence analysis and detection, and specifically relates to an electrochemiluminescence sensor enhanced based on a defect engineering strategy, a preparation method and an application thereof, and more particularly to an electrochemiluminescence sensor of a g-C3N4-NV / S-Nb2C composite material that enhances the luminescence of g-C3N4 based on a defect engineering strategy, a preparation method thereof and an application thereof in the detection of enrofloxacin (ENR). Background Art

[0002] Antibiotics are a class of chemical substances that can affect normal life activities such as cell growth and development. They are derived from microorganisms including bacteria, fungi, actinomycetes or higher animals and plants, and are secondary metabolites with anti-pathogen or other activities produced in their life activities. As the scope of antibiotic use gradually expands, the phenomenon of antibiotic abuse is also emerging in an endless stream. Since antibiotics cannot be fully absorbed and utilized, excessive antibiotics are either excreted into the environment or remain in the body of animals, and eventually act on the human body through food enrichment, causing damage to human health.

[0003] Enrofloxacin (ENR) is a low-cost and effective fluoroquinolone antibiotic that inhibits the activity of DNA gyrase and topoisomerase IV from Gram-negative bacteria. ENR is widely used to control mycoplasma infections and bacterial diseases in animal husbandry. However, the biodegradation and absorption capacity of ENR are poor and unsatisfactory. The abuse of ENR in food and feed, or improper use in animal farming, may cause residues to accumulate in the human body, causing harm to the bone and cartilage development of children and adolescents, and also disrupting the balance of intestinal microecology. Therefore, its use in food and feed should be strictly controlled. Referring to the EU veterinary drug residue limit standard, the maximum residue limit of ENR is 100 mg / kg.

[0004] At present, ENR in actual samples is mainly analyzed by various methods such as chromatography technology and enzyme immunoassay. However, the use of these analytical methods relies on expensive instruments, professional experimental operators, and requires complex pretreatment. Therefore, it is crucial to further develop new ENR detection methods with high selectivity and sensitivity.

[0005] Electrochemiluminescence (ECL), also known as electrochemiluminescence, is the product of the combination of chemiluminescence and electrochemistry. It refers to the generation of some electrogenerated substances on the electrode surface by applying a certain voltage for electrochemical reaction, and then these electrogenerated substances or between the electrogenerated substances and certain components in the system form an excited state through electron transfer, and then return from the excited state to the ground state to produce a luminescent phenomenon. Electrochemiluminescence involves a very wide range of fields and has a very broad application prospect. Electrochemiluminescence has the characteristics of high sensitivity, wide linear range, simple instruments and equipment, easy operation, intersection with many disciplines, and a wide range of analytical applications. Therefore, it has become one of the hot spots for scientific researchers.

[0006] Defect engineering is an important strategy to improve the electrochemical luminescence efficiency of photoactive materials. The place where the perfect periodic arrangement of atoms or molecules in chemical materials is disrupted or destroyed is called a crystal defect, which is simply called a defect in chemistry. Defects inevitably exist in all photoactive materials. The ECL emission of nanomaterial systems mostly originates from surface defects and can be used to reflect the surface state. Therefore, ECL can be used as a characterization method to identify surface defects. Conversely, defects can be used as a reference to screen new ECL materials, and can also be used as a modulation object for modified ECL. ECL emission is extremely sensitive to defects. Therefore, increasing the defect concentration of highly active ECL and passivating defects that are not conducive to ECL emission are the two main means to improve ECL efficiency. Summary of the invention

[0007] The present invention provides an electrochemical luminescence sensor of a g-C3N4-NV / S-Nb2C composite material based on defect engineering strategy to enhance the luminescence of g-C3N4, a preparation method thereof, and an application thereof in detecting enrofloxacin (ENR). An electrochemical sensor with high detection sensitivity, wide detection range, and low detection limit is obtained and applied to ENR detection, and the detection cost is low.

