CN110988067B - Electrochemical luminescence method for detecting diethylstilbestrol - Google Patents

Electrochemical luminescence method for detecting diethylstilbestrol Download PDF

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CN110988067B
CN110988067B CN201911281735.5A CN201911281735A CN110988067B CN 110988067 B CN110988067 B CN 110988067B CN 201911281735 A CN201911281735 A CN 201911281735A CN 110988067 B CN110988067 B CN 110988067B
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李静娴
单学凌
蒋鼎
陈智栋
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Changzhou University
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Abstract

The invention discloses an electrochemiluminescence method for detecting diethylstilbestrol, and particularly belongs to the field of electrochemiluminescence detection. The operation flow comprises the following steps: (1) immobilized amination of Ti-based MOFs (NH) with Perylenetetracarboxylic acid (PTCA)2-MIL-125); (2) preparing an electrochemiluminescence aptamer (apt) sensor; (3) diethylstilbestrol was detected by electrochemiluminescence. Wherein the dosage is apt/PTCA/NH2And the MIL-125 modified glassy carbon electrode is used as a working electrode, the Ag/AgCl electrode is used as a reference electrode, and the platinum electrode is used as an auxiliary electrode to form a traditional three-electrode system. The detection range of the method is 1.0 multiplied by 10‑15mol/L～1.0×10‑6mol/L, the lowest detection limit is 2.8 multiplied by 10‑16mol/L. The method for detecting the diethylstilbestrol has the advantages of low cost, high sensitivity, strong specificity and simple operation.

Description

Electrochemical luminescence method for detecting diethylstilbestrol
Technical Field
The invention belongs to the technical field of medical detection, and relates to an electrochemiluminescence method for detecting diethylstilbestrol.
Background
With the rapid development of scientific technology and the increasing material demand of people, a series of environmental problems are developed. Environmental Endocrine Disruptors (EDCs) are a class of chemical substances with hormone-like effects existing in natural environment, and have interfering effects on the synthesis, secretion, transport and degradation processes of endogenous hormones in organisms, thereby damaging reproductive development, immunity, Endocrine and nervous systems of the organisms and even causing Endocrine-metabolic disorders of offspring.
The Diethylstilbestrol (DES) is an artificially synthesized non-steroidal estrogen substance which can generate all pharmacological and therapeutic effects same as natural estradiol, is mainly used for functional bleeding and amenorrhea caused by low or deficient estrogen and hormone balance disorder, and can be used before stillbirth induction of labor to improve the sensitivity of the myometrium to oxytocin and the incident therapy of prostate cancer. If the female is taken with diethylstilbestrol during pregnancy, the female fetus is easy to be aborted until the adult pregnancy, mainly because diethylstilbestrol causes uterine deformity; for male fetuses, the symptoms caused by diethylstilbestrol mainly include: cryptorchidism, testicular cancer, epididymal cyst, infertility, etc. Diethylstilbestrol as an estrogen which can promote the synthesis of animal proteins is mainly present in animal foods such as pork, mutton and the like, and diethylstilbestrol is still illegally used as a growth promoter in some areas to increase the yield of animals. However, compared with natural estrogen, diethylstilbestrol is more stable in human body and is less liable to be metabolized out of human body, and diethylstilbestrol remained in animal body will enter human body through food chain, thus endangering human health. Thus, the 235 th bulletin of the ministry of agriculture of the people's republic of China in the revised "highest residual amount of veterinary drugs in animal food" stipulates that the highest content of diethylstilbestrol in all food animals and all edible tissues is undetected. In recent years, many researches have suggested that diethylstilbestrol widely existing in natural water environment not only has a lasting chronic toxicological effect on the growth, development and reproduction of various fishes, but also has an influence on the lipid metabolism process of the fishes, so that abnormal fat deposition is caused, and the quality and edible safety of the cultured fishes are influenced. Therefore, the establishment of a rapid and sensitive diethylstilbestrol residue detection method has important significance.
In the last few years, the most common methods for detecting DES are High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), capillary electrophoresis, chemiluminescence, and fluorescence. However, these methods are generally expensive, time consuming and cumbersome and have low sensitivity. Therefore, the establishment of a method for rapidly and sensitively measuring the diethylstilbestrol residual quantity has important significance.
