Preparation method of electrochemical luminescence modified electrode for detecting oxytetracycline and sensor

Abstract

The invention discloses a method for detecting oxytetracycline (OTC) based on nitrogen-doped graphdiyne (2N-GY) electrochemical luminescence sensing. The prepared 2N-GY has an electrogenerated chemiluminescence signal, the surface of the electrode is modified with an aptamer (Apt I) of carboxylated 2N-GY and OTC, and then non-specific binding sites are closed by using BSA to obtain the modified electrode. The modified electrode is used as a working electrode, an electrochemiluminescence biosensor is constructed by continuously modifying OTC and quenching a probe (PDAs-Apt II), and quantitative analysis is carried out on the OTC by detecting the change of an electrochemiluminescence signal. According to the present invention, the two OTC aptamers (Apt I and Apt II) are adopted as the recognition molecules to construct the electrochemiluminescence biosensor so as to achieve the high-specificity and high-sensitivity detection of OTC, and can be used for the actual detection application of milk, and the novel carbon material is adopted to replace the toxic light-emitting reagent, such that the detection cost is reduced, and the safety problem existing in the traditional light-emitting system is reduced.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic detection and electrochemiluminescence technology, specifically relating to a method for preparing an electrochemiluminescence modified electrode for detecting antibiotics and a sensor thereof. Background Technology

[0002] Oxytetracycline (OTC) is one of the most commonly used tetracycline antibiotics for treating infections in livestock. It is highly effective against both Gram-positive and Gram-negative bacteria and is inexpensive. However, due to its widespread use and the difficulty of its natural degradation, it easily enters the human body through the food chain (meat, milk, and eggs), causing allergies and liver and kidney damage, resulting in serious side effects on human health. Excessive residues of OTC products in the environment can also lead to antibiotic resistance. Therefore, developing sensitive and accurate OTC detection and analysis technologies is crucial.

[0003] There are currently various methods for detecting residual OTC, including high performance liquid chromatography, surface plasmon resonance, fluorescence spectroscopy, and colorimetry. However, the former is complex to operate and costly, while the latter has poor anti-interference capabilities and high background signal.

[0004] Electrochemiluminescence (ECL) is the emission of light produced by an electrochemical reaction. Combining the advantages of electrochemistry and spectroscopy, it features high sensitivity, high stability, and low background signal, and has been widely used in the detection of pesticide and veterinary drug residues. In the ECL system, Ru(bpy)3... 2+ It possesses characteristics such as good stability, good electrochemical performance, and good biocompatibility, and is often used as a luminescent probe; Ru(bpy)3 2+ The / TPrA system is widely used in ECL biosensors, but the required Ru(bpy)3 2+ The concentration is high, the linear range is not wide, and TPR is biotoxic, which can easily cause environmental pollution. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing an electrochemiluminescence modified electrode and a sensor for detecting oxytetracycline. This invention prepares and uses a novel nitrogen-doped graphyne (2N-GY) with excellent ECL performance as the luminescent material, potassium persulfate (K2S2O8) as the co-reactant, and polydopamine nanoparticles (PDAs) and OTC aptamer chains to form quenching probes. The quenching probes are introduced using a double aptamer sandwich method. When OTC is present, OTC binds to its aptamer Apt I. Subsequently, the quenching probe PDAs-Apt II is incubated on the modified electrode, where the OTC aptamer Apt II also binds to OTC. The higher the concentration of OTC, the more PDAs-Apt II are bound, and the PDAs reduce the ECL signal. By observing the changes in the ECL signal, ultrasensitive detection of OTC is achieved.

[0006] This invention prepares a novel 2N-GY luminescent material and utilizes its ECL properties to prepare a modified electrode for stable and sensitive detection of OTC.

