Working electrode for enhancing electrogenerated chemiluminescence performance based on catalyst, preparation method of working electrode and aptamer sensor for acetamiprid detection

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a working electrode for enhancing electrochemiluminescence performance based on a catalyst, a preparation method of the working electrode and an aptamer sensor for acetamiprid detection. The working electrode for enhancing the electrogenerated chemiluminescence property based on the catalyst is obtained by sequentially dispensing an activated europium-based MOF solution, a cDNA1 solution, a bovine serum albumin solution, a target mixture and a ZiF-67-cDNA2 solution on a glassy carbon electrode, according to the present invention, the nano confinement effect and the oxidation reduction mediation effect of ZIF-67 synergistically improve the electrochemiluminescence signal intensity, and an electrochemiluminescence enhancement strategy using ZIF-67 as the catalyst is provided, such that during acetamiprid detection, the enrichment and the reaction kinetics of active free radicals are significantly promoted through the ternary synergistic effect of ZIF-67, europium-based MOF and tripropylamine so as to significantly improve the detection sensitivity of acetamiprid. Therefore, the sensitivity of the sensing system is improved, and new technical support is provided for agricultural product safety monitoring.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a working electrode with catalyst-enhanced electrochemiluminescence performance, its preparation method, and an aptamer sensor for acetamiprid detection. The working electrode with catalyst-enhanced electrochemiluminescence performance is a working electrode with enhanced electrochemiluminescence performance based on a multifunctional catalyst zeolite imidazole ester framework material (ZIF-67). Background Technology

[0002] Acetamiprid (ACE) is a widely used neonicotinoid insecticide, prevalent in agricultural production. However, its residues in the environment and agricultural products accumulate through the food chain, causing neurotoxicity, cell damage, and even inflammation and apoptosis in mammals. Therefore, developing highly sensitive and selective methods for detecting acetamiprid is of great significance for ensuring food safety and human health.

[0003] Electrochemiluminescence (ECL) technology has attracted widespread attention in the field of biosensing due to its advantages of low background noise, high sensitivity, and fast response. However, traditional ECL systems often suffer from low efficiency in the formation of active intermediates and slow electron transport rates, limiting further improvements in their detection performance. Metal-organic framework (MOF) materials are widely used in the construction of sensing platforms due to their tunable pore structure, high specific surface area, and good chemical stability. Among them, europium-based MOFs (ETMs) have a rigid framework structure, which can effectively suppress nonradiative relaxation and enhance luminescence efficiency, but their electron transfer ability under electrochemical excitation is still insufficient. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a working electrode with catalyst-enhanced electrochemiluminescence performance, its preparation method, and an aptamer sensor for acetamiprid detection. The working electrode is prepared by sequentially drop-coating activated europium-based MOF solution, cDNA1 solution, bovine serum albumin solution, target mixture, and ZiF-67-cDNA2 solution onto a glassy carbon electrode. By leveraging the nano-confining effect and redox-mediated interaction of ZIF-67 to synergistically enhance the electrochemiluminescence signal intensity, a strategy using ZIF-67 as a catalyst for electrochemiluminescence enhancement is proposed. During acetamiprid detection, the ternary synergistic effect of ZIF-67 with europium-based MOF and tripropylamine significantly promotes the enrichment of active free radicals and reaction kinetics, thereby improving the sensitivity of the sensing system and providing new technological support for agricultural product safety monitoring.

[0005] The second objective of this invention is to construct an aptamer sensor using a working electrode based on catalyst-enhanced electrochemiluminescence performance. The constructed aptamer sensor is applied to the highly sensitive and specific detection of trace amounts of acetamiprid, achieving rapid analysis in the concentration range of 0.1 pM to 20 nM, with a detection limit as low as 52 fM. It also has good anti-interference ability and applicability to practical samples, thus realizing highly sensitive and accurate detection of acetamiprid.

[0006] Based on the above technical objectives, the present invention adopts the following technical solution: This invention protects a method for preparing a working electrode based on catalyst-enhanced electrochemiluminescence performance, comprising the following steps: S1. Polish the glassy carbon electrode with polishing powder, then clean it with deionized water. Place the glassy carbon electrode in a 5mM potassium ferricyanide solution and scan it at a potential of -0.2V to 0.6V to ensure that the difference in peak potential is less than 110mV. This ensures that the glassy carbon electrode surface is clean, highly active, and has a fast electron transfer rate, providing a reliable substrate for subsequent accurate electrochemical measurements.

