An electrochemical aptamer sensor for detecting streptomycin and a preparation method thereof

Abstract

The application provides an electrochemical aptamer sensor for detecting streptomycin and a preparation method thereof, and the preparation method comprises the following steps: S1, pretreating a glassy carbon electrode; S2, preparing Cu-ZIF-8@AuNPs; performing nitrogen plasma treatment on the Cu-ZIF-8@AuNPs; S3, dropping the plasma modified Cu-ZIF-8@AuNPs suspension on the pretreated glassy carbon electrode and drying; S4, modifying the plasma modified Cu-ZIF-8@AuNPs / GCE composite electrode by using streptomycin aptamer to obtain a modified electrode; S5, performing blocking treatment by using BSA, and then performing washing and drying to obtain the electrochemical aptamer sensor; the plasma modified Cu-ZIF-8@AuNPs can significantly improve the electron transfer efficiency, and can enhance the streptomycin detection sensitivity, selectivity and stability of the electrochemical aptamer sensor.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical aptamer sensor technology, specifically relating to an electrochemical aptamer sensor for detecting streptomycin and its preparation method. Background Technology

[0002] Streptomycin is an aminoglycoside antibiotic widely used for the prevention and treatment of diseases in humans, animals, and plants, and is also used as a feed additive to promote livestock and poultry growth. However, most of the streptomycin ingested by humans and animals is excreted in its original form or as metabolites, entering the environment through pharmaceutical wastewater, livestock and poultry manure, aquaculture, and agricultural emissions, leading to increasingly serious antibiotic pollution problems. To control its environmental risks, there is an urgent need to establish efficient, sensitive, and on-site streptomycin detection technologies. Currently commonly used detection methods include high-performance liquid chromatography-mass spectrometry (HPLC-MS, LC-MS), enzyme-linked immunosorbent assay (ELISA), fluorescence spectrometry, capillary electrophoresis, and ultraviolet-visible spectrophotometry. Although these methods have high sensitivity and specificity, the equipment is expensive, the operation is complex, and the sample pretreatment is cumbersome, which is not conducive to rapid on-site detection.

[0003] In recent years, electrochemical detection methods have attracted widespread attention in the field of antibiotic detection due to their advantages such as low cost, high sensitivity, rapid response, and ease of miniaturization. The performance of electrochemical detection mainly depends on the modification material of the working electrode. Currently, single modification materials have limitations in terms of sensitivity and selectivity, and combining different types of materials to form a synergistic effect has become an effective way to improve detection performance. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides an electrochemical aptamer sensor for detecting streptomycin and its preparation method. This invention combines gold nanoparticles with Cu-ZIF-8 and simultaneously treats them with nitrogen plasma. The resulting plasma-modified Cu-ZIF-8@AuNPs can significantly improve electron transfer efficiency and enhance the streptomycin detection sensitivity, selectivity, and stability of the electrochemical aptamer sensor.

[0005] The technical solution of the present invention is as follows: A method for preparing an electrochemical aptamer sensor for detecting streptomycin includes the following steps: S1. Pretreatment of glassy carbon electrode; S2. Preparation of Cu-ZIF-8@AuNPs; Cu-ZIF-8@AuNPs were subjected to nitrogen plasma treatment to obtain plasma-modified Cu-ZIF-8@AuNPs; S3. Prepare plasma-modified Cu-ZIF-8@AuNPs suspension; drop the plasma-modified Cu-ZIF-8@AuNPs suspension onto the pretreated glassy carbon electrode, and then dry it to obtain the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode. S4. The surface of the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode was modified with streptomycin aptamer to obtain the modified electrode; S5. The modified electrode is sealed with BSA, then washed and dried with PBS buffer to obtain the electrochemical aptamer sensor.

[0006] This invention utilizes plasma-modified Cu-ZIF-8@AuNPs to functionalize glassy carbon electrodes. In this composite material, Cu-ZIF-8 serves as a porous carrier with a high specific surface area, efficiently loading a large number of highly conductive gold nanoparticles (AuNPs). The superior conductivity of AuNPs significantly promotes interfacial electron transport. Simultaneously, the stable framework structure of Cu-ZIF-8 effectively inhibits the aggregation of AuNPs. Together, they construct a stable three-dimensional conductive network, enhancing not only the electrode's electron transfer efficiency but also providing abundant active sites for subsequent streptomycin aptamer immobilization, thereby amplifying the signal changes induced by streptomycin recognition. Furthermore, the copper doping in ZIF-8 allows for stronger coordination or redox reactions with specific functional groups (such as amino and guanidine groups) in the streptomycin molecule, providing additional binding sites and reaction pathways for streptomycin recognition and capture, thus contributing to improved overall sensor response performance.

