Preparation method and application of antibiotic electrochemical luminescence sensor with double enhancement strategies

Abstract

The invention belongs to the technical field of immunoassay and biosensing detection, and particularly relates to a preparation method and application of an antibiotic electrochemical luminescence sensor based on a double-enhancement strategy. According to the invention, a bifunctional metal organic gel is used as a substrate, a graphite-phase carbon nitride quantum dot is used as a luminous body, and a self-assembly split aptamer walker is combined to construct the electrochemical luminescence sensor for sensitive detection of antibiotics. The sensor has the advantages of extremely high specificity and sensitivity, wide detection range, low detection limit and strong anti-interference performance, and has important scientific significance and application value in detection of antibiotic residues in the environment.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an antibiotic electrochemiluminescence sensor with a double enhancement strategy, and belongs to the technical fields of nano functional materials, immunoassay and biosensor. Background Art

[0002] Antibiotics are a class of compounds that treat infections by inhibiting or killing bacterial growth. Their mechanisms of action include interference with cell wall synthesis (e.g., β-lactams), inhibition of protein synthesis (e.g., macrolides and aminoglycosides), and blocking nucleic acid replication (e.g., quinolones). They are widely used in human medicine and aquaculture (livestock, poultry, and aquaculture) to treat and prevent diseases caused by a variety of Gram-negative bacteria (e.g., Escherichia coli, Salmonella, and Vibrio) and Gram-positive bacteria (e.g., Staphylococcus and Streptococcus). However, long-term, extensive, and especially irregular use leads to the accumulation of antibiotics and their metabolites in animal-derived foods (e.g., meat, eggs, milk, and fish) and aquaculture environments (e.g., water bodies and soil). These residues, especially once released into the environment, exert sustained selective pressure on microbial communities, promoting the survival and proliferation of bacteria carrying or acquiring resistance genes (resistant bacteria), leading to the accumulation and spread of antibiotic-resistant bacteria (ARBs) and resistance genes (ARGs) in the environment. Resistance factors can be transmitted to humans through the environment and the food chain. This can not only cause direct toxicity or microecological imbalance, but more seriously, it can lead to a drug resistance crisis in human clinical treatment, making common infections difficult to cure, dramatically increasing the risks of medical treatments such as surgery and chemotherapy, and threatening public health with "superbugs." Therefore, the development of rapid, sensitive, and accurate antibiotic residue detection technologies is crucial for monitoring food safety, assessing environmental pollution, curbing the spread of drug resistance, and protecting human health.

[0003] There are many existing methods for detecting antibiotics, such as enzyme-linked immunosorbent assay, high-performance liquid chromatography, and ultraviolet-visible spectrophotometry. However, the above methods have disadvantages such as radioactive pollution, low sensitivity, long detection cycle, and complicated steps. Electrochemiluminescence sensor is a new analytical method that combines chemiluminescence technology with electrochemical technology. Therefore, it has the controllability of chemiluminescence technology and the sensitivity of electrochemical technology. Compared with traditional analytical methods, the electrochemiluminescence immunosensor adopted in the present invention has the advantages of high sensitivity, good selectivity, simple structure, easy operation, easy miniaturization, fast response, low background signal, and can effectively avoid false positive signals. Therefore, the antibiotic electrochemiluminescence sensor with a double enhancement strategy proposed in the present invention has a breakthrough significance for achieving ultra-high sensitivity and precise quantification of trace antibiotics, promoting on-site rapid screening, deepening environmental pollution assessment, and ultimately serving drug resistance prevention and control and human health and safety.

[0004] In the present invention, the metal organic gel serves as the substrate of the biosensor and synergistically enhances the luminescence of graphite carbon nitride quantum dots through its catalytic metal active sites, while providing a high-quality three-dimensional fiber scaffold for the immobilization of the probe. The initial signal is quenched by carrying ferrocene through a DNA hairpin containing an RNA base sequence. The designed split aptamer can co-recognize antibiotics through double strands and further assemble into a walker. By hydrolyzing the RNA bases in the hairpin DNA, the signal is restored and falls off to enter the next walking cycle. The designed split aptamer walker not only effectively avoids the leg entanglement problem caused by long aptamer chains, but also further amplifies the signal through sequential walking. The constructed "on-off-on" mode electrochemiluminescence sensor has the advantages of wide detection range, low detection limit, high sensitivity, simple operation, fast detection speed, etc. while effectively avoiding the occurrence of false positive signals. It also has good reproducibility, stability and selectivity, realizes sensitive detection of antibiotics, and is of great significance to the detection and prevention of environmental pollutants. Summary of the Invention

