CN112114018A - Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof - Google Patents
Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof Download PDFInfo
- Publication number
- CN112114018A CN112114018A CN202010932340.3A CN202010932340A CN112114018A CN 112114018 A CN112114018 A CN 112114018A CN 202010932340 A CN202010932340 A CN 202010932340A CN 112114018 A CN112114018 A CN 112114018A
- Authority
- CN
- China
- Prior art keywords
- mof
- diethylstilbestrol
- cdna
- solution
- nps
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Electrochemistry (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a sensor for detecting diethylstilbestrol by an electrochemiluminescence method based on resonance energy transfer, and a preparation method and application thereof, wherein the sensor is used as a donor Ag in energy resonance transfer3PO4/Cu‑MOF([Cu(L)(H2O)2]n) Ag NPs as acceptors in energy resonance transfer3PO4the/Cu-MOF composite material and the Ag NPs are connected through two DNA chains, wherein one is connected with an acceptor, and the other is an aptamer which is used for connecting a donor and the DNA chain connected with the acceptor and is modified on the glassy carbon electrode together); ag3PO4the/Cu-MOF composite material is made of Ag3PO4Fixing the novel Cu-MOF. The electrochemical luminescence sensor based on resonance energy transfer has high detection performanceThe detection sensitivity and the preparation method are simple, and the method has the advantages of low cost, high sensitivity and strong specificity in the application of detecting the diethylstilbestrol.
Description
Technical Field
The invention belongs to the technical field of electrochemical luminescence analysis and detection, relates to an electrochemical luminescence sensor based on resonance energy transfer, and a preparation method and application thereof, and particularly relates to silver orthophosphate (Ag)3PO4) Immobilization of Cu-based MOFs ([ Cu (L)) (H)2O)2]n) Then Ag is added3PO4the/Cu-MOF is used as a substrate material, an aptamer (apt) with specific recognition function is used as a recognition element, the aptamer and the recognition element are jointly modified on a Glassy Carbon Electrode (GCE), and then Ag-cDNA/apt/Ag is used3PO4An electrochemiluminescence analysis method for quantitatively detecting diethylstilbestrol in the fishpond water by taking/Cu-MOF/GCE as a working electrode.
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 the diethylstilbestrol during pregnancy, the female is easy to be aborted when the fetus develops to the adult pregnancy, mainly because the diethylstilbestrol causes uterine malformation; 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.
The DES detection method specified by national standards of China is liquid chromatography-tandem mass spectrometry, and the methods for DES detection mainly comprise an enzyme-linked immunosorbent assay method, a high performance liquid chromatography, a liquid chromatography-mass spectrometry combined method, an electrochemical method, a radioimmunoassay and the like. The method is accurate and sensitive, but has the defects of complex process, complicated operation, inconvenient carrying and the like, and the Electrochemiluminescence (ECL) is chemiluminescence caused by electrochemical reaction, so that the method draws wide attention due to the advantages of high sensitivity, strong reaction controllability, simple instruments and equipment and the like. Resonance energy transfer is an emerging method of molecular spectroscopic analysis, specifically the transfer of electron excitation energy between appropriate pairs of energy donors and energy acceptors. The electrochemiluminescence-resonance energy transfer (ECL-RET) combines the advantages of the electrochemiluminescence and the resonance energy transfer (ECL-RET), and is a new field with development potential. The biosensor does not need an excitation light source, has low background noise, avoids the influence of scattered light, and is widely applied to the construction of biosensors. The technical problem to be solved by the invention is how to prepare the biosensor with high detection sensitivity on diethylstilbestrol.
The invention is prepared by mixing silver orthophosphate (Ag)3PO4) Immobilization of Cu-based MOFs ([ Cu (L)) (H)2O)2]n) Then Ag is added3PO4the/Cu-MOF is used as a donor in the energy resonance transfer, and the Ag NPs are used as an acceptor in the energy resonance transfer. The donor and the acceptor are connected through two DNA chains (apt, cDNA), are jointly modified on a Glassy Carbon Electrode (GCE), and then are subjected to Ag-cDNA/apt/Ag3PO4An electrochemiluminescence analysis method for quantitatively detecting diethylstilbestrol in river water by taking/Cu-MOF/GCE as a working electrode. Due to Ag3PO4Closely combined with the novel Cu-MOF through electrostatic interaction, so that Ag3PO4The luminescence intensity of the/Cu-MOF/GCE modified electrode is high and stable, and is based on Ag3PO4The electrochemiluminescence resonance energy transfer between the/Cu-MOF and the Ag NPs and the specific combination of the aptamer and the diethylstilbestrol inhibit the transfer of resonance energy, so that the light intensity is recovered, 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 the electrochemical luminescence analysis, but also has important practical significance for the specificity detection of the diethylstilbestrol in the river water.
