Electrochemiluminescence aptasensor for sulfadimethoxine detection and detection method

Abstract

The present invention provides an electrochemiluminescence aptasensor for sulfadimethoxine detection and a detection method. In the present invention, with Ce-MOF / AuNPs as the sensing interface and Zn-TBAPy / SP as the signal probe, an aptasensor is constructed to achieve ultrasensitive detection of SDM, providing a new detection approach for SDM in food. Compared with traditional SDM detection methods, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment materials, thus providing a new analytical method for the detection of SDM.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical detection, and specifically relates to an electrochemiluminescence aptasensor and a detection method for sulfadimethoxine detection. Background Art

[0002] Sulfadimethoxine (SDM) is a kind of sulfonamide (SAs) antibiotic, belonging to broad-spectrum antibacterial drugs, which can effectively treat and prevent infectious diseases. Because of its advantages such as stable performance, broad antibacterial spectrum, and low cost, it has been widely used in the treatment and prevention of livestock and poultry diseases; in addition, it has a promoting effect on the growth of livestock and poultry, and is also used as a feed additive, directly added to animal feed or taken orally. However, the abuse or improper use of SDM antibiotics may cause environmental pollution and damage, and the increase of its residues will promote the enhancement of bacterial drug resistance, posing a potential hazard to human health. Therefore, establishing a rapid and sensitive method for detecting SDM antibiotic residues in food is of great significance for food safety control.

[0003] Currently, the methods for detecting SDM mainly include HPLC, CE,, ELISA, colorimetry, EC, and PEC. However, most of these methods require a long experimental period, complex experimental techniques, expensive equipment, and low sensitivity. Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides an electrochemiluminescence aptasensor for sulfadimethoxine detection. In the present invention, a Ce-MOF / AuNPs is used as a sensing interface and Zn-TBAPy / SP is used as a signal probe to construct an aptasensor to achieve ultrasensitive detection of SDM, providing a new detection approach for SDM in food.

[0005] Unless otherwise specified, the parts mentioned in the present invention are by weight, and the percentages are by mass percentage.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] An electrochemiluminescence aptasensor for sulfadimethoxine detection, characterized in that the construction method of the electrochemiluminescence aptasensor for sulfadimethoxine detection includes the following steps:

[0008] The dry electrode is electrochemically activated in H2SO4, then rinsed with ultrapure water and dried to obtain a clean glassy carbon electrode. The Ce-MOF / PEI and AuNPs solutions are dropped onto the surface of the clean glassy carbon electrode and dried at room temperature. Then, the dsDNA complex is dropped and incubated overnight at room temperature. Next, 6-mercapto-1-hexanol (MCH) solution is dropped and incubated for 30 - 50 min at room temperature. Then, sulfamethoxine solution is dropped and incubated for 1 - 2 h at room temperature. Then, the Zn-TBAPy / SP signal probe solution is dropped and incubated for 1 - 3 h at room temperature, thus obtaining an electrochemiluminescence aptasensor for the detection of sulfamethoxine.

[0009] The preparation method of the Zn-TBAPy / SP signal probe solution is as follows:

[0010] EDC and NHS are dissolved in MES solution to obtain MES buffer. The MES buffer is added to the prepared 0.5 - 2 mg / mL Zn-TBAPy dispersion. After stirring at room temperature for 20 - 40 min, the signal probe (SP) is added, and stirred in an ice bath for 10 - 12 h. Then, it is centrifuged, washed with water, and the precipitate is redispersed in ultrapure water to obtain the Zn-TBAPy / SP signal probe solution.

[0011] The preparation method of the Zn-TBAPy dispersion is as follows:

[0012] Zn(NO3)2·6H2O and H4TBAPy are added to the mixed solution of DMF and HNO3, and ultrasonically treated for 3 - 6 min. Subsequently, it is transferred to a round flask and heated under reflux at 110 - 130 °C for 20 - 25 h. Finally, the obtained yellow precipitate is centrifuged and washed with DMF, and placed in a vacuum drying oven at 80 - 120 °C. After drying, it is dispersed in ultrapure water to obtain the Zn-TBAPy dispersion.