[0008] In the first aspect, the present invention provides an electrochemiluminescence sensor enhanced based on a defect engineering strategy, comprising a glassy carbon electrode apt / g-C3N4-NV / S-Nb2C / GCE modified with a g-C3N4-NV / S-Nb2C complex and an aptamer with a specific recognition function; wherein the g-C3N4-NV / S-Nb2C complex and the aptamer are connected via a phosphoamide bond formed by an amino group on g-C3N4 and a phosphate group on the aptamer; and the g-C3N4-NV / S-Nb2C complex is obtained by mixed calcination of g-C3N4, thiourea and Nb2C Mxene.

[0009] In some optional embodiments, the aptamer with specific recognition function is an ENR aptamer, and the nucleotide sequences of the two chains of the aptamer are: ENR-2 chain: 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCGGC-3' and ENR-1 chain: 5'-ATC TCT GAG CCC GGG TTA TTT CAG GGG GA-3', and the combination of the two split aptamers can specifically recognize ENR.

[0010] In a second aspect, the present invention also provides a method for preparing the above-mentioned electrochemiluminescence sensor enhanced based on defect engineering strategy, comprising: A certain amount of g-C3N4, thiourea, and Nb2C Mxene were dispersed in deionized water, ultrasonically treated, and then placed in a tube furnace for calcination at 520 °C and annealed for two hours to obtain a g-C3N4-NV / S-Nb2C composite. The g-C3N4-NV / S-Nb2C composite is drop-coated on a clean glassy carbon electrode surface to obtain a g-C3N4-NV / S-Nb2C / GCE modified electrode; The ENR split aptamer ENR-1 chain was drop-coated and incubated on the g-C3N4-NV / S-Nb2C / GCE modified electrode to obtain the electrochemiluminescence sensor apt / g-C3N4-NV / S-Nb2C / GCE.

[0011] In some optional embodiments, the mass percentage of the Nb2C Mxene to the g-C3N4-NV is 2-5%, preferably 3%; the mass ratio of the thiourea to the Nb2C Mxene is 10:1.

[0012] In some optional embodiments, the g-C3N4-NV / S-Nb2C composite is applied to the glassy carbon electrode in an amount of 4.0 μL and a concentration of 0.5-2.0 mg / mL.

[0013] In some optional embodiments, the concentration of the aptamer is 3-5 μmol / L, preferably 3 μmol / L; and the drop-coating volume is 5.0 μL.

[0014] In the third aspect, the present invention also provides the use of the electrochemiluminescence sensor enhanced by the defect engineering strategy or the electrochemiluminescence sensor obtained by the preparation method of the electrochemiluminescence sensor enhanced by the defect engineering strategy in the electrochemiluminescence detection of ENR. When two split aptamers that specifically recognize ENR specifically bind to the detection object ENR, an ENR-aptamer complex is formed, which hinders electron transfer, resulting in a weakened ECL signal, and the light intensity has a linear relationship with the concentration of ENR, which is then used for quantitative detection and analysis of the ENR content.

[0015] In some optional embodiments, the application comprises the following steps: Prepare a series of ENR standard solutions with different concentrations; The g-C3N4-NV / S-Nb2C / GCE was used as the working electrode, the platinum electrode as the counter electrode, and the Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage was 300~500V, the scanning speed was 0.1~0.15V / s, and the time-luminescence intensity curve was recorded. Then, a series of ENR standard solutions containing the ENR split aptamer 2 of different concentrations were respectively added dropwise to the electrochemiluminescence sensor apt / g-C3N4-NV / S-Nb2C / GCE, and cyclic voltammetry scanning was continued within the electrochemical window range of -1.3 ~0 V, with a photomultiplier tube high voltage of 300V~500V and a scanning speed of 0.1~0.15V / s, to record the time-luminescence intensity curve before and after the binding of ENR and the aptamer; The ENR concentration in the sample solution was calculated using the linear regression equation obtained from the linear relationship between the luminescence intensity difference and the logarithmic value of the ENR concentration.

[0016] In some optional embodiments, the pH value of the PBS phosphate buffer solution containing potassium persulfate is 5-10, preferably pH 8, the concentration of PBS is 0.1 mol / L, and the concentration of potassium persulfate is 0.05 mol / L.