[0007] This invention uses 2N-GY luminescent material as the luminescent group. PDAs adsorb a large number of aptamer chains through the π-π conjugation effect to construct a quenching probe. The quenching probe is introduced using a double aptamer sandwich method. When the analyte OTC is present, OTC binds to its aptamer Apt I. PDAs-Apt II are incubated on the electrode, and Apt II also binds to OTC. The higher the concentration of OTC, the more PDAs-Apt II are bound, and the lower the ECL signal. Based on this, an electrochemiluminescence biosensor is constructed, providing a reference for the application of novel nanoluminescent materials in the field of electrochemiluminescence technology. Using K2S2O8 to replace the luminescent system containing toxic co-reactants provides a new approach to the construction of electrochemiluminescence sensors. The specific technical solution of this invention is as follows: A novel 2N-GY electrochemiluminescent material preparation method and a sensor for preparing an electrochemiluminescent modified electrode for detecting oxytetracycline, the method comprising the following steps: (1) A novel 2N-GY electrochemiluminescent material, formed by the reaction of calcium carbide (CaC2) and pyrazine; (2) The 2N-GY luminescent material was carboxylated using concentrated nitric acid (HNO3) to obtain carboxylated 2N-GY; (3) PDA nanoparticles were linked to OTC aptamers (Apt II) to obtain quenching probes: PDAs-Apt II; (4) Disperse 2N-GY in water, drop the resulting suspension onto the electrode surface, dry it, and add a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) onto the electrode surface to activate the carboxyl groups, thus obtaining a 2N-GY / GCE modified electrode. (5) Add amino-modified OTC aptamer (Apt Ⅰ) to the surface of the 2N-GY / GCE modified electrode, incubate, wash and dry to obtain Apt Ⅰ / 2N-GY / GCE, and then incubate the modified electrode with bovine serum albumin (BSA) solution. (6) A certain concentration of OTC solution was added to the surface of the BSA / AptⅠ / 2N-GY / GCE modified electrode, and after incubation, it was washed and dried. (7) A quenching probe was dropped onto the surface of the OTC / BSA / Apt Ⅰ / 2N-GY / GCE modified electrode. After cleaning and drying, the modified electrode for detecting oxytetracycline was obtained: PDAs-Apt Ⅱ / OTC / BSA / Apt Ⅰ / 2N-GY / GCE.

[0008] The 2N-GY electrochemiluminescent material described in step (1) was prepared by high-energy ball milling of 1.5 g CaC2 and 0.5 mL pyrazine under argon protection. The ball mill was used to react at 600 rpm, and the reaction was carried out continuously for 16 h using a cycle of 30 min ball milling / 15 min cooling. After washing with 1 M HNO3, ultrapure water, and anhydrous ethanol to remove impurities, the material was vacuum dried at 80 °C for 12 h to obtain 2N-GY.

[0009] The oxygen element in the 2N-GY electrochemiluminescent material described in step (1) comes from the adsorption of oxygen from the air.

[0010] The 2N-GY electrochemiluminescent material mentioned in step (1) is obtained by electrophilic substitution of the six-membered ring with alkyne-bonded carbon in CaC2, and the alkyne bond connecting the benzene ring to obtain graphdiyne doped with two nitrogen atoms in the benzene ring structure.

[0011] The electrode can be any working electrode reported in the prior art that can be used for electrochemiluminescence biosensors, such as a glassy carbon electrode (GCE).

[0012] In step (2), HNO3 is concentrated nitric acid with a mass fraction of 65% to 68%, and the ratio of 2N-GY to HNO3 is 4 mg to 1 mL.

[0013] In step (2), 2N-GY is added to HNO3 and heated at 80°C for 2-4 h to carboxylate 2N-GY.

[0014] In step (3), PDAs and OTC aptamers (Apt II) were mixed and incubated in a constant temperature shaker at 200 rpm for 2 h to obtain PDAs-Apt II. The ratio of PDAs to OTC aptamers was 500 μg / mL. -1 : 1 μM, 100 μL.

[0015] In step (4), the concentration of 2N-GY is 2 mg / mL, and 10 μL of suspension is dropped onto the electrode surface.

[0016] In step (4), the concentrations of EDC and NHS are 0.4 M and 0.1 M, respectively.

[0017] In step (4), a mixed solution of EDC and NHS was added dropwise to the surface of the 2N-GY / GCE modified electrode and incubated at room temperature for 2 h to activate the carboxyl groups.

