Method for the preparation of an electrochemiluminescent sensor for the detection of mdpv and use thereof

Abstract

The application discloses an electrochemiluminescence sensor for detecting MDPV and a preparation method and application thereof, and belongs to the field of electrochemiluminescence detection. The electrochemiluminescence aptamer sensor is formed by loading apt on the surface of a RuHPCN-222 / P-COF modified glassy carbon electrode (GCE). The detection method is that a traditional three-electrode system is formed by taking the apt / RuHPCN-222 / P-COF / GCE modified electrode as a working electrode, taking an Ag / AgCl electrode as a reference electrode and taking a platinum electrode as an auxiliary electrode. The preparation method of the modified electrode in the application is simple, the sensitivity of the modified electrode for detecting MDPV is high, the selectivity of the modified electrode is good, and the linear range of the modified electrode is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of detection of new psychoactive substances, and particularly relates to a preparation method and application of an electrochemiluminescence sensor for detecting synthetic cathinones (MDPV), and more particularly to a modified electrode apt / RuHPCN-222 / P-COF / GCE and a preparation method and application thereof. BACKGROUND

[0002] Synthetic cathinones (SC) are an important branch of new psychoactive substances (NPS), which have strong toxicity and harm after chemical modification. Among them, 3,4-methylenedioxypyrovalerone (MDPV) is a new type of NPS. The combination of this drug with alcohol contained in beverages can cause loss of consciousness, vomiting, poor coordination and balance, difficulty breathing, and loss of control. The problem of alcohol beverages mixed with drugs has seriously affected the personal health and safety of the public, so there is an urgent need for a method that can detect psychoactive substances in alcohol beverages. At present, widely used MDPV detection methods include chromatography-mass spectrometry (C-MS) and Raman spectroscopy (LRS). However, limited sensitivity and complex procedures limit their application. Therefore, there is an urgent need for a new, sensitive, and accurate MDPV detection method.

[0003] Electrochemiluminescence (ECL) has become a powerful tool for trace analysis with its low background signal, high sensitivity, and wide detection range. However, traditional ECL systems require high excitation voltage to generate strong ECL signals, and high triggering potential can cause many negative effects, such as: (1) electrochemical interference generated by poor oxidation / reduction reactions; (2) ECL emission induced by oxygen in the electrolyte can produce significant background signals, limiting sensitivity; (3) high potential can damage the stability of biological molecules, such as nucleotide sequences. Therefore, ECL systems with low triggering potential have lower background signals and higher sensitivity. SUMMARY

[0004] In view of the deficiencies of the prior art for MDPV detection, the application provides an electrochemiluminescence sensor for detecting MDPV and a preparation method and application thereof. The application utilizes RuHPCN-222 / TAPB-BTCA-COF nanocomposite to modify the surface of a glassy carbon electrode, obtains a RuHPCN-222 / P-COF / GCE modified electrode, and significantly improves the sensitivity and stability of electrochemiluminescence. The aptamer apt is loaded on the surface of the composite material modified electrode RuHPCN-222 / P-COF / GCE to prepare an electrochemiluminescence aptamer sensor, namely apt / RuHPCN-222 / P-COF / GCE. After adding the detection substance MDPV, the electrochemiluminescence of the MDPV-apt / RuHPCN-222 / P-COF / GCE electrode is quenched, and the detection of MDPV can be realized. After adding other interferents, it is found that the aptamer sensor has good specificity.

[0005] In one aspect, the application provides a modified electrode apt / RuHPCN-222 / P-COF / GCE for detecting MDPV, which is obtained by loading an aptamer apt on the surface of a composite material RuHPCN-222 / TAPB-BTCA-COF modified glassy carbon electrode; wherein the composite material RuHPCN-222 / TAPB-BTCA-COF is a photonic crystal assembled by Ru-anchored Zr-based porphyrin MOF and TAPB-BTCA-COF and formed by electrostatic interaction; and the aptamer apt has a base sequence of 5'-SH-(CH2)6-ACCTTAAGTGGGGTTGGGTGGAGTTTATGGGGT-Fc-3'.

[0006] Further, the preparation method of the composite material RuHPCN-222 / TAPB-BTCA-COF modified glassy carbon electrode comprises the following steps:

[0007] (1) Preparation of TAPB-BTCA-COF: 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-benzene tricarboxaldehyde (BTCA) with a molar ratio of 1:1 are dissolved in acetonitrile and ultrasonically mixed uniformly, then 12 M acetic acid (AcOH) is poured into the above solution, and the volume ratio of AcOH to acetonitrile is 1-1.5:5. Finally, the mixture is stirred vigorously for 10 s and left to stand at room temperature for 72 h; and TAPB-BTCA-COF (denoted as P-COF) is obtained.