[0017] In some optional embodiments, the binding time of the ENR and the aptamer in the electrochemiluminescence sensor apt / g-C3N4-NV / S-Nb2C / GCE is more than 30 minutes.

[0018] The present invention uses Nb2C MXene composite materials as co-reaction accelerators, aptamers with specific recognition functions as recognition elements, and g-C3N4-NV / S-Nb2C and apt form a phosphoamide bond through the amino group on g-C3N4 and the phosphate group of the aptamer, and co-modifies the g-C3N4 / S-Nb2C with the phosphate group on the glassy carbon electrode (GCE) to prepare a sensor for highly sensitive detection of ENR. The present invention introduces thiourea for co-calcination during the preparation of g-C3N4-NV / S-Nb2C, which not only provides nitrogen and sulfur sources, but also increases the specific surface area of ​​the composite and further enhances its electrochemical performance. The g-C3N4 (g-C3N4-NV) containing high-density nitrogen vacancies is used as a luminophore and introduced into a PBS phosphate buffer solution containing potassium persulfate, and then the apt / g-C3N4-NV / S-Nb2C / GCE modified electrode is used as a working electrode for electrochemiluminescence detection. The introduction of Nb2C MXene can greatly promote the reaction of persulfate in PBS phosphate buffer solution, thus obtaining a strong and stable ECL signal.

[0019] The electrochemiluminescent sensor provided by the present invention is an electrochemiluminescent sensor of a g-C3N4-NV / S-Nb2C composite material based on a defect engineering strategy to enhance the luminescence of g-C3N4. Compared with ordinary electrochemiluminescent sensors, the Nb2C MXene nanomaterial can promote the reaction of persulfate, thereby improving the ECL signal.

[0020] The electrochemiluminescence sensor of the g-C3N4-NV / S-Nb2C composite material provided by the present invention, which enhances the luminescence of g-C3N4 based on the defect engineering strategy, is used to detect ENR, and realizes the quantitative detection of ENR. The high-performance electrochemiluminescence sensor of g-C3N4-NV / S-Nb2C of the present invention not only has the advantages of high sensitivity, wide linear range and simple operation of electrochemiluminescence analysis, but also has important practical significance for the specific detection of ENR by using the high-performance g-C3N4-NV / S-Nb2C system ECL system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 is a simplified flow chart of the preparation of an electrochemiluminescence sensor and the detection of ENR according to an exemplary embodiment of the present invention.

[0022] Figure 2Comparison of the ECL response intensities of g-C3N4 (a), g-C3N4-NV (b), g-C3N4-NV / Nb2C (c) and g-C3N4-NV / S-Nb2C (d) composite materials.

[0023] Figure 3 This is the optimization diagram of Nb2C MXene content in g-C3N4-NV / S-Nb2C composite.

[0024] Figure 4 1 is the effect of the concentration of the ENR split aptamer 1 on the luminescence intensity according to an exemplary embodiment of the present invention.

[0025] Figure 5 FIG. 5 is the effect of the binding time between ENR and the aptamer on the luminescence intensity according to an exemplary embodiment of the present invention.

[0026] Figure 6 FIG. 5 is the effect of the pH of the PBS solution on the ECL luminescence intensity in an exemplary embodiment of the invention.

[0027] Figure 7 is a linear curve of the luminescence intensity after adding different concentrations of ENR to the three-electrode system according to an exemplary embodiment of the present invention, a ~ h: 1.0 × 10 -16 mol / L(a), 1.0 × 10 -15 mol / L(b), 1.0 ×10 -14 mol / L(c), 1.0 × 10 -13 mol / L(d), 1.0 × 10 -12 mol / L(e), 1.0×10 -11 mol / L(f),1.0×10 -10 mol / L(g),1.0×10 -9 mol / L(h).

