Electrochemiluminescent compound, preparation method and application thereof

Abstract

The present invention discloses an electrochemiluminescent compound, a preparation method thereof and an application thereof, wherein the structural formula of the electrochemiluminescent compound is: wherein n is 100-200, R1, R2, R3 and R4 are all selected from one of a Y group and a hydrogen group, and at least one of R1, R2, R3 and R4 is a Y group. The preparation method of the electrochemiluminescent compound comprises the following steps: dissolving an iridium-containing complex, N, N-diisopropylethylamine, N, N, N', N'-tetramethyl-O-(N-succinimide) urea tetrafluoroborate in N, N-dimethylformamide and mixing; adding a polyethyleneimine solution, performing an amide reaction, separating, and obtaining the electrochemiluminescent compound. The electrochemiluminescent compound (Ir-PEI) of the present invention can improve the water solubility of the iridium-containing complex, can also be used as a coreactant unit in the molecule to promote the generation of ECL signals, and can be used as an ECL active material to construct and prepare an iridium ECL reagent.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to an electrochemiluminescent compound, a preparation method thereof and an application thereof. Background Art

[0002] Tetracycline hydrochloride is a broad-spectrum antibiotic with significant therapeutic effects against many deadly bacterial infections. Its low price makes it widely used. However, due to its overuse, tetracycline residues are increasing in various foods. Tetracycline residues have been found in foods such as honey and milk. Long-term consumption of foods with excessive tetracycline residues can cause serious harm to human health, such as liver damage, tetracycline resistance, and changes in bone development. Tetracycline is also highly soluble in water, causing significant water pollution and ultimately accumulating through the food chain, causing even greater harm to the human body. Developing sensitive and rapid tetracycline detection methods is crucial for food and environmental safety. Traditional tetracycline detection methods include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS / MS), but these methods require large and expensive instrumentation.

[0003] Therefore, it is necessary to construct an economical, time-saving and highly specific method for detecting tetracycline residues.

[0004] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0005] The present invention aims to provide an electrochemiluminescent compound, which can be used to prepare an iridium ECL reagent and applied to the detection of tetracycline.

[0006] To achieve the above objectives, an embodiment of the present invention provides an electrochemiluminescent compound, the structural formula of which is:

[0007]

[0008] wherein n is 100 to 200, R1, R2, R3, and R4 are all selected from one of a Y group and a hydrogen group, and at least one of R1, R2, R3, and R4 is a Y group, and the Y group has any one of the following structural formulas:

[0009]

[0010]

[0011]

[0012] An embodiment of the present invention further provides a method for preparing the electrochemiluminescent compound as described above, comprising the following steps:

[0013] Dissolve the iridium complex, N,N-diisopropylethylamine, and N,N,N',N'-tetramethyl-O-(N-succinimidyl)urea tetrafluoroborate in N,N-dimethylformamide and mix well;

[0014] A polyethyleneimine solution is added to carry out an amide reaction, and the mixture is separated to obtain the electrochemiluminescent compound.

[0015] In one or more embodiments of the present invention, the iridium-containing complex is a bidentate ligand compound having the structural formula

[0016] Wherein, M is iridium, C^N bidentate ligand is the first ligand, N^N is the second ligand, X - is hexafluorophosphate;

[0017] The first ligand has any one of the following structural formulas:

[0018]

[0019] The second ligand has any one of the following structural formulas:

[0020]

[0021] An embodiment of the present invention provides a use of the electrochemiluminescent compound as described above in preparing an iridium ECL reagent and in detecting tetracycline.

[0022] An embodiment of the present invention provides an iridium ECL reagent, comprising a buffer solution and the electrochemiluminescent compound as described above dissolved in the buffer solution.

[0023] An embodiment of the present invention provides a method for detecting tetracycline, comprising the following steps:

[0024] preparing an iridium ECL reagent, wherein the iridium ECL reagent comprises a buffer solution and the electrochemiluminescent compound as described above dissolved in the buffer solution;

[0025] Establishing an electrochemical aptamer sensor, the electrochemical aptamer sensor comprising a three-electrode system comprising a working electrode, an Ag / AgCl reference electrode, a platinum wire electrode as a counter electrode, and the iridium ECL reagent, wherein the working electrode is a glassy carbon electrode with an aptamer loaded on its surface, the aptamer being modified with ferrocene, and wherein the aptamer is capable of binding to tetracycline and separating from the glassy carbon electrode;

[0026] Establishing a linear regression equation between the luminescence intensity of an electrochemical aptamer sensor and the tetracycline concentration, wherein the electrochemical aptamer sensor includes the iridium ECL reagent and a three-electrode system;

[0027] The working electrode is placed in a test solution containing tetracycline, removed after incubation, and rinsed. The three-electrode system corresponding to the working electrode is placed in the iridium ECL reagent, and the detection luminescence intensity of the electrochemical aptamer sensor is tested to obtain the detection luminescence intensity. The concentration of tetracycline in the test solution is determined based on the linear regression equation and the detection luminescence intensity of the electrochemical aptamer sensor.

[0028] In one or more embodiments of the present invention, the step of establishing a linear regression equation between the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration includes:

[0029] Obtain multiple sets of electrochemical aptasensors;

[0030] placing a set of the three-electrode system in one of the iridium ECL reagents to test and obtain the blank luminescence intensity of the electrochemical aptamer sensor;

[0031] Prepare tetracycline standard solutions of different concentrations, insert the working electrodes of the remaining electrochemical aptamer sensors into the tetracycline standard solutions of different concentrations, remove them after incubation, rinse them, and then place the three-electrode systems corresponding to the multiple working electrodes in the iridium ECL reagent of the corresponding electrochemical aptamer sensor to obtain the standard luminescence intensity of multiple groups of electrochemical aptamer sensors;

[0032] According to the blank luminescence intensity of the electrochemical aptamer sensor and the standard luminescence intensity of multiple groups of electrochemical aptamer sensors, a linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration was obtained.

[0033] In one or more embodiments of the present invention, the steps of preparing the working electrode include:

[0034] Grafting a carboxylic acid molecular layer onto the surface of the glassy carbon electrode;

[0035] The glassy carbon electrode grafted with a carboxylic acid molecular layer is immersed in Tris-HCl containing cDNA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to undergo an amidation reaction, and then taken out and rinsed;

[0036] The electrode was immersed in Tris-HCl containing the aptamer to perform DNA hybridization, taken out, rinsed, and dried to obtain a working electrode.