[0005] The present invention provides a preparation method and application of an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which realizes sensitive detection of antibiotics;

[0006] One of the purposes of the present invention is to provide a method for preparing an antibiotic electrochemiluminescence sensor with a double enhancement strategy;

[0007] The second purpose of the present invention is to apply the prepared antibiotic electrochemiluminescence sensor with a double enhancement strategy to the highly sensitive and specific detection of antibiotics.

[0008] The technical solutions of the present invention are as follows: 1. A method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy, comprising the following steps: (1) Polish a glassy carbon electrode with a diameter of 3.0 to 5.0 mm to a mirror surface using aluminum oxide polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) Add 6.0 μL of 1.0-3.0 mg / mL metal organic gel dispersion to the electrode surface, let it dry at room temperature, rinse the electrode surface with ultrapure water, and then let it dry. (3) Continue to add 6 μL of 1.0-5.0 mg / mL graphite phase carbon nitride quantum dot dispersion onto the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water and dry it; (4) Continue to add 6 μL of 5.0-15.0 μg / mL hairpin DNA dispersion to the electrode surface and incubate in a 4°C refrigerator for 8-14 hours; (5) Continue to add 3.0 μL of 0.8-1.2 mg / mL thioglycolic acid solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH = 7.38 phosphate buffer and dry in a refrigerator at 4°C. (6) 6.0 μL of the split aptamer walker dispersion was added dropwise to the electrode surface, rinsed with pH = 7.38 phosphate buffer, incubated at 37°C for 6 h, and rinsed again with pH = 7.38 phosphate buffer to prepare an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which was stored in a refrigerator at 4°C for future use.

[0009] 2. Graphite phase carbon nitride quantum dot dispersion, preparation steps are as follows: (1) Mix 0.3-0.5 g urea and 0.1-0.3 g sodium citrate with 15 mL ultrapure water and ultrasonicate for 15 minutes; (2) The mixed solution was transferred to a 25 mL polytetrafluoroethylene autoclave, reacted in an oven at 180° C. for 2 to 4 hours, cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, washed with ultrapure water, and centrifuged three times. The resulting supernatant was transferred to a dialysis bag, and an appropriate amount of ultrapure water was added for dialysis purification for 12 hours to obtain a graphite phase carbon nitride quantum dot precursor dispersion; (3) The graphite phase carbon nitride quantum dot precursor dispersion was freeze-dried at -60°C for 12 hours, and then dispersed into 2-10 mL of ultrapure water to obtain a graphite phase carbon nitride quantum dot dispersion of the desired concentration.

[0010] 3. Metal organic gel dispersion, preparation steps are as follows: (1) Dissolve 0.2-0.5 mg of 2,6-bis(2-benzimidazolyl)pyridine in 8-10 mL of methanol to obtain a 2,6-bis(2-benzimidazolyl)pyridine solution; (2) Add 0.5-0.8 mL of the above-prepared 2,6-bis(2-benzimidazolyl)pyridine solution to a 2 mL microcentrifuge tube, and continue to add 0.5-0.8 mL of AgNO3 solution. After thorough mixing, let it stand for 1 hour to obtain the metal organic gel, which was then stored in a refrigerator at 4°C. The AgNO3 solution was prepared by weighing 0.273 g of solid silver nitrate and adding it to 5 mL of ultrapure water to dissolve and mix. (3) The metal organic gel was diluted with 2-4 mL of ultrapure water and freeze-dried at -60°C to form a solid. The solid was then dispersed in ultrapure water to obtain metal organic gel dispersions of different concentrations. The dispersions were stored in a refrigerator at 4°C for later use.