[0028] Figure 8 is a standard curve of the logarithm of the luminescence intensity and the ENR concentration after adding ENR to the three-electrode system according to an exemplary embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any simple changes or modifications made to the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0030] The Nb2C MXene in the specific embodiment of the present invention can be purchased, and the g-C3N4 nanomaterial can be obtained by the existing method in the prior art. For example, it can be prepared by the following method: It was prepared by a self-assembly method using melamine (99%, Acros Organics) and cyanuric acid (99%, Acros Organics). 1 g of melamine and 1 g of cyanuric acid were added to 300 mL of deionized water respectively and stirred at 80 °C for about 10 minutes until completely dissolved. The melamine solution was then slowly added to the cyanuric acid solution while stirring evenly, and it was allowed to self-assemble at 80 °C for 1.5 hours. The product was centrifuged and washed twice with 80 °C deionized water to remove unassembled melamine and cyanuric acid. The filtered product was redispersed in deionized water and then allowed to stand at room temperature for 12 hours. The supernatant was removed to obtain a flocculent precipitate. The precipitate was freeze-dried for 48 hours to obtain the g-C3N4 precursor. The product was calcined at 520 °C for 2 hours under N2 atmosphere with a temperature gradient of 5 °C·min -1 , to obtain g-C3N4.

[0031] The preparation method of the electrochemiluminescent sensor according to the specific embodiment of the present invention is shown in Figure 1 ,include: g-C3N4, 20 mg of thiourea and a dispersion containing 2 mg of Nb2C Mxene were dissolved or dispersed in 1 mL of deionized water, ultrasonically treated for 5 min, and then placed in a tube furnace for calcination at 520 °C and annealed for two hours to obtain a g-C3N4-NV / S-Nb2C composite.

[0032] The g-C3N4-NV / S-Nb2C composite was drop-coated on a clean glassy carbon electrode surface to obtain a g-C3N4-NV / S-Nb2C / GCE modified electrode.

[0033] The ENR split aptamer 1 (ENR-1 chain) was drop-coated and incubated on the g-C3N4-NV / S-Nb2C / GCE modified electrode to obtain the electrochemiluminescence sensor apt / g-C3N4-NV / S-Nb2C / GCE.

[0034] In the above sensor, the aptamer sequence is as follows: The sequences of the two chains of the ENR split aptamer were: ENR-2 chain: 5′-CCC ATC AGG GGG CTA GGCTAA CAC GGT TCG GC-3′ and ENR-1 chain: 5′-ATC TCT GAG CCC GGG TTA TTT CAG GGG GA-3′, which were ordered from Sangon Biotech (Shanghai) Co., Ltd.

[0035] The specific detection method of the specific embodiment of the present invention is as follows: Step 1: Prepare a series of ENR standard solutions containing ENR split aptamer 2 (ENR-2 chain, 3 mmol / mL) at different concentrations, ranging from 1.0 × 10 -16 mol / L~1.0×10 -9 mol / L; Step 2, using the modified electrode apt / g-C3N4-NV / S-Nb2C / GCE as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system, and placing the three-electrode system in a PBS phosphate buffer solution containing potassium persulfate. The ENR standard solution containing a series of different concentrations of ENR split aptamer 2 (3mM) prepared in step 1 is added dropwise to GCE, and a cyclic voltammetry scan is performed in the electrochemical window range of -1.3~0 V, with a photomultiplier tube high voltage of 300V and a scan rate of 0.15 V / s, and the time-luminescence intensity curve is recorded. The linear relationship between the difference in luminescence intensity before and after the addition of ENR and the logarithm of the ENR concentration is established to obtain the corresponding linear regression equation; Step 3, sample testing, test according to the above step 2 and obtain the luminescence intensity, the obtained luminescence intensity is calculated using the linear regression equation obtained in step 2 to obtain the concentration of ENR in the sample.

[0036] The technical solution of the present invention is further described below in conjunction with embodiments, but the present invention is not limited to the following embodiments. Example 1

[0037] (1) Preparation of g-C3N4-NV The g-C3N4 was placed in a tube furnace and calcined at 520 °C and annealed for two hours to obtain g-C3N4-NV.

[0038] (2) Preparation of g-C3N4-NV / Nb2C The mass percentage of Nb2C Mxene and g-C3N4 is designed to be 3%. 66 mg of g-C3N4 and 400 μL of Nb2CMxene dispersion (5 mg / mL) were dispersed in 1 mL of deionized water, ultrasonicated for 5 minutes, and then placed in a tube furnace for calcination at 520 ° C and annealed for two hours to obtain about 2 mg of g-C3N4-NV / Nb2C composite.