[0037] In one or more embodiments of the present invention, the base sequence of the cDNA is the base sequence shown in SEQ ID NO.1, and the base sequence of the aptamer is the base sequence shown in SEQ ID NO.2.

[0038] In one or more embodiments of the present invention, the step of grafting a carboxylic acid molecular layer onto the surface of the glassy carbon electrode comprises:

[0039] Dissolve NaNO3 and 4-ABA (4-aminobenzoic acid) in HCl solution and stir for 3 to 60 minutes at -3 to 40°C in the dark to generate diazonium cations;

[0040] Under the condition of scanning potential of +0.6V to -0.6V (vs Ag / AgCl), cyclic voltammetry scanning was performed on the glassy carbon electrode at a scanning rate of 50mV / s to obtain a glassy carbon electrode with an electrochemically grafted carboxylic acid molecular layer.

[0041] Compared to the prior art, the electrochemiluminescent compounds according to embodiments of the present invention can be used as ECL active materials to prepare iridium ECL reagents, thereby constructing electrochemical aptamer sensors. These electrochemical aptamer sensors can be used to detect tetracycline concentrations. The electrochemiluminescent compounds of the present invention not only improve the water solubility of iridium-containing complexes, but also covalently couple PEI with a luminescent substance to form a self-enhanced ECL system. Due to the amine-rich nature of PEI, the abundant iridium (III) metal complex can be immobilized on the PEI molecular chain, thereby enhancing the luminescence intensity of Ir-PEI (the electrochemiluminescent compound of the present invention). Because intramolecular electron transfer pathways are shorter and energy losses are lower, intramolecular ECL reactions are more efficient than intermolecular reactions, significantly improving ECL efficiency. The electrochemiluminescent detection methods employed by the electrochemiluminescent compounds of the present invention offer the advantages of simplicity and sensitivity, avoiding the need to add any co-reactants to the test solution for signal amplification. The electrochemiluminescent compounds of the present invention have great potential as ECL analytical reagents and biomarker probes, and may also promote the further development of iridium-based ECL luminophores in aqueous solution analysis applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 1 is an ECL intensity-potential curve and a CV curve diagram of 20 mg / mL Ir-PEI in 0.1 M PBS solution according to one embodiment of the present invention;

[0043] Figure 2 1 is a schematic diagram of the construction process and principle of an electrochemical aptasensor according to one embodiment of the present invention;

[0044] Figure 3 is a data graph showing the electrochemiluminescence intensity difference and tetracycline concentration of the electrochemical aptamer sensor according to one embodiment of the present invention;

[0045] Figure 4is a data graph showing the electrochemiluminescence intensity difference and tetracycline concentration of the electrochemical aptamer sensor according to one embodiment of the present invention;

[0046] Figure 5 1. This is a graph showing improved anti-interference detection data of an electrochemical aptamer sensor according to one embodiment of the present invention;

[0047] Figure 6 is a luminescence intensity-time curve diagram of the electrochemical aptasensor according to one embodiment of the present invention;

[0048] Figure 7 The iridium-containing complex according to one embodiment of the present invention is in a DMSO-d6 solution. 1 HNMR spectrum;

[0049] Figure 8 is a high-resolution mass spectrum of an iridium-containing complex according to one embodiment of the present invention;

[0050] Figure 9 FT-IR spectra of the iridium-containing complex according to one embodiment of the present invention, PEI, and the electrochemiluminescent compound of the present invention. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0052] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0053] Electrochemiluminescence (ECL) is also known as electrochemiluminescence.

[0054] In existing ECL sensors, the luminescent groups of electrochemiluminescent materials emit very weak or almost no light during the electrochemical reaction without a co-reactant, making it difficult to detect the ECL signal, or the ECL signal is very weak. Therefore, existing techniques generally mix a substance containing a luminescent group with a co-reactant, leveraging the reaction between the co-reactant and the luminescent group to enhance the luminescent group's luminescence efficiency and amplify the ECL signal, facilitating detection and thus improving sensor sensitivity.

[0055] The electrochemiluminescent compound according to a preferred embodiment of the present invention can be used to prepare an iridium ECL reagent and applied to the detection of tetracycline.

[0056] To achieve the above objectives, an embodiment of the present invention provides an electrochemiluminescent compound, the structural formula of which is:

[0057]

[0058] wherein n is 100 to 200, R1, R2, R3, and R4 are all selected from a Y group and a hydrogen group, and at least one of R1, R2, R3, and R4 is a Y group, and the Y group has any of the following structures:

[0059]

[0060]

[0061]

[0062] It should be noted that the electrochemiluminescent compound of the present invention can be a block copolymer or a homopolymer, i.e., R1, R2, R3, and R4 in each polymerized unit can be the same or different. n is the average degree of polymerization. For convenience, Ir-PEI will be used below to represent the electrochemiluminescent compound of the present invention.

[0063] Among them, the structural formula of the Y group Indicates a substitution position.

[0064] Since Ir-PEI contains abundant tertiary amine units, it has good intramolecular self-enhanced ECL performance. To prove the above point, Ir-PEI was tested in 0.1M PBS buffer without adding any additional co-reactants. ECL detection was performed using a three-electrode system, with Ag / AgCl as the reference electrode, platinum wire as the auxiliary electrode, and glassy carbon electrode (GCE, Φ=3mm) as the working electrode. The Ir-PEI concentration in the ECL detection was 20mg / mL. The photomultiplier tube voltage was set to 900V, the scan rate was 50mV / s, and the following results were obtained: Figure 1 As shown ( Figure 1 (ECL intensity-potential curve and CV curve data of Ir-PEI in 0.1M PBS solution) The Ir-PEI in 0.1M PBS buffer begins to oxidize at about 1.2V. When the potential exceeds 1.4V, in the solution without adding a co-reactant, Ir-PEI begins to generate an ECL signal and then increases rapidly, indicating that Ir-PEI does have good intramolecular self-enhanced ECL performance.