[0007] S2. Add 8 μL of activated europium-based MOF solution (2 mg / mL to 5 mg / mL) to a glassy carbon electrode and dry at room temperature to obtain glassy carbon electrode I; europium-based MOF is an electroluminescent material.

[0008] The activated europium-based MOF is obtained by activating europium-based MOF materials with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (NHS).

[0009] S3. Add 8 μL of cDNA1 (5 μg / mL to 10 μg / mL) to glassy carbon electrode I. After drying at room temperature, wash with PBS buffer to remove excess cDNA1. Dry at room temperature to obtain glassy carbon electrode II. cDNA1 is complementary DNA, which is a DNA strand that can pair complementaryly with the bases of acetamiprid aptamer. The base sequence of acetamiprid aptamer is known, and the complementary DNA pairs complementaryly with its bases. The nucleotide sequence of cDNA1 is shown in SEQ ID No. 1.

[0010] S4. Add 3 μL of 1%–2% bovine serum albumin (BSA) solution to glassy carbon electrode II to block non-specific binding sites. After drying, wash away excess BSA with PBS buffer and dry at room temperature to obtain glassy carbon electrode III. EDC / NHS is a bridging agent. The 5' end of cDNA1 is modified with an amino group. After the europium-based MOF nanomaterial is activated by EDC / NHS, the amino group of cDNA1 can be linked to the carboxyl group of the activated europium-based MOF through EDC / NHS, thereby immobilizing it on the glassy carbon electrode. The target analyte is a biomolecule, which also carries amino and carboxyl groups and will also be immobilized on the glassy carbon electrode. Therefore, BSA (BSA is also a biomolecule) is needed to cover the remaining activation sites after cDNA1 binding, thereby ensuring that the subsequently added acetamiprid aptamer and analyte biomolecules will not non-specifically bind to the glassy carbon electrode.

[0011] S5. Incubate 100 μL of acetamiprid aptamer (APT) solution (5 μM~10 μM) and 100 μL of acetamiprid solution (different concentrations) on a mechanical shaker at 37 °C for 2 h to obtain a target mixture. APT stands for nucleic acid aptamer, and its function is to specifically bind to the target molecule (analyte). The acetamiprid aptamer selected is one that can only recognize and bind to the target analyte, acetamiprid. Add 8 μL of the target mixture to glassy carbon electrode III, dry at room temperature, then wash with PBS buffer to remove excess biomolecules, and dry at room temperature to obtain glassy carbon electrode IV.

[0012] S6. Add 8 μL of ZiF-67-cDNA2 solution (2 mg / mL~5 mg / mL) to glassy carbon electrode IV, dry at room temperature, wash with PBS buffer to remove unbound molecules, and dry at room temperature to obtain the working electrode based on catalyst-enhanced electrochemiluminescence performance. Like the acetamiprid aptamer, cDNA2 can also bind to cDNA1. The nucleotide sequence of cDNA2 is shown in SEQ ID No. 2. The 5' end of cDNA2 is modified with a thiol group, which is linked to the metal ionic bond of ZiF-67.

[0013] Preferably, the mass ratio of ZiF-67 to cDNA2 is 3 mg to 5 mg: 1 mL to 3 mL; further, the mass ratio of ZiF-67 to cDNA2 is 3 mg: 1 mL.

[0014] Preferably, the europium-based MOF solution is prepared according to the following steps: 90 mg–103 mg of EuCl3 and 30 mg–45 mg of tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (H4TCBPE) are dissolved in 10 mL–30 mL of N,N-dimethylformamide (DMF), followed by the addition of 0.2 mL–0.6 mL of acetic acid to the mixture, and heating at 100 °C–150 °C for 10 h–30 h. The product is then thoroughly treated with DMF and methanol. Wash and then dry at 60°C to obtain europium-based MOF material (ETM); dissolve 1 mg to 2 mg of ETM in PBS buffer, then add 200 μL to 400 μL of a solution containing 400 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 mM N-hydroxythiosuccinimide (NHS), shake for 2 h, centrifuge once, and disperse the obtained solid in 1 mL of PBS buffer.