[0011] 4. Preparation of hairpin DNA dispersion, steps are as follows: (1) 1 mL of 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide and 1 mL of 0.1-1 mol / L N-hydroxysuccinimide solution were added to 5 mL of 1-5 mol / L carboxyferrocene solution to obtain a quencher solution for use; (2) 5-10 mmol / L tris(2-carboxyethyl)phosphine was added to 1 mL of the self-designed hairpin DNA solution, and the solution was placed at room temperature for 60 minutes. The solution was then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 15 minutes. 10 μL of the quencher solution prepared in step (1) was added. The solution was incubated in a 4°C refrigerator for 8-12 hours, and then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 60 minutes to prepare a hairpin DNA dispersion.

[0012] 5. Preparation of Split Aptamer Walker Dispersion: (1) 30 μL of the self-designed split aptamer chain 1 and chain 2 solutions were heated to 95°C in a nucleic acid amplification instrument for 5 minutes, and then cooled to room temperature within 15 minutes to obtain pretreated split aptamer chain 1 and chain 2 solutions; (2) Take 10 μL of the above-mentioned pretreated split aptamer chain 1 and chain 2 solutions and 10-30 μL of 0.1 ng / mL-1 μg / mL antibiotic solution, mix the three, and then continue to add 0.25 mol / L magnesium chloride solution. Incubate at 37°C for 2 hours to prepare the split aptamer walker dispersion, and store it in a refrigerator at 4°C for use.

[0013] 6. A dual-enhancement strategy antibiotic electrochemiluminescence sensor for antibiotic detection, comprising the following steps: (1) The test was performed using an electrochemical workstation with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared antibiotic electrochemiluminescence sensor with a double enhancement strategy was used as the working electrode. The high voltage of the photomultiplier tube was set to 700 V, the scanning potential was 0 to -1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 100 mmol / L potassium persulfate at pH 5.5-8.5, the electrochemiluminescence signal intensity generated by the analyte at different concentrations was detected by an electrochemiluminescence system and the electrochemiluminescence signal intensity was recorded; (3) Record the electrochemiluminescence signal intensity corresponding to different concentrations of antibiotics; (4) Using the standard curve method, a working curve was drawn with a linear range of 0.1 ng / mL to 1 μg / mL to determine the concentration of the antibiotic in the sample to be tested.

[0014] Beneficial results of the present invention (1) The present invention successfully synthesized graphene-phase carbon nitride quantum dots. This material not only has a simple and convenient preparation method, but also has good biocompatibility and excellent electroluminescence intensity, avoiding tedious synthesis steps and harsh reaction conditions; (2) The metal-organic gel liquid used as a substrate in the present invention has excellent electrical conductivity, and its complex fiber structure allows for the fixation of more probes. Furthermore, the variable-valence silver metal acts as an active site, catalyzing the conversion of more potassium persulfate into free radicals, thereby enhancing the efficiency of electrochemiluminescence. This increases the signal change during antibiotic testing, improving detection sensitivity. Furthermore, as a substrate, the metal-organic gel has a stronger and more secure linking capability to the probes, eliminating the need for a crosslinking agent, reducing the number of steps, and avoiding the effects of the crosslinker's poor conductivity. (3) The split aptamer walker designed in the present invention can greatly improve the accuracy of specific recognition by co-recognizing antibiotics with double chains, effectively avoiding the problem of low recognition efficiency caused by the entanglement of the legs of traditional long-chain aptamers, and can further cascade amplify the electrochemiluminescence signal through sequential walking, effectively improving the sensitivity of sensor detection; (4) The antibiotic electrochemiluminescence sensor with a dual enhancement strategy prepared by the present invention has an "on-off-on" mode and can be used for the detection of antibiotics. It has the advantages of short response time, wide linear range, low detection limit, good stability and reproducibility, and can achieve simple, fast, highly sensitive and specific detection. DETAILED DESCRIPTION (The present invention will now be further described by way of specific embodiments, but is not limited thereto) Example 1. A method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy, comprising the following steps: (1) A glassy carbon electrode with a diameter of 3.0 mm was polished to a mirror surface using aluminum oxide polishing powder and then ultrasonically cleaned in anhydrous ethanol. (2) Add 6.0 μL of 1.0 mg / mL metal organic gel dispersion to the electrode surface, let it dry at room temperature, rinse the electrode surface with ultrapure water, and then let it dry. (3) Continue to add 6 μL of 1.0 mg / mL graphite phase carbon nitride quantum dot dispersion to the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water and dry it; (4) Continue to add 6 μL of 5.0 μg / mL hairpin DNA dispersion to the electrode surface and incubate in a 4°C refrigerator for 8 to 14 hours; (5) Continue to add 3.0 μL of 0.8 mg / mL thioglycolic acid solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH = 7.38 phosphate buffer and dry in a refrigerator at 4°C. (6) 6.0 μL of the split aptamer walker dispersion was added dropwise to the electrode surface, rinsed with pH = 7.38 phosphate buffer, incubated at 37°C for 6 h, and rinsed again with pH = 7.38 phosphate buffer to prepare an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which was stored in a refrigerator at 4°C for future use.