[0039] (3) Preparation of g-C3N4-NV / S-Nb2C 66 mg of g-C3N4, 20 mg of thiourea and 400 μL of Nb2C MXene dispersion (5 mg / mL) were dissolved and dispersed in 1 mL of deionized water, ultrasonicated for 5 min, and then placed in a tube furnace for calcination at 520 °C and annealed for two hours with a temperature gradient of 5 °C min -1 , and obtained g-C3N4-NV / S-Nb2C composite.

[0040] (4) Preparation of electrochemical sensors The glassy carbon electrode is cleaned by, but not limited to, the following methods: the glassy carbon electrode is polished with Al2O3 on suede in turn, ultrasonicated with anhydrous ethanol and ultrapure water for 5 min respectively to remove surface contaminants, and naturally dried for use.

[0041] Disperse g-C3N4, g-C3N4-NV, g-C3N4-NV / Nb2C and g-C3N4-NV / S-Nb2C in deionized water to prepare a suspension with a concentration of 1 mg / mL. Pipette 4.0 μL of the suspension onto the surface of a clean glassy carbon electrode and dry it at room temperature to obtain a modified electrode. The prepared modified electrode was used as the working electrode for the electrochemiluminescence test and the electrode was placed in PBS phosphate buffer solution (0.1 mol / L) for testing. A bare glassy carbon electrode was used as a control.

[0042] like Figure 2 As shown, the ECL intensity of g-C3N4 / GCE (b) is about 3000 a.u., the ECL intensity of g-C3N4-NV / GCE (c) containing nitrogen vacancies after secondary calcination is about 6000 a.u., the ECL intensity of g-C3N4-NV / Nb2C / GCE (d) without adding thiourea is about 10000 au, and the ECL signal of g-C3N4-NV / S-Nb2C / GCE (e) used in the present invention is the strongest, about 18000 a.u. Example 2

[0043] The effect of different mass percentages of Nb2C Mxene and g-C3N4 (0%, 2%, 3%, 5%, 10%) on the electrochemical luminescence intensity was investigated. Different amounts of g-C3N4 (0, 20, 40, 66, 100 mg), 20 mg of thiourea and 400 μL of Nb2C Mxene dispersion (5 mg / mL) were dissolved and dispersed in 1 mL of deionized water, ultrasonically treated for 5 minutes, and then placed in a tube furnace for calcination at 520 ° C and annealed for two hours with a temperature gradient of 5 ° C min -1 , and obtained g-C3N4-NV / S-Nb2C composite.

[0044] The modified electrode was prepared according to the method of Example 1 and electrochemiluminescence detection was performed. Figure 3 As shown in the figure, when the Nb2C Mxene content is 3%, that is, when 66 mg of g-C3N4 is weighed, the electrochemiluminescence intensity of the g-C3N4-NV / S-Nb2C composite is the highest and the performance is the most stable (Nb2C content is 3%; the mass ratio of thiourea to Nb2C Mxene is 10:1), so g-C3N4-NV / S-Nb2C is used as the modification material of the ENR sensor. Example 3

[0045] Effects of different aptamer concentrations on the ECL luminescence performance of g-C3N4-NV / S-Nb2C electrochemiluminescence sensor: A 3% g-C3N4-NV / S-Nb2C / GCE modified electrode was prepared by referring to the method of Example 2. Then, 5 μL of a tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution of the ENR split aptamer 1 with a concentration of 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, and 5 μmol / L was added dropwise to the electrode to obtain an apt / g-C3N4-NV / S-Nb2C / GCE modified electrode.

[0046] Electrochemiluminescence detection: In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage was 300 V, the scan rate was 0.15 V / s, and the luminescence intensity was recorded.