[0015] Preferably, the ZiF-67-cDNA2 solution is prepared according to the following steps: 250 mg to 291 mg of Co(NO3)2·6H2O and 500 mg to 657 mg of 2-methylimidazole are dissolved in 10 mL to 20 mL of methanol, respectively. The two solutions are mixed and stirred at room temperature for 2 h. The mixture is then thoroughly washed with methanol and dried at 60 °C to obtain ZiF-67. 3 mg to 5 mg of ZiF-67 is mixed with 1 mL to 3 mL of cDNA2 (10 μM). The mixture is shaken for 12 h and then centrifuged once. The resulting product is redispersed in 1 mL of PBS buffer.

[0016] This invention also protects a working electrode based on catalyst-enhanced electrochemiluminescence performance, which is prepared using the above-described method.

[0017] The present invention also protects an aptamer sensor for the detection of acetamiprid, which is prepared using the above-described working electrode based on catalyst-enhanced electrochemiluminescence performance.

[0018] Preferably, the aptamer sensor for acetamiprid detection includes a three-electrode system, an electrochemical workstation, and a chemiluminescence detector.

[0019] A three-electrode system consisting of a working electrode, a reference electrode, and a counter electrode based on catalyst-enhanced electrochemiluminescence performance was adopted. The three-electrode system was connected to an electrochemical workstation, which was then connected to a chemiluminescence detector. The high voltage of the photomultiplier tube was set to 800V, the cyclic voltammetric scan potential range was set to 0V~1.5V, and the scan rate was set to 0.15V / s.

[0020] Using PBS buffer (pH 6.0–8.5) containing 12 mM tripropylamine as the electrolyte, one end of the three-electrode system was placed in the electrolyte. The electrochemiluminescence signal intensity generated by acetamiprid standard solutions of different concentrations was detected by electrochemiluminescence method, and a working curve showing the relationship between acetamiprid concentration and electrochemiluminescence signal intensity was plotted. The acetamiprid sample solution to be tested was used instead of the acetamiprid standard solution for determination. Based on the working curve and the obtained electrochemiluminescence signal intensity, the concentration of acetamiprid in the acetamiprid sample solution to be tested was calculated.

[0021] The overall approach to acetamiprid detection is as follows: acetamiprid aptamers exhibit the strongest specific recognition of the analyte acetamiprid. Therefore, a target mixture is first prepared by mixing the acetamiprid aptamer and acetamiprid, resulting in an aptamer + analyte structure, along with residual acetamiprid aptamer (the amount of acetamiprid aptamer is higher than that of acetamiprid). This mixture is then dropped onto a working electrode based on catalyst-enhanced electrochemiluminescence performance. The residual acetamiprid aptamer binds to cDNA1, leaving residual cDNA1 (the amount of cDNA1 is higher than that of acetamiprid aptamer). This residual cDNA1 then binds to cDNA2 (the recognition and binding strength is: acetamiprid aptamer + acetamiprid > acetamiprid aptamer + cDNA1 > cDNA1 + cDNA2). At this point, the catalyst ZiF-67 is attached to cDNA2 (ETM reacts with tripropylamine; the addition of ZiF-67 allows for the simultaneous catalysis of both ETM and tripropylamine, enhancing the luminescence intensity), thus strengthening the ECL signal of the electrode. Based on this, the more acetamiprid the analyte, the fewer acetamiprid aptamers remain, resulting in even fewer acetamiprid aptamers binding to the catalyst-enhanced electrochemiluminescence working electrode. Consequently, more cDNA1 remains on the catalyst-enhanced electrochemiluminescence working electrode, leading to more cDNA2 attached to it and a stronger sensor signal.

[0022] Compared with the prior art, the beneficial results of this invention are as follows: 1. This invention provides a working electrode based on catalyst-enhanced electrochemiluminescence performance. The working electrode is obtained by sequentially drop-coating an activated europium-based MOF solution, a cDNA1 solution, a bovine serum albumin solution, a target mixture, and a ZiF-67-cDNA2 solution onto a glassy carbon electrode. The activated europium-based MOF provides bridging conditions with cDNA1 and serves as an electroluminescent material. cDNA1 binds to the acetamiprid aptamer during detection. Bovine serum albumin covers the remaining activation sites on the activated europium-based MOF and cDNA1, preventing interference. The target mixture provides the basis for detection. In ZiF-67-cDNA2, ZiF-67 provides the catalytic conditions during detection, and cDNA2 binds to cDNA1. In summary, the catalyst-enhanced electrochemiluminescence working electrode provides the fundamental conditions for convenient acetamiprid detection.