[0013] The preparation method of the Ce-MOF dispersion is as follows:

[0014] Ce(NO3)3·6H2O is added to ultrapure water and ultrasonically treated for 2 - 4 min. Subsequently, H3BTC is dissolved in a water - ethanol mixture (v / v = 1:1), and the H3BTC solution is dropped into the Ce(NO3)3·6H2O solution under magnetic stirring, and placed in a water bath at 50 - 70 °C for reaction for 1 - 2 h. Finally, the obtained white precipitate is centrifuged and washed several times, and placed at 50 - 70 °C for drying; then, it is dispersed in ultrapure water to obtain the Ce-MOF dispersion.

[0015] The preparation method of the Ce-MOF / PEI dispersion is as follows:

[0016] PEI was added to the Ce-MOF dispersion prepared above, stirred at room temperature for 10 - 15 h, and then centrifuged and washed to obtain the Ce-MOF / PEI dispersion.

[0017] The preparation method of the dsDNA complex is as follows:

[0018] The amino-modified capture probe (CP) and the aptamer (Apt) were mixed, heated at 80 - 100 °C for 3 - 6 min, cooled to 60 - 70 °C and then reacted for 10 - 15 min, and then naturally cooled to room temperature to finally obtain the dsDNA complex.

[0019] The preparation method of AuNPs is as follows:

[0020] 1% HAuCl4 solution was added to 100 mL of ultrapure water and boiled, and then 2.5 mL of 1% trisodium citrate solution was quickly added and boiled for another 15 min. After cooling, it was restored to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs).

[0021] An electrochemiluminescence aptasensor for the detection of sulfadimethoxine (SDM), characterized in that the construction method of the electrochemiluminescence aptasensor for the detection of sulfadimethoxine includes the following steps:

[0022] (1) Preparation of the signal probe:

[0023] 1) Zn-TBAPy: Weigh 52.36 mg of Zn(NO3)2·6H2O (0.045 mmol) and 41.08 mg of H4TBAPy (0.015 mmol), add them to a mixed solution of 10 mL of DMF and 240 μL of HNO3 (65 wt%), and ultrasonically treat for 5 min. Subsequently, transfer it to a 50 mL round-bottom flask, heat and reflux at 120 °C for 24 h. Finally, wash the obtained yellow precipitate with DMF by centrifugation several times and dry it at 100 °C in a vacuum drying oven.

[0024] 2) Zn-TBAPy / SP: Weigh 153.40 mg of EDC and 23.02 mg of NHS and dissolve them in 1 mL of MES solution to obtain a MES buffer solution. Under stirring, add the MES buffer solution to 1 mL of the 1 mg / mL Zn-TBAPy dispersion prepared in 1), stir at room temperature for 30 min, then add 200 μL of SP (3 μM), stir in an ice bath for 12 h, then centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the Zn-TBAPy / SP signal probe solution.

[0025] (2) Preparation of the substrate material:

[0026] 1) Ce-MOF: Weigh 108 mg of Ce(NO3)3·6H2O (0.25 mmol) and add it to 5 mL of ultrapure water, then ultrasonically treat for 3 min. Subsequently, weigh 52 mg of H3BTC (trimesic acid) (0.25 mmol) and dissolve it in 5 mL of a water-ethanol mixture (v / v = 1:1). Under magnetic stirring, the H3BTC solution is dropped into the Ce(NO3)3·6H2O solution, and the mixture is placed in a water bath at 60 °C and reacted for 1 h. Finally, the obtained white precipitate is centrifuged and washed several times, and then dried at 60 °C.

[0027] 2) Ce-MOF / PEI: Take 200 μL of 1% PEI and add it to 1 mL of the 1.2 mg / mL Ce-MOF dispersion prepared above. Stir at room temperature for 12 h, then centrifuge and wash to obtain the Ce-MOF / PEI dispersion.