[0007] The present invention further employs nitrogen plasma treatment to modify the surface of Cu-ZIF-8@AuNPs. This treatment can achieve slight etching on the material surface, introduce more defect sites on the Cu-ZIF-8 support and AuNPs surface, and form highly active catalytic and adsorption centers. Simultaneously, the nitrogen doping effect occurring during plasma treatment allows nitrogen atoms to form strong hydrogen bonds or electrostatic interactions with Lewis basic groups such as amino groups (-NH2, guanidinyl groups, etc.) in streptomycin molecules using their lone pair electrons. This effectively enhances the specific recognition and enrichment ability of Cu-ZIF-8@AuNPs for streptomycin. Furthermore, nitrogen plasma treatment may partially open the material's pores, making the internally loaded AuNPs and Cu active sites more easily accessible. This treatment also improves the surface hydrophilicity and wettability of the material, promoting the penetration of aqueous electrolytes and rapid ion diffusion within the pores, ensuring full utilization of all active sites, and thus improving the sensor's response speed. In summary, nitrogen plasma modification can synergistically optimize the physicochemical properties and surface state of Cu-ZIF-8@AuNPs, effectively improving their selectivity, sensitivity, and stability in streptomycin detection.

[0008] Preferably, the nitrogen plasma treatment time is 100-125 s, the discharge power is 80-100 W, and the gas pressure is 70-110 Pa. This invention optimizes the parameters such as the nitrogen plasma treatment time, discharge power, and gas pressure to achieve both effective surface chemical reactions and gentle physical etching during nitrogen plasma treatment, without causing structural damage to Cu-ZIF-8@AuNPs.

[0009] Preferably, the preparation method of Cu-ZIF-8@AuNPs includes the following steps: 1) PVP, 2-methylimidazole, ZnCl2 and CuCl2·2H2O were dissolved in methanol in sequence under stirring. After stirring for 10-24 hours, the mixture was allowed to stand for one week. Then, Cu-ZIF-8 was obtained by centrifugation, washing and drying. 2) Disperse Cu-ZIF-8 in deionized water and then ultrasonically disperse it to obtain a dispersion. While stirring, slowly add HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 20-30 minutes, then slowly add sodium citrate aqueous solution. After the addition is complete, raise the temperature to 70-80℃ and continue stirring for 4-6 hours. Then centrifuge, wash and dry to obtain Cu-ZIF-8@AuNPs.

[0010] In preparing Cu-ZIF-8@AuNPs according to this invention, an aqueous solution of HAuCl4 is first added dropwise, followed by Au... 3+ The ions will react with Cu in Cu-ZIF-8 + After the reaction occurs, tiny gold atom clusters are formed in situ on the surface of Cu-ZIF-8@AuNPs. Then, by adding an aqueous solution of sodium citrate, a weak reducing agent, the remaining Au atoms can be dissolved. 3+ The ions are slowly and continuously reduced to gold atoms, and after 4 to 6 hours of growth, a uniform and firmly attached nano-gold layer can be formed on the Cu-ZIF-8 surface.

[0011] The Cu-ZIF-8@AuNPs prepared by the above method have a stable structure, and the AuNPs are not easily detached from Cu-ZIF-8 during nitrogen plasma treatment. The highly active AuNPs can still be uniformly and stably distributed on the porous support.

[0012] Preferably, in step 1), the molar ratio of 2-methylimidazole, ZnCl2 and CuCl2·2H2O is 30~35:0.8~3.3:1.

[0013] Preferably, in step 2), the molar ratio of HAuCl4 in the Cu-ZIF-8 and HAuCl4 aqueous solution is 0.8~1.0g:1mmol; the concentration of the HAuCl4 aqueous solution is 10~20 mM; the concentration of the sodium citrate aqueous solution is 10~20 mM; the volume ratio of the HAuCl4 aqueous solution to the sodium citrate aqueous solution is 1:5~10; and the dropping time of the sodium citrate aqueous solution is 1~2h.

[0014] Preferably, the plasma-modified Cu-ZIF-8@AuNPs suspension is obtained by dispersing plasma-modified Cu-ZIF-8@AuNPs in PBS buffer; the concentration of the plasma-modified Cu-ZIF-8@AuNPs suspension is 1~2 mg / mL.