[0065] Among them, the self-enhanced ECL mechanism can be summarized as follows by the following formulas 1-4:

[0066] Ir-PEI–2e - →Ir + -PEI ·+ [1]

[0067] Ir + -PEI ·+ →Ir + -PEI · +H + [2]

[0068] Ir + -PEI · →Ir * -PEI [3]

[0069] Ir * -PEI→Ir-PEI+hv [4]

[0070] It is understood that the Ir-PEI of the present invention can be considered to be prepared by an amide reaction between an iridium-containing complex (having a carboxylic acid group in the ligand) and PEI (polyethyleneimine); wherein the molecular formula of PEI (polyethyleneimine) is: The substitution sites can be considered to be the amino groups on the side chains of the polymerized units of polyethyleneimine.

[0071] Therefore, the Ir-PEI of the present invention can improve the water solubility of the iridium-containing complex on the one hand, and on the other hand, PEI can serve as a co-reactant unit in the molecule to promote the generation of ECL signals.

[0072] The embodiments of the present invention further provide a method for preparing an electrochemiluminescent compound, which can be used to prepare the above-mentioned Ir-PEI. The preparation method comprises the following steps:

[0073] S101. Dissolve the iridium complex, N,N-diisopropylethylamine, and N,N,N',N'-tetramethyl-O-(N-succinimidyl)urea tetrafluoroborate in N,N-dimethylformamide and mix well.

[0074] Among them, the iridium-containing complex is a bidentate ligand electrochemiluminescent compound, and its structural formula is

[0075] Wherein, M is iridium, C^N bidentate ligand is the first ligand, N^N is the second ligand, X - is hexafluorophosphate; N,N-dimethylformamide can be considered as a solvent.

[0076] The first ligand has any of the following structural formulas:

[0077]

[0078] The second ligand has any of the following structural formulas:

[0079]

[0080]

[0081] S102, adding polyethyleneimine solution, carrying out amide reaction, and separating to obtain an electrochemiluminescent compound.

[0082] The separation step may specifically include: dialyzing in distilled water to remove unreacted reagents, and freeze-drying to obtain yellow solid Ir-PEI (ie, the electrochemiluminescent compound of the present invention).

[0083] In one embodiment, the polyethyleneimine solution in step S2 can be an aqueous solution of polyethyleneimine. For example, PEI (1% by mass), i.e., the polyethyleneimine solution has a polyethyleneimine mass fraction of 1%. Specifically, the polyethyleneimine solution is D110141-100g produced by Anaiji Company, which can be diluted to a desired concentration (mass fraction).

[0084] An embodiment of the present invention provides a use of the electrochemiluminescent compound as described above in preparing an iridium ECL reagent and in detecting tetracycline.

[0085] An embodiment of the present invention provides an iridium ECL reagent, comprising a buffer solution and the electrochemiluminescent compound as described above dissolved in the buffer solution.

[0086] The buffer solution may be any one of PBS buffer, TNT buffer, TES buffer, Tris-HCl buffer, SSC buffer, and TBS buffer. Specifically, the PBS buffer solution may be the PBS buffer solution sold by Haibiyuntian Biotechnology Co., Ltd., with the product number ST476.

[0087] An embodiment of the present invention provides a method for detecting tetracycline, comprising the following steps:

[0088] S210 , preparing an iridium ECL reagent, which includes a buffer solution and the electrochemiluminescent compound as described above dissolved in the buffer solution.

[0089] The electrochemiluminescent compound in step S210 is Ir-PEI. Therefore, the steps of preparing the iridium ECL reagent in step S210 may include steps S101 and S102, or may not include steps S101 and S102, i.e., Ir-PEI is prepared by another preparation method. The Ir-PEI is then mixed with a buffer solution (equivalent to a solvent) to obtain an iridium ECL reagent of a certain concentration.

[0090] To ensure test accuracy, the Ir-PEI concentration and buffer in the electrochemical aptamer sensor used in all steps of the tetracycline detection method of the present invention are the same. For example, the Ir-PEI concentration in the iridium ECL reagent can be 20 mg / mL, and the buffer used is PBS buffer (product number ST476) sold by Haibi Yuntian Biotechnology Co., Ltd., with a fixed Ir-PEI structural formula. Of course, the Ir-PEI concentration and buffer in the iridium ECL reagent can be adjusted as needed.

[0091] S220. Establish an electrochemical aptamer sensor, which includes a three-electrode system consisting of a working electrode, Ag / AgCl as a reference electrode, a platinum wire electrode as a counter electrode, and an iridium ECL reagent (i.e., the iridium ECL reagent in step S210), wherein the working electrode is a glassy carbon electrode with an aptamer loaded on its surface, and the aptamer is modified with ferrocene (Fc), wherein the aptamer can bind to tetracycline and separate from the glassy carbon electrode.

[0092] It should be noted that ferrocene (Fc) is equivalent to a quencher; that is, when the three-electrode system is inserted into the iridium ECL reagent and an electrochemiluminescence reaction occurs, Fc can quench ECL more effectively than traditional quenchers, so that the electron transfer between Fc and Ir-PEI quenches the ECL signal of Ir-PEI, thereby reducing the luminescence intensity of the electrochemical aptamer sensor.

[0093] Aptamers are substances that specifically bind to tetracycline; that is, they contain base sequences that specifically bind to tetracycline. Taking advantage of the fact that the binding strength of the aptamer to tetracycline is greater than that of the aptamer to the surface of the glassy carbon electrode, once the working electrode is immersed in a solution containing tetracycline, the aptamer specifically binds to the tetracycline and is subsequently released from the surface of the glassy carbon electrode. This is because ferrocene (Fc) is released from the glassy carbon electrode. Because the amount of ferrocene (Fc)-modified aptamer released is correlated with the tetracycline concentration, a relationship between the ECL response and tetracycline concentration can be established, enabling rapid, sensitive, and selective detection of tetracycline.

[0094] To ensure test accuracy, the three-electrode systems in the electrochemical aptamer sensors used in all steps of the tetracycline detection method of the present invention are purchased from the same batch and have consistent performance, or are custom-made and have consistent performance. Specifically, the working electrodes in the three-electrode system must have consistent performance, and the aptamers on the working electrodes must be identical in content, distribution density, and location.

[0095] Among them, Fc can quench ECL signals more effectively than traditional quenchers.