[0018] In step (5), 1 μM of amino-modified OTC aptamer (Apt I) is added dropwise onto the electrode surface, and the mixture is incubated at a constant temperature for 6 h.

[0019] In step (5), the concentration of BSA solution added to the modified electrode surface is 1 wt%. 10 μL of BSA solution is dropped onto the electrode surface and incubated at room temperature for 1 h.

[0020] In step (6), 10 μL of a 1×10⁻⁶ solution was dropped onto the surface of the BSA / AptⅠ / 2N-GY / GCE modified electrode. -7 -1×10 -12 M in OTC solution, incubated at 37°C for 1 h.

[0021] In step (7), 10 μL of a 500 μg / mL solution was dropped onto the surface of the OTC / BSA / AptⅠ / 2N-GY / GCE modified electrode. -1 Quenching probe: PDAs-Apt II, incubated at 37℃ for 1 h.

[0022] This invention provides a method for preparing 2N-GY luminescent materials.

[0023] The modified electrode for detecting oxytetracycline prepared according to the above method is also within the scope of protection of this invention. The modified electrode of this invention has 2N-GY luminescent material immobilized on its surface. It can bind Apt I through amide bonds and can also form an electrochemiluminescence system with potassium persulfate solution (K2S2O8) to provide an ECL signal.

[0024] This invention modifies the electrode surface with Apt I and uses BSA to block non-specific binding sites. When the analyte OTC is incubated on the modified electrode, OTC binds to its aptamer Apt I. When PDAs-Apt II are incubated on the modified electrode, Apt II also binds to OTC. The higher the concentration of OTC, the more PDAs-Apt II are bound. PDAs reduce the ECL signal, thereby achieving the purpose of detecting samples with unknown concentrations.

[0025] This invention also provides the application of the modified electrode for detecting oxytetracycline in the detection of oxytetracycline. This modified electrode can serve as the working electrode of an electrochemiluminescence biosensor, forming a three-electrode system with an auxiliary electrode and a reference electrode, achieving high sensitivity and high specificity in the detection of oxytetracycline.

[0026] The present invention also provides an electrochemiluminescent biosensor for detecting oxytetracycline, the electrochemiluminescent biosensor including a working electrode, the working electrode being the modified electrode for detecting oxytetracycline described above.

[0027] The electrochemiluminescence biosensor further includes an auxiliary electrode and a reference electrode, wherein the auxiliary electrode is a platinum electrode and the reference electrode is a calomel electrode.

[0028] This invention relates to an electrochemically induced biosensor that excites a luminescent substance on an electrode surface to react with its co-reactant via an electrical signal. The luminescence intensity is used as the detection signal, and OTC is specifically detected via a dual aptamer. A 2N-GY / K2S2O8 luminescent reagent system is employed. 2N-GY provides the luminescence signal, which is detected in the K2S2O8 solution as the ECL signal. PDAs-Apt II act as a quenching probe. When OTC is present, it binds to its aptamer Apt I. Simultaneously, Apt II also binds to OTC. The higher the concentration of OTC, the more PDAs-Apt II bind. The PDAs quenching probe quenches the 2N-GY ECL signal, thus allowing the detection of oxytetracycline sample concentration based on changes in the ECL signal.

[0029] The present invention also provides a method for detecting oxytetracycline, wherein the modified electrode for detecting oxytetracycline described above is used as the working electrode, a platinum electrode is used as the auxiliary electrode, a calomel electrode is used as the reference electrode, and a K2S2O8 co-reactant solution is used as the detection solution, and the content of oxytetracycline is detected by electrochemiluminescence.

[0030] In the above electrochemiluminescence method, the measurement type is intensity mode, the scanning potential is -2.5 V to 0 V, the scanning rate is 0.1 V / s, the amplification stage is 3, and the photomultiplier tube voltage is 600 V.

[0031] In the above electrochemiluminescence method, the concentration of K2S2O8 solution is 100 mM.