Disclosure of Invention
The invention aims to provide a method for detecting an electrochemiluminescence sensor, aiming at the defects of the diethylstilbestrol detection prior art. The electrochemical luminescence sensor prepared by the invention is based on perylene tetracarboxylic acid (PTCA) fixed amination Ti-based MOFs (NH)2-MIL-125), followed by PTCA/NH2MIL-125 as a substrate material, an aptamer (apt) with specific recognition function as a recognition element, and the recognition element and the aptamer are jointly modified on a Glassy Carbon Electrode (GCE), so that the sensitivity and the stability of electrochemiluminescence are remarkably improved, and the electrochemiluminescence is based on NH2The MIL-125 has good enrichment capacity on diethylstilbestrol, and the specific combination of the aptamer and diethylstilbestrol improves the selectivity on diethylstilbestrol. The electrochemical luminescence aptamer sensor based on the invention can realize the rapid detection of the diethylstilbestrol content, has simple use method and high practical value.
The scheme adopted by the invention is as follows: an electrochemiluminescence method for detecting diethylstilbestrol is characterized in that an ECL aptamer sensor apt/PTCA/NH is used2And (3) taking MIL-125/GCE as a working electrode of an electrochemiluminescence test, taking a platinum electrode as an auxiliary electrode and taking Ag/AgCl as a reference electrode to form a three-electrode system for electrochemiluminescence detection of diethylstilbestrol.
Further, the preparation method of the ECL aptamer sensor comprises the following steps:
(1)PTCA/NH2-preparation of MIL-125 composite:
adding an aminated Ti-based MOFs material and perylene tetracarboxylic acid into an ethanol solution, performing ultrasonic homogenization, stirring in a magnetic stirrer, performing centrifugal separation after stirring, washing and drying to obtain PTCA/NH2-MIL-125 composite;
(2) PTCA/NH prepared in the step (1)2Dispersing an-MIL-125 composite material in DMF to prepare PTCA/NH2-a DMF solution of MIL-125 composite;
(3) modified electrode apt/PTCA/NH2Preparation of MIL-125/GCE:
transferring the PTCA/NH prepared in step (2) by a microsyringe2Dropping DMF solution of-MIL-125 on the surface of the pretreated glassy carbon electrode, and drying at room temperature to obtain PTCA/NH2-MIL-125/GCE modified electrode, then aptamer was added dropwise to PTCA/NH2Modifying the surface of the electrode by MIL-125/GCE, and combining an aptamer and PTCA through an amide bond to obtain apt/PTCA/NH2-MIL-125/GCE modified electrode; finally, apt/PTCA/NH2And placing the-MIL-125/GCE modified electrode in a refrigerator at 4 ℃ for 6 hours to obtain the ECL aptamer sensor. The purpose of placing the sensor in the refrigerator for a period of time is to prevent the sensor from being contaminated and to maintain the sensor at an optimum performance
Further, the aminated Ti-based MOFs material in the step (1) is prepared by the following method: adding isopropyl titanate and 2-amino terephthalic acid into a mixed solution of DMF and methanol, stirring and mixing uniformly by ultrasonic, pouring the obtained yellow milky liquid into a stainless steel autoclave with a polytetrafluoroethylene lining for reaction, centrifuging, washing and drying the product after the reaction is finished to obtain yellow powdery aminated Ti-based MOFs material.
Further, the perylene tetracarboxylic acid in the step (1) is prepared by the following method: dissolving perylene tetracarboxylic dianhydride in NaOH solution, violently stirring to obtain yellow-green solution, dripping HCl solution until complete precipitation, centrifuging, repeatedly washing with deionized water, and drying to obtain dark red powdery perylene tetracarboxylic dianhydride.
Furthermore, the mass ratio of the mass of the aminated Ti-based MOFs material in the step (1) to the mass of the perylene tetracarboxylic acid is 1-3: 1-3, and the concentration of a system after ethanol is added is 0.5-1 mg/mL;
PTCA/NH in step (2)2The concentration of a DMF solution of the-MIL-125 composite material is 0.5-2 mg/mL;
PTCA/NH in step (3)2The dripping amount of the DMF solution of MIL-125 on the glassy carbon electrode is 1-3 mu L;
in the step (3), the dripping amount of the aptamer is 1-3 mu L, and the concentration of the aptamer is 1-3 mu mol/L.