[0096] The working electrode in step S220 can be purchased directly or prepared by oneself according to needs.

[0097] In one embodiment, the steps for preparing the working electrode of the present invention include:

[0098] S221. Graft a carboxylic acid molecular layer onto the surface of the glassy carbon electrode.

[0099] wherein NaNO3 and 4-ABA (4-aminobenzoic acid) are dissolved in HCl solution and stirred at -3 to 40°C in the dark for 3 to 60 minutes to generate diazonium cations;

[0100] Under the condition of scanning potential of +0.6V to -0.6V (vs Ag / AgCl), cyclic voltammetry scanning was performed on the glassy carbon electrode at a scanning rate of 50mV / s to obtain a glassy carbon electrode with an electrochemically grafted carboxylic acid molecular layer.

[0101] Specifically, the scanning cycle can be 3 cycles. For example, NaNO3 and 4-ABA (4-aminobenzoic acid) can be dissolved in HCl solution and stirred for 10 minutes at -3°C in the dark to generate diazonium cations.

[0102] During the above steps, the glassy carbon electrode may be polished and cleaned first to facilitate the subsequent grafting of carboxylic acid.

[0103] Specifically, the steps for polishing and cleaning the glassy carbon electrode can be: first, polish the glassy carbon electrode GCE with Φ=3.0 mm using 0.3 and 0.05 μm Al2O3 polishing powders, respectively, and then ultrasonically clean it in ultrapure water, ethanol and ultrapure water in turn, and blow dry it with nitrogen at room temperature.

[0104] S222. The glassy carbon electrode grafted with a carboxylic acid molecule layer is immersed in Tris-HCl containing cDNA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to undergo an amidation reaction, and is taken out and rinsed after the reaction.

[0105] The base sequence of the cDNA is shown in SEQ ID NO. 1, and the base sequence of the aptamer is shown in SEQ ID NO. 2. This arrangement utilizes the principle of complementary binding of base pairs to modify the aptamer on the surface of the glassy carbon electrode.

[0106] In one embodiment, the concentration of the cDNA solution can be 1×10 -6 mol / L; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide can be 4:1.

[0107] S223. Immerse in Tris-HCl containing the aptamer to perform DNA hybridization, take out, rinse, and dry to obtain a working electrode.

[0108] In one embodiment, the concentration of the cDNA solution is 1×10 -6 mol / L; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide was 4:1; the concentration of the aptamer solution was 1×10 -6 mol / L.

[0109] Figure 2 This can be considered as a process of establishing an electrochemical aptamer sensor, in which the first step is to obtain a glassy carbon electrode with an electrochemically grafted carboxylic acid molecular layer using 4ABA, followed by an amidation reaction using EDC NHS to fix the NH2-modified DNA on the electrode surface, and then the Fc-modified aptamer is modified onto the electrode through base complementary pairing; in the absence of tetracycline, the ECL signal is quenched off; in the presence of tetracycline, the ECL signal is restored on.

[0110] S230. Establish a linear regression equation between the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration. The electrochemical aptamer sensor includes an iridium ECL reagent and a three-electrode system.

[0111] Specifically, step S230 may include the following steps:

[0112] S231. Obtain multiple groups of electrochemical aptamer sensors.

[0113] The iridium ECL reagent in the multiple electrochemical aptamer sensors is the same as that in step S210, and the three-electrode system in the multiple electrochemical aptamer sensors is the same as that in step S220. This is to reduce the impact of other factors on subsequent detection.

[0114] S232. Place a three-electrode system in an iridium ECL reagent and test the blank luminescence intensity of the electrochemical aptamer sensor.

[0115] It can be understood that in this step, a three-electrode system is placed in an iridium ECL reagent, which is one of the multiple electrochemical aptamer sensors in step S231. The purpose is to measure the blank luminescence intensity of the electrochemical aptamer sensor, which serves as the detection background of the luminescence intensity.

[0116] The step of testing and obtaining the blank luminescence intensity of the electrochemical aptamer sensor may include:

[0117] In the electrochemical window range of 0-1.5V, the photomultiplier tube high voltage was 900V and the scan rate was 0.05V / s to perform cyclic voltammetry scanning, and the luminescence intensity-time curve was recorded. The highest value in the luminescence intensity-time curve under the test conditions can be considered as the blank luminescence intensity value of the electrochemical aptasensor.

[0118] S233. Prepare tetracycline standard solutions of different concentrations, insert the working electrodes of the remaining electrochemical aptamer sensors into the tetracycline standard solutions of different concentrations, remove them after incubation, rinse them, and then place the three-electrode systems corresponding to the multiple working electrodes into the iridium ECL reagent of the corresponding electrochemical aptamer sensor, and test to obtain the standard luminescence intensity of multiple groups of electrochemical aptamer sensors;

[0119] The remaining electrochemical aptamer sensors are all of the multiple groups of electrochemical aptamer sensors in step S231, excluding the group of electrochemical aptamer sensors in step S232. In this step, the working electrode of each electrochemical aptamer sensor is independently inserted into a tetracycline standard solution of the corresponding concentration and incubated. This step allows the aptamer on the working electrode to specifically bind to tetracycline, thereby causing the Fc-modified aptamer to detach from the surface of the glassy carbon electrode and enter the tetracycline standard solution. The amount of aptamer detached is related to the tetracycline concentration in the tetracycline standard solution: namely, the higher the concentration, the greater the aptamer detachment and the less aptamer on the working electrode.

[0120] The purpose of the flushing process is to flush away the solution attached to the surface of the working electrode to avoid affecting subsequent steps.

[0121] The three-electrode systems corresponding to the multiple working electrodes were then placed in the iridium ECL reagents of the corresponding electrochemical aptamer sensors to measure and obtain the standard luminescence intensities of multiple sets of electrochemical aptamer sensors. Specifically, each working electrode that had been reacted (also referred to as incubated) with a tetracycline standard solution of varying concentrations was placed in the iridium ECL reagent corresponding to the working electrode. Thus, each set of electrochemical aptamer sensors contained a three-electrode system and an iridium ECL reagent. Electrochemical reactions were then performed separately to obtain the standard luminescence intensities of each set of electrochemical aptamer sensors. This yielded data on the standard luminescence intensities of multiple sets of electrochemical aptamer sensors that had reacted with tetracycline standard solutions of varying concentrations.