Disclosure of Invention
The invention aims to provide an electrochemiluminescence sensor based on resonance energy transfer and a preparation method and application thereof aiming at the defects of the diethylstilbestrol detection prior art. The invention relates to an electrochemical luminescence sensor based on resonance energy transfer, which is formed by mixing Ag3PO4/Cu-MOF (silver orthophosphate (Ag)3PO4) Abbreviation of fixed Cu-based MOFs materials, wherein Cu-based MOFs materials are also abbreviated ([ Cu (L)) (H)2O)2]n) As donors in energy resonance transfer, silver nanoparticles (also called Ag NPs) act as acceptors in energy resonance transfer. The donor and the acceptor are connected through two DNA chains (one DNA chain is an aptamer, the other DNA chain is connected with the acceptor, the aptamer is used for connecting the donor and the acceptor, the aptamer and the acceptor are connected), and the donor and the acceptor are jointly modified on a Glassy Carbon Electrode (GCE), so that the sensitivity and the stability of electrochemiluminescence are remarkably improved, and the Ag-based electrochemical luminescence is based on Ag3PO4The electrochemiluminescence resonance energy transfer between the Cu-MOF and the Ag NPs and the selection of the aptamer greatly improve the selectivity and the sensitivity to a target detection object. 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 preparation method of the electrochemical luminescence sensor based on resonance energy transfer comprises the following steps:
(1)Ag3PO4preparation of/Cu-MOF composite material:
adding disodium hydrogen phosphate solution into silver nitrate solution dropwise, stirring vigorously to obtain yellow emulsion, centrifuging, washing with ultrapure water for three times, and drying to obtain yellow powder, i.e. Ag3PO4；
Mixing copper sulfate pentahydrate (CuSO)4·5H2O) and 4- (3-carboxy-1, 2, 4-H-triazol-1-yl) benzoic acid (H)2L) adding the mixture into a mixed solution of N, N-Dimethylacetamide (DMF) and acetonitrile, ultrasonically stirring and uniformly mixing, pouring the obtained blue milky liquid into a stainless steel autoclave with a polytetrafluoroethylene lining for reaction, centrifuging, washing and drying a product after the reaction is finished to obtain blue powder, namely the novel Cu-MOF.
The prepared novel Cu-MOF and the prepared Ag3PO4Adding into DMF, ultrasonically dispersing, stirring in a magnetic stirrer, centrifuging, washing with organic solvent and deionized water, and drying to obtain yellow gray powder, i.e. Ag3PO4a/Cu-MOF composite material and Ag3PO4the/Cu-MOF composite material is dispersed in DMF for use;
(2) preparation of Ag NPs-cDNA colloidal solution:
Tris-H containing cDNA3PO4Adding the solution into 50% Ag NPs water solution, standing for reaction, centrifuging, collecting precipitate, and adding Tris-H3PO4Controlling the pH value to be 7.5, performing ultrasonic treatment, and oscillating at room temperature to prepare an Ag NPs-cDNA colloidal solution, and storing the Ag NPs-cDNA colloidal solution at 4 ℃ for later use; the cDNA is the DNA chain for connecting the receptor;
(3) preparing an electrochemical luminescence sensor based on resonance energy transfer:
applying PBS buffer solution containing N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) on clean bare glassy carbon electrode, and obtaining activated glassy carbon after at least 1 hourAn electrode, followed by mixing the Ag prepared in step (1)3PO4Dripping DMF dispersion liquid of/Cu-MOF composite material on the activated glassy carbon electrode to obtain Ag3PO4a/Cu-MOF/GCE modified electrode; then, PBS buffer containing NHS and EDC was applied to Ag3PO4Activating carboxyl in the novel Cu-MOF on the/Cu-MOF/GCE modified electrode; finally, Tris-H containing aptamers was sequentially applied dropwise3PO4The solution and the Ag NPs-cDNA colloidal solution are used for obtaining the electrochemical luminescence sensor based on resonance energy transfer; the aptamer is the DNA chain for connecting the donor.