Still further, the mass ratio of the aminated Ti-based MOFs material in the step (1) to the perylenetetracarboxylic acid is any one of 1:1, 2:1, 3:1, 1:2 and 1:3, the system concentration after adding ethanol is any one of 05mg/mL, 0.75mg/mL and 1mg/mL, and the PTCA/NH in the step (2)2Concentration of DMF solution of MIL-125 composite material of 0.5mg/mL, 1mg/mL, 1.5mg/mL or 2mg/mL, PTCA/NH in step (3)2The dropping amount of the DMF solution of MIL-125 on the glassy carbon electrode is 1 muL, 1.5 muL, 2 muL, 2.5 muL or 3 muL, the dropping amount of the aptamer in the step (3) is any one of 1 muL, 2 muL or 3 muL, and the aptamer concentration is any one of 1 mumol/L, 1.5 mumol/L, 2 mumol/L, 2.5 mumol/L or 3 mumol/L.
Still further, the pretreatment method of the glassy carbon electrode comprises the following steps: polishing the glassy carbon electrode, respectively performing ultrasonic treatment by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying.
Further, the method comprises the following specific steps:
step 1, preparing a product containing K2S2O8PBS buffer solution of (4);
step 2, drawing a standard curve
Modified electrode apt/PTCA/NH2-MIL-125/GCE is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and the three-electrode system is placed in a chamber containing diethylstilbestrol with different concentrations and 0.01-0.1 mol/L K mol/prepared in the step (1)2S2O8Is soaked in PBS buffer solution and is added with 0.05mol/L of K prepared in the step (1)2S2O80.1mol/L PBS buffer solution with pH of 7.5 as blank solution,
in the electrochemical window range of-1.6-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 600-800V and a scanning speed of 0.025-0.125V/s, recording a potential-luminous intensity curve, and establishing a linear relation between a luminous intensity difference before and after adding diethylstilbestrol and a diethylstilbestrol concentration logarithm value to obtain a corresponding linear regression equation;
step 3, sample detection
And (3) carrying out pretreatment and then adjusting the pH value in the sample detection, testing according to the step 2 to obtain the luminous intensity, and calculating the luminous intensity by using the linear regression equation obtained in the step 2 to obtain the concentration of the diethylstilbestrol in the sample.
Still further, the PBS buffer solution in step 1 contains 0.05mol/LK2S2O8The pH value of the PBS buffer solution is 7.5, the concentration of the PBS is 0.1mol/L, and the modified electrode apt/PTCA/NH2the-MIL-125/GCE soaking time is 80 min.
Compared with the prior art, the invention has the following technical effects: the invention is characterized in that perylene tetracarboxylic acid (PTCA) is fixed to aminate Ti-based MOFs (NH)2-MIL-125), followed by PTCA/NH2MIL-125 as a substrate material with specificityThe recognition aptamer (apt) is recognition element, which is modified on Glassy Carbon Electrode (GCE) together with apt/PTCA/NH2-MIL-125/GCE is a working electrode, and the electrochemiluminescence analysis method is used for quantitatively detecting diethylstilbestrol in milk. Due to PTCA and NH2MIL-125 is tightly bound by electrostatic interaction, so PTCA/NH2The luminous intensity of the-MIL-125/GCE modified electrode is high and stable, and is based on NH2The MIL-125 has good enrichment capacity on the diethylstilbestrol, and the specific binding of the aptamer and the diethylstilbestrol causes the fluorescence quenching of the modified electrode, and the light intensity has a linear relation with the concentration of the diethylstilbestrol. The invention not only has the advantages of high sensitivity, strong specificity, wide linear range, simple instrument and the like of electrochemical luminescence analysis, but also has important practical significance for detecting the diethylstilbestrol in the fishpond water. Compared with the common electrochemical luminescence sensor, PTCA is used as a co-reaction promoter to promote NH2Light intensity NH of MIL-1252MIL-125 as a co-reaction promoter to boost the light intensity of PTCA; the addition of an aptamer allows the sensor to specifically detect diethylstilbestrol.
Drawings
FIG. 1 is a graph of ECL-potential of diethylstilbestrol at various concentrations in example 1.