[0122] The steps of testing and obtaining the standard luminescence intensity of multiple groups of electrochemical aptamer sensors may include:

[0123] Each three-electrode system was immersed in the corresponding iridium ECL reagent and the following electrochemical reaction was carried out. Cyclic voltammetry was performed within the electrochemical window range of 0-1.5 V, with a photomultiplier tube high voltage of 900 V and a scan rate of 0.05 V / s. The luminescence intensity-time curve was recorded. The highest value in the luminescence intensity-time curve under these test conditions can be considered as the standard luminescence intensity value of this group of electrochemical aptamer sensors.

[0124] Then, a new group of electrochemical aptamer sensors is selected to repeat the above steps to obtain multiple groups of standard luminescence intensities of electrochemical aptamer sensors. The standard luminescence intensity of each group of electrochemical aptamer sensors corresponds to the data of the reaction with tetracycline standard solutions of different concentrations.

[0125] In order to ensure the accuracy of the test, the contact time between the working electrode and the solution containing tetracycline in all steps of the method for detecting tetracycline of the present invention (whether the above steps or all subsequent steps) is the same, for example, the incubation time between the working electrode and the solution containing tetracycline is 120 minutes.

[0126] In one embodiment, the steps of preparing tetracycline standard solutions of different concentrations may include:

[0127] Accurately weigh a certain amount of tetracycline, prepare a 0.5 mg / L solution with water, and then dilute it with 0.1 mol / L PBS buffer solution with a pH of 7.4 to obtain a series of tetracycline standard solutions with different concentrations ranging from 5 ng / mL to 5 mg / mL.

[0128] S234. Obtain a linear regression equation between the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration based on the blank luminescence intensity of the electrochemical aptamer sensor and the standard luminescence intensity of multiple groups of electrochemical aptamer sensors.

[0129] Specifically, a linear relationship was established between the difference in luminescence intensity before and after the electrochemical aptamer sensor bound to tetracycline and the concentration of tetracycline in the tetracycline standard solution, and the corresponding linear regression equation was obtained.

[0130] The luminescence intensity value of the electrochemical aptamer sensor before binding to tetracycline can be considered as the blank luminescence intensity value of the electrochemical aptamer sensor in step S232; the luminescence intensity value of the electrochemical aptamer sensor after binding to tetracycline can be considered as the standard luminescence intensity value of each group of electrochemical aptamer sensors in step S233; the luminescence intensity difference is the difference between the standard luminescence intensity value of each group of electrochemical aptamer sensors in S233 and the blank luminescence intensity value of the electrochemical aptamer sensors in step S232. The luminescence intensity difference is used as the ordinate, and the tetracycline concentration value in the tetracycline standard solution corresponding to the standard luminescence intensity value of each group of electrochemical aptamer sensors in S233 is used as the abscissa to obtain the corresponding linear regression equation, which is the linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration.

[0131] It can be understood that the luminescence intensity value of the electrochemical aptamer sensor in the linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration in the above steps is the difference between the standard luminescence intensity value of the electrochemical aptamer sensor and the blank luminescence intensity value of the electrochemical aptamer sensor.

[0132] S240. Place the working electrode in a test solution containing tetracycline, take it out after incubation, rinse it, place the three-electrode system corresponding to the working electrode in an iridium ECL reagent, test and obtain the detection luminescence intensity of the electrochemical aptamer sensor, and determine the concentration of tetracycline in the test solution based on the linear regression equation and the detection luminescence intensity of the electrochemical aptamer sensor.

[0133] The working electrode in step S240 is a working electrode from a new set of electrochemical aptamer sensors, i.e., one that has not reacted with tetracycline. Furthermore, this working electrode is identical to the working electrodes from the other sets of electrochemical aptamer sensors in the aforementioned steps (electrodes that have not undergone any testing or detection steps).

[0134] By substituting the detected luminescence intensity value of the electrochemical aptamer sensor into the linear regression equation, the corresponding tetracycline concentration value can be obtained, which is the concentration value of tetracycline in the test solution.

[0135] In one embodiment, the step of testing and obtaining the luminescence intensity of multiple groups of electrochemical aptamer sensors may include:

[0136] Each three-electrode system was immersed in the corresponding iridium ECL reagent and subjected to the following electrochemical reaction. Cyclic voltammetry was performed within the electrochemical window of 0-1.5 V, using a photomultiplier tube at 900 V and a scan rate of 0.05 V / s. The luminescence intensity-time curve was recorded. The highest value in the luminescence intensity-time curve under these test conditions was considered the detected luminescence intensity value of the electrochemical aptasensor. The following, in conjunction with specific examples, details the electrochemiluminescent compounds of the invention, their preparation methods, and applications, as well as the method for detecting tetracycline of the invention.

[0137] Example 1

[0138] (1) Preparation of iridium ECL reagent:

[0139] 10 μmol of a metal iridium (III) complex with 2-(2,4-difluorophenyl)pyridine as the primary ligand and 4-(2,2'-bipyridin-4-yl)butyric acid electrochemiluminescent compound as the secondary ligand, 4 mg of N,N-diisopropylethylamine, and 4 mg of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate were dissolved in 1 mL of N,N-dimethylformamide and stirred for 4 hours. 40 μL of polyethyleneimine (D110141-100g, manufactured by Anaiji, diluted to 1% (mass fraction)) solution was added and mixed. The mixture was then dialyzed in distilled water for 24 hours to remove unreacted reagents. After dialysis, yellow solid Ir-PEI was obtained by freeze-drying. Ir-PEI was dissolved in PBS buffer (product number ST476, sold by Haibi Yuntian Biotechnology Co., Ltd.) to obtain an iridium ECL reagent with an Ir-PEI concentration of 20 mg / mL.

[0140] (2) Polishing and cleaning of glassy carbon electrode GCE:

[0141] First, the glassy carbon electrode (GCE, Φ = 3.0 mm) was polished with 0.3 and 0.05 μm Al2O3 polishing powder, respectively, and then ultrasonically cleaned in ultrapure water, ethanol and ultrapure water in sequence, and dried with nitrogen at room temperature.