[0015] Preferably, step S4 specifically includes the following steps: adding streptomycin aptamer solution to the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode and letting it stand at 4°C for 6~15h to obtain the modified electrode; The streptomycin aptamer is a thiolated streptomycin aptamer; the sequence of the thiolated streptomycin aptamer is 5'-TAG GGA ATT CGT CGA CGG ATC CGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGTCGT CTG CAG GTC GAC GCA TGC GCC G-3'SH C6.

[0016] Preferably, the streptomycin aptamer solution is prepared by mixing streptomycin aptamer and TE buffer; the concentration of the streptomycin aptamer solution is 0.5~4 μM.

[0017] Preferably, the pretreatment of the glassy carbon electrode specifically includes the following steps: grinding and polishing the glassy carbon electrode with nano-alumina powder; rinsing the electrode surface with deionized water; ultrasonicating in ethanol; cleaning the electrode; and then air-drying the cleaned glassy carbon electrode at room temperature.

[0018] The beneficial technical effects of this invention are as follows: 1. This invention relates to an electrochemical aptamer sensor constructed based on plasma-modified Cu-ZIF-8@AuNPs nanocomposite material and streptomycin aptamer. By combining gold nanoparticles with Cu-ZIF-8 and simultaneously treating with nitrogen plasma, the resulting plasma-modified Cu-ZIF-8@AuNPs has added electrochemical active sites and specific recognition sites, which can effectively improve electron transfer efficiency and enhance the sensitivity, selectivity and stability of the electrochemical aptamer sensor for streptomycin detection, providing a new technical route for rapid and accurate detection of streptomycin.

[0019] 2. By optimizing the nitrogen plasma treatment process parameters and the preparation method of Cu-ZIF-8@AuNPs, this invention enables effective surface chemical reaction and physical etching of the nanocomposite material during plasma modification of Cu-ZIF-8@AuNPs without causing structural damage to Cu-ZIF-8@AuNPs, thus demonstrating excellent and stable plasma modification effect. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] Example 1: This embodiment provides a method for preparing an electrochemical aptamer sensor for detecting streptomycin, comprising the following steps: S1. Pretreatment of glassy carbon electrode: Select a glassy carbon electrode with a diameter of 3 mm. First, use alumina powder with an average particle size of 0.05 μm to grind and polish the glassy carbon electrode on chamois leather to remove surface residues and improve surface smoothness. Then, thoroughly rinse the alumina powder off the electrode surface with deionized water, then sonicate in ethanol, and then clean with deionized water. Allow the cleaned glassy carbon electrode to air dry naturally at room temperature to obtain the pretreated glassy carbon electrode for later use.

[0022] S2. First, prepare Cu-ZIF-8@AuNPs: Dissolve 0.75 g PVP K30, 1.32 g 2-methylimidazole, 0.204 g ZnCl2, and 0.085 g CuCl2·2H2O in 100 mL of methanol under stirring. Stir at room temperature for 12 h, let the solution stand at room temperature for one week, centrifuge (10000 r, 5 min), wash 5 times with methanol, discard the supernatant, and dry at room temperature to obtain Cu-ZIF-8; take 0.20 g Cu-ZIF-8 and disperse it in 200 mL of deionized water, then sonicate to obtain a dispersion; while stirring, slowly add 20 mL of 10 mM HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 30 min, then slowly add 100 mL of 10 mM sodium citrate aqueous solution (completed within 1 h), then heat to 80℃ and continue stirring for 4 h, then centrifuge (10000 r, 5 min). The product was repeatedly washed with deionized water and then freeze-dried to obtain Cu-ZIF-8@AuNPs.

[0023] 50 mg of Cu-ZIF-8@AuNPs was evenly spread in a quartz boat and placed in a plasma treatment device for nitrogen plasma treatment. The pressure in the plasma treatment device was controlled at 70 Pa, the discharge power was 100 W, and the nitrogen plasma treatment time was 100 s to obtain plasma-modified Cu-ZIF-8@AuNPs. S3. Add the plasma-modified Cu-ZIF-8@AuNPs to PBS buffer, disperse evenly, and prepare a plasma-modified Cu-ZIF-8@AuNPs suspension with a concentration of 1 mg / mL. Store at 4 ℃ for later use.

[0024] 6 µL of plasma-modified Cu-ZIF-8@AuNPs suspension was dropped onto the pretreated glassy carbon electrode and then dried in air to form a plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode.