[0023] This invention relates to a working electrode based on catalyst-enhanced electrochemiluminescence performance. It uses a europium-based metal-organic framework (Eu-MOFs, ETM) as the luminescent matrix and introduces the zeolite imidazolium ester framework ZIF-67 as a multifunctional catalyst. In the detection of acetamiprid, a ternary synergistic reaction system of ETM / ZIF-67 / tripropylamine is constructed. (Tripropylamine acts as a co-reactant, reacting with the luminescent material ETM to generate an ECL signal. The addition of catalyst ZIF-67 simultaneously catalyzes both ETM and tripropylamine, enhancing the luminescence intensity; hence the term "ternary synergistic.") The zeolite imidazolium ester framework material (ZIF-67) possesses abundant porous structures and reversible Co... 2+ / Co 3+ The redox center can simultaneously catalyze ETM and tripropylamine because: firstly, it enriches tripropylamine through nanoconfinement, shortening the mass transfer distance of the tripropylamine reactive radical; secondly, it acts as an electron mediator, accelerating interfacial charge transfer via Co. 2+ / Co 3+ Reversible valence state conversion promotes the electrochemical oxidation process of ETM and tripropylamine, significantly reduces the reaction energy barrier, and enhances the electrochemiluminescence efficiency, thereby overcoming the problem of insufficient electron transfer capability of ETM under electrochemical excitation in existing technologies.

[0024] 2. This invention uses ZIF-67 as a multifunctional catalyst, which enriches the tripropylamine active intermediate through its nano-confinement effect, while utilizing Co... 2+ / Co 3+ Reversible valence state transitions promote electron transfer processes, significantly enhancing the electrochemiluminescence efficiency of ETM and effectively improving the sensor's detection sensitivity.

[0025] 3. Based on the above design of the working electrode with catalyst-enhanced electrochemiluminescence performance, this invention constructs an aptamer sensor that achieves highly sensitive detection of acetamiprid (ACE), with a linear range of 0.1 pmol / L to 20 nmol / L and a detection limit as low as 52 fmol / L. It also exhibits good specificity, reproducibility, and stability, making it suitable for rapid detection of pesticide residues in agricultural products and environmental samples.

[0026] The aptamer sensor constructed in this invention achieves high-performance detection of acetamiprid, with the advantages of simple operation, fast response, and good selectivity. It exhibits a good linear relationship in the concentration range of 0.1 pM to 20 nM and is suitable for high-precision analysis of trace amounts of acetamiprid in complex samples. Attached Figure Description

[0027] Figure 1 This is a scanning electron microscope image of the ETM obtained based on Example 2.

[0028] Figure 2 The image shows a scanning electron microscope image of ZiF-67 obtained based on Example 3.

[0029] Figure 3 The image shows the ECL signal of the acetamiprid ECL sensor obtained in Example 4.

[0030] Figure 4 The graph shows the operating curve of the acetamiprid ECL sensor obtained in Example 4.

[0031] Figure 5 ECL signal diagram of aptamer sensor obtained by replacing acetamiprid standard solution with dichlorvos, malathion, etc. as interfering substances. Detailed Implementation

[0032] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention is further described below with reference to specific embodiments. However, the embodiments are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0033] This invention employs an electrochemiluminescence enhancement strategy using ZIF-67 as a catalyst. In the detection of acetamiprid, the ternary synergistic effect of ZIF-67 with europium-based MOF and tripropylamine significantly promotes the enrichment of active free radicals and reaction kinetics, thereby enhancing the sensitivity of the sensing system and solving the problem of insufficient electron transfer capability under electrochemical excitation.

[0034] This invention uses activated europium-based MOF, cDNA1, bovine serum albumin, target mixture, and ZiF-67-cDNA2 as raw materials to achieve specificity, reproducibility, and stability detection of acetamiprid.

[0035] The technical solution of the present invention will be studied using the following embodiments. The specific research methods and results are shown below: Example 1 A method for preparing a working electrode based on catalyst-enhanced electrochemiluminescence performance includes the following steps: S1. Polish the glassy carbon electrode with polishing powder, then clean it with deionized water, and then place the glassy carbon electrode in a 5mM potassium ferricyanide solution and scan it at a potential of -0.2V to 0.6V to make the difference in peak potential less than 110mV.