[0015] Example 2. A method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy, comprising the following steps: (1) A glassy carbon electrode with a diameter of 4.0 mm was polished to a mirror surface using alumina polishing powder and then ultrasonically cleaned in anhydrous ethanol; (2) Add 6.0 μL of 2.0 mg / mL metal organic gel dispersion to the electrode surface, let it dry at room temperature, rinse the electrode surface with ultrapure water, and then let it dry. (3) Continue to add 6 μL, 3.0 mg / mL graphite phase carbon nitride quantum dot dispersion to the electrode surface, dry at room temperature, rinse the electrode surface with ultrapure water and dry; (4) Continue to add 6 μL of 10.0 μg / mL hairpin DNA dispersion to the electrode surface and incubate in a 4°C refrigerator for 8 to 14 hours; (5) Continue to add 3.0 μL of 0.8-1.2 mg / mL thioglycolic acid solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH = 7.38 phosphate buffer and dry in a refrigerator at 4°C. (6) 6.0 μL of the split aptamer walker dispersion was added dropwise to the electrode surface, rinsed with pH = 7.38 phosphate buffer, incubated at 37°C for 6 h, and rinsed again with pH = 7.38 phosphate buffer to prepare an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which was stored in a refrigerator at 4°C for future use.

[0016] Example 3. A method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy, comprising the following steps: (1) A glassy carbon electrode with a diameter of 5.0 mm was polished to a mirror surface using aluminum oxide polishing powder and then ultrasonically cleaned in anhydrous ethanol. (2) Add 6.0 μL of 3.0 mg / mL metal organic gel dispersion to the electrode surface, let it dry at room temperature, rinse the electrode surface with ultrapure water, and then let it dry. (3) Continue to add 6 μL of 5.0 mg / mL graphite phase carbon nitride quantum dot dispersion onto the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water and dry it; (4) Continue to add 6 μL of 15.0 μg / mL hairpin DNA dispersion to the electrode surface and incubate in a 4°C refrigerator for 8 to 14 hours; (5) Continue to add 3.0 μL of 1.2 mg / mL thioglycolic acid solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH = 7.38 phosphate buffer and dry in a refrigerator at 4°C. (6) 6.0 μL of the split aptamer walker dispersion was added dropwise to the electrode surface, rinsed with pH = 7.38 phosphate buffer, incubated at 37°C for 6 h, and rinsed again with pH = 7.38 phosphate buffer to prepare an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which was stored in a refrigerator at 4°C for future use.

[0017] Example 4. Graphite phase carbon nitride quantum dot dispersion, preparation steps are as follows: (1) Mix 0.3 g urea and 0.1 g sodium citrate with 15 mL ultrapure water and sonicate for 15 minutes; (2) The mixed solution was transferred to a 25 mL polytetrafluoroethylene autoclave, reacted in an oven at 180°C for 2 hours, cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, washed with ultrapure water, and centrifuged three times. The resulting supernatant was transferred to a dialysis bag, and an appropriate amount of ultrapure water was added for dialysis purification for 12 hours to obtain a graphite phase carbon nitride quantum dot precursor dispersion; (3) The graphite phase carbon nitride quantum dot precursor dispersion was freeze-dried at -60°C for 12 hours and then dispersed into 2 mL of ultrapure water to obtain a graphite phase carbon nitride quantum dot dispersion of the desired concentration.