Further, novel Cu-MOF and Ag3PO4The mass ratio of (A) to (B) is 2: 1; step (3) Ag3PO4The concentration of the/Cu-MOF dispersion is 3.0 mg/mL; the concentration of the aptamer is 2.0 mu mol/L, and the concentration of the Ag NPs-cDNA colloidal solution is 2 mu mol/L; ag3PO4The volume ratio of the/Cu-MOF dispersion liquid to the aptamer solution to the Ag NPs-cDNA colloidal solution is 5:2: 3.
Preferably, the method comprises the following steps: the amount of the novel Cu-MOF in the step (1) is 10.0mg, Ag3PO4The amount of the added (1) is 5.0mg, and the volume of the solvent DMF is 10 mL; ag3PO4The dripping amount of the/Cu-MOF is 5.0 mu L, and the concentration is 3.0 mg/mL; the amount of the aptamer was 2.0. mu.L at a concentration of 2.0. mu. mol/L, and the amount of the Ag NPs/cDNA colloidal solution was 3.0. mu.L at a concentration of 2. mu. mol/L.
The application of the electrochemical luminescence sensor based on resonance energy transfer in detecting diethylstilbestrol by an electrochemical luminescence method is that the diethylstilbestrol aptamer nucleotide sequence is as follows:
aptamer 5' -GGC GAT GGG GTA GGG GGT GTG GAG GGG CCG GAC GGA GGG G NH2-3'；
cDNA：SH-(CH)2-5'-CCC-CTC-CGT-CCG-GCC-CCT-CCA-3'；
Further, the specific detection method is as follows:
Step 2, using the electrochemical luminescence sensor based on resonance energy transfer as a working electrode, using a platinum electrode as an auxiliary electrode, using Ag/AgCl as a reference electrode to form a three-electrode system, placing the three-electrode system in the standard solution containing a series of diethylstilbestrol with different concentrations prepared in the step 1 for soaking for a certain time, and using the standard solution containing K2S2O8The PBS buffer solution is used as a blank solution, cyclic voltammetry scanning is carried out on the solution at a photomultiplier tube high pressure of 800V and a scanning speed of 0.1V/s within an electrochemical window range of-1.8-0V, a potential-luminescence intensity curve is recorded, a linear relation between a luminescence intensity difference value before and after the addition of diethylstilbestrol and a diethylstilbestrol concentration logarithm value is established, and a corresponding linear regression equation is obtained;
and 3, detecting the sample, filtering the sample to remove impurities, adjusting the pH value to 8, testing according to the step 2, obtaining 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.
Preferably, the PBS buffer solution in step 1 contains 0.1mol/LK2S2O8The pH value of the PBS buffer solution is 8, the concentration of the PBS buffer solution is 0.1mol/L, and the soaking time of the electrochemical luminescence sensor based on the resonance energy transfer is 50 min.
Compared with the common electrochemical luminescence sensor, the electrochemical luminescence sensor for detecting the diethylstilbestrol and the preparation method thereof have the following two remarkable advantages: ag3PO4The light intensity of the novel Cu-MOF is improved by serving as a co-reaction promoter; the addition of the aptamer enables the sensor to specifically detect diethylstilbestrol, and the preparation of the donor-receptor is more beneficial to the improvement of the detection sensitivity of diethylstilbestrol; the introduction of the resonance energy transfer system leads the sensor to have higher sensitivity and stronger specificity, and the minimum detection limit is 7.2 multiplied by 10-13mol/L。
Drawings
Fig. 1 is a schematic flow chart of the preparation of the sensor and the detection of diethylstilbestrol in the present invention.
FIG. 2 is a graph of ECL-potential curves for various concentrations of diethylstilbestrol.
Wherein the concentration of the diethylstilbestrol is as follows according to the peak value of the curve from top to bottom: 1.0X 10-4mol/L(a)、1.0×10-5mol/L(b)、1.0×10-6mol/L(c)、1.0×10-7mol/L(d)、1.0×10-8mol/L(e)、1.0×10-9mol/L(f)、1.0×10-10mol/L (g), and 1.0X 10-11mol/L(h)、1.0×10-12mol/L(i)。
Fig. 3 is a standard curve of the difference in the luminescence intensity before and after addition of diethylstilbestrol and the logarithm of the diethylstilbestrol concentration.