[0025] S4. Add thiolized streptomycin aptamer (5'-TAG GGA ATT CGT CGA CGG ATCCGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCCG-3'SH C6) to TE buffer, mix to prepare a 3.0 μM streptomycin aptamer solution, and store at 4°C. 10 µL of streptomycin aptamer solution was added to the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode and allowed to stand at 4 °C for 12 h to ensure that the aptamer was firmly bound, thus obtaining the modified electrode. S5. The modified electrode was blocked with 10 μL of 0.1 mg / mL BSA at 25 °C for 20 min to reduce nonspecific adsorption. Finally, it was rinsed with PBS solution and dried to obtain an electrochemical aptamer sensor for detecting streptomycin.

[0026] Example 2: This embodiment provides a method for preparing an electrochemical aptamer sensor for detecting streptomycin, comprising the following steps: S1. Pretreatment of glassy carbon electrode: Select a glassy carbon electrode with a diameter of 3 mm. First, use alumina powder with an average particle size of 0.05 μm to grind and polish the glassy carbon electrode on chamois leather to remove surface residues and improve surface smoothness. Then, thoroughly rinse the alumina powder off the electrode surface with deionized water, then sonicate in ethanol, and then clean with deionized water. Allow the cleaned glassy carbon electrode to air dry naturally at room temperature to obtain the pretreated glassy carbon electrode for later use.

[0027] S2. First, prepare Cu-ZIF-8@AuNPs: Dissolve 0.75 g PVP K30, 1.42 g 2-methylimidazole, 0.215 g ZnCl2, and 0.085 g CuCl2·2H2O in 100 mL of methanol under stirring. Stir at room temperature for 12 h, let the solution stand at room temperature for one week, centrifuge (10000 r, 5 min), wash 5 times with methanol, discard the supernatant, and dry at room temperature to obtain Cu-ZIF-8; take 0.16 g Cu-ZIF-8 and disperse it in 200 mL of deionized water, then sonicate to obtain a dispersion; while stirring, slowly add 20 mL of 10 mM HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 20 min, then slowly add 100 mL of 20 mM sodium citrate aqueous solution (completed within 1 h), then heat to 70℃ and continue stirring for 6 h, then centrifuge (10000 r, 5 min). The product was repeatedly washed with deionized water and then freeze-dried to obtain Cu-ZIF-8@AuNPs.

[0028] 50 mg of Cu-ZIF-8@AuNPs was evenly spread in a quartz boat and placed in a plasma treatment device for nitrogen plasma treatment. The pressure in the plasma treatment device was controlled at 110 Pa, the discharge power was 80 W, and the nitrogen plasma treatment time was 125 s to obtain plasma-modified Cu-ZIF-8@AuNPs. S3. Add the plasma-modified Cu-ZIF-8@AuNPs to PBS buffer, disperse evenly, and prepare a plasma-modified Cu-ZIF-8@AuNPs suspension with a concentration of 2 mg / mL. Store at 4 ℃ for later use.

[0029] 6 µL of plasma-modified Cu-ZIF-8@AuNPs suspension was dropped onto the pretreated glassy carbon electrode and then dried in air to form a plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode.

[0030] S4. Add thiolized streptomycin aptamer (5'-TAG GGA ATT CGT CGA CGG ATCCGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCCG-3'SH C6) to TE buffer, mix to prepare a 2.0 μM streptomycin aptamer solution, and store at 4°C. 10 µL of streptomycin aptamer solution was added to the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode and allowed to stand at 4 °C for 12 h to ensure that the aptamer was firmly bound, thus obtaining the modified electrode. S5. The modified electrode was blocked with 10 μL of 0.1 mg / mL BSA at 25 °C for 20 min to reduce nonspecific adsorption. Finally, it was rinsed with PBS solution and dried to obtain an electrochemical aptamer sensor for detecting streptomycin.

[0031] Example 3: This embodiment provides a method for preparing an electrochemical aptamer sensor for detecting streptomycin, comprising the following steps: S1. Pretreatment of glassy carbon electrode: Select a glassy carbon electrode with a diameter of 3 mm. First, use alumina powder with an average particle size of 0.05 μm to grind and polish the glassy carbon electrode on chamois leather to remove surface residues and improve surface smoothness. Then, thoroughly rinse the alumina powder off the electrode surface with deionized water, then sonicate in ethanol, and then clean with deionized water. Allow the cleaned glassy carbon electrode to air dry naturally at room temperature to obtain the pretreated glassy carbon electrode for later use.

