Electrochemiluminescence biosensor for detecting diethylstilbestrol as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to an electrochemiluminescence biosensor for detecting diethylstilbestrol as well as a preparation method and application of the electrochemiluminescence biosensor for detecting diethylstilbestrol. The preparation method comprises the following steps: dispensing a ZnRuMOF solution on a glassy carbon electrode (GCE), and drying; depositing gold nanoparticles on the surface of the glassy carbon electrode; dripping H5, and hatching at 37 DEG C; mCH is added for a reaction, and PBS washing is carried out to remove excessive MCH; and finally, dropwise adding the activated Cas 12a-crRNA compound on a glassy carbon electrode, and hatching to obtain the Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemical luminescence biosensor which is used as a working electrode for electrochemical luminescence test. According to the diethylstilbestrol detection method, the target object can be efficiently, sensitively and rapidly detected, the analysis simplicity and the detection accuracy are remarkably improved, and an innovative and sensitive detection platform is provided for diethylstilbestrol analysis.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to an electrochemiluminescent biosensor for detecting diethylstilbestrol and a preparation method and application 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 combined by 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 has been 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 and interfere with the body's hormone 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 drops the Cas12a complex 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 purpose, the technical solution adopted by the present invention is as follows:

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

[0007] (1) Weigh 1-2 mg ZnRuMOF material and prepare 1-2 mg mL 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 to react for 0.5-1 h, 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) 100-300 μL of oxalic acid and 2-4 mL of hydrochloric acid are added to a mixed solution of 45 mL of N,N-dimethylformamide (DMF) and water, followed by adding 10-20 mg of Ru(bpy)3Cl2·6H2O and 85-115 mg of Zn(NO3)2; the mixed system is subjected to ultrasonic oscillation 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 h to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol and ultrapure water, respectively, and vacuum dried at 60° C. overnight to obtain a ZnRuMOF material.

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

[0013] 1) Take 15-25 μL of the mixed solution of MB / H1 / ssDNA+aptamer, mix it with 5-15 μL of target solution of different concentrations, 5-15 μL of H2 and 5-15 μL of 2U T7 endonuclease, and shake it at 35-40°C for 1.5-2.5h to obtain a reaction solution containing orange short-chain DNA 1; the concentration range of diethylstilbestrol in the target solution is 1 fg·mL -1 To 10 ng·mL -1 ;

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

[0015] 3) Adsorbing and dispersing the reaction solution obtained in step 2) by a magnet to obtain a layered supernatant and a precipitate; taking 15-20 μL of the supernatant and mixing it with 5-15 μL of 100 nM crRNA and 5-15 μL of 100 nM Cas12a, and then reacting 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 mixed solution is: 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 min 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 mixed solution, and 20-30 μL of H1 are mixed, and shaken at 35-40°C for 1.5-2.5h to obtain MB / H1 / ssDNA+aptamer mixed solution, which is stored at 4°C for standby 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: 30-50 μL 5 mg·mL-1 activated MB is mixed with 15-25 μL H3, and the mixture is shaken at 35-40°C for 1.5-2.5h.

[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 h. After the reaction is completed, the MB is washed three times with PBS buffer and prepared into 500 μL of a 5 mg·mL-1 MB solution with PBS buffer solution, which is stored at 4°C for later use.

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

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

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

[0023] (1) Detecting the response intensity of the electrochemiluminescent 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 ;

[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 the diethylstilbestrol concentration, and obtain the corresponding linear regression equation;

[0026] (3) Actual sample testing: The 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 electrochemiluminescent 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, the ZnRuMOF material has the advantages of high electrocatalytic activity, large specific surface area, good biocompatibility, etc., and can be used as a substrate material to form orderly channels to form Ru(bpy)3 2+ The coating can significantly improve the stability of ruthenium bipyridine. The present invention constructs an amplification reaction system by combining ZnRuMOF with the highly specific CRISPR-Cas12a system and DNA Walker mechanism, 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 improves the simplicity of analysis and the accuracy of detection, and provides an innovative and efficient detection platform for diethylstilbestrol analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The schematic diagram is a principle diagram of the ultrasensitive detection of diethylstilbestrol by the electrochemiluminescent biosensor of the present invention;