[0047] like Figure 4 As shown in the figure, as the concentration of ENR split aptamer 1 increased from 1 μmol / L to 3 μmol / L, the ECL signal gradually weakened. When the concentration of split aptamer 1 reached 3 μmol / L, the ECL signal tended to be stable, indicating that the adsorption of split aptamer 1 on GCE reached saturation. Example 4

[0048] Effect of the difference in binding time between ENR and aptamer on the ECL luminescence performance of g-C3N4-NV / S-Nb2C electrochemiluminescence sensor: The apt / g-C3N4-NV / S-Nb2C / GCE modified electrode (ENR split aptamer 2 concentration 3 μmol / L) was prepared by referring to the method in Example 3. The preparation concentration was 1.0×10 -10 mol / L ENR standard solution containing ENR split aptamer 2 (3 μmol / L) was added dropwise to the apt / g-C3N4-NV / S-Nb2C / GCE modified electrode for 10, 20, 30, 40, and 50 min. Electrochemiluminescence detection: In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage was 300 V, the scan rate was 0.15 V / s, and the luminescence intensity was recorded.

[0049] As the binding time of split aptamer 1 and split aptamer 2 to ENR increased from 10 min to 30 min, the ECL response showed a downward trend and stabilized after 30 min ( Figure 5 ), indicating that the binding of target ENR to the two split aptamers reached stability. Therefore, we selected 30 min as the optimal binding time. Example 5

[0050] Effects of PBS phosphate buffer solutions with different pH values ​​on the luminescence properties of g-C3N4-NV / S-Nb2C electrochemiluminescence sensor ECL (1) The apt / g-C3N4-NV / S-Nb2C / GCE modified electrode was prepared by referring to the method in Example 4.

[0051] (2) Preparation of PBS phosphate buffer solution containing persulfate: 8.4758 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and 0.2028 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) were added to 250 mL of ultrapure water containing potassium persulfate (0.05 mol / L) to dissolve, and the pH values ​​were adjusted to 5, 6, 7, 8, 9, and 10, respectively, to obtain PBS phosphate buffer solutions containing potassium persulfate at different pH values, and finally stored for later use; (3) Electrochemical detection: The modified electrode apt / g-C3N4-NV / S-Nb2C / GCE was used as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in PBS phosphate buffer solutions containing potassium persulfate with pH values ​​of 5, 6, 7, 8, 9, and 10, respectively. In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage was 300 V, the scan rate was 0.15 V / s, and the luminescence intensity was recorded.

[0052] like Figure 6 As shown, with the increase of pH value, the ECL response gradually increases, but as the pH increases from 8 to 10, the ECL intensity decreases, so the pH value of 8 is preferred. Example 6

[0053] An electrochemiluminescence sensor based on g-C3N4-NV / S-Nb2C composite material with enhanced luminescence of g-C3N4 based on defect engineering strategy was constructed to detect enrofloxacin (ENR). The specific preparation process and the brief process of ENR detection can be found in Figure 1 .

[0054] Preparation of electrochemiluminescence sensor for high-sensitivity detection of ENR: (1) Preparation of g-C3N4-NV / S-Nb2C 66 mg of g-C3N4, 20 mg of thiourea and 400 μL of Nb2C Mxene dispersion (5 mg / mL) were dissolved and dispersed in 1 mL of deionized water, ultrasonically treated for 5 minutes, and then placed in a tube furnace for calcination at 520 °C and annealed for two hours to obtain about 2 mg of g-C3N4-NV / S-Nb2C composite.

[0055] (2) Preparation of g-C3N4-NV / S-Nb2C electrochemical sensor for ENR detection The glassy carbon electrode is cleaned by, but not limited to, the following methods: the glassy carbon electrode is polished on suede with Al2O3 in turn, ultrasonicated with anhydrous ethanol and ultrapure water for 5 min respectively to remove surface pollutants, and dried naturally for use.

[0056] At room temperature, 6.0 μL of 0.01 M PBS buffer (pH 7.4) containing 0.005 M NHS and 0.01 M EDC was dropped onto a clean bare glassy carbon electrode for 1 h to activate the electrode. 4 μL of 1 mg / mL g-C3N4-NV / S-Nb2C was modified on a clean GCE surface to obtain g-C3N4-NV / S-Nb2C / GCE. Then, the modified electrode was incubated for 8 hours with 5 μL of 3 μmol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution of ENR aptamer 1, to obtain apt / g-C3N4-NV / S-Nb2C / GCE modified electrode.