Detailed Description
The invention is described in more detail below with reference to the following examples:
example 1:
(1)Ag3PO4preparation of/Cu-MOF composite material:
to AgNO3To an aqueous solution of (10mL, 0.6M) was added dropwise Na2HPO4Aqueous solution (10mL, 0.2M), stirred for 1h, centrifuged and washed three times with deionized water, then dried under vacuum at 60 ℃ to give a yellow precipitate, Ag3PO4。
0.0116g of 4- (3-carboxy-1, 2, 4-H-triazol-1-yl) benzoic acid (H)2L) (purchased from: jinan chemostat science Co., Ltd.), 0.0125g of copper sulfate pentahydrate (CuSO)4·5H2O) adding the mixture into a mixed solution of 4mL of acetonitrile and 2mL of deionized water, stirring and mixing uniformly by ultrasonic, pouring the obtained light blue milky liquid into a 50mL high-pressure kettle, heating for 72h at 95 ℃ in a vacuum drying oven, after the kettle is cooled, washing for three times by using the deionized water and ethanol respectively, centrifuging, and drying at 60 ℃ to obtain light blue powder, namely the novel Cu-MOF.
10mg of the novel Cu-MOF material prepared above was dissolved in 10mL of ultrapure water and then sonicated for 360min to obtain a suspension of an aqueous Cu-MOF solution, 2.5mL of LAgNO was added3Is slowly added to a continuously stirred aqueous suspension of Cu-MOFThe above mixed suspension was stirred in a magnetic stirrer at a stirring rate of 1080rpm for 720min, and then 2.5ml of Na was added2HPO4The aqueous solution was added dropwise to the above mixture, and stirred for 30 min. After stirring, centrifugally separating, washing with ethanol and deionized water, and finally drying the washed product in a vacuum drying oven at 60 ℃ to obtain yellow-green powder.
(2) Preparing silver nano particles:
1.7mL of a 1% aqueous solution of trisodium citrate was added to a three-necked round bottom flask with a reflux condenser containing 100mL of deionized water and stirred rapidly, and the mixture was boiled for 15min through a heating mantle, and then 2mL of a 1% citrate solution was added to the reaction solution. The reaction solution was kept under reflux for 1 hour under vigorous mechanical stirring, then cooled to room temperature, and then centrifuged at 6000r/min for 7min, and collected. The resulting Ag NPs solution was stored in a refrigerator at 4 ℃ until use.
(3) Preparation of Ag NPs-cDNA:
200 μ LAg aqueous solution was taken and 100 μ L of 2 μ M prepared Tris-H containing cDNA was added thereto3PO4(pH 7.5), shaking for 16H to allow full reaction, centrifuging at 10000rpm for 10min, and collecting the precipitate and dissolving in 200 μ L Tris-H3PO4Carrying out ultrasonic treatment on the obtained colloidal solution for 10min, oscillating for 1h at room temperature to obtain Ag-cDNA, and storing the Ag-cDNA at 4 ℃ for later use.
(4) Electrochemical luminescence sensor based on resonance energy transfer
AgNPs-cDNA/apt/Ag3PO4Preparation of/Cu-MOF/GCE:
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, naturally airing the polished glassy carbon electrode for later use, and dripping 5 mu L of 0.01M PBS buffer solution (pH 7.5) containing 0.005M NHS and 0.01M EDC on a clean bare glassy carbon electrode for 1 hour at room temperature so as to activate the electrode. Subsequently, 5. mu.L of 3mg/mL Ag was added dropwise3PO4the/Cu-MOF DMF solution is put on GCE to obtain Ag3PO4the/Cu-MOF/GCE modified electrode. Then, 5. mu.L of 0 containing 0.005M NHS and 0.01M EDC was applied again.01M PBS buffer (pH 7.5) in Ag3PO4the/Cu-MOF/GCE modifies the electrode in order to activate the carboxyl groups in the Cu-MOF for attachment to the amino groups in the subsequent apt. Finally, 2 mu L of apt Tris-H is sequentially dripped3PO4The solution (2 mu M) and 3 mu L of Ag NPs/cDNA colloidal solution are added to obtain Ag NPs-cDNA/apt/Ag3PO4the/Cu-MOF/GCE modified electrode. Finally, the Ag NPs-cDNA/apt/Ag3PO4And placing the/Cu-MOF/GCE modified electrode in a refrigerator at 4 ℃ for 6 hours to obtain the electrochemical luminescence sensor based on resonance energy transfer.
In the above sensor, the aptamer and cDNA sequences are as follows: (purchased from Biotechnology engineering (Shanghai) Ltd.)