[0018] Example 5. Graphite phase carbon nitride quantum dot dispersion, preparation steps are as follows: (1) Mix 0.4 g urea and 0.2 g sodium citrate with 15 mL ultrapure water and sonicate for 15 minutes; (2) The mixed solution was transferred to a 25 mL polytetrafluoroethylene autoclave, reacted in an oven at 180°C for 3 hours, cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, washed with ultrapure water, and centrifuged three times. The resulting supernatant was transferred to a dialysis bag, and an appropriate amount of ultrapure water was added for dialysis purification for 12 hours to obtain a graphite phase carbon nitride quantum dot precursor dispersion; (3) The graphite phase carbon nitride quantum dot precursor dispersion was freeze-dried at -60°C for 12 hours and then dispersed into 5 mL of ultrapure water to obtain a graphite phase carbon nitride quantum dot dispersion of the desired concentration.

[0019] Example 6. Graphite phase carbon nitride quantum dot dispersion, the preparation steps are as follows: (1) Mix 0.5 g urea and 0.3 g sodium citrate with 15 mL ultrapure water and sonicate for 15 minutes; (2) The mixed solution was transferred to a 25 mL polytetrafluoroethylene autoclave, reacted in an oven at 180°C for 4 hours, cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, washed with ultrapure water, and centrifuged three times. The resulting supernatant was transferred to a dialysis bag, and an appropriate amount of ultrapure water was added for dialysis purification for 12 hours to obtain a graphite phase carbon nitride quantum dot precursor dispersion; (3) The graphite phase carbon nitride quantum dot precursor dispersion was freeze-dried at -60°C for 12 hours and then dispersed into 10 mL of ultrapure water to obtain a graphite phase carbon nitride quantum dot dispersion of the desired concentration.

[0020] Example 7. Metal organic gel dispersion, the preparation steps are as follows: (1) Dissolve 0.2 mg of 2,6-bis(2-benzimidazolyl)pyridine in 8 mL of methanol to obtain a 2,6-bis(2-benzimidazolyl)pyridine solution; (2) 0.5 mL of the prepared 2,6-bis(2-benzimidazolyl)pyridine solution was added to a 2 mL microcentrifuge tube, and 0.5 mL of AgNO3 solution was added. After thorough mixing, the mixture was allowed to stand for 1 hour to obtain the metal organic gel, which was then stored in a refrigerator at 4°C. The AgNO3 solution was prepared by weighing 0.273 g of solid silver nitrate and adding it to 5 mL of ultrapure water to dissolve and mix. (3) The metal organic gel was diluted with 2 mL of ultrapure water and freeze-dried at -60°C to form a solid. The solid was then dispersed in ultrapure water to obtain metal organic gel dispersions of different concentrations. The dispersions were stored in a refrigerator at 4°C for later use.

[0021] Example 8. Metal organic gel solution, the preparation steps are as follows: (1) Dissolve 0.3 mg of 2,6-bis(2-benzimidazolyl)pyridine in 9 mL of methanol to obtain a 2,6-bis(2-benzimidazolyl)pyridine solution; (2) 0.7 mL of the prepared 2,6-bis(2-benzimidazolyl)pyridine solution was added to a 2 mL microcentrifuge tube, and 0.7 mL of AgNO3 solution was added. After thorough mixing, the mixture was allowed to stand for 1 hour to obtain the metal organic gel, which was then stored in a refrigerator at 4°C. The AgNO3 solution was prepared by weighing 0.273 g of solid silver nitrate and adding it to 5 mL of ultrapure water to dissolve and mix. (3) The metal organic gel was diluted with 3 mL of ultrapure water and freeze-dried at -60°C to form a solid. The solid was then dispersed in ultrapure water to obtain metal organic gel dispersions of different concentrations. The dispersions were stored in a refrigerator at 4°C for later use.

[0022] Example 9. Metal organic gel solution, the preparation steps are as follows: (1) Dissolve 0.5 mg of 2,6-bis(2-benzimidazolyl)pyridine in 10 mL of methanol to obtain a 2,6-bis(2-benzimidazolyl)pyridine solution; (2) 0.8 mL of the prepared 2,6-bis(2-benzimidazolyl)pyridine solution was added to a 2 mL microcentrifuge tube, and 0.8 mL of AgNO3 solution was added. After thorough mixing, the mixture was allowed to stand for 1 hour to obtain the metal organic gel, which was then stored in a refrigerator at 4°C. The AgNO3 solution was prepared by weighing 0.273 g of solid silver nitrate and adding it to 5 mL of ultrapure water to dissolve and mix. (3) The metal organic gel was diluted with 4 mL of ultrapure water and freeze-dried at -60°C to form a solid. The solid was then dispersed in ultrapure water to obtain metal organic gel dispersions of different concentrations. The dispersions were stored in a refrigerator at 4°C for later use.