[0032] The above method for detecting oxytetracycline includes the following steps: (1) Prepare standard solutions of oxytetracycline at different concentrations for later use; (2) The modified electrode for detecting oxytetracycline prepared above was incubated with oxytetracycline standard solutions of different concentrations at 37°C for 1 h. (3) The modified electrode for detecting oxytetracycline prepared above was incubated with PDAs-Apt II at 37°C for 1 h; (4) Remove the modified electrode and combine it with the auxiliary electrode and the reference electrode. Use K2S2O8 solution as the detection solution to detect the electrochemiluminescence intensity signal. (5) Plot a standard curve with the logarithm of OTC concentration on the x-axis and electrochemiluminescence intensity on the y-axis; (6) The modified electrode for detecting oxytetracycline was incubated with the sample solution at 37°C for 1 h, and then incubated with PDAs-AptⅡ at 37°C for 1 h. The electrode was then used with an auxiliary electrode and a reference electrode, and K2S2O8 solution was used as the detection solution to detect the electrochemiluminescence intensity signal. The electrochemiluminescence intensity was then incorporated into the standard curve to obtain the OTC concentration in the sample.

[0033] To construct an ECL sensor with higher sensitivity, stronger specificity, higher safety, and wider detection range, 2N-GY luminescent material was used as the luminescent reagent, an OTC dual aptamer was used as the specific recognition molecule, and K2S2O8 was used as the co-reactant. This ECL biosensor was constructed for the detection of oxytetracycline, providing a new strategy for food safety control and meeting the urgent need for rapid detection of antibiotic residues in food. This invention constructs an electrochemiluminescence sensor for the detection of oxytetracycline with high specificity, high sensitivity, high safety, and a wide detection range. Compared with existing technologies, this invention has the following advantages: 1. This invention prepares a novel luminescent material: 2N-GY, and verifies its ECL signal; 2. This invention uses 2N-GY and K2S2O8 as the luminescent system, replacing the traditional toxic luminescent reagent system, which greatly improves the safety of the biosensor; 3. This invention is the first to use 2N-GY as the luminescent material to construct an electrochemiluminescence biosensor, providing a reference for the development of novel carbon-based luminescent materials in the field of electrochemiluminescence technology. Verification showed that using the 2N-GY and K2S2O8 luminescent system, quantitative analysis of OTC drugs was performed by detecting the electrochemiluminescence signal, with a detection range of 1×10⁻⁶. -7 -1×10 -12 M has a detection limit as low as 0.86 pM, which has the advantages of low detection limit and wide detection range. Attached Figure Description

[0034] Figure 1 SEM images and structural diagrams of 2N-GY at different magnifications; Figure 2 A schematic diagram illustrating the construction process of an ECL biosensor based on the novel luminescent material 2N-GY and the quenching probe PDAs-Apt II; Figure 3 Figure 1 shows the optimized experimental results of the concentrations of PDAs (quencher), Apt II, and T sequences in Apt II during the construction of the ECL biosensor, as well as the incubation time of PDAs and Apt II, and the incubation time of 2N-GY and Apt I. Figure 4 ECL images (A) and calibration curves (B) of ECL concentration versus ECL intensity were obtained from 0.01M PBS containing 100 mM K2S2O8 at different concentrations of OTC from 1 pM to 100 nM. Figure 5 Stability of ECL biosensors; Figure 6 Specificity of ECL biosensors. Detailed Implementation

[0035] The present invention will be further described below through specific embodiments. The following description is merely exemplary and does not limit its content. Unless otherwise specified, all terms or methods not described in detail below are prior art.

[0036] In the following embodiments, 2N-GY was obtained using the method described in the literature "Site-Defined High-Loading Tellurium Single-Atom Nanozymes Anchored on Checkerboard-Patterned Graphyne for Sensor Array Construction" (Jianing Xia, Jian Guo, Zhen Li, Saichao Cao, Ya Tang, Hongbin Zhao,* and Daixin Ye*, Site-Defined High-Loading Tellurium Single-Atom Nanozymes Anchored on Checkerboard-Patterned Graphyne for Sensor Array Construction. Small, 2025, 21:2501797).