[0032] S2. First, prepare Cu-ZIF-8@AuNPs: Dissolve 0.75 g PVP K30, 1.32 g 2-methylimidazole, 0.204 g ZnCl2, and 0.085 g CuCl2·2H2O in 100 mL of methanol under stirring. Stir at room temperature for 12 h, let the solution stand at room temperature for one week, centrifuge (10000 r, 5 min), wash 5 times with methanol, discard the supernatant, and dry at room temperature to obtain Cu-ZIF-8; take 0.20 g Cu-ZIF-8 and disperse it in 200 mL of deionized water, then sonicate to obtain a dispersion; while stirring, slowly add 20 mL of 10 mM HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 25 min, then slowly add 200 mL of 10 mM sodium citrate aqueous solution (completed within 2 h), then heat to 75℃ and continue stirring for 4 h, then centrifuge (10000 r, 5 min). The product was repeatedly washed with deionized water and then freeze-dried to obtain Cu-ZIF-8@AuNPs.

[0033] 50 mg of Cu-ZIF-8@AuNPs was evenly spread in a quartz boat and placed in a plasma treatment device for nitrogen plasma treatment. The pressure in the plasma treatment device was controlled at 110 Pa, the discharge power was 90 W, and the nitrogen plasma treatment time was 115 s to obtain plasma-modified Cu-ZIF-8@AuNPs. S3. Add the plasma-modified Cu-ZIF-8@AuNPs to PBS buffer, disperse evenly, and prepare a plasma-modified Cu-ZIF-8@AuNPs suspension with a concentration of 1.5 mg / mL. Store at 4 ℃ for later use.

[0034] 6 µL of plasma-modified Cu-ZIF-8@AuNPs suspension was dropped onto the pretreated glassy carbon electrode and then dried in air to form a plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode.

[0035] S4. Add thiolized streptomycin aptamer (5'-TAG GGA ATT CGT CGA CGG ATCCGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCCG-3'SH C6) to TE buffer, mix to prepare a 3.5 μM streptomycin aptamer solution, and store at 4°C. 10 µL of streptomycin aptamer solution was added to the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode and allowed to stand at 4 °C for 12 h to ensure that the aptamer was firmly bound, thus obtaining the modified electrode. S5. The modified electrode was blocked with 10 μL of 0.1 mg / mL BSA at 25 °C for 20 min to reduce nonspecific adsorption. Finally, it was rinsed with PBS solution and dried to obtain an electrochemical aptamer sensor for detecting streptomycin.

[0036] Comparative Example 1: Unlike Example 1, ZIF-8@AuNPs were used instead of Cu-ZIF-8@AuNPs, and nitrogen plasma modification was not performed.

[0037] The preparation method of the electrochemical aptamer sensor for detecting streptomycin in this comparative example specifically includes the following steps: S1. Pretreatment of glassy carbon electrode: Select a glassy carbon electrode with a diameter of 3 mm. First, use alumina powder with an average particle size of 0.05 μm to grind and polish the glassy carbon electrode on chamois leather to remove surface residues and improve surface smoothness. Then, thoroughly rinse the alumina powder off the electrode surface with deionized water, then sonicate in ethanol, and then clean with deionized water. Allow the cleaned glassy carbon electrode to air dry naturally at room temperature to obtain the pretreated glassy carbon electrode for later use.

[0038] S2. First, prepare ZIF-8@AuNPs: Dissolve 0.75 g PVP K30, 1.32 g 2-methylimidazole, and 0.204 g ZnCl2 sequentially in 100 mL of methanol under stirring. Stir at room temperature for 12 h, allow the solution to stand at room temperature for one week, centrifuge (10000 r, 5 min), wash 5 times with methanol, discard the supernatant, and dry at room temperature to obtain ZIF-8; take 0.20 g ZIF-8 and disperse it in 200 mL of deionized water, then sonicate to obtain a dispersion; while stirring, slowly add 20 mL of 10 mM HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 30 min, then slowly add 100 mL of 10 mM sodium citrate aqueous solution (completed within 1 h), then heat to 80℃ and continue stirring for 4 h, then centrifuge (10000 r, 5 min). (r, 5 min), and repeatedly washed with deionized water, the resulting product was freeze-dried to obtain ZIF-8@AuNPs.

[0039] S3. Add ZIF-8@AuNPs to PBS buffer, disperse evenly, and prepare a ZIF-8@AuNPs suspension with a concentration of 1 mg / mL. Store at 4 ℃ for later use.