Aptamer 5' -GGC GAT GGG GTA GGG GGT GTG GAG GGG CCG GAC GGA GGG G NH2-3'；
cDNA：SH-(CH)2-5'-CCC-CTC-CGT-CCG-GCC-CCT-CCA-3'；
(5) Drawing of standard curve
Preparing a diethylstilbestrol standard solution, adding a preset amount of diethylstilbestrol aqueous solution into a solution containing K2S2O8PBS buffer solution (specifically, containing 0.1mol/L of K)2S2O80.1mol/L PBS buffer solution of pH 8) to obtain a series of standard solutions of diethylstilbestrol at different concentrations;
an electrochemical luminescence sensor Ag NPs-cDNA/apt/Ag based on resonance energy transfer3PO4taking/Cu-MOF/GCE as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode to form a three-electrode system, soaking the three-electrode system in the standard solution containing diethylstilbestrol with a series of different concentrations for 50min, and adding K containing 0.1mol/L2S2O8The luminescence intensity was measured using 0.1mol/L PBS buffer solution (pH 8) as a blank solution.
The three-electrode system was placed in a series of diethylstilbestrol concentrations (1.0X 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、1.0×10-6mol/L、1.0×10-5mol/L and 1.0X 10-4mol/L) contains 0.1mol/L of K2S2O8In the 0.1mol/LPBS buffer solution with the pH of 8, within an electrochemical window range of-1.8-0V, 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), establishing a linear relation between a luminescence intensity difference value before and after adding diethylstilbestrol and a diethylstilbestrol concentration logarithm value, and obtaining a corresponding linear regression equation as follows: delta IECL12306.68388+958.7303LogC (mol/L), the correlation coefficient (R) is 0.9982. The detection range of the linear regression equation is 1.0 multiplied by 10-12～1.0×10-4mol/L, the lowest detection limit is 7.2 multiplied by 10-13mol/L。
(6) Detection of samples
Adding a certain amount of filtered and impurity-removed river water into K with the concentration of 0.1mol/L2S2O8The pH of the sample solution (5) is 0.1mol/LPBS buffer solution, and the concentration of diethylstilbestrol in the sample to be detected is calculated according to the linear regression equation corresponding to the step (5), and the results are shown in Table 1.
Comparative example 1
(1)Ag3PO4Preparation of/Cu-MOF/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. Transfer 5.0. mu.L of 3.0mg/mLAg with a microsyringe3PO4DMF solution (Ag) of/Cu-MOF composite3PO4The preparation method of the/Cu-MOF composite material is as in example 1) and the Ag is obtained by dripping the composite material on the surface of a clean glassy carbon electrode and drying the composite material at room temperature3PO4the/Cu-MOF/GCE modified electrode is used as a working electrode for an electrochemiluminescence test.
(2) Drawing of standard curve
With Ag3PO4the/Cu-MOF/GCE is used as a working electrode, the platinum electrode is used as an auxiliary electrode, the Ag/AgCl is used as a reference electrode, and a three-electrode system is formed and used for detecting a series of diethylstilbestrol standard solutions with different concentrations. The detection method was the same as in example 1.
The results are shown in Table 1.
Comparative example 2:
(1) preparation of AgNPs/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. 5.0 μ LAg of aqueous solution of NPs (AuNPs aqueous solution was prepared as in example 1) was pipetted onto the clean glassy carbon electrode surface and dried at room temperature to obtain Ag NPs/GCE modified electrode as the working electrode for electrochemiluminescence testing.
(2) Drawing of standard curve
And forming a three-electrode system by using an Ag NPs/GCE modified electrode as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode, and detecting a series of diethylstilbestrol standard solutions with different concentrations. The detection method was the same as in example 1.
The results are shown in Table 1.
Comparative example 3
Comparative example 3 differs from example 1 in that: ag3PO4Taking the MOF as a donor to prepare AgNPs-cDNA/apt/Ag3PO4the/MOF/GCE sensor is used as a working electrode, and the detection method is the same as that of example 1.
The detection result shows that when the sensor prepared in the comparative example 3 is used for detecting diethylstilbestrol, the light intensity is very low, and diethylstilbestrol is difficult to detect.
Comparative example 4
Comparative example 4 is different from example 1 in that: the Ag NPs-cDNA/apt/Cu-MOF/GCE sensor prepared by using Cu-MOF as a donor is used as a working electrode, and the detection method is the same as that of example 1.
The detection result shows that when the sensor prepared in the comparative example 4 is used for detecting diethylstilbestrol, resonance transfer is difficult to occur, and diethylstilbestrol is difficult to detect.