Wherein the concentration of the diethylstilbestrol is as follows according to the height of the peak value of the curve from left to right in sequence: 1.0X 10-15mol/L(a)、1.0×10-14mol/L(b)、1.0×10-13mol/L(c)、1.0×10-12mol/L(d)、1.0×10-11mol/L(e)、1.0×10-10mol/L(f)、1.0×10-9(g)、1.0×10-8(h)、1.0×10-7(i) And 1.0X 10-6mol/L(j)。
FIG. 2 is a standard curve showing the difference in the luminescence intensity between before and after addition of diethylstilbestrol and the logarithm of the diethylstilbestrol concentration in example 1 (C in the abscissa is the concentration of DES).
FIG. 3 is NH2-MIL-125(A)、PTCA(B)、PTCA/NH2Scanning electron microscopy of MIL-125(C, D). In order to determine the size and morphology of the prepared composite, characterization was performed using a Scanning Electron Microscope (SEM). SEM in FIG. 3(A) showsTo NH2the-MIL-125 nano-crystal is homogeneous nano-particle, is a cubic disc structure with the average diameter of 300-500 nm, has a relatively rough surface and is consistent with the literature. The SEM in FIG. 3(B) shows that the synthesized PTCA is in the shape of a short rod. PTCA/NH2SEM images of MIL-125 Complex, as shown in FIGS. 3(C), 3(D), it can be seen that PTCA converts NH2MIL-125 is bound and, through electrostatic interactions, the two bond well.
Detailed Description
The invention is described in more detail below with reference to the following examples:
the following examples and comparative examples are provided to illustrate the detection of diethylstilbestrol in fish pond water, but the method of the present invention is not limited to the detection of diethylstilbestrol in fish pond water, diethylstilbestrol in milk, and diethylstilbestrol in other environments.
Example 1:
an electrochemiluminescence method for detecting diethylstilbestrol comprises the following specific steps:
(1) immobilized amination of Ti-based MOFs (NH) with Perylenetetracarboxylic acid (PTCA)2MIL-125) preparation of composite material:
adding 2.4mL of isopropyl titanate and 2.2g of 2-aminoterephthalic acid into a mixed solution of 20mL of DMF and 20mL of methanol, stirring and mixing uniformly by ultrasonic, pouring the obtained yellow milky liquid into a 50mL autoclave, heating for 48h at 150 ℃ in a vacuum drying oven, after the autoclave is cooled, washing for three times by using methanol and DMF respectively, centrifuging, and drying at 60 ℃ to obtain yellow powder, namely NH2-MIL-125。
Dissolving 0.1g of perylene tetracarboxylic dianhydride (PTCDA) in 10mL of 0.1M NaOH, stirring for 2h at 80 ℃ to obtain a yellow-green solution, then dripping 1.0M HCl until complete precipitation, centrifuging, washing with deionized water for three times, and drying at 60 ℃ to obtain dark red powder, namely PTCA.
15mg of NH prepared above2adding-MIL-125 and 5mg PTCA into 20mL ethanol solution, performing ultrasonic treatment for 1h, stirring in a magnetic stirrer at a stirring speed of 1080rpm for 24h, performing centrifugal separation after stirring, washing with ethanol and deionized water respectively, and vacuum drying the washed productDrying in a cabinet at 60 ℃ gave a pale red powder and 10mg of the composite was dispersed in 10mL of DMF for use.
(2) Modified electrode apt/PTCA/NH2Preparation of MIL-125/GCE:
polishing glassy carbon electrode, sequentially performing ultrasonic treatment with nitric acid, anhydrous ethanol and deionized water, naturally drying, and transferring 2.0 μ L of 1.0mg/mL PTCA/NH with microsyringe2dropping-MIL-125 on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain PTCA/NH2-MIL-125/GCE modified electrode, followed by dropwise addition of 2.0. mu.L of 2.0. mu. mol/L recognition molecule apt to PTCA/NH2Surface of MIL-125/GCE, aptamer and PTCA binding by amide bond to get apt/PTCA/NH2-MIL-125/GCE. Finally, apt/PTCA/NH2And placing the-MIL-125/GCE modified electrode in a refrigerator at 4 ℃ for 6 hours to obtain the ECL aptamer sensor.