[0040] 6 µL of ZIF-8@AuNPs suspension was dropped onto the pretreated glassy carbon electrode and then dried in air to form a ZIF-8@AuNPs / GCE composite electrode.

[0041] S4. Add thiolized streptomycin aptamer (5'-TAG GGA ATT CGT CGA CGG ATCCGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCCG-3'SH C6) to TE buffer, mix, and prepare a streptomycin aptamer solution with a concentration of 3.0 μM. 10 µL of streptomycin aptamer solution was added to the ZIF-8@AuNPs / GCE composite electrode and allowed to stand at 4℃ for 12 h to ensure that the aptamer was firmly bound, thus obtaining the modified electrode. S5. The modified electrode was blocked with 10 μL of 0.1 mg / mL BSA at 25 °C for 20 min to reduce nonspecific adsorption. Finally, it was rinsed with PBS solution and dried to obtain an electrochemical aptamer sensor for detecting streptomycin.

[0042] Comparative Example 2: Unlike Example 1, Cu-ZIF-8@AuNPs are not modified with nitrogen plasma.

[0043] The preparation method of the electrochemical aptamer sensor for detecting streptomycin in this comparative example specifically includes the following steps: S1. Pretreatment of glassy carbon electrode: Select a glassy carbon electrode with a diameter of 3 mm. First, use alumina powder with an average particle size of 0.05 μm to grind and polish the glassy carbon electrode on chamois leather to remove surface residues and improve surface smoothness. Then, thoroughly rinse the alumina powder off the electrode surface with deionized water, then sonicate in ethanol, and then clean with deionized water. Allow the cleaned glassy carbon electrode to air dry naturally at room temperature to obtain the pretreated glassy carbon electrode for later use.

[0044] S2. First, prepare Cu-ZIF-8@AuNPs: Dissolve 0.75 g PVP K30, 1.32 g 2-methylimidazole, 0.204 g ZnCl2, and 0.085 g CuCl2·2H2O in 100 mL of methanol under stirring. Stir at room temperature for 12 h, let the solution stand at room temperature for one week, centrifuge (10000 r, 5 min), wash 5 times with methanol, discard the supernatant, and dry at room temperature to obtain Cu-ZIF-8. 0.20 g of Cu-ZIF-8 was dispersed in 200 mL of deionized water and then ultrasonically dispersed to obtain a dispersion. While stirring, 20 mL of 10 mM HAuCl4 aqueous solution was slowly added dropwise to the dispersion. After the addition was complete, the mixture was stirred for 30 min. Then, 100 mL of 10 mM sodium citrate aqueous solution was slowly added dropwise (completed within 1 h). The mixture was then heated to 80 °C and stirred continuously for 4 h. After centrifugation (10000 r, 5 min), the mixture was washed repeatedly with deionized water. The resulting product was freeze-dried to obtain Cu-ZIF-8@AuNPs.

[0045] S3. Add Cu-ZIF-8@AuNPs to PBS buffer, disperse evenly, and prepare a Cu-ZIF-8@AuNPs suspension with a concentration of 1 mg / mL. Store at 4 ℃ for later use.

[0046] 6 µL of Cu-ZIF-8@AuNPs suspension was dropped onto the pretreated glassy carbon electrode and then dried in air to form a Cu-ZIF-8@AuNPs / GCE composite electrode.

[0047] S4. Add thiolized streptomycin aptamer (5'-TAG GGA ATT CGT CGA CGG ATCCGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGT CTG CAG GTC GAC GCA TGC GCCG-3'SH C6) to TE buffer, mix, and prepare a streptomycin aptamer solution with a concentration of 3.0 μM. 10 µL of streptomycin aptamer solution was added to the Cu-ZIF-8@AuNPs / GCE composite electrode and allowed to stand at 4℃ for 12 h to ensure that the aptamer was firmly bound, thus obtaining the modified electrode. S5. The modified electrode was blocked with 10 μL of 0.1 mg / mL BSA at 25 °C for 20 min to reduce nonspecific adsorption. Finally, it was rinsed with PBS solution and dried to obtain an electrochemical aptamer sensor for detecting streptomycin.

[0048] Comparative Example 3: Unlike Example 1, when Cu-ZIF-8@AuNPs were subjected to nitrogen plasma treatment, the gas pressure in the plasma treatment equipment was controlled at 70 Pa, the discharge power was 100 W, and the nitrogen plasma treatment time was 160 s.