[0028] 3) AuNPs: Take 1 mL of 1% HAuCl4 solution and add it to 100 mL of ultrapure water and boil. Then quickly add 2.5 mL of 1% sodium citrate solution and continue boiling for 15 min. After cooling, adjust the volume back to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs).

[0029] (3) Preparation of dsDNA:

[0030] Mix 500 μL of amino-modified capture probe (CP) (3 μM) and 500 μL of aptamer (Apt) (3 μM), heat at 90 °C for 5 min, immediately cool to 65 °C and react for 10 min, then naturally cool to room temperature to finally obtain the dsDNA complex.

[0031] (4) Construction of an electrochemiluminescence aptasensor for sulfadimethoxine detection:

[0032] 1) Treat the SDM-binding aptamer (Apt), complementary strand (CP), and signal strand (SP) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store for later use;

[0033] 2) Immerse the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min, then rinse it thoroughly with ultrapure water and set aside for later use;

[0034] 3) Polish the electrode obtained in step 2) to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders respectively, then ultrasonically treat the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water respectively, and dry for later use;

[0035] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0036] 5) Gradually drop 10 μL of the Ce-MOF / PEI and AuNPs solution onto the surface of the cleaned glassy carbon electrode obtained in step 4), and dry at room temperature;

[0037] 6) Drop 10 μL of the prepared dsDNA onto the electrode prepared in step 5) and incubate overnight at room temperature;

[0038] 7) Drop 10 μL of 1% 6-mercapto-1-hexanol (MCH) solution onto the electrode obtained in step 6) and incubate at room temperature for 40 min;

[0039] 8) Drop 10 μL of SDM solutions with different concentrations onto the electrode obtained in step 7) and incubate at room temperature for 1.5 h;

[0040] 9) Drop 10 μL of the Zn-TBAPy / SP solution onto the electrode prepared in step 8) and incubate at room temperature for 2 h to obtain an electrochemiluminescence aptasensor for SDM detection.

[0041] The present invention also provides a method for detecting SDM using the above-mentioned electrochemiluminescence aptasensor.

[0042] The method for detecting SDM using the above-mentioned electrochemiluminescence aptasensor is characterized by including the following steps:

[0043] 1) Drop SDM with different concentrations, the target substance, onto the electrode of the sensor;

[0044] 2) Characterize the electrode in a 0.1 M PBS (pH = 7.0) solution containing 50 mM K2S2O8 and measure its luminescence intensity value;

[0045] 3) Draw a working curve according to the linear relationship between the luminescence intensity obtained in step 1) and the logarithm value of the SDM concentration;

[0046] 4) Detect the sample to be measured using the sensor, and calculate the SDM concentration of the sample to be measured through the working curve prepared in step 2) based on the obtained current value.

[0047] Compared with the prior art, the preparation method and application of an electrochemiluminescence aptasensor for detecting SDM of the present invention have the outstanding characteristics that:

[0048] The present invention for the first time constitutes a binary ECL system by the co-reaction of Zn-TBAPy and K2S2O8, realizing signal amplification and improving the sensitivity of the sensor. The present invention prepares a pyrene-based metal-organic framework (Zn-TBAPy) luminescent body, and then loads a large number of signal probes (SP) through the bonding effect between carboxylation and amino groups, and finally prepares a signal probe solution of Zn-TBAPy / SP. The present invention uses Ce-MOF / AuNPs as a sensing interface to capture a large number of dsDNA strands to construct an aptasensor. By the above means, the prepared electrochemiluminescence aptasensor is successfully used for the ultrasensitive detection of SDM. Compared with the traditional SDM detection method, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment materials, thus providing a new analytical method for the detection of SDM.

[0049] The beneficial effects of the present invention are as follows:

[0050] 1) Through the luminescent body of pyrene-based metal-organic framework (Zn-TBAPy), the characteristics of low luminescence efficiency and poor luminescence stability of pyrene ligands can be greatly improved, thereby improving the sensor performance to achieve signal amplification and at the same time improving the detection sensitivity.

[0051] 2) Using Ce-MOF / AuNPs as the base material has a large specific surface area and good conductivity, and immobilizes the dsDNA strand on the electrode.