[0037] Example 1 (1) Preparation of 2N-GY 1.5 g CaC2 and 0.5 mL pyrazine were prepared by high-energy ball milling under argon protection. The ball mill was operated at 600 rpm, and the reaction was carried out continuously for 16 h using a 30 min ball milling / 15 min cooling cycle. After washing with 1 M HNO3, ultrapure water, and anhydrous ethanol to remove impurities, the mixture was dried under vacuum at 80 °C for 12 h to obtain 2N-GY. (2) Carboxylation of 2N-GY Add 4 mg of 2N-GY to 1 mL of concentrated nitric acid and heat at 80 °C for 4 h. Pour the reaction mixture into ice water, collect the precipitate by centrifugation, and wash three times with water to obtain carboxylated 2N-GY. Redisperse it in 2 mL of ultrapure water and store at 4 °C for later use. (3) Preparation of PDAs-Apt II PDAs (100 μL, 500 μg mL) -1The mixture was mixed with Oxy aptamer (Apt II, 100 μL, 1 μM) and shaken at 200 rpm for 2 h at 37 °C. The product was centrifuged (8000 × 5), purified with distilled water, and redispersed in 100 μL of 0.01 MPBS (pH=7.4). (4) Construction of PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE ECL biosensor: Before modifying the glassy carbon electrode (GCE), the electrode surface was first polished with 0.3 μm and 0.05 μm alumina powder, then ultrasonically cleaned with ethanol and ultrapure water for 2 min respectively, and dried with nitrogen. 5 μL of 2 mg / mL 2N-GY luminescent material was dropped onto the pretreated electrode surface, and the prepared 2N-GY / GCE modified electrode was allowed to dry naturally in air. Add 10 μL of a mixed solution of 10 mg / mL 0.4 M EDC and 0.1 M NHS to the electrode surface and incubate at room temperature for 2 h; gently rinse with ultrapure water and dry. Add 10 μL of a mixed solution of 1 μM Apt I to the electrode surface and incubate at room temperature for 6 h; gently rinse with ultrapure water and air dry. The modified electrode was sealed with 10 μL of 1% BSA solution at room temperature for 1 h to block non-specific binding sites, and then gently rinsed with ultrapure water to remove unbound BSA molecules. The obtained PDAs-Apt Ⅱ / OTC / BSA / Apt Ⅰ / 2N-GY / GCE biosensor was incubated with standard concentration of OTC at 37℃ for 1 h, gently rinsed with ultrapure water and air-dried. Finally, 10 μL of PDAs-Apt II was added to the electrode, incubated at 37 °C for 1 h, gently rinsed with ultrapure water, and then air-dried. (5) Characterization of 2N-GY luminescent materials 2N-GY was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (see [link to SEM]). Figure 1 ); (6) Condition Optimization In the experiment, the concentrations of quenchers PDAs, Apt II, the number of T sequences in Apt II, the incubation time of PDAs and Apt II, and the incubation time of 2N-GY and Apt I were optimized. Quenching probes were prepared by preparing PDAs at concentrations of 0.1, 0.3, 0.5, 0.7 and 0.9 mg / mL according to the above steps, and were ready for use. The OTC / BSA / Apt Ⅰ / 2N-GY / GCE modified electrode was incubated with quenching probes prepared from PDAs of different concentrations at 37℃ for 1 h, with all other steps and conditions being the same, to finally form a PDAs-Apt Ⅱ / OTC / BSA / Apt Ⅰ / 2N-GY / GCE modified electrode. The modified electrode was then used as the working electrode, and together with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode), a three-electrode system was constructed. A 0.01M PBS solution containing 100 mM K2S2O8 was used as the detection solution to construct an ECL biosensor. The working electrode, auxiliary electrode, and reference electrode were immersed in a K₂S₂O₈ detection solution. Electrochemiluminescence (ECL) was used, with a scanning potential ranging from -2.5 V to 0 V, a scanning rate of 0.1 V / s, amplification stage of 3, and a photomultiplier tube voltage of 600 V to obtain the ECL signal (see...). Figure 3 ); Similarly, under the same conditions, by varying the concentration of Apt II, the number of T sequences in Apt II, the incubation time of PDAs with Apt II, or the incubation time of 2N-GY with Apt I, the working electrode, auxiliary electrode, and reference electrode were immersed in the K2S2O8 detection solution. Electrochemiluminescence was used with a scan potential of -2.5 V to 0 V, a scan rate of 0.1 V / s, a photomultiplier tube voltage of 600 V, and amplification stage of 3 to obtain the ECL signal (see...). Figure 3 ); (7) Determination of standard curve and detection limit Prepare OTC standard solutions with concentrations of 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM for later use; Using a BSA / Apt I / 2N-GY / GCE modified electrode as the working electrode, it was first incubated with OTC standard solutions of different concentrations at 37℃ for 1 h. Then, the OTC / BSA / Apt I / 2N-GY / GCE modified electrode was incubated with PDAs-Apt II at 37℃ for 1 h to form a PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE modified electrode. The modified electrode was then used as the working electrode, and together with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode), a three-electrode system was constructed. A 0.01 