[0142] (3) Construction steps of electrochemical aptasensor:

[0143] 12 mg of NaNO₃ and 27 mg of 4-ABA (4-aminobenzoic acid) were dissolved in 20 mL of HCl and stirred at -3°C in the dark for 10 minutes to generate diazonium cations. Cyclic voltammetry was performed on a glassy carbon electrode (GCE) at a scan rate of 50 mV / s over a potential range of +0.6 V to -0.6 V (vs Ag / AgCl) for three cycles to obtain a GCE electrochemically grafted with a carboxylic acid molecular layer. The modified GCE was washed with distilled water and ethanol and then immersed in 80 μL of 0.01 M Tris-HCl containing 1 μM cDNA, 40 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 10 mM N-hydroxysuccinimide for 3 hours for amidation. The cDNA sequence shown in SEQ ID NO. 1 was modified at the 5' end with NH2-(CH2)6-. The modified sequence is: 5'-NH2-(CH2)6-GTTGG TCCCACCACG TGGAG CTCGG ATCCA CGCGCAG-3'. The base sequence shown in SEQ ID NO. 1 is 5'-GTTGG TCCCACCACG TGGAG CTCGG ATCCACGCGCAG-3'. After the reaction, the GCE was gently rinsed with 0.01 M PBS buffer. The electrode was then immersed in 80 μL of 0.01 M Tris-HCl containing 1 μM aptamer for 3 hours for DNA hybridization. The aptamer is a modified version of the base sequence shown in SEQ ID NO. 2: 5'-Fc-CGTAC GGAAT TCGCT AGCCC CCCGG CAGGC CACGG CTTGG GTTGG TCCCACTGCG CGTGGATCCG AGCTC CACGT G-3', where Fc is ferrocene. The base sequence shown in SEQ ID NO. 2 is 5'-CGTACGGAAT TCGCT AGCCC CCCGG CAGGC CACGG CTTGG GTTGG TCCCA CTGCG CGTGG ATCCG AGCTCCACGT G-3'. After the reaction, the glassy carbon electrode was gently rinsed with 0.01 M PBS buffer and then air-dried to obtain a working electrode.

[0144] An electrochemical aptasensor was constructed using a three-electrode system consisting of a working electrode, Ag / AgCl as a reference electrode, a platinum wire electrode as a counter electrode, and an iridium ECL reagent.

[0145] (4) Linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration:

[0146] The three-electrode system of an electrochemical aptamer sensor was placed in the corresponding iridium ECL reagent. Cyclic voltammetry was performed in the electrochemical window range of 0-1.5 V with a photomultiplier tube high voltage of 900 V and a scan rate of 0.05 V / s. The luminescence intensity-time curve of the electrochemical aptamer sensor under these conditions was recorded. Figure 1 The ECL intensity-potential curve and CV curve of 20 mg / mL Ir-PEI in 0.1 M PBS solution are shown, and the blank luminescence intensity value of the electrochemical aptamer sensor is obtained.

[0147] A certain amount of tetracycline was accurately weighed and prepared into a 0.5 mg / L solution with water. This solution was then diluted with 0.1 mol / L PBS buffer (pH 7.4) to obtain a series of tetracycline standard solutions with different concentrations: 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 100 ng / mL, 500 ng / mL, and 5000 ng / mL. The working electrodes of multiple sets of electrochemical aptasensors were then immersed in the tetracycline standard solutions of varying concentrations for 120 minutes before being removed and rinsed. The three-electrode systems corresponding to the multiple working electrodes were placed in the corresponding iridium ECL reagent, and cyclic voltammetry was performed in the electrochemical window range of 0-1.5V with a photomultiplier tube high voltage of 900V and a scan rate of 0.05V / s to obtain the luminescence intensity-time curves of the multiple groups of electrochemical aptamer sensors under this condition, and then the standard luminescence intensity values ​​of the multiple groups of electrochemical aptamer sensors were obtained. According to the standard luminescence intensity values ​​of the multiple groups of electrochemical aptamer sensors and the blank luminescence intensity value of the electrochemical aptamer sensors, the following were obtained: Figure 3 The data graph shown is Figure 3 The vertical axis in the figure is the electrochemiluminescence intensity difference (i.e., the difference between the standard luminescence intensity value of the electrochemical aptamer sensor and the blank luminescence intensity value of the electrochemical aptamer sensor). It can be seen that the data graph of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration is a broken line. The linear regression equation obtained by the above number has a large error in the experiment. When the concentration of tetracycline is 5ng / mL to 100ng / mL, the following equation is obtained: Figure 4 From the data graph shown, it can be seen that the linear relationship between the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration is good. The linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration obtained based on this data is: △I ECL= 3.35099C + 0.5849 (ng / mL), where C is the tetracycline concentration. The correlation coefficient (R) is 0.999, indicating a good linear relationship with a small error. The above linear regression equation represents the linear regression equation between the luminescence intensity of the electrochemical aptasensor and the tetracycline concentration (the detection range of tetracycline is 5 ng / mL to 100 ng / mL).

[0148] Therefore, in order to improve the accuracy of detection, the detection range of the method for detecting tetracycline of the present invention is preferably 5 ng / mL to 100 ng / mL. According to the above linear regression equation, the minimum detection limit of the method for detecting tetracycline of the present invention is 2.73 ng / mL.

[0149] (5) Sample testing

[0150] Honey and lake water samples were taken separately. 1 g of honey was weighed and mixed with ethanol solution, and the honey was dissolved using an ultrasonic instrument. The sample solution was diluted with 0.01 M PBS buffer at a volume ratio of 1:20 and centrifuged at 8000 rpm for 15 min. The supernatant was collected for further use. A series of tetracycline concentrations (0, 20, 50, 100 ng / mL) were added to the obtained solution. 100 mL of lake water was taken from Shihu (Jiangsu, China). The lake water was first centrifuged at a speed of 10000 rpm, and then the supernatant was filtered with a microporous filter membrane (0.45 mm, China Biotechnology Co., Ltd.) to remove impurity particles. The filtered sample was diluted 10 times with 0.01 M PBS buffer. Finally, a series of tetracycline concentrations (0, 20, 50, 100 ng / mL) were added to the obtained solution. The test was carried out under the same electrochemiluminescence test conditions as step (4), and the luminescence intensity of the electrochemical aptamer sensor was recorded. The obtained linear regression equation was used to calculate the concentration of tetracycline in the sample to be tested, and the results are listed in Table 1.