[0052] 2) The aptamer is highly specific for the recognition of the target, which can improve the selectivity of the sensor, thus providing a new research direction and analytical method for the detection of trace SDM.

[0053] 4) The materials involved can be synthesized under laboratory conditions, with simple operation, low price of raw materials, and extremely small usage amount each time, reducing the experimental cost.

[0054] 5) The entire detection and analysis method has clear and simple steps, high sensitivity, and rapid signal response.

[0055] 6) The electrochemical aptasensor prepared by this method can provide a new method for the detection of SDM; the electrochemiluminescence aptasensor prepared by this invention can also be applied to other aspects such as the determination of food and drugs, biological samples, and environmental monitoring. Description of the Drawings

[0056] Figure 1 It is a voltage-electrochemiluminescence intensity diagram obtained by scanning different modified electrodes in 0.1 M PBS (pH 7.0) containing 50 mM K2S2O8 in the voltage range from 0 to -2V at a scanning rate of 300 mV / s.

[0057] Figure 2Cyclic voltammetry characterization diagrams of different modified electrodes obtained in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a scanning rate of 100 mV / s in the voltage range from -0.2 to 0.6V.

[0058] Figure 3 Results of detecting different concentrations of SDM by the sensor of the present invention. Among them, Figure A is a time-electrochemiluminescence intensity diagram of the sensor scanning 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL of SDM respectively in 0.1 M PBS (pH 7.0) containing 50 mM K2S2O8; Figure B is a calibration curve of the electrochemiluminescence intensity of the sensor versus the logarithm of different concentrations of SDM.

[0059] Figure 4 Results of sensor stability detection, which is a time-electrochemiluminescence intensity diagram obtained after continuously scanning 11 cycles of the sensor incubated with 10 pg / mL SDM;

[0060] Figure 5 Results of reproducibility obtained by scanning 5 sensors of the same batch and 3 sensors of different batches incubated with 1 ng / mL SDM under the same conditions.

[0061] Figure 6 Specific detection diagram of the aptamer sensor, where the interfering substances are erythromycin (ERY, 1 ng / mL), oxytetracycline hydrochloride (OTC, 1 ng / mL), chloramphenicol (CAP, 1 ng / mL), and sulfamethazine (SMZ, 1 ng / mL). Detailed implementation manners

[0062] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0063] The main chemical reagents used in the embodiments of the present invention are as follows:

[0064] The standards of sulfadimethoxine (SDM), 6-mercapto-1-hexanol (MCH), and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from J&K Scientific Ltd (Beijing, China); zirconium chloride (ZrCl4), lauric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); 2-aminoterephthalic acid (NH2-BDC), 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) were purchased from Merck (Shanghai, China); zinc nitrate hexahydrate (Zn(NO3)2·6H2O); chloroauric acid (HAuCl4) were purchased from Sigma (USA); N,N-dimethylformamide (DMF) and potassium persulfate (K2S2O8) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); poly(ethyleneimine) (PEI) was purchased from Alfa Aesar Co (USA).

[0065] The aptamers involved were synthesized by Sangon Biotech Co., Ltd. Shanghai, and the specific sequences are as follows:

[0066] Aptamer: 5’-TTTGAGGGCAACGAGTGTTTATAGA-3’

[0067] Capture probe: 5’-NH2-C6-TTTTCTATAAACACTCGTTGCCCTC-3’

[0068] Signal probe: 5’-NH2-C6-GAGGGCAACGAGTGTTTATAGAAAA-3’

[0069] The equipment and technical parameters used:

[0070] Instrument: The time / voltage-electrochemiluminescence intensity was measured using an MPI-E type electrochemiluminescence workstation (Xi'an, China). Cyclic voltammetry (CV) measurements were carried out using a Metrohm Autolab B.V. electrochemical workstation (Modular Instruments, Switzerland). Electrochemiluminescence detection employed a three-electrode system: the modified glassy carbon electrode (4 mm in diameter) served as the working electrode, the platinum wire as the counter electrode, and silver-silver chloride (saturated KCl) as the reference electrode. Electrochemical detection used a three-electrode system: the modified glassy carbon electrode (4 mm in diameter) was the working electrode, the platinum wire was the counter electrode, and the saturated calomel electrode (SCE) was the reference electrode. The pH value was monitored with a pH meter (S210 SevenCompact, Mettler-Toledo, Shanghai, China). The electrochemiluminescence three-electrode system was scanned at 300 mV / s in 0.1 M PBS (pH 7.0) containing 50 mM K2S2O8. The electrochemical three-electrode system was scanned at 100 mV / s in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0071] Example 1 Preparation of signal probe and substrate material.

[0072] The operations were carried out according to the following steps:

[0073] (1) Preparation of signal probe:

[0074] 1) Zn-TBAPy: Weigh 52.36 mg of Zn(NO3)2·6H2O (0.045 mmol) and 41.08 mg of H4TBAPy (0.015 mmol), add them to a mixture of 10 mL of DMF and 240 μL of HNO3 (65 wt%), and ultrasonically treat for 5 min. Subsequently, transfer it to a 50 mL round-bottom flask and heat under reflux at 120 °C for 24 h. Finally, wash the obtained yellow precipitate with DMF by centrifugation several times and dry it in a vacuum drying oven at 100 °C.

[0075] 2) Zn-TBAPy / SP: Weigh 153.40 mg of EDC and 23.02 mg of NHS and dissolve them in 1 mL of MES solution to obtain MES buffer. Under stirring, add the MES buffer to 1 mL of the 1 mg / mL Zn-TBAPy dispersion prepared in 1). After stirring at room temperature for 30 min, add 200 μL of SP (3 μM), stir in an ice bath for 12 h, then centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the Zn-TBAPy / SP signal probe solution.

[0076] (2) Preparation of substrate material:

[0077] 1) Ce-MOF: Weigh 108 mg of Ce(NO3)3·6H2O (0.25 mmol) and add it to 5 mL of ultrapure water, then ultrasonically treat for 3 min. Subsequently, weigh 52 mg of H3BTC (trimesic acid) (0.25 mmol) and dissolve it in 5 mL of a water-ethanol mixture (v / v = 1:1). While stirring magnetically, add the H3BTC solution dropwise to the Ce(NO3)3·6H2O solution, and place it in a water bath at 60 °C for reaction for 1 h. Finally, centrifuge and wash the obtained white precipitate several times, and dry it at 60 °C.

[0078] 2) Ce-MOF / PEI: Take 200 μL of 1% PEI and add it to 1 mL of the 1.2 mg / mL Ce-MOF dispersion prepared above, stir at room temperature for 12 h, then centrifuge and wash to obtain the Ce-MOF / PEI dispersion.

[0079] 3) AuNPs: Take 1 mL of 1% HAuCl4 solution and add it to 100 mL of ultrapure water and boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. After cooling, restore the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs).

[0080] (3)Preparation of dsDNA:

[0081] Mix 500 μL of the amino-modified capture probe (CP) (3 μM) and 500 μL of the aptamer (Apt) (3 μM), heat at 90 °C for 5 min, immediately cool to 65 °C and react for another 10 min, then naturally cool to room temperature to finally obtain the dsDNA complex.

[0082] (4)Construction of an electrochemiluminescent aptasensor for SDM detection, which is operated according to the following steps:

[0083] 1) Treat the SDM-binding aptamer (Apt), complementary strand (CP), and signal strand (SP) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store for later use;

[0084] 2) Immerse the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min, then rinse it thoroughly with ultrapure water and store for later use;

[0085] 3) Polish the electrode obtained in step 2) to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders respectively, then ultrasonically treat the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water respectively, and dry it for later use;

[0086] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0087] 5) Gradually drop 10 μL of the Ce-MOF / PEI and AuNPs solution onto the surface of the cleaned glassy carbon electrode obtained in step 4), and dry at room temperature;

[0088] 6) Drop 10 μL of the prepared dsDNA onto the electrode prepared in step 5) and incubate overnight at room temperature;