[0036] S2. Add 8 μL of activated europium-based MOF solution (2 mg / mL) dropwise onto the glassy carbon electrode and dry at room temperature to obtain glassy carbon electrode I.

[0037] The europium-based MOF material was prepared according to the following steps: 103 mg of EuCl3 and 45 mg of tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (H4TCBPE) were dissolved together in 30 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Then, 0.6 mL of acetic acid was added to the mixture, and the mixture was heated at 120 °C for 24 h. The product was thoroughly washed with DMF and methanol, and then dried at 60 °C to obtain the europium-based MOF material.

[0038] The activated europium-based MOF solution was prepared as follows: 2 mg of europium-based MOF material was dissolved in PBS buffer, and then 400 μL of a solution containing 400 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 mM N-hydroxythiosuccinimide (NHS) was added. The mixture was shaken for 2 h, centrifuged once, and the resulting solid was dispersed in 1 mL of PBS buffer.

[0039] S3. Add 8 μL of 10 μg / mL cDNA1 to glassy carbon electrode I, dry at room temperature, then wash with PBS buffer to remove excess cDNA1, and dry at room temperature to obtain glassy carbon electrode II.

[0040] S4. Add 3 μL of 1% bovine serum albumin solution to glassy carbon electrode II to block non-specific binding sites. After drying, wash away excess BSA with PBS buffer and dry at room temperature to obtain glassy carbon electrode III.

[0041] S5. 100 μL of 8 μM acetamiprid aptamer and 100 μL of 0.1 pM to 20 nM acetamiprid were incubated on a mechanical shaker at 37 °C for 2 h to obtain a target mixture. The target mixture was then stored in a refrigerator at 4 °C. 8 μL of target mixtures of different concentrations were dropped onto different glassy carbon electrodes III. After drying at room temperature, the electrodes were washed with PBS buffer to remove excess biomolecules and dried at room temperature to obtain different glassy carbon electrodes IV.

[0042] S6. Add 8 μL of 3 mg / mL ZiF-67-cDNA2 solution to different glassy carbon electrodes IV, dry at room temperature, wash with PBS buffer to remove unbound molecules, and dry at room temperature to obtain the working electrode based on catalyst-enhanced electrochemiluminescence performance.

[0043] The ZiF-67-cDNA2 solution was prepared as follows: 291 mg of Co(NO3)2·6H2O and 657 mg of 2-methylimidazole were dissolved in 20 mL of methanol, respectively. The two solutions were then mixed and stirred at room temperature for 2 h. The mixture was then thoroughly washed with methanol and dried at 60 °C to obtain ZiF-67. 3 mg of ZiF-67 was mixed with 1 mL of cDNA2 (10 μM), shaken for 12 h, and then centrifuged once more. The resulting product was redispersed in 1 mL of PBS buffer.

[0044] The sequence of cDNA1 is shown in SEQ ID No. 1, the sequence of cDNA2 is shown in SEQ ID No. 2, and the sequence of acetamiprid aptamer DNA is shown in SEQ ID No. 3. The 5' end of cDNA1 is modified with an amino group, and the 5' end of cDNA2 is modified with a thiol group.

[0045] The sequence of cDNA1 (SEQ ID No. 1): 5'-TCTTCATAATATGGTGTCAGCCCAACGATTAACCCTTAGC-3'.

[0046] The sequence of cDNA2 (SEQ ID No. 2): 5'-GACACCACATTATACACAACACACCCAC-3'.

[0047] The sequence of acetamiprid aptamer DNA (SEQ ID No. 3): CTGACACCATATTATGAAGA.

[0048] A method for preparing an aptamer sensor for detecting acetamiprid includes the following steps: S1. The three-electrode system of the electrochemical workstation was used for testing. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the counter electrode, and the working electrode based on the catalyst-enhanced electrochemiluminescence performance was used to form the three-electrode system. The electrochemical workstation and the chemiluminescence detector were connected together. The high voltage of the photomultiplier tube was set to 800V, the cyclic voltammetric scan potential range was 0V~1.5V, and the scan rate was 0.15V / s.