[0057] (3) Drawing of standard curve A series of ENR standard solutions containing ENR split aptamer 2 at different concentrations were prepared; The modified electrode apt / g-C3N4-NV / S-Nb2C / GCE was used as a working electrode, a platinum electrode was used as an auxiliary electrode, and Ag / AgCl was used as a reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. Within the electrochemical window range of -1.3 to 0 V, the photomultiplier tube high voltage was 300 V, the scanning rate was 0.15 V / s, and the time-luminescence intensity curve (T-ECL) was recorded; Then, a series of ENR standard solutions containing ENR split aptamer 2 with different concentrations were added dropwise to the modified electrode apt / g-C3N4-NV / S-Nb2C / GCE, and incubated for more than 30 min to obtain ENR / apt / g-C3N4-NV / S-Nb2C / GCE corresponding to a series of ENR standard solutions with different concentrations. Continue to use ENR / apt / g-C3N4-NV / S-Nb2C / GCE as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system is placed in a PBS phosphate buffer solution containing potassium persulfate. In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage is 300V, the scan rate is 0.15V / s, and the time-luminescence intensity curve (T-ECL) is recorded; see Figure 7 , a ~ h: 1.0 × 10 -16 mol / L(a), 1.0 × 10 -15 mol / L(b), 1.0 ×10 -14 mol / L(c), 1.0 × 10 -13 mol / L(d), 1.0 × 10 -12 mol / L(e), 1.0×10 -11 mol / L(f),1.0×10 -10 mol / L(g),1.0×10 -9 mol / L(h).

[0058] The linear relationship between the difference in luminescence intensity before and after the addition of ENR and the logarithmic value of the ENR concentration was established, see Figure 8 , the corresponding linear regression equation is: I ECL =493.43 lgC (mol / L) + 11591.88, correlation coefficient (R 2 ) is 0.9836. The detection range of the linear regression equation is 1.0×10 -16 ~1.0×10 -9 mol / L, the minimum detection limit is 2.37×10 -16 mol / L.

[0059] (4) Sample testing The three-electrode system was immersed in filtered lake water for 20 min, and electrochemiluminescence detection was performed according to the above standard solution detection method. The samples were tested three times in parallel, and the concentration of ENR in the test samples was calculated according to the regression equation corresponding to the above standard curve. The results are listed in Table 1. Comparative Example 1

[0060] Comparative Example 1 is different from Example 6 in that: the apt / g-C3N4-NV / S-Nb2C / GCE modified electrode is used as the working electrode, the platinum electrode is used as the auxiliary electrode, and the Ag / AgCl is used as the reference electrode to form a three-electrode system, and the three-electrode system is placed in a PBS phosphate buffer solution without potassium persulfate, and the detection method is the same as Example 6. The detection results show that when the sensor prepared in Comparative Example 1 is used for ENR detection, the light intensity is very low, and it is difficult to achieve ENR detection. The results are listed in Table 1.

[0061] Table 1 Determination results of ENR in river water

[0062] Remark: a It is the average of three measurements.

[0063] As shown in Table 1, the sample was tested three times in parallel, the relative standard deviation was less than 5%, and the spike recovery range was 98% to 105%. The above results show that ENR cannot be detected by using apt / g-C3N4-NV / S-Nb2C / GCE modified but placed in PBS phosphate buffer solution without potassium persulfate. The apt / g-C3N4-NV / S-Nb2C / GCE electrochemiluminescence sensor provided by the specific embodiment of the present invention is effective and feasible for detecting ENR, and the operation is simple and flexible, the detection range is wide, and the detection limit is low.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An electrochemiluminescence sensor, characterized in that: It includes a glassy carbon electrode apt / g-C3N4-NV / S-Nb2C / GCE modified with a g-C3N4-NV / S-Nb2C complex and an aptamer with a specific recognition function, and an electrolyte; wherein the g-C3N4-NV / S-Nb2C complex is obtained by mixed calcination of g-C3N4, thiourea and Nb2C Mxene; and the electrolyte is a phosphate buffer solution containing potassium persulfate.