[0151] Example 2

[0152] (1) Preparation of iridium ECL reagent:

[0153] 5 mg of a metal iridium (III) complex with 2,2'-bipyridine-4-butyric acid as the second ligand and 2-(2,4-difluorophenyl)pyridine as the first ligand, 6 mg of N,N-diisopropylethylamine and 6 mg of N,N,N',N'-tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate were dissolved in 2 mL of N,N-dimethylformamide and stirred for 3 hours. 25 μL of polyethyleneimine (D110141-100 g produced by Anaiji, diluted to 1% (mass fraction)) solution was added and mixed. The mixture was then dialyzed in distilled water for 24 hours to remove the unreacted reagent. After dialysis, yellow solid Ir-PEI was obtained by freeze-drying. The Ir-PEI was dissolved in PBS buffer (PBS buffer sold by Haibi Yuntian Biotechnology Co., Ltd. with product number ST476) to obtain an iridium ECL reagent with an Ir-PEI concentration of 20 mg / mL.

[0154] Steps (2), (3), (4), and (5) are the same as steps (2), (3), (4), and (5) of Example 1.

[0155] Example 3

[0156] (1) Preparation of iridium ECL reagent:

[0157] 20 mg of a metal iridium (III) complex with 2,2'-bipyridine-4-butyric acid as the second ligand and 2-(2,4-difluorophenyl)pyridine as the first ligand, 10 mg of N,N-diisopropylethylamine and 10 mg of N,N,N',N'-tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate were dissolved in 5 mL of N,N-dimethylformamide and stirred for 3 hours. 40 μL of polyethyleneimine (D110141-100 g produced by Anaiji, diluted to 1% (mass fraction)) solution was added and mixed. The mixture was then dialyzed in distilled water for 24 hours to remove the unreacted reagent. After dialysis, yellow solid Ir-PEI was obtained by freeze drying. The Ir-PEI was dissolved in PBS buffer (PBS buffer sold by Haibi Yuntian Biotechnology Co., Ltd. with product number ST476) to obtain an iridium ECL reagent with an Ir-PEI concentration of 20 mg / mL.

[0158] Steps (2), (3), (4), and (5) are the same as steps (2), (3), (4), and (5) of Example 1.

[0159] Table 1: Determination results of tetracycline in actual samples

[0160]

[0161] Note: The recovery amount a is the average value of three measurements in Examples 1, 2 and 3.

[0162] As shown in Table 1, the sample was tested three times in parallel, the relative standard deviation was less than 5%, and the spiked recovery ranged from 98.6% to 103.5%. The present invention is feasible for detecting tetracycline residues in actual samples.

[0163] In order to prove the existence of the electrochemiluminescent compound (Ir-PEI) of the present invention, firstly, according to Figure 7 The iridium-containing complex of Example 1 shown in DMSO-d6 solution 1 H NMR spectrum, and Figure 8 The high-resolution mass spectrum of the iridium-containing complex of Example 1 can prove that the structural formula of the iridium-containing complex in Example 1 is

[0164] Then, the iridium complex (herein referred to as Ir-COOH), PEI and yellow solid Ir-PEI in Example 1 were characterized by FT-IR spectroscopy (Fourier transform infrared spectroscopy), and the following results were obtained: Figure 9 The infrared data shown. Among them, the iridium complex (herein referred to as Ir-COOH) corresponds to Figure 9 Curve a and PEI correspond to Figure 9 Curve c in the figure and the yellow solid Ir-PEI correspond to Figure 9 Curve b in Figure 9 As shown by the middle curve a, 2927cm -1 The absorption peak at 1706cm is the stretching vibration peak of -OH in the Ir-COOH complex. -1 and 1647cm -1 The peak at belongs to the C=O stretching vibration of Ir-COOH and Ir-PEI ( Figure 9 Middle curve b), 1289cm -1 The absorption peaks at 3269cm are typical stretching vibrations of the CN functional groups in Ir-PEI. These two absorption peaks well prove the existence of amide bonds in Ir-PEI. -1 and 3287cm -1 The stretching vibration absorption peak of the NH functional group is ( Figure 9 Middle curve c), at 1114cm -1 、1463cm -1 、1638cm -1 The absorption peak at is a typical stretching vibration absorption peak of CN in the PEI structure. The infrared data provide strong evidence that our material has been successfully synthesized, that is, the iridium complex reacts with PEI to form an amide bond, thus forming Ir-PEI.

[0165] In order to further verify the accuracy and selectivity of the method for detecting tetracycline of the present invention, the electrochemical aptamer sensor of the present invention was subjected to anti-interference detection. Six sets of electrochemical aptamer sensors were taken, and six working electrodes thereof were incubated in 5 μg / mL chlortetracycline (CHL), oxytetracycline (OTC), neomycin (NEO), kanamycin (KAN) and doxycycline (DOX) interferors, as well as in 100 ng / mL tetracycline (TET) standard solution for 120 min. Then, the detection was carried out according to the detection method in Example 1. The detection results are shown in FIG. Figure 5 .

[0166] from Figure 5 It can be seen that the electrochemical aptamer sensor in the method for detecting tetracycline of the present invention has a selective recognition effect on tetracycline, and even a 50-fold concentration of interfering substances after mixing has little effect on the detection of tetracycline. Therefore, the electrochemical aptamer sensor can achieve interference-resistant and selective detection of tetracycline. In other words, the method for detecting tetracycline of the present invention can achieve interference-resistant and selective detection of tetracycline.

[0167] At the same time, in the step (4) of Example 1, the linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration was obtained. When the tetracycline concentration was 40 ng / mL, the luminescence intensity-time curve of the electrochemical aptamer sensor was obtained. The working electrode in the electrochemical aptamer sensor was immersed in a tetracycline standard solution with a concentration of 40 ng / mL, and the incubation time was 120 min. Then it was taken out and rinsed. The three-electrode system corresponding to the working electrode was placed in the corresponding iridium ECL reagent. In the electrochemical window range of 0 to 1.5 V, the photomultiplier tube was set at a high voltage of 900 V and a scan rate of 0.05 V / s to perform cyclic voltammetry scanning (16 consecutive cycles) to obtain the luminescence intensity-time curve of the electrochemical aptamer sensor under this condition. From Figure 6 It can be seen that the ECL intensity of Ir-PEI in the electrochemical aptamer sensor did not decrease significantly, indicating that this iridium ECL reagent has good stability in the electrochemical aptamer sensor.