(3) Drawing of standard curve
Modified electrode apt/PTCA/NH2-MIL-125/GCE as working electrode, platinum electrode as auxiliary electrode, Ag/AgCl as reference electrode to form three-electrode system, and placing the three-electrode system in the chamber containing a series of diethylstilbestrol and K with different concentrations2S2O8In PBS buffer solution for 80min, and adding K with a concentration of 0.05mol/L2S2O8The luminescence intensity was measured using 0.1mol/L PBS buffer solution (pH 7.5) as a blank solution.
The three-electrode system was placed in a series of diethylstilbestrol concentrations (1.0X 10)-15mol/L、1.0×10-14mol/L、1.0×10-13mol/L、1.0×10-12mol/L、1.0×10-11mol/L、1.0×10-10mol/L、1.0×10-9、1.0×10-8、1.0×10-7And 1.0X 10-6mol/L) contains 0.05mol/L of K2S2O8In 0.1mol/L PBS buffer solution with pH of 7.5, within an electrochemical window range of-1.6-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 800V and a sweeping speed of 0.1V/s, recording a potential-luminescence intensity curve (E-ECL), and establishing a luminescence intensity difference value before and after adding diethylstilbestrol and a diethylstilbestrol concentration logarithm valueLinear relation, the corresponding linear regression equation is obtained as: delta IECL16655.2957+1031.2687LogC (mol/L), the correlation coefficient (R) is 0.9965. The detection range of the linear regression equation is 1.0 multiplied by 10-15～1.0×10-6mol/L, the lowest detection limit is 2.8 multiplied by 10-16mol/L。
(4) Detection of samples
Adding a certain amount of treated fishpond water into a mixture containing 0.05mol/L K2S2O8The pH of the sample solution is 7.5, and the concentration of diethylstilbestrol in the sample to be detected is calculated according to the linear regression equation corresponding to the step (3) when the sample solution is used for electrochemical luminescence detection, and the results are shown in Table 1. The pretreatment of the sample and the purpose of adjusting the pH value are as follows: in order that the pH value does not affect the detection effect of the sensor.
Comparative example 1
(1)apt/NH2Preparation of-MIL-125/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. 2.0. mu.L of 1.0mg/mL NH was pipetted using a microsyringe2Dropping DMF solution of-MIL-125 material on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain NH2-MIL-125/GCE modified electrode. Next, 2.0. mu.L of 2.0. mu. mol/L of the recognition molecule apt was added dropwise to NH2Surface of MIL-125/GCE, obtaining apt/NH2MIL-125/GCE modified electrode as working electrode for electrochemiluminescence test.
(2) Drawing of standard curve
At apt/NH2-MIL-125/GCE modified electrode as working electrode, platinum electrode as auxiliary electrode, Ag/AgCl as reference electrode to form three-electrode system, and K containing 0.05mol/L2S2O80.1mol/L PBS buffer solution with pH of 7.5 as blank solution, and the three-electrode system was placed in a series of diethylstilbestrol concentrations (1.0X 10)-15mol/L、1.0×10-14mol/L、1.0×10-13mol/L、1.0×10-12mol/L、1.0×10-11mol/L、1.0×10-10mol/L、1.0×10-9mol/L、1.0×10-8mol/L、1.0×10-7mol/L and 1.0X 10-6mol/L) contains 0.05mol/L of K2S2O8In the 0.1mol/L PBS buffer solution with the pH of 7.5, within an electrochemical window range of-1.6-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 800V and a sweeping speed of 0.1V/s, recording an E-ECL curve, and establishing a linear relation between a luminous intensity difference before and after adding diethylstilbestrol and a diethylstilbestrol concentration logarithm value to obtain a corresponding linear regression equation.
(3) Detection of samples
Adding a certain amount of treated fishpond water into a solution containing K of 0.05mol/L2S2O8The pH of the sample solution is 7.5, and the concentration of diethylstilbestrol in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemical luminescence detection in a 0.1mol/L PBS buffer solution, and the result is shown in Table 1.
Comparative example 2
(1) Preparation of apt/PTCA/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. And 2.0 mu L of 1.0mg/mL DMF solution of PTCA material is dripped on the surface of a clean glassy carbon electrode by a micro-sampler and dried at room temperature to obtain the PTCA/GCE modified electrode. Then, 2.0 μ L of 2.0 μmol/L recognition molecule apt is dripped on the surface of PTCA/GCE to obtain the apt/PTCA/GCE modified electrode which is used as a working electrode for electrochemical luminescence test.