M PBS solution containing 100 mM K2S2O8 was used as the detection solution to construct an ECL biosensor. The working electrode, auxiliary electrode, and reference electrode were immersed in the K2S2O8 detection solution. Electrochemiluminescence was used to detect the ECL signal by scanning at a potential of -2.5 V to 0 V, a scanning rate of 0.1 V / s, a photomultiplier tube voltage of 600 V, and amplification stage of 3. The reaction principle between the working electrode and the detection solution is as follows: S2O8 2- + e - → SO4 2- + SO4 ·- (1) 2N-GY + SO4 ·- → SO4 2- + 2N-GY ·+ (2) 2N-GY + e - → 2N-GY ·- (3) 2N-GY ·+ + 2N-GY ·- → 2N-GY* (4) or, 2N-GY ·+ + e - → 2N-GY* (5) 2N-GY* → 2N-GY + light (6) The working electrode, PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE, reacts with the co-reactant K2S2O8 under a high voltage of 600V, generating an ECL signal. Apt I and BSA are incubated on the 2N-GY / GCE modified electrode, respectively. The hindrance effect of DNA and protein reduces electron transfer efficiency, thus decreasing the ECL signal. When OTC is incubated with the BSA / Apt I / 2N-GY / GCE modified electrode, OTC, being a small molecule antibiotic, has little effect on reducing the ECL signal. Finally, when PDAs-Apt II is incubated with the OTC / BSA / Apt I / 2N-GY / GCE modified electrode, PDAs quench the ECL signal, causing a change in the ECL signal intensity. The ECL signal intensity is inversely proportional to the OTC concentration (see...). Figure 4 ); At 37℃, optimized concentrations of PDAs, Apt II, the number of T sequences in Apt II, incubation times of PDAs and Apt II, and incubation times of 2N-GY and Apt I were used to modify OTC standard solutions of different concentrations (100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM). The ECL intensity of the PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE modified electrode was plotted on the ordinate, and the logarithm of the OTC concentration was plotted on the abscissa (see [link to standard curve]). Figure 4 ); As shown by the standard curve, the ECL signal is inversely proportional to the logarithm of the OTC concentration, and their linear relationship is I. ECL=5658.921-1637.121LgC OTC (R 2 =0.9961), the detection limit of this ECL sensor is 0.86 pM; (8) Stability test The BSA / Apt I / 2N-GY / GCE modified electrode was incubated with 10 pM OTC solution, and then incubated with PDAs-Apt II at 37 °C for 1 h to form a PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE modified electrode. This modified electrode was then used as the working electrode, along with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode) to construct a three-electrode system. A 0.01 M PBS solution containing 100 mM K₂S₂O₈ was used as the detection solution to construct an ECL biosensor. The working electrode, auxiliary electrode, and reference electrode of the modified 10 pM OTC were immersed in a K2S2O8 detection solution. Electrochemiluminescence (ECL) was used to detect the OTC signal. The scanning potential ranged from -2.5 V to 0 V, the scanning rate was 0.1 V / s, the photomultiplier tube voltage was 600 V, and the amplification stage was 3. Ten consecutive scanning cycles were performed to obtain a relatively stable ECL curve. The relative standard deviation (RSD) of the ECL signal was only 1.53%, indicating that the ECL immunosensor has good stability in detecting OTC. Figure 5 ); (9) Specificity test Prepare standard solutions of chloramphenicol (CAP), chlortetracycline (CHL), streptomycin (STR), kanamycin (KAN), tetracycline (TET), and natamycin (NAt) at concentrations of 100 nM for later use; Using a BSA / Apt I / 2N-GY / GCE modified electrode as the working electrode, different antibiotic standard solutions were incubated at 37℃ for 1 h. Then, a PDAs-Apt II modified electrode was incubated at 37℃ for 1 h to form a PDAs-Apt II / antibiotics / BSA / Apt I / 2N-GY / GCE modified electrode. This modified electrode was then used as the working electrode, together with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode) to construct a three-electrode system. A 0.01 M PBS solution containing 100 mM K₂S₂O₈ was used as the detection solution to construct an ECL immunosensor. The working electrode, auxiliary electrode, and reference electrode were immersed in the K2S2O8 detection solution. Electrochemiluminescence was used to detect the ECL signal by scanning at a potential of -2.5 V to 0 V, a scanning rate of 0.1 V / s, a photomultiplier tube voltage of 600 V, and amplification stage of 3. At 37°C, an ECL sensor was used to detect working electrodes containing 100 nM of different antibiotics. Different ECL signals were obtained using a PDAs-Apt II / antibiotics / BSA / Apt I / 2N-GY / GCE modified electrode. A bar graph was plotted with different antibiotic types on the x-axis and their ECL signals on the y-axis (see...). Figure 6 As can be seen from the figure, the response to the target containing OTC is the lowest, while the signal strength to other interfering objects is very high. The results indicate that the ECL sensor of this invention exhibits good specificity for OTC.