[0168] Compared with the prior art, the electrochemiluminescent compound of the present invention, its preparation method and application have the following beneficial effects:

[0169] (1) The electrochemiluminescent compound (Ir-PEI) prepared by the present invention is used as an ECL active material to construct an iridium ECL reagent, and the ECL signal of the iridium ECL reagent is greatly enhanced compared with the monomeric iridium (III) complex.

[0170] (2) The present invention utilizes the excellent ECL performance of the iridium ECL reagent and the specificity of the aptamer to construct an electrochemical aptamer sensor. The sensor exhibits a good linear range (5 ng / mL to 100 ng / mL) and a low detection limit (2.73 ng / mL).

[0171] (3) The constructed electrochemical aptasensor was used to detect tetracycline in honey and lake water samples, and the results were consistent with the current national standards for monitoring tetracycline residues in actual samples.

[0172] (4) Compared with traditional detection methods, the method for detecting tetracycline of the present invention has the advantages of simple operation, simple equipment, small amount of reagents, and low economic cost.

[0173] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An electrochemiluminescent compound, characterized in that Its structural formula is: ； wherein n is 100 to 200, R1, R2, R3, and R4 are all selected from one of a Y group and a hydrogen group, and at least one of R1, R2, R3, and R4 is a Y group, and the Y group has any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 2. A method for preparing an electrochemiluminescent compound according to claim 1, characterized in that: The following steps are involved: Dissolve the iridium complex, N,N-diisopropylethylamine, and N,N,N',N'-tetramethyl-O-(N-succinimidyl)urea tetrafluoroborate in N,N-dimethylformamide and mix well; A polyethyleneimine solution is added to carry out an amide reaction, and the mixture is separated to obtain the electrochemiluminescent compound.

3. The method for preparing the electrochemiluminescent compound according to claim 2, wherein: The iridium-containing complex is a bidentate ligand compound, and its structural formula is ; Wherein, M is iridium, C^N bidentate ligand is the first ligand, N^N is the second ligand, X - is hexafluorophosphate; The first ligand has any one of the following structural formulas: 、 、 、 、 、 、 ； The second ligand has any one of the following structural formulas: 、 、 、 。 4. Use of the electrochemiluminescent compound according to claim 1 in preparing an iridium ECL reagent and in detecting tetracycline.

5. An iridium ECL reagent, characterized in that The method comprises a buffer solution and the electrochemiluminescent compound according to claim 1 dissolved in the buffer solution.

6. A method for detecting tetracycline, characterized in that, The following steps are involved: preparing an iridium ECL reagent, the iridium ECL reagent comprising a buffer solution and the electrochemiluminescent compound according to claim 1 dissolved in the buffer solution; Establishing an electrochemical aptamer sensor, the electrochemical aptamer sensor comprising a three-electrode system comprising a working electrode, an Ag / AgCl reference electrode, a platinum wire electrode as a counter electrode, and the iridium ECL reagent, wherein the working electrode is a glassy carbon electrode with an aptamer loaded on its surface, the aptamer being modified with ferrocene, and wherein the aptamer is capable of binding to tetracycline and separating from the glassy carbon electrode; Establishing a linear regression equation between the luminescence intensity of an electrochemical aptamer sensor and the tetracycline concentration, wherein the electrochemical aptamer sensor includes the iridium ECL reagent and a three-electrode system; The working electrode is placed in a test solution containing tetracycline, removed after incubation, and rinsed. The three-electrode system corresponding to the working electrode is placed in the iridium ECL reagent, and the detection luminescence intensity of the electrochemical aptamer sensor is tested to obtain the detection luminescence intensity. The concentration of tetracycline in the test solution is determined based on the linear regression equation and the detection luminescence intensity of the electrochemical aptamer sensor.

7. The method for detecting tetracycline according to claim 6, wherein The steps for establishing a linear regression equation between the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration include: Obtain multiple sets of electrochemical aptasensors; placing a set of the three-electrode system in one of the iridium ECL reagents to test and obtain the blank luminescence intensity of the electrochemical aptamer sensor; Prepare tetracycline standard solutions of different concentrations, insert the working electrodes of the remaining electrochemical aptamer sensors into the tetracycline standard solutions of different concentrations, remove them after incubation, rinse them, and then place the three-electrode systems corresponding to the multiple working electrodes in the iridium ECL reagent of the corresponding electrochemical aptamer sensor to obtain the standard luminescence intensity of multiple groups of electrochemical aptamer sensors; According to the blank luminescence intensity of the electrochemical aptamer sensor and the standard luminescence intensity of multiple groups of electrochemical aptamer sensors, a linear regression equation of the luminescence intensity of the electrochemical aptamer sensor and the tetracycline concentration was obtained.

8. The method for detecting tetracycline according to claim 6, wherein The preparation steps of the working electrode include: Grafting a carboxylic acid molecular layer onto the surface of the glassy carbon electrode; The glassy carbon electrode grafted with a carboxylic acid molecular layer was immersed in Tris-HCl containing cDNA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for amidation reaction, and then removed and rinsed; Immerse in Tris-HCl containing aptamers for DNA hybridization, remove, rinse, and dry to obtain a working electrode; The base sequence of the cDNA is the base sequence shown in SEQ ID NO.1, and the base sequence of the aptamer is the base sequence shown in SEQ ID NO.

2.

9. The method for detecting tetracycline according to claim 8, wherein The steps of grafting a carboxylic acid molecular layer onto the surface of the glassy carbon electrode include: Dissolve NaNO3 and 4-aminobenzoic acid in HCl solution and stir for 3-60 min in the dark at -3-40°C to generate diazonium cations; Under the conditions of scanning potential of +0.6V ~ -0.6V and using silver / silver chloride as reference electrode, cyclic voltammetry scanning was carried out on the glassy carbon electrode at a scanning rate of 50mV / s to obtain a glassy carbon electrode with an electrochemically grafted carboxylic acid molecular layer.

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