[0049] Comparative Example 4: Unlike Example 1, when Cu-ZIF-8@AuNPs were subjected to nitrogen plasma treatment, the gas pressure in the plasma treatment equipment was controlled at 70 Pa, the discharge power was 135 W, and the nitrogen plasma treatment time was 100 s.

[0050] Comparative Example 5: Unlike Example 1, when Cu-ZIF-8@AuNPs were subjected to nitrogen plasma treatment, the gas pressure in the plasma treatment equipment was controlled at 70 Pa, the discharge power was 50 W, and the nitrogen plasma treatment time was 100 s.

[0051] Performance testing: 1. Two electrochemical aptamer sensors were prepared using the methods described in Examples 1-3 and Comparative Examples 1-5. One electrochemical aptamer sensor was directly tested for its detection effect on streptomycin, while the other electrochemical aptamer sensor was stored at 4 °C for 12 days, and then the detection effect of the stored electrochemical aptamer sensor on streptomycin was tested.

[0052] The specific testing method was as follows: The prepared electrochemical aptamer sensor was immersed in a 0.1 μM streptomycin solution and treated at 25°C for 30 min to allow the streptomycin to specifically bind to the aptamer on the electrode surface. The electrode was then transferred to a potassium ferrocyanide-potassium ferrocyanide mixed solution and tested using differential pulse voltammetry (DPV). The DPV detection parameters were: initial voltage -0.2 V, ending voltage 0.4 V, pulse width 1 mV, pulse amplitude 50 mV, and pulse period 0.2 s. The peak current of the electrochemical aptamer sensor was recorded.

[0053] The method for preparing the 0.1 μM streptomycin solution includes: weighing 0.05817 g of streptomycin and adding it to 10 ml of PBS solution to prepare a 1 mM streptomycin solution, then diluting it to a 0.1 μM solution and storing it at 4 ℃.

[0054] The preparation of the potassium ferricyanide-potassium ferrocyanide mixed solution involves adding 0.16462 g of potassium ferricyanide, 0.21195 g of potassium ferrocyanide and 0.75 g of KCl sequentially to 100 ml of distilled water, stirring, and storing in a brown glass bottle at 4 ℃.

[0055] The specific detection results of the peak current of the electrochemical aptamer sensor are shown in Table 1. Table 1 Peak current of electrochemical aptamer sensors As shown in Table 1, the peak currents of the electrochemical aptamer sensors in Examples 1-3 are all higher than those in the comparative examples, indicating that the plasma-modified Cu-ZIF-8@AuNPs used in the electrochemical aptamer sensors of these examples possess higher electrochemical activity. Furthermore, Table 1 shows that even after 12 days, the peak currents of the electrochemical aptamer sensors in Example 1 remained at 94.7% of their original values, those in Example 2 remained at 95.2%, and those in Example 3 remained at 95.8%, demonstrating the excellent stability of the electrochemical aptamer sensors. A comparison between Example 1 and Comparative Examples 1-2 shows that copper doping of ZIF-8 and nitrogen plasma treatment of Cu-ZIF-8@AuNPs both improve the stability and peak current of the electrochemical aptamer sensors. A comparison of Examples 1, 2, and 3-4 shows that if the time or discharge power is slightly too high during nitrogen plasma treatment, it can negatively impact the structure of Cu-ZIF-8@AuNPs, affecting the peak current and the stability of the electrochemical aptamer sensor. A comparison of Examples 1 and 5 shows that if the time and power during nitrogen plasma treatment are too low, the modification effect will be weakened.

[0056] 3. After centrifuging the milk samples to remove interfering substances, the samples were filtered through a 0.22 μm sterile membrane and diluted 10 times with PBS buffer solution. Then, streptomycin was added to prepare milk test samples containing different concentrations of streptomycin. The electrochemical aptamer sensors in Example 1 and Comparative Examples 1-4 were used to detect the milk test samples containing different concentrations of streptomycin. The detection results of streptomycin in the milk test samples are shown in Tables 2-6.

[0057] Table 2. Streptomycin detection results of the electrochemical aptamer sensor in Example 1 Table 3. Streptomycin detection results of the electrochemical aptamer sensor in Comparative Example 1 Table 4. Streptomycin detection results of the electrochemical aptamer sensor in Comparative Example 2 Table 5. Streptomycin detection results of the electrochemical aptamer sensor in Comparative Example 3. Table 6. Streptomycin detection results of the electrochemical aptamer sensor in Comparative Example 4 As shown in Tables 2-6, the electrochemical aptamer sensor in Example 1 of this invention can effectively quantify streptomycin in milk samples, and the detection results are superior to those of the electrochemical aptamer sensors in Comparative Examples 1-4. The electrochemical aptamer sensor in this invention possesses excellent selectivity, anti-interference ability, and stability, enabling accurate detection of streptomycin.