TABLE 1 measurement results of diethylstilbestrol in fishpond water
Remarking:athe 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 102%. The above results show that AgNPs-cDNA/apt/Ag is not used3PO4Modification of/Cu-MOF/GCE with AgNPs alone or with Ag3PO4The Cu-MOF modified glassy carbon electrode can not detect the diethylstilbestrol, and the method is feasible for detecting the diethylstilbestrol in the river 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 (7)
1. A sensor for detecting diethylstilbestrol by an electrochemiluminescence method based on resonance energy transfer is characterized in that: including as donor Ag in energy resonance transfer3PO4Composite material of/Cu-MOF, Ag NPs as acceptors in energy resonance transfer, said Ag3PO4the/Cu-MOF composite material and the Ag NPs are connected through two DNA chains and are jointly modified on the glassy carbon electrode, wherein one DNA chain is an aptamer (apt), and the other DNA chain is connected with a receptor to obtain the AgNPs-cDNA/apt/Ag3PO4A/Cu-MOF/GCE sensor.
2. The sensor for detecting diethylstilbestrol by an electrochemiluminescence method based on resonance energy transfer as claimed in claim 1, wherein: the aptamers are shown below:
aptamer nucleotidesSequence 5' -GGC GAT GGG GTA GGG GGT GTG GAG GGG CCG GAC GGA GGG G NH2-3'；
cDNA sequence: SH- (CH)2-5'-CCC-CTC-CGT-CCG-GCC-CCT-CCA-3'。
3. The method for preparing a sensor for detecting diethylstilbestrol by an electrochemiluminescence method based on resonance energy transfer as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1)Ag3PO4preparation of/Cu-MOF composite material:
separately preparing Ag3PO4And novel Cu-MOF, novel Cu-MOF and Ag to be produced3PO4Adding into solvent, ultrasonically dispersing, stirring in magnetic stirrer, centrifuging, washing with organic solvent and deionized water, and drying to obtain yellow gray powder, i.e. Ag3PO4a/Cu-MOF composite material and Ag3PO4the/Cu-MOF composite material is dispersed in DMF for use;
(2) preparation of Ag NPs-cDNA colloidal solution:
Tris-H containing cDNA3PO4Adding the solution into 50% Ag NPs water solution, standing for reaction, centrifuging, collecting precipitate, and adding Tris-H3PO4Controlling the pH value to be 7.5, performing ultrasonic treatment, and oscillating at room temperature to prepare an Ag NPs-cDNA colloidal solution, and storing the Ag NPs-cDNA colloidal solution at low temperature for later use; the cDNA is a DNA chain for connecting a receptor;
(3) preparing an electrochemical luminescence sensor based on resonance energy transfer:
dripping PBS buffer solution containing NHS and EDC on a clean bare glassy carbon electrode to obtain an activated glassy carbon electrode, and then, preparing the Ag prepared in the step (1)3PO4Dripping DMF dispersion liquid of/Cu-MOF composite material on the activated glassy carbon electrode to obtain Ag3PO4a/Cu-MOF/GCE modified electrode; then, PBS buffer containing NHS and EDC was applied to Ag3PO4Activating carboxyl in the novel Cu-MOF on the/Cu-MOF/GCE modified electrode; finally, inSub-instillation of aptamer (apt) -containing Tris-H3PO4The solution and the Ag NPs-cDNA colloidal solution are used for obtaining the electrochemical luminescence sensor Ag NPs-cDNA/apt/Ag based on resonance energy transfer3PO4Cu-MOF/GCE; the aptamer is a DNA strand for linking a donor.
4. The method for preparing a sensor for detecting diethylstilbestrol by an electrochemiluminescence method based on resonance energy transfer as claimed in claim 3, wherein the method comprises the following steps: step (1) novel Cu-MOF and Ag3PO4The mass ratio of (A) to (B) is 2: 1; step (3) Ag3PO4The concentration of the/Cu-MOF dispersion is 3.0 mg/mL; the concentration of the aptamer is 2.0 mu mol/L, and the concentration of the AgNPs-cDNA colloidal solution is 2 mu mol/L; ag3PO4The volume ratio of the/Cu-MOF dispersion liquid to the aptamer solution to the Ag NPs-cDNA colloidal solution is 5:2: 3.
