An electrochemiluminescence biosensor for detecting diethylstilbestrol and its preparation method and application

Abstract

The present invention belongs to the field of biosensor technology, specifically an electrochemiluminescent biosensor for detecting diethylstilbestrol, its preparation method, and application. The preparation method comprises drop-coating a ZnRuMOF solution onto a glassy carbon electrode (GCE) and drying it; depositing gold nanoparticles on the surface of the GCE; drop-coating H5 and incubating it at 37°C; adding MCH for reaction, washing with PBS to remove excess MCH; and finally, drop-coating the activated Cas 12a-crRNA complex onto the GCE and incubating it. This yields a Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescent biosensor, which serves as a working electrode for electrochemiluminescence testing. The present invention can efficiently, sensitively, and rapidly detect target substances, significantly improving analytical simplicity and detection accuracy, and providing an innovative and sensitive detection platform for diethylstilbestrol analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to an electrochemiluminescence biosensor for detecting diethylstilbestrol, a preparation method thereof, and applications thereof. Background Art

[0002] Electrochemiluminescence (ECL) technology is an advanced method that combines chemiluminescence and electrochemical analysis. With its excellent high sensitivity, low background noise, wide linear response range and good reaction controllability, it has become an important tool in the field of analysis. Since ECL does not rely on external excitation light sources and can effectively reduce background interference, it is very suitable for in situ field detection. When constructing ECL sensors, the introduction of high-performance nanomaterials, especially metal-organic frameworks (MOFs), is a key strategy to improve detection sensitivity. MOFs are composed of metal ions or clusters and organic ligands through coordination bonds. They have a highly ordered pore structure, which gives them excellent physical and chemical properties and are widely used in sensor optimization and performance enhancement.

[0003] Diethylstilbestrol (DES) is a synthetic non-steroidal estrogen that was once widely used in animal husbandry and aquaculture as a feed additive to promote animal growth and improve feed conversion rate. However, DES can enter the human body through the food chain, disrupting the body's hormonal balance. Long-term exposure may even cause serious health problems such as cancer. The presence of DES poses a potential threat to food safety and environmental health. Therefore, accurate and efficient detection of DES in food and the environment has become an important measure to protect public health. Summary of the Invention

[0004] The present invention is directed to the above problems and provides an electrochemiluminescent biosensor for detecting diethylstilbestrol and its preparation method and application. The present invention adds a target solution at the beginning of DNA amplification, obtains a Cas12a complex through a series of amplifications, and then adds the Cas12a complex dropwise on a working electrode for detection. The present invention detects diethylstilbestrol by constructing an electrochemiluminescent sensor based on multiple signal amplification. In the presence of diethylstilbestrol, signal amplification is achieved by chain displacement amplification and biological enzyme synergy, thereby providing more DNA chains for subsequent activation of the CRISPR-Cas12a system. As the activation degree of the CRISPR-Cas12a system increases, the ECL response intensity also increases, and the ECL signal intensity is positively correlated with the concentration of diethylstilbestrol, thereby achieving quantitative detection of diethylstilbestrol.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing an electrochemiluminescence biosensor for detecting diethylstilbestrol comprises the following steps:

[0007] (1) Weigh 1-2 mg of ZnRuMOF material and prepare 1-2 mg mL of chitosan solution. -1 The ZnRuMOF solution was then drop-coated on the glassy carbon electrode with 5-15 μL of ZnRuMOF solution and dried at 35-45°C for 2-4 hours. Gold nanoparticles were deposited on the surface of the glassy carbon electrode. 5-15 μL of H5 was then drop-coated and incubated in a dark room at 35-45°C for 3-5 hours.

[0008] (2) Add 6-10 μL MCH and react for 0.5-1 h, then wash with PBS to remove excess MCH; take 5-15 uL of the activated Cas12a-crRNA complex and drop it on the glassy carbon electrode, incubate it at 25-30 ° C for 50-70 min, and obtain the Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor, which is used as the working electrode for electrochemiluminescence test.

[0009] In the present invention, preferably, the preparation process of the ZnRuMOF material is:

[0010] 1) Add 100-300 μL of oxalic acid and 2-4 mL of hydrochloric acid to a mixture of 45 mL of N,N-dimethylformamide (DMF) and water, followed by the addition of 10-20 mg of Ru(bpy)3Cl2·6H2O and 85-115 mg of Zn(NO3)2; ultrasonically oscillate the mixture for 2 to 8 minutes to obtain a uniformly dispersed mixed solution;

[0011] 2) The mixed solution was transferred to a round-bottom flask and refluxed at 60°C for 22-24 hours to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol, and ultrapure water, respectively, and dried in vacuo at 60°C overnight to obtain the ZnRuMOF material.

[0012] In the present invention, preferably, the preparation process of the Cas 12a-crRNA complex is:

[0013] 1) 15-25 μL of the MB / H1 / ssDNA+aptamer mixture was mixed with 5-15 μL of target solution of varying concentrations, 5-15 μL of H2, and 5-15 μL of 2U T7 endonuclease. The mixture was shaken at 35-40°C for 1.5-2.5 hours to obtain a reaction solution containing orange short-chain DNA 1. The concentration of diethylstilbestrol in the target solution ranged from 1 fg mL -1 to 10 ng·mL -1 ;

[0014] 2) Take 15-25 μL of the reaction solution obtained in step 1) and mix it with 15-25 μL of MB / H3 mixture, 5-15 μL of H4, and 5-15 μL of 2U T7 endonuclease. Oscillate at 35-40°C for 1.5-2.5 hours to obtain a reaction solution containing yellow short-chain DNA 2;

[0015] 3) The reaction solution obtained in step 2) was adsorbed and dispersed by a magnet to obtain a layered supernatant and a precipitate; 15-20 μL of the supernatant was mixed with 5-15 μL of 100 nM crRNA and 5-15 μL of 100 nM Cas12a, and then reacted at 35-40 ° C for 60 to 70 minutes to obtain an activated Cas12a-crRNA complex.

[0016] In the present invention, preferably, the preparation process of the MB / H1 / ssDNA+aptamer mixture is as follows: 15-25 μL of single-stranded DNA is mixed with 15-25 μL of diethylstilbestrol aptamer solution, annealed at 85-95°C for 3-7 minutes in TE buffer, and then slowly cooled to room temperature to obtain ssDNA+aptamer double-stranded DNA; then 40-60 μL of 5 mg·mL-1 activated MB, 20-30 μL of ssDNA+aptamer double-stranded DNA mixture, and 20-30 μL of H1 are mixed, and the mixture is shaken at 35-40°C for 1.5-2.5 hours to obtain MB / H1 / ssDNA+aptamer mixture, which is stored at 4°C for use.

[0017] In the present invention, preferably, the H1, H2, H3, H4 and H5 DNA chains are all annealed before use, and the specific steps are: annealing the H1, H2, H3, H4 and H5 chain hairpin structure DNA solutions in TE buffer at 90°C for 5 minutes, and then slowly cooling to room temperature, wherein the amount and concentration of each of the five chain hairpin structure DNA solutions H1, H2, H3, H4 and H5 are 100 μL 5 μM.

[0018] In the present invention, preferably, the preparation process of the MB / H3 mixed solution is: mix 30-50 μL 5 mg·mL-1 activated MB with 15-25 μL H3, and shake at 35-40°C for 1.5-2.5 hours.

[0019] In the present invention, preferably, the activation preparation process of the magnetic beads MB is as follows: 500 μL of MB is placed in 1 mL of a mixture of 10 mM EDC and 20 mM NHs and reacted at 37 ° C for 1 hour. After the reaction is completed, the MB is washed three times with PBS buffer and prepared with PBS buffer solution to prepare 500 μL of MB solution with a concentration of 5 mg·mL-1, which is stored at 4 ° C for use.

[0020] In the present invention, preferably, in the mixture of N-dimethylformamide and water in step (1), the mass ratio of N-dimethylformamide to water is 2:1.

[0021] The present invention also provides an electrochemiluminescence biosensor for detecting diethylstilbestrol prepared by the above-mentioned preparation method.

[0022] In addition, the present invention also provides an application of an electrochemiluminescence biosensor in detecting diethylstilbestrol, characterized in that the specific steps are as follows:

[0023] (1) Detecting the response intensity of the electrochemiluminescence biosensor containing target solutions with different diethylstilbestrol concentrations, where the concentration range of diethylstilbestrol in the target solution is 1 fg·mL -1 to 10 ng·mL -1 ;

[0024] (2) Drawing of the standard curve: The prepared Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor was used as the working electrode, the platinum wire electrode was used as the auxiliary electrode, and the Ag|AgCl electrode was used as the reference electrode to form a three-electrode system; the three-electrode system was placed in a 0.01 M PBS buffer solution containing 20 mM triethylamine and pH 7.4 for measurement;

[0025] In the electrochemical window range of 0.3-1.3 V, the photomultiplier tube high voltage was 500 V and the scan rate was 0.1 V·s -1 , perform cyclic voltammetry scanning, record the potential-luminescence intensity curve, establish a linear relationship between the luminescence intensity difference and the logarithm of diethylstilbestrol concentration, and obtain the corresponding linear regression equation;

[0026] (3) Actual sample testing: Actual sample testing is calculated according to the linear regression equation in step (2) above.

[0027] By adopting the above technical solution, the beneficial effects of the present invention are:

[0028] (1) The electrochemiluminescence biosensor prepared by the method of the present invention has a detection process different from the previous electrochemical method for detecting diethylstilbestrol. The electrochemical biosensor of the present invention introduces nucleic acid amplification, uses diethylstilbestrol as a promoter, and triggers nucleic acid cascade amplification through diethylstilbestrol, thereby realizing sensitive and highly selective detection of diethylstilbestrol in milk.

[0029] (2) In the electrochemiluminescent biosensor of the present invention, ZnRuMOF material has the advantages of high electrocatalytic activity, large specific surface area, good biocompatibility, etc., and can be used as a base material to form ordered channels to form Ru(bpy)3 2+ The coating can significantly improve the stability of ruthenium bipyridine. The present invention combines ZnRuMOF with the highly specific CRISPR-Cas12a system and DNA Walker mechanism to construct an amplification reaction system. That is, by introducing the DNA Walker mechanism and strand displacement reaction for signal amplification, the intensity of the detection signal is significantly enhanced, thereby effectively reducing the detection limit of diethylstilbestrol.

[0030] Therefore, the multiple signal amplification electrochemiluminescence biosensor designed in the present invention can detect the target object efficiently, sensitively and quickly, significantly improving the simplicity of analysis and the accuracy of detection, and providing an innovative and efficient detection platform for diethylstilbestrol analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the principle of ultrasensitive detection of diethylstilbestrol by the electrochemiluminescence biosensor of the present invention;

[0032] Figure 2 CV diagram and impedance diagram of the electrochemiluminescent biosensor of the present invention;

[0033] Figure 3 The stability graph, reproducibility graph and specificity graph of the electrochemiluminescence biosensor of the present invention are shown;

[0034] Figure 4 Graph showing the ECL response values ​​and standard curves of the electrochemiluminescence biosensor at different concentrations of diethylstilbestrol in the present invention. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1

[0037] A method for preparing a Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor for detecting diethylstilbestrol comprises the following steps:

[0038] (1) Add 200uL oxalic acid (0.75mol·L -1 ) and 3 mL HCl (1 mol·L -1 ) was added to a mixture of 45 mL of NN dimethylformamide (DMF) and water (2:1), followed by the addition of 15 mg of Ru(bpy)3Cl2·6H2O and 100 mg of Zn(NO3)2. After ultrasonic oscillation for 5 minutes, a uniform mixed solution was obtained. The mixed solution was transferred to a round-bottom flask and refluxed at 60°C for 24 hours to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol and ultrapure water, respectively, and dried in vacuo at 60°C overnight to obtain the ZnRuMOF material.

[0039] (2) Preparation of Cas 12a-crRNA complex: 20 μL of MB / H1 / ssDNA+aptamer mixture, 10 μL of target solution of different concentrations, 10 μL of H2 and 10 μL of 2U T7 endonuclease were mixed and shaken at 37°C for 2 h to obtain a reaction solution containing orange short-chain DNA 1. The concentration range of diethylstilbestrol in the target solution was 1 fg mL -1 to 10 ng·mL -1 . Take 20 μL of the reaction solution containing the orange short-chain DNA 1, 20 μL of the MB / H3 mixture, 10 μL of H4, and 10 μL of 2U T7 nuclease, mix them, and shake them at 37°C for 2 hours to obtain a reaction solution containing the yellow short-chain DNA 2. The reaction solution containing the yellow short-chain DNA 2 is adsorbed and dispersed by a magnet to obtain a layered supernatant and precipitate. Take 20 μL of the supernatant and 10 μL of 100nM crRNA and 10 μL of 100nM Cas12a, react at 37°C for 60 minutes, and obtain an activated Cas12a-crRNA complex.

[0040] The preparation process of MB / H1 / ssDNA+aptamer mixture is as follows: 20 μL single-stranded DNA (4 μM) and 20 μL diethylstilbestrol aptamer (4 μM) are annealed in TE buffer at 90°C for 5 min, and then gradually cooled to room temperature to obtain a mixture of ssDNA / aptamer double-stranded DNA; then 50 μL 5 mg mL -1Activated magnetic beads (MB), 25 μL of ssDNA+aptamer double-stranded DNA mixture, and 25 μL of H1 were mixed and shaken at 37°C for 2 h to obtain MB / H1 / ssDNA+aptamer mixture, which was stored at 4°C for use.

[0041] The activation process of magnetic beads MB is as follows: carboxylated MB is thoroughly shaken and mixed before use, washed with PBS three times, and the final concentration is 5 mg mL -1 Specifically, 500 μL of MB was placed in 1 mL of a mixture of 10 mM EDC and 20 mM NHs and reacted at 37°C for 1 h. After the reaction, the MB was washed three times with PBS buffer and diluted to 500 μL with PBS buffer at a concentration of 5 mg mL -1 The MB solution was stored at 4°C until use.

[0042] MB / H3 was prepared by combining 50 μL of activated magnetic beads with 25 μL of 4 μM hairpin H3 via an amide bond reaction at 37°C for 2 hours. The beads were then rinsed with PBS and stored at 4°C until use.

[0043] In addition, the H1, H2, H3, H4 and H5 DNA chains were annealed before use. The specific steps were as follows: the H1, H2, H3, H4 and H5 chain hairpin structure DNA solutions were annealed in TE buffer at 90°C for 5 minutes, and then slowly cooled to room temperature. The amount and concentration of each of the five chain hairpin structure DNA solutions H1, H2, H3, H4 and H5 were 100 μL and 5 μM.

[0044] (3) The GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in sequence, dried with nitrogen, and then drop-coated with 10 μL of ZnRuMOF solution (1 mg ZnRuMOF material was weighed and prepared with 1% chitosan solution to form 1 mg mL -1 The ZnRuMOF solution was prepared and dried at 37°C. Gold nanoparticles (AuNPs) were deposited on the electrode surface by electrodeposition. 10 μL of H5 was dropwise applied and incubated in the dark at 37°C for 4 h. 8 μL of 1 mM MCH was added and reacted for 1 h. The excess MCH was washed away. 10 μL of the activated Cas 12a-crRNA complex was dropwise added to the electrode and incubated at 25°C for 60 min. This resulted in a Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor, which served as the working electrode for electrochemiluminescence testing.

[0045] When the electrochemiluminescence biosensor of the present invention is applied to detect diethylstilbestrol, the specific steps are as follows:

[0046] (1) Detecting the response intensity of the electrochemiluminescence biosensor containing target solutions with different diethylstilbestrol concentrations, wherein the concentration range of diethylstilbestrol in the target solution is 1 fg·mL -1 to 10 ng·mL -1 ;

[0047] (2) Drawing of standard curve

[0048] The prepared Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor was used as the working electrode, the platinum wire electrode was used as the auxiliary electrode, and the Ag|AgCl electrode was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in a 0.01 M PBS buffer solution containing 20 mM triethylamine and pH 7.4.

[0049] Cyclic voltammetry was performed within the electrochemical window range of 0.3-1.3 V, with a photomultiplier tube high voltage of 500 V and a scan rate of 0.1 V / s. The potential-luminescence intensity curve was recorded, and a linear relationship between the luminescence intensity difference and the logarithm of the diethylstilbestrol concentration was established, resulting in the corresponding linear regression equation.

[0050] (3) Actual sample testing

[0051] The actual sample milk test is calculated according to the linear regression equation in the above step (2).

[0052] Example 2

[0053] A method for preparing a Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor for detecting diethylstilbestrol comprises the following steps:

[0054] (1) Add 150 μL of oxalic acid (0.75 mol·L -1 ) and 4 mL HCl (1 mol·L -1 ) was added to a mixture of 45 mL of NN dimethylformamide (DMF) and water (2:1), followed by the addition of 20 mg of Ru(bpy)3Cl2·6H2O and 100 mg of Zn(NO3)2. After ultrasonic oscillation for 5 minutes, a uniform mixed solution was obtained. The mixed solution was transferred to a round-bottom flask and refluxed at 60°C for 24 hours to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol and ultrapure water, respectively, and dried in vacuo at 60°C overnight to obtain the ZnRuMOF material.

[0055] (2) Preparation of Cas 12a-crRNA complex: 20 μL of MB / H1 / DNA / aptamer mixture, 15 μL of target solution with different concentrations, 15 μL of H2 and 15 μL of 2U T7 endonuclease were mixed and shaken at 37°C for 2 h to obtain a reaction solution containing orange short-chain DNA 1. The concentration of diethylstilbestrol in the target solution ranged from 1 fg mL -1 to 10 ng·mL -1 . Take 25 μL of the reaction solution containing the orange short-chain DNA 1, 20 μL of the MB / H3 mixture, 15 μL of H4, and 15 μL of 2U T7 nuclease, mix them, and shake them at 37°C for 2 hours to obtain a reaction solution containing the yellow short-chain DNA 2. The reaction solution containing the yellow short-chain DNA 2 is adsorbed and dispersed by a magnet to obtain a layered supernatant and precipitate. Take 20 μL of the supernatant and 15 μL of 100 nM crRNA and 15 μL of 100 nM Cas12a, react at 37°C for 60 minutes, and obtain an activated Cas12a-crRNA complex.

[0056] The preparation process of MB / H1 / ssDNA+aptamer mixture is as follows: 20 μL single-stranded DNA (4 μM) and 20 μL diethylstilbestrol aptamer (4 μM) are annealed in TE buffer at 90°C for 5 min, and then gradually cooled to room temperature to obtain a mixture of ssDNA+aptamer double-stranded DNA; then 50 μL 5 mg·mL -1 Activated magnetic beads (MB), 25 μL of ssDNA+aptamer double-stranded DNA mixture, and 25 μL of H1 were mixed and shaken at 37°C for 2 h to obtain MB / H1 / ssDNA+aptamer mixture, which was stored at 4°C for use.

[0057] (3) The glassy carbon electrode (GCE) polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in sequence, dried with nitrogen, and then drop-coated with 10 μL of ZnRuMOF solution (1 mg ZnRuMOF material was weighed and prepared with 1% chitosan solution to form 1 mg mL -1 The ZnRuMOF solution was prepared and dried at 37°C. Gold nanoparticles (AuNPs) were deposited on the electrode surface by electrodeposition. 10 μL of H5 was dropwise applied and incubated in the dark at 37°C for 4 h. 8 μL of 1 mM MCH was added and reacted for 1 h. The excess MCH was washed away. 10 μL of the activated Cas 12a-crRNA complex was added dropwise to the electrode and incubated at 25°C for 60 min. This resulted in the Cas12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor, which served as the working electrode for electrochemiluminescence testing.

[0058] The rest are the same as in Example 1.

[0059] The nucleotide sequence used in the present invention is as follows:

[0060] Aptamer sequence: 5'-GGC GAT GGG GTA GGG GGT GTG GAG GGG CCG GAG GGA GGG G-3'

[0061] ssDNA (red line) sequence: 5'-H2N—TTT TTT TTT AAC CCC TCC CTC CGG CCC CCA-3'

[0062] H1 sequence: 5'-H2N—TTT GGT TGG GGG CCG GAG GGT ACG CGG CCC CCA TCC-3'

[0063] H2 sequence: 5'-GGG CCG GAG ATA ATC CAA CCC CCT CCG GCC CCC AAC C-3'

[0064] H3 sequence: 5'-H2N—TTT TTT TTA ATC CAA CCG GGG GGG GGT TGG ATT ATC-3'

[0065] H4 sequence: 5'-GAT AAT CCA ACC CCC CCC TGA GGA TTT AGA GGA GCG TGT GGT TGGATT-3'

[0066] H5 sequence: 5'-SH--CTT CAT GAT TCC TAC CTA CAA GTA GGA ATC TCT CTT TTT TTTTT-Fc-3'

[0067] crRNA sequence: 5'-UAA UUU CUA CUA AGU GAU GAU AAU CCA ACC ACA CGC UCC UC-3'

[0068] Comparative Example 1

[0069] Preparation method of a Cas 12a-crRNA / MCH / AuNPs / ZnRuMOF / GCE biosensor:

[0070] (1) The GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in sequence, dried with nitrogen, and then drop-coated with 10 μL of ZnRuMOF solution and dried at 37°C. Gold nanoparticles (AuNPs) were deposited on the electrode surface by electrodeposition. 8 μL of 1 mM MCH was added and reacted for 1 h, and the excess MCH was washed away. 10 μL of the activated Cas 12a-crRNA complex was dropped onto the electrode and incubated at 25°C for 60 min to obtain the Cas 12a-crRNA / MCH / AuNPs / ZnRuMOF / GCE biosensor, which was used as the working electrode for electrochemiluminescence testing.

[0071] The rest are the same as in Example 1.

[0072] Comparative Example 2

[0073] Preparation method of a Cas 12a-crRNA / MCH / H5 / ZnRuMOF / GCE biosensor:

[0074] A GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and then deionized water. After drying with nitrogen, 10 μL of ZnRuMOF solution was drop-coated and dried at 37°C. 10 μL of H5 was drop-coated and incubated in the dark at 37°C for 4 hours. 8 μL of 1 mM MCH was added and reacted for 1 hour. Excess MCH was removed by washing. 10 μL of the activated Cas 12a-crRNA complex was then added to the electrode and incubated at 25°C for 60 minutes. This yielded a Cas 12a-crRNA / MCH / H5 / ZnRuMOF / GCE biosensor, which served as the working electrode for electrochemiluminescence measurements.

[0075] The rest are the same as in Example 1.

[0076] Comparative Example 3

[0077] Preparation method of a Cas 12a-crRNA / MCH / H5 / AuNPs / GCE biosensor:

[0078] Alumina-polished GCE was ultrasonically treated with anhydrous ethanol and then deionized water. After drying with nitrogen, gold nanoparticles (AuNPs) were deposited on the electrode surface via electrodeposition. 10 μL of H5 was drop-coated and incubated in the dark at 37°C for 4 hours. 8 μL of 1 mM MCH was added and reacted for 1 hour. Excess MCH was removed by washing. 10 μL of the activated Cas12a-crRNA complex was then drop-coated on the electrode and incubated at 25°C for 60 minutes. This yielded a Cas12a-crRNA / MCH / H5 / AuNPs / GCE biosensor, which served as the working electrode for electrochemiluminescence analysis.

[0079] Comparative Example 3 differs from Example 1 in that, due to the lack of luminescent material, the electrochemiluminescence intensity does not respond and diethylstilbestrol cannot be detected. All other aspects are the same as Example 1.

[0080] The applicant used the biosensors prepared in the embodiment of the present invention and comparative examples 1 and 2 to detect diethylstilbestrol in milk (target), and the results are shown in Table 1.

[0081] Table 1 Determination results of diethylstilbestrol in milk

[0082]

[0083]

[0084] Note: a is the average of three measurements.

[0085] As shown in Table 1, only the electrochemiluminescent biosensor according to the embodiment of the present invention can detect diethylstilbestrol in milk, while neither Comparative Examples 1 nor 2 can detect diethylstilbestrol in milk. Furthermore, the electrochemiluminescent biosensor according to the embodiment of the present invention has a high recovery rate for diethylstilbestrol and a small relative standard deviation, demonstrating that the electrochemiluminescent biosensor according to the embodiment of the present invention can achieve sensitive and highly selective detection of diethylstilbestrol in milk.

[0086] In order to verify the reaction activity of the working electrode of the electrochemiluminescent biosensor of the present invention, a CV test was performed on the working electrode of the sensor. Figure 2 As shown in Figure A, when ZnRuMOF material (curve c) is added to the bare glassy carbon electrode (curve a), the peak potential of the electrode increases. When AuNPs are deposited on the electrode surface by electrodeposition, the peak potential of the electrode decreases due to the good conductivity of AuNPs. With the subsequent loading of DNA chains (curve d) and the closure of the active sites of MCH (curve f), the peak potential of the electrode gradually increases. When the activated Cas12a-crRNA system is modified on the electrode surface (curve e), the short DNA chain with ferrocene is detached from the electrode, and the peak potential of the electrode decreases. The working electrode of the sensor is subjected to EIS testing, as shown in Figure 5. Figure 2 As shown in Figure B, when ZnRuMOF material (curve c) is added to the bare glassy carbon electrode (curve a), the peak redox current decreases significantly. When AuNPs are deposited on the electrode surface by electrodeposition (curve b), the peak redox current increases due to the good conductivity of AuNPs. With the subsequent loading of DNA chains (curve d) and the closure of the active sites of MCH (curve f), the peak redox current decreases significantly. When the activated Cas12a-crRNA system is modified on the electrode surface (curve e), the short DNA chain with ferrocene is separated from the electrode, and the peak redox current increases significantly.

[0087] The applicant also analyzed the performance of the electrochemiluminescent biosensor of the present invention:

[0088] The electrochemiluminescence biosensor was used to detect different concentrations of diethylstilbestrol in the target. Figure 3 As the concentration of diethylstilbestrol increased from 1 fg·mL -1 Increased to 10 ng·mL -1 ( Figure 3 The diethylstilbestrol concentrations of the eight samples in A were 1 fg·mL -1 , 10fg·mL -1 , 100fg·mL -1 , 1pg·mL -1 , 10 pg·mL -1 , 100 pg·mL -1 , 1ng·mL -1 , 10 ng·mL -1 Since the detection limit is low enough, if no detection is found, it is assumed that no diethylstilbestrol is present). The intensity of the ECL reaction gradually increases, and there is a strong linear correlation between the ECL intensity and the logarithm of the diethylstilbestrol concentration ( Figure 3 B), the fitting regression equation is: I = 1504.5lgC (ng·mL -1 )+11267.4. Based on 3σ calculation, the detection limit of diethylstilbestrol is 0.109 fg·mL -1 . Figure 4 A shows the ECL response obtained by performing 10 consecutive cycles of ECL scanning. We observed that the ECL intensity did not show significant changes, and the RSD value of the ECL intensity was 0.4%. Figure 4 As shown in B, five groups of 1 ng mL -1 The reproducibility of the ECL strategy was verified by the relative standard deviation (RSD) of diethylstilbestrol, which was 1.6%, indicating that the ECL sensing platform has good reproducibility. Figure 4 As shown in Figure C, five interfering substances were selected for testing: estrone (E1), estradiol (E2), estriol (E3), testosterone (testred), and bisphenol A (BPA). The results showed that the ECL response intensity of diethylstilbestrol was significantly higher than that of the blank sample and all five interfering substances, demonstrating that the electrochemiluminescence sensor of the present invention has excellent selectivity and can effectively distinguish the target substance from common interfering substances, demonstrating its high specificity in practical applications. The electrolyte of the biosensor constructed above is a 0.01M PBS buffer solution containing 20mM triethylamine and a pH of 7.4.

[0089] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing an electrochemiluminescence biosensor for detecting diethylstilbestrol, characterized in that: The steps include: (1) Weigh 1-2 mg of ZnRuMOF material and prepare 1-2 mg mL of chitosan solution. -1 The ZnRuMOF solution was then drop-coated on the glassy carbon electrode with 5-15 μL of ZnRuMOF solution and dried at 35-45°C for 2-4 hours. Gold nanoparticles were deposited on the surface of the glassy carbon electrode. 5-15 μL of H5 was then drop-coated and incubated in a dark room at 35-45°C for 3-5 hours. (2) Add 6-10 μL MCH and react for 0.5-1 h, then wash with PBS to remove excess MCH; take 5-15 uL of the activated Cas12a-crRNA complex and drop it on the glassy carbon electrode, incubate it at 25-30 ° C for 50-70 min, and obtain the Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor, which is used as the working electrode for electrochemiluminescence test.

2. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 1, wherein: The preparation process of the ZnRuMOF material is as follows: 1) Add 100-300 μL of oxalic acid and 2-4 mL of hydrochloric acid to a mixture of 45 mL of N,N-dimethylformamide and water, followed by the addition of 10-20 mg of Ru(bpy)3Cl2·6H2O and 85-115 mg of Zn(NO3)2; ultrasonically oscillate the mixture for 2 to 8 minutes to obtain a uniformly dispersed mixed solution; 2) The mixed solution was transferred to a round-bottom flask and refluxed at 60°C for 22-26 hours to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol, and ultrapure water, respectively, and dried in vacuo at 60°C overnight to obtain the ZnRuMOF material.

3. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 1, wherein: The preparation process of the Cas 12a-crRNA complex is: 1) 15-25 μL of the MB / H1 / DNA / aptamer mixture was mixed with 5-15 μL of target solution of varying concentrations, 5-15 μL of H2, and 5-15 μL of 2U T7 endonuclease. The mixture was shaken at 35-40°C for 1.5-2.5 hours to obtain a reaction solution containing orange short-chain DNA 1. The concentration of diethylstilbestrol in the target solution ranged from 1 fg mL -1 to 10 ng·mL -1 ; 2) Take 15-25 μL of the reaction solution obtained in step 1) and mix it with 15-25 μL of MB / H3 mixture, 5-15 μL of H4, and 5-15 μL of 2U T7 endonuclease. Oscillate at 35-40°C for 1.5-2.5 hours to obtain a reaction solution containing yellow short-chain DNA 2; 3) The reaction solution obtained in step 2) was adsorbed and dispersed by a magnet to obtain a layered supernatant and a precipitate; 15-20 μL of the supernatant was mixed with 5-15 μL of 100 nM crRNA and 5-15 μL of 100 nM Cas12a, and then reacted at 35-40 ° C for 60 to 70 minutes to obtain an activated Cas12a-crRNA complex.

4. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 3, wherein: The preparation process of the MB / H1 / ssDNA+aptamer mixture is as follows: 15-25 μL of single-stranded DNA and 15-25 μL of diethylstilbestrol aptamer solution are mixed, annealed at 85-95°C for 3-7 minutes in TE buffer, and then slowly cooled to room temperature to obtain ssDNA+aptamer double-stranded DNA; then 40-60 μL of 5 mg·mL -1 Mix the activated MB, 20-30 μL of ssDNA+aptamer double-stranded DNA mixture, and 20-30 μL of H1, and shake at 35-40°C for 1.5-2.5 hours to obtain the MB / H1 / ssDNA+aptamer mixture, which is then stored at 4°C for later use.

5. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 3, wherein: The H1, H2, H3, H4 and H5 DNA chains were all annealed before use. The specific steps were as follows: the H1, H2, H3, H4 and H5 chain hairpin structure DNA solutions were annealed in TE buffer at 90°C for 5 minutes, and then slowly cooled to room temperature. The amount and concentration of each of the five chain hairpin structure DNA solutions H1, H2, H3, H4 and H5 were 100 μL and 5 μM.

6. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 3, wherein: The preparation process of the MB / H3 mixture is as follows: 30-50 μL of 5 mg mL -1 The activated MB was mixed with 15-25 μL H3 and shaken at 35-40° C. for 1.5-2.5 h.

7. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 3, characterized in that: MB activation was prepared as follows: 500 μL MB was placed in 1 mL of a mixture of 10 mM EDC and 20 mM NHs and reacted at 37°C for 1 h. After the reaction, the MB was washed three times with PBS buffer and diluted to 500 μL with PBS buffer at a concentration of 5 mg mL -1 The MB solution was stored at 4°C until use.

8. The method for preparing the electrochemiluminescence biosensor for detecting diethylstilbestrol according to claim 2, wherein: In the mixture of N,N-dimethylformamide and water in step (1), the mass ratio of N,N-dimethylformamide to water is 2:

1.

9. An electrochemiluminescence biosensor for detecting diethylstilbestrol prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the electrochemiluminescence biosensor according to claim 9 for detecting diethylstilbestrol, characterized in that: The specific steps are as follows: (1) Detecting the response intensity of the electrochemiluminescence biosensor containing target solutions with different diethylstilbestrol concentrations, where the concentration range of diethylstilbestrol in the target solution is 1 fg·mL -1 to 10 ng·mL -1 ; (2) Drawing of standard curve The prepared Cas12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescence biosensor was used as the working electrode, the platinum wire electrode as the auxiliary electrode, and the Ag|AgCl electrode as the reference electrode to construct a three-electrode system; the three-electrode system was placed in a 0.01 M PBS buffer solution containing 20 mM triethylamine and pH 7.4 for measurement; Cyclic voltammetry was performed within the electrochemical window range of 0.3-1.3 V, with a photomultiplier tube high voltage of 500 V and a scan rate of 0.1 V / s. The potential-luminescence intensity curve was recorded, and a linear relationship between the luminescence intensity difference and the logarithm of the diethylstilbestrol concentration was established, resulting in the corresponding linear regression equation. (3) Actual sample testing The actual sample detection is calculated according to the linear regression equation in the above step (2).

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