[0023] Example 10 Preparation of hairpin DNA dispersion, the steps are as follows: (1) 1 mL of 1 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide and 1 mL of 0.1 mol / L N-hydroxysuccinimide solution were added to 5 mL of 1 mol / L carboxyferrocene solution to obtain a quencher solution for use; (2) 5 mmol / L tris(2-carboxyethyl)phosphine was added to 1 mL of the self-designed hairpin DNA solution, and the solution was placed at room temperature for 60 minutes. The solution was then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 15 minutes. 10 μL of the quencher solution prepared in step (1) was added. The solution was incubated in a 4°C refrigerator for 8 hours, and then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 60 minutes to prepare a hairpin DNA dispersion.

[0024] Example 11 Preparation of hairpin DNA dispersion, the steps are as follows: (1) 1 mL of 3 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide and 1 mL of 0.5 mol / L N-hydroxysuccinimide solution were added to 5 mL of 3 mol / L carboxyferrocene solution to obtain a quencher solution for use; (2) 5 mmol / L tris(2-carboxyethyl)phosphine was added to 1 mL of the self-designed hairpin DNA solution, and the solution was placed at room temperature for 60 minutes. The solution was then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 15 minutes. 10 μL of the quencher solution prepared in step (1) was added. The solution was incubated in a 4°C refrigerator for 9 hours, and then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 60 minutes to prepare a hairpin DNA dispersion.

[0025] Example 12 Preparation of hairpin DNA dispersion, the steps are as follows: (1) 1 mL of 5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide and 1 mL of 1 mol / L N-hydroxysuccinimide solution were added to 5 mL of 5 mol / L carboxyferrocene solution to obtain a quencher solution for use; (2) 10 mmol / L tris(2-carboxyethyl)phosphine was added to 1 mL of the self-designed hairpin DNA solution, and the solution was placed at room temperature for 60 minutes. The solution was then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 15 minutes. 10 μL of the quencher solution prepared in step (1) was added. The solution was incubated in a 4°C refrigerator for 12 hours, and then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 60 minutes to prepare a hairpin DNA dispersion.

[0026] Example 13 Split aptamer walker dispersion, prepared as follows: (1) 30 μL of the self-designed split aptamer chain 1 and chain 2 solutions were heated to 95°C in a nucleic acid amplification instrument for 5 minutes, and then cooled to room temperature within 15 minutes to obtain pretreated split aptamer chain 1 and chain 2 solutions; (2) Take 10 μL of the above-mentioned pretreated split aptamer chain 1 and chain 2 solutions and 10 μL of 0.1 ng / mL to 1 μg / mL antibiotic solution, mix the three, and then continue to add 0.25 mol / L magnesium chloride solution. Incubate at 37°C for 2 hours to prepare the split aptamer walker dispersion, and store it in a refrigerator at 4°C for use.

[0027] Example 14 Split aptamer walker dispersion, prepared as follows: (1) 30 μL of the self-designed split aptamer chain 1 and chain 2 solutions were heated to 95°C in a nucleic acid amplification instrument for 5 minutes, and then cooled to room temperature within 15 minutes to obtain pretreated split aptamer chain 1 and chain 2 solutions; (2) Take 10 μL of the above-mentioned pretreated split aptamer chain 1 and chain 2 solutions and 20 μL of 0.1 ng / mL to 1 μg / mL antibiotic solution, mix the three, and then continue to add 0.25 mol / L magnesium chloride solution. Incubate at 37°C for 2 hours to prepare the split aptamer walker dispersion, and store it in a refrigerator at 4°C for use.

[0028] Example 15 Split aptamer walker dispersion, prepared as follows: (1) 30 μL of the self-designed split aptamer chain 1 and chain 2 solutions were heated to 95°C in a nucleic acid amplification instrument for 5 minutes, and then cooled to room temperature within 15 minutes to obtain pretreated split aptamer chain 1 and chain 2 solutions; (2) Take 10 μL of the above-mentioned pretreated split aptamer chain 1 and chain 2 solutions and 30 μL of 0.1 ng / mL to 1 μg / mL antibiotic solution, mix the three, and then continue to add 0.25 mol / L magnesium chloride solution. Incubate at 37°C for 2 hours to prepare the split aptamer walker dispersion, and store it in a refrigerator at 4°C for use.