5. Use of a resonance energy transfer based sensor according to claim 1 for the electrochemiluminescence detection of diethylstilbestrol, wherein: the detection method comprises the following specific steps:
(1) containing potassium persulfate (K)2S2O8) Preparing a Phosphate (PBS) buffer solution;
(2) contains diethylstilbestrol with different concentrations and 0.1mol/LK2S2O8Preparing a PBS buffer solution;
accurately weighing a certain amount of diethylstilbestrol, and preparing 1.0 × 10 with ethanol-4Adding a certain amount of diethylstilbestrol solution into the solution with the concentration of 0.1mol/LK2S2O80.1mol/LpH ═ 8 in PBS buffer to give a series of standard solutions of diethylstilbestrol at various concentrations ranging from 1.0X 10-12mol/L～1.0×10-4mol/L；
(3) Drawing of standard curve
Modified electrode Ag NPs-cDNA/apt/Ag3PO4Forming a three-electrode system by using/Cu-MOF/GCE as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode, and placing the three-electrode system in the one-containing systemDiethylstilbestrol was present at various concentrations and at a concentration of 0.1mol/L K2S2O8The PBS buffer solution is soaked for a certain time and then K with the concentration of 0.1mol/L2S2O8The luminescence intensity was measured using 0.1mol/L PBS buffer solution with pH 8 as a blank solution;
in the electrochemical window range of-1.8-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 800V and a scanning speed of 0.1V/s, recording a potential-luminous intensity curve (E-ECL), 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;
(4) actual sample detection
And (4) carrying out pretreatment and then adjusting the pH value in the actual sample detection, and calculating according to the linear regression equation in the step (3).
6. The electrochemiluminescence method for detecting diethylstilbestrol based on resonance energy transfer according to claim 5, characterized in that: the PBS buffer solution in the step (3) contains 0.1mol/L K2S2O8The pH value of the PBS buffer solution is 8, the concentration of the PBS buffer solution is 0.1mol/L, and the Ag-cDNA/apt/Ag of the modified electrode3PO4The soaking time of the/Cu-MOF/GCE is 50 min.
7. The electrochemiluminescence method for detecting diethylstilbestrol based on resonance energy transfer according to claim 5, characterized in that: the lowest detection limit is 7.2 multiplied by 10-13mol/L。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010932340.3A CN112114018B (en) | 2020-09-08 | 2020-09-08 | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010932340.3A CN112114018B (en) | 2020-09-08 | 2020-09-08 | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112114018A true CN112114018A (en) | 2020-12-22 |
| CN112114018B CN112114018B (en) | 2022-09-27 |
Family
ID=73803304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010932340.3A Active CN112114018B (en) | 2020-09-08 | 2020-09-08 | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112114018B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114113265A (en) * | 2021-12-14 | 2022-03-01 | 郑州轻工业大学 | Aptamer sensor and preparation method thereof |
| CN114858878A (en) * | 2022-04-19 | 2022-08-05 | 常州大学 | Sensor for detecting tetracycline by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802391A (en) * | 2018-08-07 | 2018-11-13 | 福建师范大学 | One kind being based on TiO2The Resonance energy transfer type electrochemical luminescence of mesomorphic induction and the immuno-sensing method to ovarian cancer markers |
| CN110220860A (en) * | 2019-05-21 | 2019-09-10 | 山东师范大学 | A method of based on aptamers and colloidal gold colorimetric detection estrogen chaff interferent |
| CN110988067A (en) * | 2019-12-13 | 2020-04-10 | 常州大学 | Electrochemical luminescence method for detecting diethylstilbestrol |
| CN111198221A (en) * | 2020-02-17 | 2020-05-26 | 常州大学 | Electrochemical luminescence sensor based on resonance energy transfer and preparation method and application thereof |
| CN111579620A (en) * | 2020-05-19 | 2020-08-25 | 西北师范大学 | Silver-based MOF (Metal organic framework) derivative nanomaterial, preparation of modified electrode of silver-based MOF derivative nanomaterial and application of silver-based MOF derivative nanomaterial as superoxide anion electrochemical sensor |
-
2020
- 2020-09-08 CN CN202010932340.3A patent/CN112114018B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802391A (en) * | 2018-08-07 | 2018-11-13 | 福建师范大学 | One kind being based on TiO2The Resonance energy transfer type electrochemical luminescence of mesomorphic induction and the immuno-sensing method to ovarian cancer markers |
| CN110220860A (en) * | 2019-05-21 | 2019-09-10 | 山东师范大学 | A method of based on aptamers and colloidal gold colorimetric detection estrogen chaff interferent |