[0089] 7) Drop 10 μL of 1% 6-mercapto-1-hexanol (MCH) solution onto the electrode obtained in step 6) and incubate at room temperature for 40 min;

[0090] 8) Drop 10 μL of SDM solutions with different concentrations onto the electrode obtained in step 7) and incubate at room temperature for 1.5 h;

[0091] 9) Drop 10 μL of the Zn-TBAPy / SP signal probe solution onto the electrode prepared in step 8) and incubate at room temperature for 2 h, thus obtaining an electrochemiluminescence aptasensor for SDM detection.

[0092] Example 2 Detection of SDM Using an Electrochemiluminescence Aptasensor

[0093] Detect SDM using the electrochemiluminescence aptasensor constructed in Example 1, and operate according to the following steps:

[0094] I. Plot the working curve

[0095] 1) Respectively place the modified electrodes in steps 5) to 9) of the construction process of the electrochemiluminescence aptasensor for SDM detection in Example 1 in 0.1 M PBS (pH = 7.0) containing 50 mM K2S2O8 for characterization, and measure their electrochemiluminescence response signals. The results are as Figure 1 :

[0096] (a) SDM / MCH / dsDNA / AuNPs / Ce-MOF / GCE

[0097] (b) tracer label / SDM / MCH / dsDNA / AuNPs / Ce-MOF / GCE.

[0098] 2) Respectively place the modified electrodes in steps 5) to 8) of the construction process of the electrochemiluminescence aptasensor for SDM detection in Example 1 in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV characterization. Measure their current response signals. The results are asFigure 2 As shown: (a) bare glassy carbon electrode; (b) dropwise addition of Ce-MOF / PEI; (c) dropwise addition of AuNPs; (d) dropwise addition of dsDNA solution; (e) dropwise addition of MCH for blocking; (f) dropwise addition of SDM.

[0099] 3) Dropwise add 10 μL of target SDM with different concentrations onto the electrode of the aptasensor prepared in Example 1, and measure their luminescence intensity respectively. As Figure 3 shown in A: the concentrations from a to h are successively: 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL.

[0100] 3) According to the linear relationship between the obtained luminescence intensity value and the logarithm value of the SDM concentration, plot the working curve (as Figure 3 shown in B). The measurement results show that the luminescence intensity response value has a good linear relationship with the logarithm value of the SDM concentration in the range of 10 fg mL -1 -100 ng mL -1 The linear correlation coefficient is 0.9977, and the detection limit is 0.26 fg mL -1 ; The results are as Figure 3 shown in B.

[0101] II. Sensor stability test: After continuously performing 11 cycles of ECL measurement on the sensor prepared in Example 1 under the optimal conditions, its luminescence intensity only decreased by 3.45% (as Figure 4 shown), indicating that the sensor has good stability.

[0102] III. Sensor reproducibility test: After performing ECL measurement on the sensors prepared in Example 1 by incubating the same concentration of SDM (1 ng / mL) with different glassy carbon electrodes (as Figure 5 shown), the relative standard deviation (RSD) of the within-batch difference is 4.26%, and the relative standard deviation (RSD) of the between-batch difference is 3.72%, indicating that the sensor has good reproducibility.

[0103] IV. Sensor specificity test: To study the specificity of the proposed aptasensor, the interfering substances that may exist in milk are used: the interfering substances are erythromycin (ERY, 1 ng / mL), oxytetracycline hydrochloride (OTC, 1 ng / mL), chloramphenicol (CAP, 1 ng / mL), sulfamethazine (SMZ, 1 ng / mL). Measure the electrochemiluminescence intensity response values of different interfering substances under the same concentration and conditions in 0.1 M PBS (pH = 7.0) containing 50 mM K2S2O8. The results show (as Figure 6 shown) that the proposed aptasensor based on the highly specific reaction of SDM has good specificity.