(2) Drawing of standard curve
An apt/PTCA/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L2S2O80.1mol/L PBS buffer solution with pH of 7.5 as blank solution, and the three-electrode system was placed in a series of diethylstilbestrol concentrations (1.0X 10)-15mol/L、1.0×10-14mol/L、1.0×10-13mol/L、1.0×10-12mol/L、1.0×10-11mol/L、1.0×10-10mol/L、1.0×10- 9mol/L、1.0×10-8mol/L、1.0×10-7mol/L and 1.0X 10-6mol/L) contains 0.05mol/L of K2S2O8Soaking the solution in 0.1mol/L PBS buffer solution with the pH of 7.5 for 80min, performing cyclic voltammetry scanning at the high voltage of a photomultiplier tube and the sweeping speed of 0.1V/s within the electrochemical window range of-1.6-0V, recording an E-ECL curve, and establishing a linear relation between the luminous intensity difference before and after adding diethylstilbestrol and the logarithm value of the diethylstilbestrol concentration to obtain a corresponding linear regression equation.
(3) Detection of samples
Adding a certain amount of the treated fishpond water into a mixture containing 0.05mol/L of K2S2O8The pH of the sample solution is 7.5, and the concentration of diethylstilbestrol in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemical luminescence detection in a 0.1mol/L PBS buffer solution, and the result is shown in Table 1.
TABLE 1 measurement results of diethylstilbestrol in fishpond water
Figure GDA0002367192760000101
Figure GDA0002367192760000111
Remarking:ais the average of three determinations
As shown in Table 1, the samples were tested in parallel for 3 times, the relative standard deviation was less than 5%, and the recovery rate of spiking ranged from 96% to 104%. The above results show that apt/PTCA/NH is not used2-MIL-125 composite modification with apt/NH alone2MIL-125 or apt/PTCA modified glassy carbon electrode can not detect diethylstilbestrol, and the method is feasible for detecting diethylstilbestrol in fishpond water.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be equivalent or changed within the technical scope of the present invention.

Claims (8)

1. An electrochemiluminescence method for detecting diethylstilbestrol, which is characterized in that: the method is characterized in that an ECL aptamer sensor apt/PTCA/NH is used2MIL-125/GCE is used as a working electrode of an electrochemiluminescence test, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode, and a three-electrode system is formed to detect diethylstilbestrol by electrochemiluminescence;
by K2S2O80.1mol/L PBS buffer solution with pH =7.5 as electrolyte solution, cyclic voltammetric scanning is carried out, and detection is realized based on the linear relation between the difference of luminescence intensity before and after addition of the diethylstilbestrol and the logarithmic value of the concentration of the diethylstilbestrol.
2. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 1, characterized in that: the preparation method of the ECL aptamer sensor comprises the following steps:
（1）PTCA/NH2-preparation of MIL-125 composite:
adding an aminated Ti-based MOFs material and perylene tetracarboxylic acid into an ethanol solution, performing ultrasonic homogenization, stirring in a magnetic stirrer, performing centrifugal separation after stirring, washing and drying to obtain PTCA/NH2-MIL-125 composite;
(2) PTCA/NH prepared in the step (1)2Dispersing an-MIL-125 composite material in DMF to prepare PTCA/NH2-a DMF solution of MIL-125 composite;
(3) modified electrode apt/PTCA/NH2Preparation of MIL-125/GCE:
transferring the PTCA/NH prepared in step (2) by a microsyringe2Dropping DMF solution of-MIL-125 on the surface of the pretreated glassy carbon electrode, and drying at room temperature to obtain PTCA/NH2-MIL-125/GCE modified electrode, then aptamer was added dropwise to PTCA/NH2Modifying the surface of the electrode with MIL-125/GCE, and combining an aptamer and PTCA through an amide bond to obtain apt/PTCA/NH2-MIL-125/GCE modified electrode; finally, apt/PTCA/NH2-MIL-125/GCE modified electrodePlacing in a refrigerator at 4 ℃ for 6h to obtain the ECL aptamer sensor.
3. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 2, characterized in that:
the aminated Ti-based MOFs material in the step (1) is prepared by the following method: adding isopropyl titanate and 2-amino terephthalic acid into a mixed solution of DMF and methanol, stirring and mixing uniformly by ultrasonic, pouring the obtained yellow emulsion into a stainless steel autoclave with a polytetrafluoroethylene lining for reaction, and after the reaction is finished, centrifuging, washing and drying the product to obtain a yellow powdery aminated Ti-based MOFs material;
and/or, the perylene tetracarboxylic acid in the step (1) is prepared by the following method: dissolving perylene tetracarboxylic dianhydride in NaOH solution, violently stirring to obtain yellow-green solution, dripping HCl solution until complete precipitation, centrifuging, repeatedly washing with deionized water, and drying to obtain dark red powdery perylene tetracarboxylic dianhydride.
4. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 2, characterized in that:
the mass ratio of the aminated Ti-based MOFs material to the perylene tetracarboxylic acid in the step (1) is 1-3: 1-3, and the concentration of a system after ethanol is added is 0.5-1 mg/mL;
PTCA/NH in step (2)2The concentration of a DMF solution of the-MIL-125 composite material is 0.5-2 mg/mL;
PTCA/NH in step (3)2The dripping amount of the DMF solution of MIL-125 on the glassy carbon electrode is 1-3 mu L;
in the step (3), the dripping amount of the aptamer is 1-3 mu L, and the concentration of the aptamer is 1-3 mu mol/L.
5. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 4, wherein: in the step (1), the mass ratio of the aminated Ti-based MOFs material to the perylene tetracarboxylic acid is any one of 1:1, 2:1, 3:1, 1:2 and 1:3, and the system concentration after ethanol is added is 05mg/mAny one of L, 0.75mg/mL or 1mg/mL, PTCA/NH in step (2)2Concentration of DMF solution of MIL-125 composite material of 0.5mg/mL, 1mg/mL, 1.5mg/mL or 2mg/mL, PTCA/NH in step (3)2The amount of the DMF solution of MIL-125 applied to the glassy carbon electrode is 1. mu.L, 1.5. mu.L, 2. mu.L, 2.5. mu.L or 3. mu.L, the amount of the aptamer applied in the step (3) is any one of 1. mu.L, 2. mu.L or 3. mu.L, and the aptamer concentration is any one of 1. mu. mol/L, 1.5. mu. mol/L, 2. mu. mol/L, 2.5. mu. mol/L or 3. mu. mol/L.
6. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 2, characterized in that: the pretreatment method of the glassy carbon electrode comprises the following steps: polishing the glassy carbon electrode, respectively performing ultrasonic treatment by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying.
7. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 2, wherein the method comprises the following specific steps:
step 1, preparing a product containing K2S2O8PBS buffer solution of (4);
step 2, drawing a standard curve
Modified electrode apt/PTCA/NH2-MIL-125/GCE is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and the three-electrode system is placed in a chamber containing diethylstilbestrol with different concentrations and 0.01-0.1 mol/L K mol/prepared in the step (1)2S2O8In PBS buffer solution, and soaking with 0.05mol/L of K prepared in the step (1)2S2O80.1mol/L PBS buffer of pH =7.5 as a blank solution,
in the electrochemical window range of-1.6-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 600-800V and a scanning speed of 0.025-0.125V/s, recording a potential-luminous intensity curve, and establishing a linear relation between a luminous intensity difference before and after adding diethylstilbestrol and a diethylstilbestrol concentration logarithm value to obtain a corresponding linear regression equation;
step 3, sample detection
And (3) carrying out pretreatment and then adjusting the pH value in the sample detection, testing according to the step 2 to obtain the luminous intensity, and calculating the luminous intensity by using the linear regression equation obtained in the step 2 to obtain the concentration of the diethylstilbestrol in the sample.
8. The electrochemiluminescence method for detecting diethylstilbestrol according to claim 7, wherein: the PBS buffer solution in the step 1 contains 0.05mol/LK2S2O8The pH value of the PBS buffer solution is 7.5, the concentration of the PBS is 0.1mol/L, and the modified electrode apt/PTCA/NH2the-MIL-125/GCE soaking time is 80 min.
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