[0029] Example 16 An antibiotic electrochemiluminescent sensor with a double enhancement strategy is used to detect enrofloxacin, and the steps are as follows: (1) The test was performed using an electrochemical workstation with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared antibiotic electrochemiluminescence sensor with a double enhancement strategy was used as the working electrode. The high voltage of the photomultiplier tube was set to 700 V, the scanning potential was 0 to -1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 100 mmol / L potassium persulfate at pH 5.5-8.5, the electrochemiluminescence signal intensity generated by the analyte at different concentrations was detected by an electrochemiluminescence system and the electrochemiluminescence signal intensity was recorded; (3) Record the electrochemiluminescence signal intensity corresponding to different concentrations of antibiotics; (4) Using the standard curve method, a working curve was drawn with a linear range of 0.1 ng / mL to 1 μg / mL to determine the concentration of the antibiotic in the sample to be tested.

[0030] Example 17 An antibiotic electrochemiluminescent sensor with a double enhancement strategy is used to detect kanamycin, and the steps are as follows: Kanamycin in the sample was detected according to the method of Example 16. The linear range was 0.1 ng / mL to 1 μg / mL, and the concentration of kanamycin in the sample was obtained.

[0031] Example 18 An antibiotic electrochemiluminescence sensor with a double enhancement strategy is used to detect norfloxacin, and the steps are as follows: Norfloxacin in the sample was detected according to the method of Example 16, and the linear range was 0.1 ng / mL to 1 μg / mL, and the concentration of kanamycin in the sample was obtained.

Claims

1. A method for preparing an antibiotic electrochemiluminescent sensor with a double enhancement strategy, characterized in that: The following steps are involved: (1) Polish a glassy carbon electrode with a diameter of 3.0 to 5.0 mm to a mirror surface using aluminum oxide polishing powder and clean it by ultrasonic cleaning in anhydrous ethanol; (2) Add 6.0 μL of 1.0-3.0 mg / mL metal organic gel dispersion to the electrode surface, let it dry at room temperature, rinse the electrode surface with ultrapure water, and then let it dry. (3) Continue to add 6 μL of 1.0-5.0 mg / mL graphite phase carbon nitride quantum dot dispersion onto the electrode surface, dry it at room temperature, rinse the electrode surface with ultrapure water and dry it; (4) Continue to add 6 μL of 5.0-15.0 μg / mL hairpin DNA dispersion to the electrode surface and incubate in a 4°C refrigerator for 8-14 hours; (5) Continue to add 3.0 μL of 0.8-1.2 mg / mL thioglycolic acid solution to the electrode surface to block nonspecific active sites on the electrode surface. Rinse the electrode surface with pH = 7.38 phosphate buffer and dry in a refrigerator at 4°C. (6) 6.0 μL of the split aptamer walker dispersion was added dropwise to the electrode surface, rinsed with pH = 7.38 phosphate buffer, incubated at 37°C for 6 h, and rinsed again with pH = 7.38 phosphate buffer to prepare an antibiotic electrochemiluminescence sensor with a double enhancement strategy, which was stored in a refrigerator at 4°C for future use.

2. The method for preparing an antibiotic electrochemiluminescence sensor with a dual enhancement strategy according to claim 1, wherein the preparation of the metal organic gel dispersion is characterized by: Here are the steps: (1) Dissolve 0.2-0.5 mg of 2,6-bis(2-benzimidazolyl)pyridine in 8-10 mL of methanol to obtain a 2,6-bis(2-benzimidazolyl)pyridine solution; (2) Add 0.5-0.8 mL of the above-prepared 2,6-bis(2-benzimidazolyl)pyridine solution to a 2 mL microcentrifuge tube, and continue to add 0.5-0.8 mL of AgNO3 solution. After thorough mixing, let it stand for 1 hour to obtain the metal organic gel, which was then stored in a refrigerator at 4°C. The AgNO3 solution was prepared by weighing 0.273 g of solid silver nitrate and adding it to 5 mL of ultrapure water to dissolve and mix. (3) The metal organic gel was diluted with 2-4 mL of ultrapure water and freeze-dried at -60°C to form a solid. The solid was then dispersed in ultrapure water to obtain metal organic gel dispersions of different concentrations. The dispersions were stored in a refrigerator at 4°C for later use.