[0049] S2. Place one end of the three-electrode system in 10 mL of electrolyte at pH 7.5. The electrolyte consists of 1 / 15 M Na2HPO4, 1 / 15 M KH2PO4, 12 mM tripropylamine and water. The electrochemiluminescence signal intensity generated by different concentrations of acetamiprid standard solutions is detected by electrochemiluminescence method, and the working curve is plotted.

[0050] S3. Replace the acetamiprid standard solution with the acetamiprid sample solution for determination. Calculate the concentration of acetamiprid in the acetamiprid sample solution based on the working curve and the obtained electrochemiluminescence signal intensity.

[0051] The aptamer sensor for detecting acetamiprid, prepared in Example 1, was used in the following study. The specific research methods and results are shown below: Figure 3 This is an ECL signal diagram from an acetamiprid ECL sensor; Figure 3 The results show that the acetamiprid ECL sensor constructed in this invention has good detection results for acetamiprid, with a detection range of 0.1 pM to 20 nM.

[0052] Figure 4 The results show that I = 32075.88 + 2117.54lgc(R) 2 =0.9927), the linear range for acetamiprid detection is 0.1 pM-20 nM, and the detection limit is 52 fM.

[0053] Figure 5 The results show that the acetamiprid ECL sensor of the present invention has the performance of specific detection of acetamiprid. The detection steps are completely consistent with the acetamiprid detection steps, except that acetamiprid is replaced with other interfering substances.

[0054] Example 2 A method for preparing a working electrode based on catalyst-enhanced electrochemiluminescence performance includes the following steps: S1. Polish the glassy carbon electrode with polishing powder, then clean it with deionized water, and then place the glassy carbon electrode in a 5mM potassium ferricyanide solution and scan it at a potential of -0.2V to 0.6V to make the difference in peak potential less than 110mV.

[0055] S2. Add 8 μL of activated europium-based MOF solution at 3 mg / mL to the glassy carbon electrode and dry at room temperature to obtain glassy carbon electrode I.

[0056] The europium-based MOF material was prepared according to the following steps: 103 mg of EuCl3 and 45 mg of tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (H4TCBPE) were dissolved together in 30 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Then, 0.6 mL of acetic acid was added to the mixture, and the mixture was heated at 100 °C for 30 h. The product was thoroughly washed with DMF and methanol, and then dried at 60 °C to obtain the europium-based MOF material.

[0057] The activated europium-based MOF solution was prepared as follows: 2 mg of europium-based MOF material was dissolved in PBS buffer, and then 200 μL of a solution containing 400 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 mM of N-hydroxythiosuccinimide (NHS) was added. The mixture was shaken for 2 h, centrifuged once, and the resulting solid was dispersed in 1 mL of PBS buffer.

[0058] S3. Add 8 μL of cDNA1 at 5 μg / mL to glassy carbon electrode I, dry at room temperature, then wash with PBS buffer to remove excess cDNA1, and dry at room temperature to obtain glassy carbon electrode II.

[0059] S4. Add 3 μL of 1.5% bovine serum albumin solution to glassy carbon electrode II to block non-specific binding sites. After drying, wash away excess BSA with PBS buffer and dry at room temperature to obtain glassy carbon electrode III.

[0060] S5. 100 μL of 8 μM acetamiprid aptamer and 100 μL of 0.1 pM to 20 nM acetamiprid were incubated on a mechanical shaker at 37 °C for 2 h to obtain a target mixture. The target mixture was then stored in a refrigerator at 4 °C. 8 μL of target mixtures of different concentrations were dropped onto different glassy carbon electrodes III. After drying at room temperature, the electrodes were washed with PBS buffer to remove excess biomolecules and dried at room temperature to obtain different glassy carbon electrodes IV.

[0061] S6. Add 8 μL of 2 mg / mL ZiF-67-cDNA2 solution to different glassy carbon electrodes IV, dry at room temperature, wash with PBS buffer to remove unbound molecules, and dry at room temperature to obtain the working electrode based on catalyst-enhanced electrochemiluminescence performance.

[0062] The ZiF-67-cDNA2 solution was prepared as follows: 291 mg of Co(NO3)2·6H2O and 657 mg of 2-methylimidazole were dissolved in 20 mL of methanol, respectively. The two solutions were then mixed and stirred at room temperature for 2 h. The mixture was then thoroughly washed with methanol and dried at 60 °C to obtain ZiF-67. 4 mg of ZiF-67 was mixed with 2 mL of cDNA2 (10 μM), shaken for 12 h, and then centrifuged once more. The resulting product was redispersed in 1 mL of PBS buffer.