Claims

1. An electrochemiluminescence aptasensor for sulfamethoxydiazine detection, characterized in that, The construction method of an electrochemiluminescence aptasensor for sulfadimethoxine detection includes the following steps: Electrochemically activate the dried electrode in H2SO4, then rinse it with ultrapure water and dry it in air to obtain a clean glassy carbon electrode. Drop Ce-MOF / PEI and AuNPs solutions onto the surface of the clean glassy carbon electrode and dry it at room temperature. Then drop the dsDNA complex and incubate it overnight at room temperature. Then drop 6-mercapto-1-hexanol solution and incubate it for 30 - 50 min at room temperature. Then drop sulfadimethoxine solution and incubate it for 1 - 2 h at room temperature. Then drop Zn-TBAPy / SP signal probe solution and incubate it for 1 - 3 h at room temperature to obtain the electrochemiluminescence aptasensor for sulfadimethoxine detection. The preparation method of the Zn-TBAPy / SP signal probe solution is as follows: EDC and NHS are dissolved in MES solution to obtain MES buffer. Add the MES buffer to the prepared Zn-TBAPy dispersion at a concentration of 0.5 - 2 mg / mL, stir at room temperature for 20 - 40 min, then add signal probe SP, stir in an ice bath for 10 - 12 h, then centrifuge, wash with water, and redisperse the precipitate in ultrapure water to obtain the Zn-TBAPy / SP signal probe solution. The preparation method of the Zn-TBAPy dispersion is as follows: Zn(NO3)2·6H2O and H4TBAPy are added to a mixed solution of DMF and HNO3, ultrasonically treated for 3 - 6 min, then transferred to a round flask and heated under reflux at 110 - 130 °C for 20 - 25 h. Finally, the obtained yellow precipitate is centrifuged and washed with DMF and placed in a vacuum drying oven at 80 - 120 °C. After drying, add ultrapure water to disperse it to obtain the Zn-TBAPy dispersion. The preparation method of the Ce-MOF dispersion is as follows: Ce(NO3)3·6H2O is added to ultrapure water and ultrasonically treated for 2 - 4 min. Then, H3BTC is dissolved in a mixture of water and ethanol with a volume ratio of 1:1, and the H3BTC solution is dropped into the Ce(NO3)3·6H2O solution under magnetic stirring and reacted in a water bath at 50 - 70 °C for 1 - 2 h. Finally, the obtained white precipitate is centrifuged and washed several times and dried at 50 - 70 °C. Then add ultrapure water to disperse it to obtain the Ce-MOF dispersion. The preparation method of the Ce-MOF / PEI dispersion is as follows: Add PEI to the above-prepared Ce-MOF dispersion and stir at room temperature for 10 - 15 h, then centrifuge and wash to obtain the Ce-MOF / PEI dispersion. The preparation method of the dsDNA complex is as follows: Mix the amino-modified capture probe CP and aptamer Apt, heat at 80 - 100 °C for 3 - 6 min, cool to 60 - 70 °C and react for 10 - 15 min, then naturally cool to room temperature to finally obtain the dsDNA complex. The preparation method of AuNPs is as follows: Add 1% HAuCl4 solution to 100 mL of ultrapure water and boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. After cooling, restore the volume to the original with ultrapure water to obtain a transparent wine-red solution, which is the gold nanoparticles AuNPs.

2. The electrochemiluminescence aptamer sensor according to claim 1, wherein It includes the following steps: (1) Preparation of signal probe: 1) Zn-TBAPy: Weigh 52.36 mg of Zn(NO3)2·6H2O and 41.08 mg of H4TBAPy and add them to a mixed solution of 10 mL of DMF and 240 μL of HNO3 with a concentration of 65 wt%. Sonicate for 5 min; subsequently, transfer it to a 50 mL round-bottom flask and heat under reflux at 120 °C for 24 h; finally, wash the obtained yellow precipitate with DMF by centrifugation several times and dry it at 100 °C in a vacuum drying oven. 2) Zn-TBAPy / SP: Weigh 153.40 mg of EDC and 23.02 mg of NHS and dissolve them in 1 mL of MES solution to obtain MES buffer. Under stirring, add the MES buffer to 1 mL of the 1 mg / mL Zn-TBAPy dispersion prepared in step 1). After stirring at room temperature for 30 min, add 200 μL of SP with a concentration of 3 μM, stir in an ice bath for 12 h, then centrifuge and wash with water. Redisperse the precipitate in 1 mL of ultrapure water to obtain the Zn-TBAPy / SP signal probe solution. (2) Preparation of substrate material: 1) Ce-MOF: Weigh 108 mg of Ce(NO3)3·6H2O and add it to 5 mL of ultrapure water, sonicate for 3 min. Subsequently, weigh 52 mg of H3BTC and dissolve it in a mixture of 5 mL of water and ethanol with a volume ratio of 1:

1. Under magnetic stirring, drop the H3BTC solution into the Ce(NO3)3·6H2O solution and react in a water bath at 60 °C for 1 h; finally, wash the obtained white precipitate by centrifugation several times and dry it at 60 °C. 2) Ce-MOF / PEI: Take 200 μL of 1% PEI and add it to 1 mL of the 1.2 mg / mL Ce-MOF dispersion prepared above. Stir at room temperature for 12 h, then centrifuge and wash to obtain the Ce-MOF / PEI dispersion. 3) AuNPs: Take 1 mL of 1% HAuCl4 solution and add it to 100 mL of ultrapure water and boil. Then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 min. After cooling, restore the volume to the original with ultrapure water to obtain a transparent wine-red solution, which is the gold nanoparticles AuNPs. (3) Preparation of dsDNA: Mix 500 μL of the amino-modified capture probe CP with a concentration of 3 μM and 500 μL of the aptamer Apt with a concentration of 3 μM, heat at 90 °C for 5 min, immediately cool to 65 °C and react for 10 min, then naturally cool to room temperature. Finally, obtain the dsDNA complex.

3. The electrochemiluminescence aptamer sensor according to claim 1, characterized in that, The construction method of an electrochemiluminescence aptasensor for sulfadimethoxine detection includes the following steps: 1) Treat the SDM-binding aptamer Apt, complementary strand CP, and signal strand SP with 20 mM Tris-HCl buffer at pH = 7.4 at room temperature and store for later use; 2) Immerse the glassy carbon electrode in a piranha washing solution with a volume ratio of 98% H2SO4 / 30% H2O2 of 3:1 for 30 min, then rinse thoroughly with ultrapure water and store for later use; 3) Polish the electrode obtained in step 2) to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders respectively, then ultrasonically treat the electrode in the order of ultrapure water, absolute ethanol, and ultrapure water respectively, and dry for later use; 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry; 5) Gradually drop 10 μL each of the Ce-MOF / PEI and AuNPs solutions onto the surface of the clean glassy carbon electrode obtained in step 4), and dry at room temperature; 6) Drop 10 μL of the prepared dsDNA onto the electrode prepared in step 5) and incubate overnight at room temperature; 7) Drop 10 μL of 1% 6-mercapto-1-hexanol solution onto the electrode obtained in step 6) and incubate for 40 min at room temperature; 8) Drop 10 μL of SDM solutions with different concentrations onto the electrode obtained in step 7) and incubate for 1.5 h at room temperature; 9) Drop 10 μL of the Zn-TBAPy / SP solution onto the electrode prepared in step 8) and incubate for 2 h at room temperature to obtain an electrochemiluminescence aptasensor for SDM detection.

4. A method for detecting SDM using the electrochemiluminescence aptasensor according to any one of claims 1-3, characterized in that, Including the following steps: 1) Drop SDM with different concentrations, the target substance, onto the electrode of the sensor; 2) Place the electrode in a 0.1 M PBS solution containing 50 mM K2S2O8 at pH = 7.0 for characterization and measure its luminescence intensity value; 3) Draw a working curve according to the linear relationship between the luminescence intensity obtained in step 2) and the logarithm of the SDM concentration; 4) Detect the sample to be tested with the sensor, and calculate the SDM concentration of the sample to be tested through the working curve prepared in step 3) based on the obtained current value.

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