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

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

[0034] Figure 4 The graph is a graph showing the ECL response value and the standard curve of the electrochemiluminescence biosensor at different concentrations of diethylstilbestrol in the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 electrochemiluminescent 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 NN dimethylformamide (DMF) and water (2:1), and then 15 mg Ru(bpy)3Cl2·6H2O and 100 mg Zn(NO3)2 were added. After ultrasonic oscillation for 5 min, a uniform mixed solution was obtained; the mixed solution was transferred to a round-bottom flask and refluxed at 60 °C for 24 h to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol and ultrapure water respectively, and vacuum dried at 60 °C overnight to obtain 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 orange short-chain DNA 1, 20 μL of MB / H3 mixture, 10 μL of H4, and 10 μL of 2U T7 endonuclease, and oscillate at 37°C for 2 hours to obtain a reaction solution containing yellow short-chain DNA 2. The reaction solution containing 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 mixed solution is as follows: 20 μL single-stranded DNA (4 uM) and 20 μL diethylstilbestrol aptamer (4 uM) are annealed at 90°C for 5 min in TE buffer solution, and then gradually cooled to room temperature to obtain a ssDNA / aptamer double-stranded DNA mixed solution; then 50 μL 5 mg·mL -1Activated magnetic beads (MB), 25 μL of a mixture of ssDNA+aptamer double-stranded DNA, and 25 μL of H1 were mixed and shaken at 37°C for 2 h to obtain a MB / H1 / ssDNA+aptamer mixture, which was stored at 4°C for future use.

[0041] The activation process of magnetic beads MB is as follows: carboxylated MB is fully 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 a mixture of 1 mL of 10 mM EDC and 20 mM NHs and reacted at 37 °C for 1 h. After the reaction was completed, the MB was washed three times with PBS buffer and prepared into 500 μL with PBS buffer solution at a concentration of 5 mg mL -1 The MB solution was stored at 4°C until use.

[0042] The preparation process of MB / H3 is as follows: 50uL of activated magnetic beads and 25uL of 4uM hairpin H3 are linked by amide bond reaction at 37°C for 2h. The magnetic beads are rinsed with PBS and stored at 4°C for future 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 at 90°C for 5 min in TE buffer, respectively, and then slowly cooled to room temperature, wherein the amount and concentration of each of the five chain hairpin structure DNA solutions H1, H2, H3, H4 and H5 were 100 μL 5 μM.

[0044] (3) The GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in turn, dried with nitrogen, and then drop-coated with 10 μL of ZnRuMOF solution (1 mg of ZnRuMOF material was weighed and prepared with 1% chitosan solution to form 1 mg mL -1 ZnRuMOF solution) and dried at 37°C. Gold nanoparticles AuNPs were deposited on the electrode surface by electrodeposition. 10 μL of H5 was drop-coated and incubated in a dark room at 37°C for 4 h. 8 μL of 1 mM MCH was added to react for 1 h, and the excess MCH was removed by washing; 10 uL of the activated Cas 12a-crRNA complex was dropped on the electrode and incubated at 25°C for 60 min to obtain the Cas 12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescent biosensor, which was used as the working electrode for electrochemiluminescence test.

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

[0046] (1) Detecting the response intensity of an electrochemiluminescent 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 electrochemiluminescent 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] In the electrochemical window range of 0.3-1.3V, the photomultiplier tube high voltage was 500V, the scanning rate was 0.1V / s, cyclic voltammetry scanning was performed, the potential-luminescence intensity curve was recorded, and the linear relationship between the luminescence intensity difference and the logarithm of the diethylstilbestrol concentration was established to obtain 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 step (2) above.

[0052] Example 2

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

[0054] (1) Add 150uL oxalic acid (0.75mol·L -1 ) and 4 mL HCl (1 mol·L -1 ) was added to a mixture of 45 mL NN dimethylformamide (DMF) and water (2:1), and then 20 mg Ru(bpy)3Cl2·6H2O and 100 mg Zn(NO3)2 were added. 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 vacuum dried at 60°C overnight to obtain ZnRuMOF material.

[0055] (2) Preparation of Cas 12a-crRNA complex: 20 μL of MB / H1 / DNA / aptamer mixture, 15 μL of target solution of 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 range of diethylstilbestrol in the target solution was 1 fg mL -1 To 10 ng·mL -1 . Take 25 μL of the reaction solution containing orange short-chain DNA 1, 20 μL of MB / H3 mixture, 15 μL of H4, and 15 μL of 2U T7 nuclease, and oscillate at 37°C for 2 hours to obtain a reaction solution containing yellow short-chain DNA 2. The reaction solution containing 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 mixed solution is as follows: 20 μL single-stranded DNA (4 uM) and 20 μL diethylstilbestrol aptamer (4 uM) are annealed at 90°C for 5 min in TE buffer solution, and then gradually cooled to room temperature to obtain a mixed solution 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 future use.

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

[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] A preparation method of 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 turn, blown dry with nitrogen, and then 10 μL of ZnRuMOF solution was drop-coated and dried at 37°C. Gold nanoparticles AuNPs were deposited on the electrode surface by electrodeposition. 8 μL of 1 mM MCH was added to react for 1 h, and the excess MCH was washed to remove it; 10 uL of the activated Cas 12a-crRNA complex was dropped on 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 test.

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

[0072] Comparative Example 2

[0073] A method for preparing a Cas 12a-crRNA / MCH / H5 / ZnRuMOF / GCE biosensor:

[0074] The GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in turn, blown dry with nitrogen, and then 10 μL of ZnRuMOF solution was drop-coated and dried at 37 °C. 10 μL of H5 was drop-coated and incubated in a dark room at 37 °C for 4 h. 8 μL of 1 mM MCH was added to react for 1 h, and the excess MCH was washed to remove it; 10 uL of the activated Cas 12a-crRNA complex was dropped on the electrode and incubated at 25 °C for 60 min to obtain the Cas 12a-crRNA / MCH / H5 / ZnRuMOF / GCE biosensor, which was used as the working electrode for electrochemiluminescence test.

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

[0076] Comparative Example 3

[0077] A method for preparing a Cas 12a-crRNA / MCH / H5 / AuNPs / GCE biosensor:

[0078] The GCE polished with alumina powder was ultrasonically treated with anhydrous ethanol and deionized water in turn, and gold nanoparticles AuNPs were deposited on the electrode surface by electrodeposition after being blown dry with nitrogen. 10 μL of H5 was applied dropwise and incubated in a dark room at 37 ° C for 4 h. 8 μL of MCH with a concentration of 1 mM was added to react for 1 h, and the excess MCH was washed away; 10 uL of the activated Cas12a-crRNA complex was added dropwise to the electrode and incubated at 25 ° C for 60 min to obtain the Cas 12a-crRNA / MCH / H5 / AuNPs / GCE biosensor, which was used as the working electrode for electrochemiluminescence test.

[0079] The difference between Comparative Example 3 and Example 1 is that due to the lack of luminescent material, the electrochemical luminescence intensity has no response and diethylstilbestrol cannot be detected. The rest is 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] According to the results in Table 1, only the electrochemiluminescent biosensor of the embodiment of the present invention can detect diethylstilbestrol in milk, while Comparative Example 1 and Comparative Example 2 cannot detect diethylstilbestrol in milk. In addition, the recovery rate of diethylstilbestrol detected by the electrochemiluminescent biosensor of the embodiment of the present invention is high, and the relative standard deviation is small, indicating that the electrochemiluminescent biosensor of the embodiment of the present invention can realize 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 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 test, as shown in 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 is significantly reduced. 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 is significantly reduced. 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 is significantly increased.

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

[0088] The electrochemiluminescent 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 Increase to 10 ng·mL -1 ( Figure 3 The concentrations of diethylstilbestrol in the eight samples in A were 1 fg·mL -1 , 10fg·mL -1 , 100fg·mL -1 , 1pg·mL -1 , 10pg·mL -1 , 100pg·mL -1 , 1ng·mL -1 , 10ng·mL -1 , since the detection limit is low enough, if it cannot be detected, it is assumed that there is no diethylstilbestrol). 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. According to 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 obvious 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 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, indicating that the electrochemiluminescence sensor of the present invention has excellent selectivity and can effectively distinguish the target from common interfering substances, reflecting its high specificity in practical applications. Among them, the electrolyte of the biosensor constructed above is a 0.01M PBS buffer solution containing 20mM triethylamine and pH 7.4.

[0089] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes 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 electrochemiluminescent biosensor for detecting diethylstilbestrol, characterized in that: The steps include: (1) Weigh 1-2 mg ZnRuMOF material and prepare 1-2 mg mL 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 to react for 0.5-1 h, 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 electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 1, characterized in that: The preparation process of the ZnRuMOF material is as follows: 1) 100-300 μL of oxalic acid and 2-4 mL of hydrochloric acid are added to a mixed solution 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; the mixed system is subjected to ultrasonic oscillation 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 h to obtain an orange-red precipitate, which was then washed twice with DMF, ethanol and ultrapure water respectively, and vacuum dried at 60° C. overnight to obtain a ZnRuMOF material.

3. The method for preparing the electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 1, characterized in that: The preparation process of the Cas 12a-crRNA complex is: 1) Take 15-25 μL of the mixed solution of MB / H1 / DNA / aptamer, mix it with 5-15 μL of target solution of different concentrations, 5-15 μL of H2 and 5-15 μL of 2U T7 endonuclease, and shake it at 35-40°C for 1.5-2.5h to obtain a reaction solution containing orange short-chain DNA 1; the concentration range of diethylstilbestrol in the target solution is 1 fg·mL -1 To 10 ng·mL -1 ; 2) 15-25 μL of the reaction solution obtained in step 1) was mixed with 15-25 μL of MB / H3 mixed solution, 5-15 μL of H4 and 5-15 μL of 2U T7 endonuclease, and shaken at 35-40° C. for 1.5-2.5 h to obtain a reaction solution containing yellow short-chain DNA 2; 3) Adsorbing and dispersing the reaction solution obtained in step 2) by a magnet to obtain a layered supernatant and a precipitate; taking 15-20 μL of the supernatant and mixing it with 5-15 μL of 100 nM crRNA and 5-15 μL of 100 nM Cas12a, and then reacting at 35-40 ° C for 60 to 70 minutes to obtain an activated Cas12a-crRNA complex.

4. The method for preparing the electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 3, characterized in that: The preparation process of the MB / H1 / ssDNA+aptamer mixed solution 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 The activated MB, 20-30 μL ssDNA+aptamer double-stranded DNA mixture, and 20-30 μL H1 were mixed and shaken at 35-40°C for 1.5-2.5 hours to obtain a MB / H1 / ssDNA+aptamer mixture, which was stored at 4°C for future use.

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

6. The method for preparing the electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 3, characterized in that: The preparation process of the MB / H3 mixed solution 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 electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 3, characterized in that: The activation preparation of the magnetic beads MB is as follows: 500 μL MB is 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 is completed, the MB is washed three times with PBS buffer and prepared into 500 μL with PBS buffer solution at a concentration of 5 mg mL -1 The MB solution was stored at 4°C until use.

8. The method for preparing the electrochemiluminescent biosensor for detecting diethylstilbestrol according to claim 2, characterized in that: In the mixture of NNN dimethylformamide and water in step (1), the mass ratio of NNN dimethylformamide to water is 2:

1.

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

10. Use of the electrochemiluminescent biosensor according to claim 9 for detecting diethylstilbestrol, characterized in that: The specific steps are as follows: (1) Detecting the response intensity of the electrochemiluminescent 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 ; (2) Drawing of standard curve The prepared Cas12a-crRNA / MCH / H5 / AuNPs / ZnRuMOF / GCE electrochemiluminescent 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 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; In the electrochemical window range of 0.3-1.3V, the photomultiplier tube high voltage was 500V, the scanning rate was 0.1V / s, cyclic voltammetry scanning was performed, the potential-luminescence intensity curve was recorded, and the linear relationship between the luminescence intensity difference and the logarithm of the diethylstilbestrol concentration was established to obtain 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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