3. The method for preparing an antibiotic electrochemiluminescence sensor with a dual enhancement strategy according to claim 1, wherein the preparation of the graphite phase carbon nitride quantum dot dispersion is characterized by: Here are the steps: (1) Mix 0.3-0.5 g urea and 0.1-0.3 g sodium citrate with 15 mL ultrapure water and ultrasonicate for 15 minutes; (2) The mixed solution was transferred to a 25 mL polytetrafluoroethylene autoclave, reacted in an oven at 180° C. for 2 to 4 hours, cooled to room temperature, centrifuged at 10,000 rpm for 10 minutes, washed with ultrapure water, and centrifuged three times. The resulting supernatant was transferred to a dialysis bag, and an appropriate amount of ultrapure water was added for dialysis purification for 12 hours to obtain a graphite phase carbon nitride quantum dot precursor dispersion; (3) The graphite phase carbon nitride quantum dot precursor dispersion was freeze-dried at -60°C for 12 hours, and then dispersed into 2-10 mL of ultrapure water to obtain a graphite phase carbon nitride quantum dot dispersion of the desired concentration.

4. The method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy according to claim 1, wherein the hairpin DNA dispersion is prepared by: Here are the steps: (1) 1 mL of 1-5 mol / L 1-ethyl-3-dimethylaminopropylcarbodiimide and 1 mL of 0.1-1 mol / L N-hydroxysuccinimide solution were added to 5 mL of 1-5 mol / L carboxyferrocene solution to obtain a quencher solution for use; (2) 5-10 mmol / L tris(2-carboxyethyl)phosphine was added to 1 mL of the self-designed hairpin DNA solution, and the solution was placed at room temperature for 60 minutes. The solution was then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 15 minutes. 10 μL of the quencher solution prepared in step (1) was added. The solution was incubated in a 4°C refrigerator for 8-12 hours, and then heated to 95°C in a nucleic acid amplifier and maintained for 5 minutes. The solution was then cooled to room temperature within 60 minutes to prepare a hairpin DNA dispersion.

5. The method for preparing an antibiotic electrochemiluminescent sensor with a dual enhancement strategy according to claim 1, wherein the preparation of the split aptamer walker dispersion is characterized in that: Here are the steps: (1) 30 μL of the self-designed split aptamer chain 1 and chain 2 solutions were heated to 95°C in a nucleic acid amplification instrument for 5 minutes, and then cooled to room temperature within 15 minutes to obtain pretreated split aptamer chain 1 and chain 2 solutions; (2) Take 10 μL of the above-mentioned pretreated split aptamer chain 1 and chain 2 solutions and 10-30 μL of 0.1 ng / mL-1 μg / mL antibiotic solution, mix the three, and then continue to add 0.25 mol / L magnesium chloride solution. Incubate at 37°C for 2 hours to prepare the split aptamer walker dispersion, and store it in a refrigerator at 4°C for use.

6. An antibiotic electrochemiluminescence sensor with a dual enhancement strategy prepared by the preparation method according to claim 1, for use in antibiotic detection, characterized in that: Here are the steps: (1) The test was performed using an electrochemical workstation with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared antibiotic electrochemiluminescence sensor with a double enhancement strategy was used as the working electrode. The high voltage of the photomultiplier tube was set to 700 V, the scanning potential was 0 to -1.5 V, and the scanning rate was 0.1 V / s. (2) In 10 mL of phosphate buffer solution containing 100 mmol / L potassium persulfate at pH 5.5-8.5, the electrochemiluminescence signal intensity generated by the analyte at different concentrations was detected by an electrochemiluminescence system and the electrochemiluminescence signal intensity was recorded; (3) Record the electrochemiluminescence signal intensity corresponding to different concentrations of antibiotics; (4) Using the standard curve method, a working curve was drawn with a linear range of 0.1 ng / mL to 1 μg / mL to determine the concentration of the antibiotic in the sample to be tested.

7. The antibiotic according to claim 1, 2, 3, 4, 5, or 6, wherein: The antibiotic is selected from one of the following: enrofloxacin, kanamycin, and norfloxacin.