[0058] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, and for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. A method for preparing an electrochemical aptamer sensor for detecting streptomycin, characterized in that, Includes the following steps: S1. Pretreatment of glassy carbon electrode; S2. Preparation of Cu-ZIF-8@AuNPs; Cu-ZIF-8@AuNPs were subjected to nitrogen plasma treatment to obtain plasma-modified Cu-ZIF-8@AuNPs; S3. Prepare plasma-modified Cu-ZIF-8@AuNPs suspension; drop the plasma-modified Cu-ZIF-8@AuNPs suspension onto the pretreated glassy carbon electrode, and then dry it to obtain the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode. S4. The surface of the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode was modified with streptomycin aptamer to obtain the modified electrode; S5. The modified electrode is sealed with BSA, then washed and dried with PBS buffer to obtain the electrochemical aptamer sensor.

2. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 1, characterized in that, The nitrogen plasma treatment time is 100~125s, the discharge power is 80~100W, and the pressure is 70~110Pa.

3. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 1, characterized in that, The preparation method of Cu-ZIF-8@AuNPs includes the following steps: 1) PVP, 2-methylimidazole, ZnCl2 and CuCl2·2H2O were dissolved in methanol in sequence under stirring. After stirring for 10-24 hours, the mixture was allowed to stand for one week, and then centrifuged, washed and dried to obtain Cu-ZIF-8. 2) Disperse Cu-ZIF-8 in deionized water and then ultrasonically disperse it to obtain a dispersion. While stirring, slowly add HAuCl4 aqueous solution to the dispersion. After the addition is complete, stir for 20-30 minutes, then slowly add sodium citrate aqueous solution. After the addition is complete, raise the temperature to 70-80℃ and continue stirring for 4-6 hours. Then centrifuge, wash and dry to obtain Cu-ZIF-8@AuNPs.

4. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 3, characterized in that, In step 1), the molar ratio of 2-methylimidazole, ZnCl2 and CuCl2·2H2O is 30~35:0.8~3.3:

1.

5. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 3, characterized in that, In step 2), the molar ratio of HAuCl4 in the Cu-ZIF-8 and HAuCl4 aqueous solution is 0.8~1.0g:1mmol; the concentration of the HAuCl4 aqueous solution is 10~20 mM; the concentration of the sodium citrate aqueous solution is 10~20 mM; the volume ratio of the HAuCl4 aqueous solution to the sodium citrate aqueous solution is 1:5~10; and the dropping time of the sodium citrate aqueous solution is 1~2h.

6. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 1, characterized in that, The plasma-modified Cu-ZIF-8@AuNPs suspension was obtained by dispersing plasma-modified Cu-ZIF-8@AuNPs in PBS buffer; the concentration of the plasma-modified Cu-ZIF-8@AuNPs suspension was 1~2 mg / mL.

7. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 1, characterized in that, Step S4 specifically includes the following steps: adding streptomycin aptamer solution to the plasma-modified Cu-ZIF-8@AuNPs / GCE composite electrode and letting it stand at 4℃ for 6~15h to obtain the modified electrode; The streptomycin aptamer is a thiolated streptomycin aptamer; the sequence of the thiolated streptomycin aptamer is 5'-TAG GGA ATT CGT CGA CGG ATC CGG GGT CTG GTG TTC TGC TTT GTT CTG TCG GGT CGTCTG CAG GTC GAC GCA TGC GCC G-3'SH C6.

8. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 7, characterized in that, The streptomycin aptamer solution was prepared by mixing streptomycin aptamer and TE buffer; the concentration of the streptomycin aptamer solution was 0.5~4 μM, and the streptomycin aptamer solution was stored at 4°C.

9. The method for preparing the electrochemical aptamer sensor for detecting streptomycin as described in claim 1, characterized in that, The pretreatment of glassy carbon electrodes includes the following steps: grinding and polishing the glassy carbon electrodes with nano-alumina powder; rinsing the electrode surface with deionized water; ultrasonicating in ethanol; cleaning the electrode; and then air-drying the cleaned glassy carbon electrodes at room temperature.

10. An electrochemical aptamer sensor for detecting streptomycin, prepared by the method described in any one of claims 1 to 8.

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