[0038] Example 2 Preparation of sample test solutions: Milk samples were diluted 10-fold with PBS, and 20% (v / v) acetic acid was added to precipitate proteins. The samples were then centrifuged at 5000 rpm for 15 min, and the supernatant was filtered through a 0.22 µm filter membrane to collect the filtrate. Finally, OTC solutions of different concentrations were added to the filtrate as standards to detect the OTC content in the milk. The BSA / Apt I / 2N-GY / GCE modified electrode prepared in step (3) of Example 1 was incubated with milk sample test solutions containing different concentrations of OTC at 37°C for 1 h. Then, the modified electrode was incubated with PDAs-Apt II at 37°C for 1 h to form a PDAs-Apt II / OTC / BSA / Apt I / 2N-GY / GCE modified electrode. The modified electrode was then used as the working electrode, and together with a platinum electrode (auxiliary electrode) and a saturated calomel electrode (reference electrode), an ECL biosensor was constructed. The working electrode, auxiliary electrode, and reference electrode were immersed in a K2S2O8 detection solution. Electrochemiluminescence (ECL) was used for detection at a scanning potential of -2.5 V to 0 V, a scanning rate of 0.1 V / s, a photomultiplier tube voltage of 600 V, and amplification stage of 3. The ECL signal was obtained as the detection value. Each sample was tested three times, and its OTC recovery concentration, recovery rate, and standard deviation were calculated. Relative standard deviation (RSD) = Standard deviation of recovery / Average recovery × 100%; The test results are shown in Table 1 below. Table 1. Results of OTC spiked recovery in milk samples:

[0039] As can be seen from the results in the table above, the method of the present invention has a high recovery rate, a wide detection range, and a low detection limit, which can meet the requirements for use.

Claims

1. A method for preparing a novel 2N-GY electrochemiluminescent material and an electrochemiluminescent modified electrode for detecting oxytetracycline, characterized in that: The following steps are adopted: (1) Preparation of 2N-GY luminescent materials; (2) Carboxylating the 2N-GY luminescent material to obtain carboxylated 2N-GY; (3) PDA nanoparticles were linked to the OTC aptamer Apt II to obtain the quenching probe: PDA-Apt II; (4) Disperse the 2N-GY luminescent material obtained in step (2) in water to obtain a suspension, drop the suspension onto the surface of a glassy carbon electrode (GCE), dry it, and add a mixed solution of EDC and NHS to activate the carboxyl groups to obtain the modified electrode 2N-GY / GCE. (5) OTC aptamer Apt I was dropped onto the surface of the 2N-GY / GCE modified electrode prepared in step (4), and after incubation, the electrode surface was cleaned to obtain the Apt I / 2N-GY / GCE modified electrode. Then, the modified electrode was incubated with BSA solution to obtain the BSA / Apt I / 2N-GY / GCE modified electrode. (6) Add OTC solution to the surface of the BSA / AptⅠ / 2N-GY / GCE modified electrode prepared in step (5), incubate, and then wash and dry. (7) Disperse the PDAs-Apt II prepared in step (3) in 0.01 M PBS, add PDAs-Apt II to the surface of the OTC / BSA / Apt I / 2N-GY / GCE modified electrode prepared in step (6), wash and dry to obtain the modified electrode for detecting OTC.

2. The preparation method according to claim 1, characterized in that: In step (1), 2N-GY was prepared; the ratio of CaC2 to pyrazine was 1.5 g : 0.5 mL. Under argon protection, CaC2 and pyrazine were reacted in a ball mill at 600 rpm, using a cyclic mode of 30 min ball milling / 15 min cooling for 16 h. After washing with 1 M HNO3, ultrapure water, and anhydrous ethanol to remove impurities, the mixture was vacuum dried at 80 °C for 12 h to obtain 2N-GY.

3. The preparation method according to claim 1, characterized in that: The preparation method according to claim 1 in step (1) is characterized in that: in step (2), concentrated nitric acid is used to carboxylate 2N-GY; wherein the mass fraction of concentrated nitric acid is 65% to 68%, and the ratio of 2N-GY to HNO3 is 4 mg: 1 mL. 2N-GY is added to concentrated HNO3 and heated at 80°C for 4 h to carboxylate 2N-GY.

4. The preparation method according to claim 1, characterized in that: Step (2) of the preparation method according to claim 1 is characterized in that: the processing method of step (3) is as follows: PDAs and OTC aptamers (Apt II) are mixed and incubated in a constant temperature shaker at 200 rpm for 2 h to obtain PDAs-Apt II, wherein the amount of PDAs to OTC aptamers is 500 μg / mL. -1 : 1 μM, 100 μL.

5. The preparation method according to claim 1, characterized in that: In step (4), the concentration of the suspension is 2 mg / mL. 10 μL of the suspension is dropped onto the electrode surface and dried. In step (4), the concentrations of EDC and NHS are 0.4 M and 0.1 M, respectively. 10 μL of the mixture is dropped onto the electrode surface, incubated at room temperature for 2 h, and then washed. In step (5), the concentration of OTC aptamer (Apt I) is 1 μM. 10 μL of Apt I is dropped onto the electrode surface, and then the modified electrode is incubated with 1 wt% BSA solution at room temperature for 1 h. In step (6), 10 μL of OTC solution is dropped onto the electrode surface. In step (7), the concentration of PDAs-Apt II is 2 mg / mL. 10 μL of the suspension is dropped onto the electrode surface.

6. The preparation method according to claim 1, characterized in that: The electrode is a glassy carbon electrode.

7. An electrode prepared by the method for preparing the 2N-GY luminescent material according to any one of claims 1-6 and the electrochemiluminescent modified electrode for detecting oxytetracycline.

8. An electrochemiluminescent biosensor for detecting oxytetracycline, characterized in that: It includes a working electrode, wherein the working electrode is a modified electrode prepared by any one of the preparation methods of claims 1-6 or a modified electrode as described in claim 7.

9. A method for detecting oxytetracycline, characterized in that: Using the modified electrode for detecting oxytetracycline as described in claim 7 as the working electrode, a platinum electrode as the auxiliary electrode, a calomel electrode as the reference electrode, and a K2S2O8 co-reactant solution as the detection solution with a concentration of 100 mM, the scanning potential for detecting oxytetracycline content is -2.5 V to 0 V, and the scanning rate is 0.1 V / s. The content of oxytetracycline is detected by electrochemiluminescence.

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