[0063] The sequence of cDNA1 is shown in SEQ ID No. 1, the sequence of cDNA2 is shown in SEQ ID No. 2, and the sequence of acetamiprid aptamer DNA is shown in SEQ ID No. 3. The 5' end of cDNA1 is modified with an amino group, and the 5' end of cDNA2 is modified with a thiol group.

[0064] Example 3 A method for preparing a working electrode based on catalyst-enhanced electrochemiluminescence performance includes the following steps: S1. Polish the glassy carbon electrode with polishing powder, then clean it with deionized water, and then place the glassy carbon electrode in a 5mM potassium ferricyanide solution and scan it at a potential of -0.2V to 0.6V to make the difference in peak potential less than 110mV.

[0065] S2. Add 8 μL of activated europium-based MOF solution (5 mg / mL) dropwise onto the glassy carbon electrode and dry at room temperature to obtain glassy carbon electrode I.

[0066] The europium-based MOF material was prepared according to the following steps: 103 mg of EuCl3 and 45 mg of tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (H4TCBPE) were dissolved together in 30 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Then, 0.6 mL of acetic acid was added to the mixture, and the mixture was heated at 150 °C for 10 h. The product was thoroughly washed with DMF and methanol, and then dried at 60 °C to obtain the europium-based MOF material.

[0067] The activated europium-based MOF solution was prepared as follows: 2 mg of europium-based MOF material was dissolved in PBS buffer, and then 300 μL of a solution containing 400 mM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 mM of N-hydroxythiosuccinimide (NHS) was added. The mixture was shaken for 2 h, centrifuged once, and the resulting solid was dispersed in 1 mL of PBS buffer.

[0068] S3. Add 8 μL of cDNA1 at 8 μg / mL to glassy carbon electrode I, dry at room temperature, then wash with PBS buffer to remove excess cDNA1, and dry at room temperature to obtain glassy carbon electrode II.

[0069] S4. Add 3 μL of 2% bovine serum albumin solution to glassy carbon electrode II to block non-specific binding sites. After drying, wash away excess BSA with PBS buffer and dry at room temperature to obtain glassy carbon electrode III.

[0070] S5. 100 μL of 8 μM acetamiprid aptamer and 100 μL of 0.1 pM to 20 nM acetamiprid were incubated on a mechanical shaker at 37 °C for 2 h to obtain a target mixture. The target mixture was then stored in a refrigerator at 4 °C. 8 μL of target mixtures of different concentrations were dropped onto different glassy carbon electrodes III. After drying at room temperature, the electrodes were washed with PBS buffer to remove excess biomolecules and dried at room temperature to obtain different glassy carbon electrodes IV.

[0071] S6. Add 8 μL of 5 mg / mL ZiF-67-cDNA2 solution to different glassy carbon electrodes IV, dry at room temperature, wash with PBS buffer to remove unbound molecules, and dry at room temperature to obtain the working electrode based on catalyst-enhanced electrochemiluminescence performance.

[0072] The ZiF-67-cDNA2 solution was prepared as follows: 291 mg of Co(NO3)2·6H2O and 657 mg of 2-methylimidazole were dissolved in 20 mL of methanol, respectively. The two solutions were then mixed and stirred at room temperature for 2 h. The mixture was then thoroughly washed with methanol and dried at 60 °C to obtain ZiF-67. 5 mg of ZiF-67 was mixed with 3 mL of cDNA2 (10 μM), shaken for 12 h, and then centrifuged once more. The resulting product was redispersed in 1 mL of PBS buffer.

[0073] The sequence of cDNA1 is shown in SEQ ID No. 1, the sequence of cDNA2 is shown in SEQ ID No. 2, and the sequence of acetamiprid aptamer DNA is shown in SEQ ID No. 3. The 5' end of cDNA1 is modified with an amino group, and the 5' end of cDNA2 is modified with a thiol group.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a working electrode based on catalyst-enhanced electrochemiluminescence performance, characterized in that, Includes the following steps: An activated europium-based MOF solution was dropped onto a glassy carbon electrode and dried at room temperature to obtain glassy carbon electrode I. Among them, the activated europium-based MOF is obtained by activating europium-based MOF material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide; cDNA1 solution was dropped onto glassy carbon electrode I. cDNA1 was used for base complementary pairing with acetamiprid aptamer. The nucleotide sequence of cDNA1 is shown in SEQ ID No.