2. The electrochemiluminescence sensor according to claim 1, characterized in that The specific preparation method of the g-C3N4-NV / S-Nb2C composite is as follows: g-C3N4, thiourea, and Nb2C Mxene are uniformly dispersed or dissolved in deionized water to obtain a mixed solution, the mixed solution is calcined at 520° C., and annealed for two hours to obtain the g-C3N4-NV / S-Nb2C composite.

3. The electrochemiluminescence sensor according to claim 2, characterized in that: The mass percentage of the Nb2C Mxene to the g-C3N4-NV is 2-5%, and the mass ratio of the thiourea to the Nb2C Mxene is 10:

1.

4. The electrochemiluminescence sensor according to claim 1, characterized in that: The pH value of the phosphate buffer solution containing potassium persulfate is 5-10, the concentration of sulfate is 0.1 mol / L, and the concentration of potassium persulfate is 0.05 mol / L.

5. The method for preparing the electrochemiluminescence sensor according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: dispersing a g-C3N4-NV / S-Nb2C complex in deionized water to prepare a suspension; drop-coating the suspension on the surface of a clean glassy carbon electrode, drying, and obtaining a g-C3N4-NV / S-Nb2C / GCE modified electrode; drop-coating and incubating an aptamer with a specific recognition function on the g-C3N4-NV / S-Nb2C / GCE modified electrode, and obtaining apt / g-C3N4-NV / S-Nb2C / GCE.

6. The method for preparing an electrochemiluminescence sensor according to claim 5, characterized in that: The aptamer with specific recognition function is an enrofloxacin aptamer; the nucleotide sequences of the two chains of the aptamer are: ENR-2 chain: 5'-CCCATC AGG GGG CTA GGC TAA CAC GGT TCG GC-3' and ENR-1 chain: 5'-ATC TCT GAG CCC GGG TTATTT CAG GGG GA-3'; the ENR-1 chain solution is modified on the electrode, and the ENR-2 chain solution is added to the solution to be tested.

7. The method for preparing an electrochemiluminescence sensor according to claim 6, characterized in that: The concentration of the ENR-1 chain solution is 3-5 μmol / L; the concentration of the ENR-2 chain solution is 3-5 μmol / L; and / or the concentration of the suspension is 0.5-2.0 mg / mL.

8. Use of the electrochemiluminescent sensor according to any one of claims 1 to 4 or the electrochemiluminescent sensor obtained by the preparation method according to any one of claims 5 to 7 in the detection of electrochemiluminescent enrofloxacin.

9. Use of the electrochemiluminescence sensor according to claim 8 in the detection of enrofloxacin, characterized in that: The following steps are involved: A series of enrofloxacin standard solutions with different concentrations were prepared; The electrochemiluminescent sensor apt / g-C3N4-NV / S-Nb2C / GCE modified with ENR-1 chain was used as the working electrode, the platinum electrode as the counter electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in a phosphate buffer solution containing potassium persulfate. In the electrochemical window range of -1.3~0 V, the photomultiplier tube high voltage was 300~500V, the scanning speed was 0.1~0.15V / s, and the time-luminescence intensity curve was recorded. Then, a series of enrofloxacin standard solutions containing ENR-2 chains of different concentrations were respectively added dropwise onto the apt / g-C3N4-NV / S-Nb2C / GCE, and cyclic voltammetry scanning was continued within the electrochemical window range of -1.3 to 0 V, with a photomultiplier tube high voltage of 300 to 500 V and a scanning speed of 0.1 to 0.15 V / s, to record the time-luminescence intensity curve before and after the binding of enrofloxacin to the aptamer; The ENR concentration in the sample solution was calculated using the linear regression equation obtained from the linear relationship between the luminescence intensity difference and the logarithmic value of the enrofloxacin concentration.

10. Use of the electrochemiluminescence sensor according to claim 9 in the detection of enrofloxacin, characterized in that: The binding time of enrofloxacin and the aptamer in the electrochemiluminescence sensor apt / g-C3N4-NV / S-Nb2C / GCE is more than 30 minutes.

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