| CN110988067A (en) * | 2019-12-13 | 2020-04-10 | 常州大学 | Electrochemical luminescence method for detecting diethylstilbestrol |
| CN111198221A (en) * | 2020-02-17 | 2020-05-26 | 常州大学 | Electrochemical luminescence sensor based on resonance energy transfer and preparation method and application thereof |
| CN111579620A (en) * | 2020-05-19 | 2020-08-25 | 西北师范大学 | Silver-based MOF (Metal organic framework) derivative nanomaterial, preparation of modified electrode of silver-based MOF derivative nanomaterial and application of silver-based MOF derivative nanomaterial as superoxide anion electrochemical sensor |
Non-Patent Citations (6)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114113265A (en) * | 2021-12-14 | 2022-03-01 | 郑州轻工业大学 | Aptamer sensor and preparation method thereof |
| CN114113265B (en) * | 2021-12-14 | 2023-08-08 | 郑州轻工业大学 | Aptamer sensor and preparation method thereof |
| CN114858878A (en) * | 2022-04-19 | 2022-08-05 | 常州大学 | Sensor for detecting tetracycline by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof |
| CN114858878B (en) * | 2022-04-19 | 2023-10-27 | 常州大学 | Sensor for detecting tetracycline by electrochemical luminescence method based on resonance energy transfer, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112114018B (en) | 2022-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Electrochemiluminescent competitive immunosensor based on polyethyleneimine capped SiO2 nanomaterials as labels to release Ru (bpy) 32+ fixed in 3D Cu/Ni oxalate for the detection of aflatoxin B1 | |
| Wu et al. | Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles | |
| CN106970061B (en) | Carbon dots/copper nano-cluster compound ratio fluorescent dopamine probe preparation method | |
| CN112114018B (en) | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and application thereof | |
| CN108918855B (en) | Preparation method and application of signal 'on-off' type electrochemiluminescence sensor based on AgNCs as luminophor | |
| Liu et al. | Highly efficient intramolecular electrochemiluminescence energy transfer for ultrasensitive bioanalysis of aflatoxin M1 | |
| Guo et al. | Mn 2+-doped NaYF 4: Yb/Er upconversion nanoparticle-based electrochemiluminescent aptasensor for bisphenol A | |
| Zhang et al. | Electrochemiluminescence immunosensor for highly sensitive detection of 8-hydroxy-2′-deoxyguanosine based on carbon quantum dot coated Au/SiO2 core–shell nanoparticles | |
| Ai et al. | An “on-off” electrochemiluminescence immunosensor for PIVKA-II detection based on the dual quenching of CeO2–Au-g-C3N4 hybrids by Ag nanocubes-VB2 | |
| Qian et al. | Simultaneous detection of multiple DNA targets by integrating dual‐color graphene quantum dot nanoprobes and carbon nanotubes | |
| CN110988067B (en) | Electrochemical luminescence method for detecting diethylstilbestrol | |
| Hu et al. | Ultrasensitive electrochemiluminescent brombuterol immunoassay by applying a multiple signal amplification strategy based on a PAMAM-gold nanoparticle conjugate as the bioprobe and Ag@ Au core shell nanoparticles as a substrate | |
| Zhou et al. | Ultrasensitive electrochemiluminescent detection of cardiac troponin I based on a self-enhanced Ru (II) complex | |
| CN111198221A (en) | Electrochemical luminescence sensor based on resonance energy transfer and preparation method and application thereof | |
| Sha et al. | Electroluminescent aptasensor based on RuSiO2 nanoparticles for detection cytochrome c using ferrocene as quenching probe | |
| CN110596060A (en) | Construction method and application of fluorescence sensor in spectral analysis for detecting prostate specific antigen | |
| CN112098484A (en) | Sensor for detecting acetamiprid based on electrochemical luminescence method, preparation method and application | |
| CN109490264A (en) | Based on the homogeneous label-free detection method of the luminous both-end complementary nucleic acid aptamers probe of aggregation and aflatoxin B1 | |
| CN111220672B (en) | Detection of Hg based on energy resonance transfer2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor | |
| CN109085158B (en) | Used for cancer cell and H2O2Construction of paper-based sensors for detection | |
| CN110823977B (en) | Hg detection method2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor | |
| CN110687177B (en) | Preparation method of electrochemical luminescence sensor for detecting procalcitonin | |
| Chen et al. | Sensitive detection of enrofloxacin using an electrochemiluminescence immunosensor based on gold-functionalized C 60 and Au@ BSA nanoflowers | |
| CN114235907B (en) | Electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 and detection method | |
| CN113514512B (en) | g-C based 3 N 4 Preparation and application of electrochemical luminescence sensor of CoPd |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |