Electrochemiluminescence aptamer sensor based on CoOOH-cDNA / Apt / AgNWs / Tb-A-COF / GCE working electrode

Abstract

The invention relates to the technical field of electrochemical luminescence detection, in particular to an electrochemical luminescence aptamer sensor based on a CoOOH-cDNA / Apt / AgNWs / Tb (at) A-COF / GCE working electrode. The iBF in a sample to be detected is detected by utilizing LC-MS / MS, GC-MS, UHPLC and HPLC-MS, so that the cost is high, the speed is low, and the sensitivity is relatively low. Aiming at the technical problem, the invention provides the CoOOH-cDNA / Apt / AgNWs / Tb-coated A-COF / GCE working electrode, the working electrode takes Tb-coated A-COF as a donor and CoOOH NSs as a receptor, the donor and the receptor are combined and modified on a glassy carbon electrode (GCE) through an aptamer and cDNA, then the CoOOH-cDNA / Apt / AgNWs / Tb-coated A-COF / GCE is taken as the working electrode, and iBF in a sample to be detected is quantitatively detected. According to the invention, through a signal "on-off-on" (on-off-on) mode, the consistency of detection results is significantly improved, the generation of false positive or false negative signals is reduced, and the detection accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, and particularly to an electrochemiluminescent aptasensor based on a CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode. Background Art

[0002] Fentanyl and its analogues are synthetic opioid drugs that have become leading analgesics and anaesthetics due to their short onset time and faster absorption by the human body. Fentanyl causes respiratory and central nervous system depression in a dose-dependent manner. Fentanyl analogues emerging as new psychoactive substances are usually synthesized by modifying the isobutyryl group or substituting the phenethyl moiety of fentanyl. Currently, there are various strategies for detecting iBF, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography (UHPLC), and high performance liquid chromatography-mass spectrometry (HPLC-MS). However, these methods have disadvantages such as high cost and the need for specialized operations, which will limit their application in actual detection. Therefore, it is urgent to find a simple, rapid, and sensitive detection method.

[0003] Electrochemiluminescence (ECL) is a method developed by combining the advantages of chemiluminescence and electrochemistry, and has been widely used in fields such as bioanalysis and surface analysis due to its unique advantages. It has the advantages of high sensitivity, low background, easy control, and short detection time, and does not require the introduction of an external light source. Compared with photoluminescence methods, it can effectively avoid the interference of the background light source and improve the detection sensitivity through a high signal-to-noise ratio, so as to achieve the purpose of trace detection of the target substance. In the development of highly sensitive electrochemiluminescent sensors, the use of single-signal switching modes such as "signal-on" or "signal-off" modes has been very common. However, ECL sensors relying on single-signal switching modes may generate inaccurate signals, which limits their practical application in analysis, especially for complex biosensors at low concentrations. Therefore, a new complex output signal mode, that is, the "On-Off-On" strategy using multi-signal switching, can greatly enhance selectivity and specificity. The ECL sensing platform based on the "on-off-on" strategy provides a new way for accurately determining the target due to its minimum background signal and higher sensitivity.

[0004] In the present invention, a novel Tb@A-COF is used as the donor of this mechanism, and CoOOH NSs is used as the quencher. The donor and the acceptor are modified on a glassy carbon electrode (GCE) through an aptamer and cDNA binding. Then, CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE is used as the working electrode to quantitatively detect iBF in beer samples. Since the specific binding ability of the aptamer to the analyte is much greater than its binding ability to Tb@A-COF, the addition of the analyte iBF causes the receptor connected to the aptamer to fall off from the electrode surface, inhibiting the quenching effect, thereby restoring the ECL signal, and the signal has a linear relationship with the concentration of iBF. The present invention not only has the advantages of high sensitivity, strong specificity, wide linear range and simple instrument in electrochemiluminescence analysis, but also has important practical significance for the detection of iBF in beer beverages. Summary of the Invention

[0005] The problems existing in the prior art are that using LC-MS / MS, GC-MS, UHPLC and HPLC-MS to detect iBF in the sample to be tested has high cost, slow speed and low sensitivity. To solve the above technical problems, the present invention provides a CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode, and its preparation method includes the following steps:

[0006] (1) Modify the surface of a clean glassy carbon electrode to obtain a Tb@A-COF coating, and obtain a Tb@A-COF / GCE electrode;

[0007] (2) Modify the surface of the Tb@A-CO / GCE electrode to obtain an AgNWs coating, and obtain an AgNWs / Tb@A-COF / GCE electrode;

[0008] (3) Modify the surface of the AgNWs / Tb@A-COF / GCE electrode to obtain an Apt coating, and obtain an Apt / AgNWs / Tb@A-COF / GCE electrode;

[0009] (4) Modify the surface of the Apt / AgNWs / Tb@A-COF / GCE electrode to obtain a CoOOH-cDNA coating, and obtain a CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode.

[0010] Preferably, the Tb@A-COF coating is a coating formed by coating a dispersion of Tb@A-COF in DMF on the electrode surface and drying. The Tb@A-COF is formed by doping the metal element Tb into A-COF, and the A-COF is a product formed by the condensation reaction of the ligand ETBC and piperazine through the condensation of aldehyde group (-CHO) and secondary amino group (R2NH).

[0011] Preferably, the preparation method of Tb@A-COF comprises the following steps:

[0012] (1) Add 120.21 mg of ETBC and 50.07 mg of piperazine into an ampoule containing 5 mL of toluene. After ultrasonic treatment until the lumpy piperazine is completely dissolved, place the ampoule in a liquid nitrogen bath for freeze degassing for at least 20 min. After cooling to room temperature, seal it. Then place the ampoule alone in an oven at 120 °C and let it stand still for 72 h without interference. After the reaction ends, a yellow solid product is obtained. The obtained yellow solid product is washed with anhydrous dichloromethane and then placed in a vacuum dryer at 60 °C to obtain A-COF;

[0013] (2) Add 2 mg of A-COF into 3 mL of a 5 mmol / L Tb(NO3)3·6H2O solution with DMF as the solvent. After ultrasonic treatment until the solid is completely dissolved, stir at room temperature for at least 24 h. After the reaction ends, the obtained solid product is washed successively with DMF and deionized water and then dried to obtain Tb@A-COF.

[0014] Preferably, the AgNWs coating is a coating formed by coating and drying a solution of AgNWs in an aqueous solution on the electrode surface. The preparation method of the AgNWs comprises the following steps:

[0015] (1) Prepare PVP-coated silver nanowires;

[0016] (2) Remove the PVP coating layer from the PVP-coated silver nanowires to obtain AgNWs.

[0017] Preferably, step (1) comprises the following steps:

[0018] (1) Add 0.8325 g of PVP to 40 mL of ethylene glycol and heat and stir at 120 °C for 0.5 h, then naturally cool to room temperature to obtain a PVP coating solution;

[0019] (2) Dissolve 0.2123 g of silver nitrate in 10 mL of ethylene glycol, and while stirring, drop it into the PVP coating solution. After dropping,

[0020] add 125 μL of 0.1 M aqueous NaCl solution to the reaction system, stir at room temperature for 30 min, then quickly raise the temperature to 160 °C at a heating rate of 5 - 8 °C / min, and keep stirring and reacting until the color of the reaction system turns gray-green. After the reaction ends, let the reaction solution naturally cool to room temperature, then separate the solid and liquid to obtain a solid product. The obtained solid product is washed successively with ethanol and deionized water to obtain PVP-coated silver nanowires.

[0021] Preferably, step (2) comprises the following steps:

[0022] 0.1 g of NaBH4 was added to a mixed solution formed by 5 mL of water and ethanol in a volume ratio of 1:1 to obtain a NaBH4 solution; 1 g of PVP-coated silver nanowires was added to the NaBH4 solution and maintained for at least 60 s to remove the PVP wrapped outside the AgNWs. After that, the solid product was obtained by filtration, and then the obtained solid product was washed with ethanol and water at least 3 times successively. After drying, AgNWs were obtained.

[0023] Preferably, the Apt coating is a coating formed by first dispersing 78.99 μg of Apt into 74.8 μL of a 10 mM Tris-HCl buffer solution to form a mixed solution, and diluting it to 10 μM with a 10 mM Tris-HCl buffer solution to obtain an Apt ligand solution, which is coated on the electrode surface and dried.

[0024] The Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid. The concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 1.0 mmol / L.

[0025] Preferably, the CoOOH-cDNA coating is a coating formed by dispersing CoOOH-cDNA into 122.9 μL of a 10 mM Tris-HCl buffer solution to form a mixed solution, and diluting it to 10 μM with a 10 mM Tris-HCl buffer solution, and then coating the mixed solution on the electrode surface and drying.

[0026] The Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid. The concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 1.0 mmol / L.

[0027] Preferably, the preparation method of the CoOOH-cDNA includes the following steps:

[0028] (1) 625 μL of a 1.0 M NaOH aqueous solution was mixed with 2.5 mL of a 10.0 mM CoCl2·6H2O aqueous solution, and then ultrasonically mixed evenly. Then, 125 μL of a 0.9 M NaClO aqueous solution was added to the mixed solution, and ultrasonic reaction was carried out for at least 15 min. Finally, the reaction solution was centrifuged, washed with water, and vacuum dried to obtain CoOOH NSs.

[0029] (2) Add the cDNA to a Tris-HCl buffer solution with a concentration of 10 mM, and the concentration of cDNA in the Tris-HCl buffer solution is 2 μM. Then, add 1 mg of CoOOH NSs to 1 mL of the Tris-HCl buffer solution to form a solution, and then incubate it on a shaker at room temperature for at least 7 h. After the incubation is completed, the incubation solution is successively subjected to solid-liquid separation, washed with water, and dried to obtain CoOOH-cDNA.

[0030] An electrochemiluminescence aptasensor, when detecting iBF in a solution to be measured, uses a three-electrode system, and the working electrode of the three-electrode system is the above-mentioned CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode.

[0031] Preferably, the reference electrode of the three-electrode system is an Ag / AgCl electrode, and the counter electrode is a platinum electrode.

[0032] The present invention has the following beneficial effects:

[0033] (1) The present invention successfully obtains a CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode by using the resonance energy transfer of the Schottky junction formed between terbium metal-doped covalent organic framework and silver nanowires and the antenna effect between lanthanide metals and covalent organic frameworks. When this working electrode is used as the working electrode of the three-electrode system of the electrochemiluminescence aptasensor, it can perform trace detection of iBF in solutions to be measured such as beer, and the detection range is 1.0×10 -15 -1.0×10 - 7 g / L, and the lowest detection limit can reach 8.97×10 -16 g / L;

[0034] (2) The present invention adopts a signal "on-off-on" mode: first, the signal is reduced by introducing a quenching probe, and then the signal is restored through the reaction of the target substance to detect iBF by electrochemiluminescence response. This effectively avoids the influence of factors such as the environment, background current, and the shedding of electrode surface modification materials, reduces the fluctuations in the single-signal mode, improves the consistency of the detection results, reduces the generation of false positive or false negative signals, and improves the accuracy of the detection;

[0035] (3) The operation of detecting iBF in the present invention is simple, has good selectivity, high sensitivity, and a wide detection range, and has important significance for promoting the application of aptasensors in actual detection. Description of the Drawings

[0036] Figure 1: It is the process flow chart for the preparation of the electrochemiluminescence aptamer sensor and the detection of iBF in Example 1 of the present invention.

[0037] Figure 2 : It is the ECL response diagram of the electrochemiluminescence aptamer sensor obtained in Example 1 after binding with different concentrations of iBF. The concentrations of iBF from a to h are as follows: (a) 1.0×10 -15 g / L; (b) 1.0×10 -14 g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (g) 1.0×10 -9 g / L; (h) 1.0×10 - 8 g / L; (i) 1.0×10 -7 g / L.

[0038] Figure 3 : It is the standard curve of the difference (ΔECL) between the stable luminescence intensities (referring to the luminescence intensity when the ECL signal stabilizes) of the electrochemiluminescence aptamer sensor obtained in Example 1 before and after adding iBF and the logarithm of different iBF concentrations;

[0039] Figure 4 : It is the SEM diagram of the A-COF obtained in Example 1.

[0040] Figure 5 : It is the diagram showing the change of ECL response of different modified working electrodes with time in Example 1. (a) Apt / Tb@A-COF / AgNWs / GCE; (b) iBF / CoOOH-cDNA / Apt / Tb@A-COF / AgNWs / GCE; (c) CoOOH-cDNA / Apt / Tb@A-COF / AgNWs / GCE. Detailed implementation manners

[0041] The present invention will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are only illustrative examples of the implementation manners of the present invention, rather than limiting the scope of the present invention.

[0042] In the following embodiments of the present invention, Apt and cDNA are obtained by centrifuging the Apt sample solution and the cDNA sample solution (vortex for 5 min before opening the lid, and then centrifuge at 4000 rpm for 15 min) and drying. The Apt sample solution and the cDNA sample solution are both purchased from Sangon Biotech (Shanghai) Co., Ltd. The cDNA sequence is 5'-(NH2C6)-ACAAGCAACTCCGTCACCAAT-3';

[0043] The DNA sequence of Apt is 5'-COOH-ACAAAATGGCAGCATTGGTGACGGAGTTGCTTGT-3', and cDNA forms base complementary pairing with Apt.

[0044] In Example 1 of the present invention, the Tb@A-COF dispersion is a dispersion formed by Tb@A-COF in DMF. The preparation method of Tb@A-COF is as follows:

[0045] (1) Add 120.21 mg of 4’,4’,4’,4’-(1,2-ethylenediyl)tetrakis[1,1’-biphenyl]-4-carbaldehyde (ETBC, CAS No. 1624970-54-2) and 50.07 mg of piperazine into an ampoule bottle containing 5 mL of toluene. After ultrasonic treatment until the lumpy piperazine is completely dissolved, place the ampoule bottle in a liquid nitrogen bath for freeze degassing for 20 min. After cooling to room temperature, seal it. Then place the ampoule bottle alone in an oven at 120 °C and let it stand still for 72 h without interference. After the reaction is completed, a yellow solid product is obtained. The obtained yellow solid product is washed with anhydrous dichloromethane and then placed in a vacuum dryer at 60 °C to obtain A-COF;

[0046] (2) Add 2 mg of A-COF to 3 mL of a 5 mmol / L Tb(NO3)3·6H2O solution with DMF as the solvent. After ultrasonic treatment until the solid is completely dissolved, stir at room temperature for 24 h. After the reaction is completed, the obtained solid product is washed successively with DMF and deionized water and then dried to obtain Tb@A-COF.

[0047] In Example 1 of the present invention, the AgNWs dispersion is a solution formed by dispersing AgNWs in deionized water. The preparation method of AgNWs is as follows:

[0048] (1) Add 0.8325 g of PVP to 40 mL of ethylene glycol and heat and stir at 120 °C for 0.5 h, and then naturally cool to room temperature to obtain a PVP coating solution;

[0049] (2) Dissolve 0.2123 g of silver nitrate in 10 mL of ethylene glycol, and while stirring, drop it into the PVP coating solution. After the dropping is completed, add 125 μL of 0.1 M aqueous NaCl solution to the reaction system, stir at room temperature for 30 min, then quickly heat up to 160 °C at a heating rate of 5 - 8 °C / min, and stir the reaction at a constant temperature until the color of the reaction system turns gray-green. After the reaction ends, let the reaction solution cool naturally to room temperature, then separate the solid and liquid to obtain a solid product. The obtained solid product is washed successively with ethanol and deionized water to obtain PVP-coated silver nanowires;

[0050] (3) Add 0.1 g of NaBH4 to a 5 mL mixed solution of water and ethanol formed in a volume ratio of 1:1 to obtain a NaBH4 solution;

[0051] (4) Add 1 g of PVP-coated silver nanowires to the NaBH4 solution and keep it for 60 s to remove the PVP wrapped outside the AgNW. Then, obtain a solid product by filtration, and then wash the obtained solid product successively with ethanol and water 3 times, and dry it to obtain AgNWs.

[0052] The Apt dispersion in Example 1 of the present invention is a dispersion obtained by first forming a mixed solution by dispersing 78.99 μg of Apt into 74.8 μL of 0.01 M Tris-HCl buffer solution, and then diluting the mixed solution to 10 μM with 10 mM Tris-HCl buffer solution. The 10 mM Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid. The concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 1.0 mmol / L.

[0053] The CoOOH-cDNA dispersion in Example 1 of the present invention is a dispersion obtained by first forming a mixed solution by dispersing 77.77 μg of CoOOH-cDNA into 122.9 μL of 10 mM Tris-HCl buffer solution, and then diluting the mixed solution to 10 μM with 10 mM Tris-HCl buffer solution. The 10 mM Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid. The concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 1.0 mmol / L. The preparation method of the CoOOH-cDNA is as follows:

[0054] (1) Mix 625 μL of 1.0 M NaOH aqueous solution with 2.5 mL of 10.0 mM CoCl₂·6H₂O aqueous solution, then ultrasonically mix evenly. Then, add 125 μL of 0.9 M NaClO aqueous solution to the mixed solution and ultrasonically react for 15 min. Finally, after centrifugation, washing with water, and vacuum drying of the reaction solution, CoOOH NSs are obtained;

[0055] (2) Add 77.77 μg of cDNA to 1229 μL of 10 mM Tris-HCl buffer solution to form a cDNA Tris-HCl buffer solution with a concentration of 10 μM, and then dilute it to a concentration of 2 μM with Tris-HCl buffer solution. Then, add 1 mg of CoOOH NSs to 1 mL of Tris-HCl buffer solution to form a solution, and then incubate it on a shaker at room temperature for 7 h. After the incubation is completed, the incubation solution is successively subjected to solid-liquid separation, washed three times with water, and dried to obtain CoOOH-cDNA.

[0056] The preparation method of the iBF standard solution in the present invention is as follows: Dilute with 10 mM Tris-HCl buffer solution to obtain a series of iBF standard solutions with different concentrations. The concentrations of iBF in the Tris-HCl buffer solution are respectively (a) 1.0×10 -15 g / L; (b) 1.0×10 -14 g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (f) 1.0×10 -9 g / L; (h) 1.0×10 -8 g / L; (i) 1.0×10 -7 g / L.

[0057] Example 1

[0058] A CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode, and its preparation method is as follows:

[0059] (1) Take 5 μL of 1 mg / mL Tb@A-COF dispersion liquid and evenly coat it on the surface of a clean glassy carbon electrode and dry it naturally to obtain a Tb@A-COF / GCE electrode;

[0060] (2) Take 5 μL of 0.5 mg / mL AgNWs dispersion liquid and evenly coat it on the surface of the Tb@A-CO / GCE electrode and dry it naturally to obtain an AgNWs / Tb@A-COF / GCE electrode;

[0061] (3) Take 5 μL of the Apt dispersion and evenly coat it on the surface of the AgNWs / Tb@A-COF / GCE electrode and let it dry naturally to obtain the Apt / AgNWs / Tb@A-COF / GCE electrode;

[0062] (4) Take 5 μL of the CoOOH-cDNA dispersion and evenly coat it on the surface of the Apt / AgNWs / Tb@A-COF / GCE electrode and let it dry naturally to obtain the CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode.

[0063] The process flow chart of the preparation of the electrochemiluminescence aptasensor and the detection of iBF in Example 1 of the present invention is as shown in the attached Figure 1 description.

[0064] The ECL response diagram of the electrochemiluminescence aptasensor obtained in Example 1 after binding with different concentrations of iBF is as shown in the attached Figure 2 description, where the concentrations of iBF from a to h are in turn: (a) 1.0×10 -15 g / L; (b) 1.0×10 -14 g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (g) 1.0×10 -9 g / L; (h) 1.0×10 -8 g / L; (i) 1.0×10 -7 g / L.

[0065] The standard curve of the difference in luminescence intensity (ΔECL) between the electrochemiluminescence aptasensor obtained in Example 1 before and after the addition of iBF and the logarithm of different iBF concentrations is as shown in the attached Figure 3 description;

[0066] The SEM diagram of the A-COF obtained in Example 1 is as shown in the attached Figure 4 description.

[0067] The diagram showing the change of the ECL response of different modified working electrodes with time in Example 1 is as shown in the attached Figure 5 description, (a) Apt / Tb@A-COF / AgNWs / GCE; (b) iBF / CoOOH-cDNA / Apt / Tb@A-COF / AgNWs / GCE; (c) CoOOH-cDNA / Apt / Tb@A-COF / AgNWs / GCE.

[0068] Comparative Example 1 was the same as Example 1, except that the working electrode in Comparative Example 1 was prepared according to the following steps:

[0069] (1) Take 5 μL of a Tb@A-COF dispersion with a mass concentration of 1 mg / mL and evenly coat it on the surface of a clean glassy carbon electrode and allow it to dry naturally to obtain a Tb@A-CO / GCE electrode;

[0070] (3) Take 5 μL of an Apt dispersion and evenly coat it on the surface of the Tb@A-COF / GCE electrode and allow it to dry naturally to obtain an Apt / Tb@A-COF / GCE electrode;

[0071] (4) Take 5 μL of a CoOOH-cDNA dispersion and evenly coat it on the surface of the Apt / Tb@A-COF / GCE electrode and allow it to dry naturally to obtain a CoOOH-cDNA / Apt / Tb@A-COF / GCE working electrode.

[0072] Comparative Example 2 was the same as Example 1, except that the preparation method of the working electrode in Comparative Example 2 was as follows:

[0073] (1) Take 5 μL of an A-COF dispersion with a mass concentration of 1 mg / mL and evenly coat it on the surface of a clean glassy carbon electrode and allow it to dry naturally to obtain an A-COF / GCE electrode;

[0074] (2) Take 5 μL of an AgNWs dispersion with a mass concentration of 1 mg / mL and evenly coat it on the surface of the A-CO / GCE electrode and allow it to dry naturally to obtain an AgNWs / A-COF / GCE electrode;

[0075] (3) Take 5 μL of an Apt dispersion and evenly coat it on the surface of the AgNWs / A-COF / GCE electrode and allow it to dry naturally to obtain an Apt / AgNWs / A-COF / GCE electrode;

[0076] (4) Take 5 μL of a CoOOH-cDNA dispersion and evenly coat it on the surface of the Apt / AgNWs / A-COF / GCE electrode and allow it to dry naturally to obtain a CoOOH-cDNA / Apt / AgNWs / A-COF / GCE working electrode. The A-COF dispersion is a dispersion formed by A-COF in DMF.

[0077] Specific Application

[0078] Application 1

[0079] Using the CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode obtained in Example 1 as the working electrode of the electrochemiluminescence aptasensor, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode, the three electrodes form a three-electrode system. Using a PBS buffer solution with a pH of 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scanning is performed.

[0080] Drawing of the standard curve:

[0081] Modify 5 μL of iBF solutions with different concentrations on the surface of the working electrode obtained in Example 1 (the concentrations of the iBF solutions are respectively (a) 1.0×10 -15 g / L; (b) 1.0×10 -14 g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (f) 1.0×10 -9 g / L; (h) 1.0×10 -8 g / L; (i) 1.0×10 -7 g / L). After the coating is naturally dried, let it stand and react for 100 min to allow the cDNA on the surface of the working electrode to bind to iBF. Then, with the Ag / AgCl reference electrode and the platinum counter electrode, a three-electrode system is formed. Using a PBS buffer solution with a pH of 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans are respectively performed, and the luminescence intensity-time curves are respectively recorded. Using the corresponding relationship between the difference in the stable luminescence intensity (ΔECL) before and after the electrochemiluminescence aptasensor binds to iBF and the logarithm of the iBF concentration in the iBF standard solution, the corresponding linear regression equation is obtained: ΔI ECL = 56479.20 + 3544.45 log c (g / L), the detection range is 1.0×10 -15 ~1.0×10 -7 g / L, the correlation coefficient R 2 = 0.996, and the detection limit is 8.97×10 -16 g / L.

[0082] Take 100 μL of a certain brand of beer beverage and place it in 10 mL of a PBS buffer solution with a concentration of 100 mM to obtain a dilution. Divide the dilution into several equal portions. Take 5 μL of the dilution and modify it on the surface of the working electrode obtained in Example 1. After the coating dries naturally, let it stand and react for 100 min to allow the cDNA on the surface of the working electrode to bind to iBF. Then, using Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode, form a three-electrode system. Using a PBS buffer solution with a pH of 7.4 and containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, perform cyclic voltammetry scanning and record the luminescence intensity-time curve. Substitute the maximum luminescence intensity corresponding to the working electrode into the linear regression equation; ΔI ECL = 56479.20 + 3544.45 log c (g / L), and the content of iBF in 5 μL of the dilution can be calculated. Subsequently, the content of iBF in this brand of beer beverage can be calculated.

[0083] Application 2

[0084] Use the CoOOH-cDNA / Apt / Tb@A-COF / GCE working electrode obtained in Comparative Example 1 as the working electrode of the electrochemiluminescence aptasensor. Use Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode. The three of them form a three-electrode system. Using a PBS buffer solution with a pH of 7.4 and containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, perform cyclic voltammetry scanning.

[0085] Drawing of the standard curve:

[0086] Modify 5 μL of iBF solutions with different concentrations on the surface of the working electrode obtained in Comparative Example 1 (the concentrations of the iBF solutions are respectively (a) 1.0×10 -15 g / L; (b) 1.0×10 -14 g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (f) 1.0×10 -9 g / L; (h) 1.0×10 -8 g / L; (i) 1.0×10 -7g / L). After the coating was naturally dried, it was left to react for 100 min to allow the cDNA on the working electrode surface to bind to iBF. Then, an Ag / AgCl reference electrode and a platinum counter electrode were used to form a three-electrode system. Using a PBS buffer solution with a pH of 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans were performed respectively, and the luminescence intensity-time curves were recorded respectively. By using the corresponding relationship between the difference in stable luminescence intensity (ΔECL) before and after the electrochemical luminescence aptasensor binds to iBF and the logarithm of the iBF concentration in the iBF standard solution, the test results showed that ΔECL before and after the electrochemical luminescence aptasensor binds to iBF did not change, and no change in the iBF concentration was detected (when only Tb@A-COF was present, the ECL intensity would gradually decrease, a strong and stable ECL signal could not be obtained, the ECL quenching signal of CoOOH could not be obtained, and the ECL signal could not be restored after the addition of iBF, so there was no linear relationship).

[0087] Take 100 μL of a certain brand of beer beverage and place it in 10 mL of a 100 mM PBS buffer solution to obtain a dilution, and divide the dilution into several equal parts. Take 5 μL of the dilution and modify it on the surface of the working electrode obtained in Comparative Example 1. After the coating was naturally dried, it was left to react for 100 min to allow the cDNA on the working electrode surface to bind to iBF. Then, using Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode, a three-electrode system was formed. Using a PBS buffer solution with a pH of 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans were performed, and the luminescence intensity-time curve was recorded.

[0088] Application 3

[0089] Using the CoOOH-cDNA / Apt / AgNWs / A-COF / GCE working electrode obtained in Comparative Example 2 as the working electrode of the electrochemical luminescence aptasensor, with Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode, the three formed a three-electrode system. Using a PBS buffer solution with a pH of 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans were performed.

[0090] Drawing of the standard curve:

[0091] Modify 5 μL of iBF solutions with different concentrations on the surface of the working electrode obtained in Comparative Example 1 (the concentrations of the iBF solutions were respectively (a) 1.0×10 -15 g / L; (b) 1.0×10 -14g / L; (c) 1.0×10 -13 g / L; (d) 1.0×10 -12 g / L; (e) 1.0×10 -11 g / L; (f) 1.0×10 -10 g / L; (f) 1.0×10 -9 g / L; (h) 1.0×10 -8 g / L; (i) 1.0×10 -7 g / L). After the coating is naturally dried, let it stand and react for 100 min to make the cDNA on the working electrode surface bind to iBF. Then, with an Ag / AgCl reference electrode and a platinum counter electrode, a three-electrode system is formed. Using a PBS buffer solution with pH 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans are carried out respectively, and the luminescence intensity-time curves are recorded respectively. Using the corresponding relationship between the difference in stable luminescence intensity (ΔECL) before and after the electrochemical luminescence aptasensor binds to iBF and the logarithm of the iBF concentration in the iBF standard solution, the corresponding standard curve is plotted. The test results show that ΔECL before and after the electrochemical luminescence aptasensor binds to iBF does not change, the change in iBF concentration is not detected, and the corresponding linear regression equation is not obtained (when only A-COF is present, the ECL intensity is very low, lower than the ECL signal intensity quenched by CoOOH, and a strong and stable ECL signal cannot be obtained. After adding iBF, the ECL signal cannot be restored either, so there is no linear relationship).

[0092] Take 100 μL of a certain brand of beer beverage and place it in 10 mL of a PBS buffer solution with a concentration of 100 mM to obtain a dilution solution, and divide the dilution solution into several equal parts. Take 5 μL of the dilution solution and modify it on the surface of the working electrode obtained in Comparative Example 1. After the coating is naturally dried, let it stand and react for 100 min to make the cDNA on the working electrode surface bind to iBF. Then, using Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode, a three-electrode system is formed. Using a PBS buffer solution with pH 7.4 containing 14 mM TPrA as the electrolyte, within the electrochemical window range of 0 V - 1.4 V, with a photomultiplier tube high voltage of 800 V and a scan rate of 0.3 V / s, cyclic voltammetry scans are carried out, and the luminescence intensity-time curve is recorded.

[0093] Detect iBF in the beer dilution solution according to the methods of Applications 1 - 3. The only difference among the three is the type of the working electrode, and the test conditions are all the same. The measured results are shown in Table 1.

[0094] Table 1

[0095]

[0096] As can be seen from the test data in Table 1, the detection limit of the working electrode obtained in Example 1 is lower, and trace iBF in the beer dilution can be accurately tested. The working electrodes obtained in Comparative Examples 1-2 respectively failed to detect iBF with a concentration of 1 ng·L -1 in the beer dilution. It can be seen that the Schottky junction resonance energy transfer formed between the terbium metal-doped covalent organic framework and silver nanowires on the electrode surface and the antenna effect between the lanthanide metal and the covalent organic framework are of great importance to the sensitivity of the working electrode for detecting iBF and cannot be ignored.

[0097] Inspired by the ideal embodiments of the present invention described above, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode, characterized in that: The preparation method comprises the following steps: (1) A Tb@A-COF coating is obtained on the surface of a clean glassy carbon electrode to obtain a Tb@A-COF / GCE electrode; (2) The surface of Tb@A-COF / GCE electrode was modified to obtain AgNWs coating to obtain AgNWs / Tb@A-COF / GCE electrode; (3) Apt coating was obtained on the surface of AgNWs / Tb@A-COF / GCE electrode to obtain Apt / AgNWs / Tb@A-COF / GCE electrode; (4) The surface of the Apt / AgNWs / Tb@A-COF / GCE electrode was modified to obtain a CoOOH-cDNA coating to obtain a CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode.

2. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 1, characterized in that: The Tb@A-COF coating is a coating formed by coating a dispersion of Tb@A-COF in DMF on an electrode surface and drying the dispersion. The Tb@A-COF is formed by doping the metal element Tb in A-COF. The A-COF is a product formed by a condensation reaction between the ligand ETBC and piperazine to form an aldehyde group (-CHO) and a secondary amine group (R2NH).

3. A CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 2, characterized in that: The preparation method of Tb@A-COF comprises the following steps: (1) 120.21 mg ETBC and 50.07 mg piperazine were added to an ampoule containing 5 mL toluene, and after ultrasonication until the blocky piperazine was completely dissolved, the ampoule was placed in a liquid nitrogen bath for freeze degassing for at least 20 min, cooled to room temperature and sealed, and then the ampoule was placed in an oven at 120° C. and allowed to stand for 72 h without interference. After the reaction was completed, a yellow solid product was obtained, and the obtained yellow solid product was washed with anhydrous dichloromethane and then dried in vacuum at 60° C. to obtain A-COF; (2) 2 mg of A-COF was added to 3 mL of a 5 mmol / L Tb(NO3)3·6H2O solution with DMF as the solvent, and ultrasonicated until the solid was completely dissolved. The mixture was stirred at room temperature for at least 24 h. After the reaction was completed, the solid product was washed with DMF and deionized water in turn and then dried to obtain Tb@A-COF.

4. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 1, characterized in that: The AgNWs coating is a coating formed by coating a solution of AgNWs in an aqueous solution on an electrode surface and drying the solution. The preparation method of the AgNWs comprises the following steps: (1) Preparation of PVP-coated silver nanowires; (2) The PVP coating layer in the PVP-coated silver nanowires was removed to obtain AgNWs.

5. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 4, characterized in that: Step (1) comprises the following steps: (1) 0.8325 g of PVP was added to 40 mL of ethylene glycol and heated and stirred at 120 °C for 0.5 h, and then naturally cooled to room temperature to obtain a PVP coating solution; (2) 0.2123 g of silver nitrate was dissolved in 10 mL of ethylene glycol, and the mixture was added dropwise to the PVP coating solution while stirring. After the addition was completed, 125 μL of 0.1 M NaCl aqueous solution was added to the reaction system, and the mixture was stirred at room temperature for 30 min. The mixture was then rapidly heated to 160° C. at a heating rate of 5-8° C. / min. The reaction was stirred at a constant temperature until the color of the reaction system changed to gray-green. The reaction was completed. After the reaction solution was naturally cooled to room temperature, a solid product was obtained after solid-liquid separation. The solid product was washed with ethanol and deionized water in turn to obtain PVP-coated silver nanowires.

6. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 4, characterized in that: Step (2) comprises the following steps: 0.1 g of NaBH4 was added to a mixed solution of 5 mL of water and ethanol in a volume ratio of 1:1 to obtain a NaBH4 solution; about 1 g of PVP-coated silver nanowires was added to the NaBH4 solution and maintained for at least 60 seconds to remove the PVP wrapped around the outside of the AgNW, and then a solid product was obtained by filtration. The solid product was then washed with ethanol and water at least 3 times in sequence, and AgNWs were obtained after drying.

7. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 1, characterized in that: The Apt coating is formed by dispersing Apt into a 10 mM Tris-HCl buffer solution to form a mixed solution, which is coated on the electrode surface and dried; The 10 mM Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and EDTA, wherein the concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of EDTA is 1.0 mmol / L.

8. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 1, characterized in that: The CoOOH-cDNA coating is formed by dispersing CoOOH-cDNA into a 0.01M Tris-HCl buffer solution to form a mixed solution, coating the electrode surface and drying the solution; The 10 mM Tris-HCl buffer solution contains KCl, NaCl, MgCl2, and EDTA, wherein the concentration of KCl is 0.2 mol / L, the concentration of NaCl is 0.1 mol / L, the concentration of MgCl2 is 5.0 mmol / L, and the concentration of EDTA is 1.0 mmol / L.

9. The CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to claim 1, characterized in that: The preparation method of the CoOOH-cDNA comprises the following steps: (1) 625 μL of 1.0 M NaOH aqueous solution and 2.5 mL of 10.0 mM CoCl2·6H2O aqueous solution were mixed and then ultrasonically mixed. Then, 125 μL of 0.9 M NaClO aqueous solution was added to the mixed solution and ultrasonically reacted for at least 15 min. Finally, the reaction solution was centrifuged, washed with water, and vacuum dried to obtain CoOOH NSs. (2) cDNA was added to a 10 mM Tris-HCl buffer solution, the concentration of cDNA in the Tris-HCl buffer solution was 2 μM, and then 1 mg of CoOOH NSs was added to 1 mL of Tris-HCl buffer solution to form a solution, which was then incubated on a shaker at room temperature for at least 7 h. After the incubation, the incubation solution was sequentially subjected to solid-liquid separation, water washing, and drying to obtain CoOOH-cDNA.

10. An electrochemiluminescent aptamer sensor, characterized in that: When detecting iBF in the solution to be tested, a three-electrode system is used, and the working electrode of the three-electrode system is the CoOOH-cDNA / Apt / AgNWs / Tb@A-COF / GCE working electrode according to any one of claims 1 to 9.

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