1. The 5' end of cDNA1 was modified with amino groups. After drying at room temperature, glassy carbon electrode II was obtained. Bovine serum albumin solution was added dropwise to glassy carbon electrode II to block the non-specific binding sites on glassy carbon electrode II, and dried at room temperature to obtain glassy carbon electrode III; The acetamiprid aptamer was mixed with acetamiprid and incubated to obtain a target mixture; the target mixture was dropped onto glassy carbon electrode III and dried at room temperature to obtain glassy carbon electrode IV. ZiF-67-cDNA2 solution was dropped onto glassy carbon electrode IV and dried at room temperature to obtain a working electrode based on catalyst-enhanced electrochemiluminescence performance. The ZiF-67-cDNA2 solution was obtained by mixing ZiF-67 and cDNA2 solution, centrifuging to obtain a solid, and then dispersing it in PBS buffer. cDNA2 is used for base pairing with cDNA1. The nucleotide sequence of cDNA2 is shown in SEQ ID No.

2. The 5' end of cDNA2 is modified with a thiol group.

2. The method for preparing the working electrode based on catalyst-enhanced electrochemiluminescence performance according to claim 1, characterized in that, The mass ratio of ZiF-67 to cDNA2 solution is 3 mg to 5 mg: 1 mL to 3 mL; the cDNA2 solution contains 10 μM cDNA2.

3. The method for preparing the working electrode based on catalyst-enhanced electrochemiluminescence performance according to claim 1, characterized in that, The mass ratio of activated europium MOF, cDNA1 to ZiF-67-cDNA2 is 2 mg~5 mg: 5 μg~10 μg: 2 mg~5 mg.

4. The method for preparing the working electrode based on catalyst-enhanced electrochemiluminescence performance according to claim 1, characterized in that, The volume ratio of activated europium MOF solution to bovine serum albumin solution was 8:

3. The mass fraction of solute in bovine serum albumin solution was 1%~2%, and the amount of europium MOF in activated europium MOF solution was 2mg / mL~5mg / mL.

5. The method for preparing the working electrode based on catalyst-enhanced electrochemiluminescence performance according to claim 1, characterized in that, Europium-based MOF materials are prepared according to the following steps: EuCl3 and tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene were dissolved together in N,N-dimethylformamide to obtain a mixture. Acetic acid was then added to the mixture, and the mixture was heated at 100℃~150℃ for 10h~30h to obtain europium-based MOF materials. The mass ratio of EuCl3 to tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene is 90~103:30~45.

6. The method for preparing the working electrode based on catalyst-enhanced electrochemiluminescence performance according to claim 5, characterized in that, The activation method is as follows: europium-based MOF material is mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide in PBS buffer and shaken for 2 hours.

7. A working electrode based on catalyst-enhanced electrochemiluminescence performance, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 6.

8. An aptamer sensor for detecting acetamiprid, characterized in that, The working electrode based on catalyst-enhanced electrochemiluminescence performance as described in claim 7 was prepared.

9. The aptamer sensor for acetamiprid detection according to claim 8, characterized in that, Aptamer sensors used for acetamiprid detection include three-electrode systems, electrochemical workstations, and chemiluminescence detectors; A three-electrode system consisting of a working electrode, a reference electrode, and a counter electrode based on catalyst-enhanced electrochemiluminescence performance was adopted. The three-electrode system was connected to an electrochemical workstation, which was then connected to a chemiluminescence detector. The high voltage of the photomultiplier tube was set to 800V, the cyclic voltammetric scan potential range was set to 0V~1.5V, and the scan rate was set to 0.15V / s. Using PBS buffer containing tripropylamine at pH 6.0–8.5 as the electrolyte, one end of the three-electrode system was placed in the electrolyte. The electrochemiluminescence signal intensity generated by acetamiprid standard solutions of different concentrations was detected by electrochemiluminescence method, and a working curve of the relationship between acetamiprid concentration and electrochemiluminescence signal intensity was plotted. The acetamiprid sample solution to be tested was used instead of the acetamiprid standard solution for determination. Based on the working curve and the obtained electrochemiluminescence signal intensity, the concentration of acetamiprid in the acetamiprid sample solution to be tested was calculated.

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