[0008] (2) Preparation of RuHPCN-222: ZrCl4, benzoic acid and H2O were dissolved in DMF under magnetic stirring for 5 minutes. Then, tetrakis(4-carboxyphenyl) porphyrin (TCPP) was further added to the above solution, and magnetic stirring was kept at room temperature. The resulting uniform solution was transferred to a polytetrafluoroethylene-lined stainless steel autoclave, and then heated at 120 DEG C for 24 hours. The product was separated by centrifugation at 10000 rpm for 5 minutes, and further purified with ethanol several times and dried to obtain a purple solid, which was HPCN-222, wherein the mass ratio of ZrCl4, tetrakis(4-carboxyphenyl) porphyrin and benzoic acid was 1:1:25; the volume ratio of H2O and DMF was 1:10;

[0009] Subsequently, HPCN-222 and RuCl3 aqueous solution were weighed and added to DMF and ultrasonically treated at room temperature for 10 minutes. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, and heated at 80 DEG C for 4 hours. Subsequently, RuHPCN-222 was separated by centrifugation at 10000 rpm for 5 minutes, and then purified with ethanol several times and dried to obtain a black purple solid, which was RuHPCN-222; wherein the mass ratio of HPCN-222 to Ru in the RuCl3 aqueous solution was 5:1.

[0010] (3) Preparation of modified electrode RuHPCN-222 / P-COF / GCE: First, the P-COF prepared in step (1) was evaporated and self-assembled on a glassy carbon electrode, and then the RuHPCN-222 prepared in step (2) was dispersed in DMF, ultrasonically treated to obtain a RuHPCN-222 dispersion, and then dropped on the surface of the above prepared GCE and naturally dried to obtain the modified electrode RuHPCN-222 / P-COF / GCE.

[0011] Further, the cleaning method of the clean glassy carbon electrode in step (3) includes polishing the glassy carbon electrode, and ultrasonically cleaning with nitric acid solution, ethanol solution and ultrapure water in sequence, and blowing dry at room temperature.

[0012] Further, the concentration of the DMF dispersion of RuHPCN-222 is 1 mg / mL, and the drop coating amount of the DMF dispersion of RuHPCN-222 is 4 μL.

[0013] The application also provides a preparation method of the above modified electrode apt / RuHPCN-222 / P-COF / GCE for detecting MDPV, comprising the following steps:

[0014] The aptamer apt is loaded on the surface of the RuHPCN-222 / P-COF composite material modified glassy carbon electrode through a Ru-S bond, and the modified electrode apt / RuHPCN-222 / P-COF / GCE for detecting MDPV is prepared by natural air drying.

[0015] Further, the specific method for loading the aptamer apt on the surface of the RuHPCN-222 / P-COF composite material modified glassy carbon electrode is as follows: first, the aptamer apt is added to a Tris-HCl buffer solution containing KCl, NaCl, MgCl2 and ethylenediaminetetraacetic acid, and an aptamer solution with an aptamer concentration of 0.5-5 μM is prepared; then, the aptamer solution is taken and drop-coated on the surface of the RuHPCN-222 / P-COF composite material modified glassy carbon electrode, and after natural air drying, 4 μL of 6-mercapto-1-hexanol (MCH) is drop-coated to block the non-specific recognition sites; finally, the sensor is used as a sensing element for electrochemiluminescence testing.

[0016] The application also provides a method for detecting MDPV based on the modified electrode apt / RuHPCN-222 / P-COF / GCE, which comprises the following steps:

[0017] The electrochemiluminescence aptamer sensor (apt / RuHPCN-222 / P-COF / GCE) as described above is used as a working electrode, Ag / AgCl is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to form a three-electrode system, MDPV in a sample is quantitatively fixed to the surface of the sensor, and the generated electrochemiluminescence signal is used for detection.

[0018] Further, the specific steps are as follows:

[0019] Step 1: Preparation of a PBS buffer solution containing K2S2O8:

[0020] A PBS buffer solution containing 0.05 mol / L K2S2O8 is prepared using a PBS buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L;

[0021] Step 2: Preparation of MDPV standard solutions with different concentrations:

[0022] An MDPV solution is prepared, and then the MDPV solution is diluted with a Tris-HCl buffer solution to obtain a series of MDPV standard solutions with different concentrations, and the concentration of MDPV in the standard solution ranges from 1.0×10 -14 to 1.0×10 -6 g / L;

[0023] Step 3: Drawing of a standard curve:

[0024] The same amount of MDPV standard solution of different concentrations prepared according to step 2 is modified on the modified electrode apt / RuHPCN-222 / P-COF / GCE and reacted for the same time, so that the modified electrode is combined with MDPV, then MDPV / apt / RuHPCN-222 / P-COF / GCE is used as a working electrode, Ag / AgCl is used as a reference electrode, and platinum is used as a counter electrode to form a three-electrode system, PBS buffer solution containing K2S2O8 in step 1 is used as an electrolyte, the electrochemical window range is -1.0~0 V, the high voltage of the photomultiplier tube is 800 V, the scanning speed is 0.1 V / s, the cyclic voltammetry scanning is carried out, the luminescence intensity-time curve is recorded, the linear relationship between the luminescence intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after combining MDPV and the logarithmic value of the MDPV concentration in the MDPV standard solution is established, and the corresponding linear regression equation is obtained.

[0025] Step 4, detection of MDPV in the sample

[0026] The sample is first pretreated and diluted with a Tris-HCl buffer solution, then modified on the surface of the electrochemiluminescence aptamer sensor according to step 3 for the same reaction time, so that the electrochemiluminescence aptamer sensor combines MDPV in the sample, then the luminescence intensity is detected by the method of step 3, and the concentration of MDPV in the sample is calculated according to the linear regression equation.

[0027] The beneficial effects of the present application are:

[0028] The present application designs an electrochemiluminescence aptamer sensor based on a photonic crystal composite material of Ru-anchored Zr-based porphyrin MOF (PCN-222) and COF self-assembly. The two materials are combined through electrostatic interaction during synthesis, and high-efficiency and stable electrochemiluminescence performance can be obtained at a low trigger potential. The present application fully utilizes the unique advantages of aptamers and low-potential electrochemiluminescence sensors, and successfully realizes the sensitive detection of MDPV through the quenching effect of MDPV on the ECL signal intensity of the system. Experimental results show that the sensing platform can specifically recognize and detect MDPV. The detection range of the present application is 1.0x10 -14 ~1.0x10 -6 g / L, and the lowest detection limit is 4.79x10 -15 g / L. The present application has the advantages of simple operation, good selectivity, high sensitivity, wide detection range, and is of great significance for promoting the application of aptamer sensors in actual detection. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A brief flowchart of the preparation of the electrochemiluminescence aptamer sensor in the present application and the detection of MDPV.

[0030] Figure 2 ECL signal diagram of different modified electrodes, wherein the concentration of MDPV is 1.0 x 10 -13 g / L.

[0031] Figure 3 ECL response diagram of the electrochemiluminescence aptamer sensor constructed in Example 1 after being combined with different concentrations of MDPV, wherein the concentration of MDPV from a to i is as follows: (a) 1.0 x 10 -6 g / L; (b) 1.0 x 10 -7 g / L; (c) 1.0 x 10 -8 g / L; (d) 1.0 x 10 -9 g / L; (e) 1.0 x 10 -10 g / L; (f) 1.0 x 10 -11 g / L; (g) 1.0 x 10 -12 g / L; (h) 1.0 x 10 -13 g / L; (i) 1.0 x 10 -14 g / L.

[0032] Figure 4 The standard curve of the difference (ΔECL) of the luminescence intensity before and after the addition of MDPV in Example 1 and the logarithmic value of the concentration of MDPV.

[0033] Figure 5 The field emission scanning electron microscope diagram of TAPB-BTCA-COF prepared in Example 1.

[0034] Figure 6 The transmission electron microscope diagram of RuHPCN-222 prepared in Example 1. DETAILED DESCRIPTION

[0035] The present application is not limited to the following specific embodiments, and those skilled in the art can implement the present application in other various specific embodiments according to the disclosure of the present application, or fall within the protection scope of the present application as long as the design structure and ideas of the present application are adopted with simple changes or modifications. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0036] The application will be further described in connection with the embodiments: in the following embodiments, the aptamer apt containing the base sequence of 5'-SH-(CH2)6-ACCTTAAGTGGGGTTGGGTGGAGTTTATGGGGT-Fc-3' is purchased from Shengong Bioengineering (Shanghai) Co., Ltd. The method for loading the aptamer apt on the surface of the RuHPCN-222 / P-COF composite material modified glassy carbon electrode is as follows: first, the aptamer is added to a Tris-HCl buffer solution containing KCl, NaCl, MgCl2 and ethylenediaminetetraacetic acid to prepare an aptamer solution, then the aptamer solution is taken and dropped on the surface of the RuHPCN-222 / P-COF composite material modified glassy carbon electrode, and after natural airing, 4 μL of 6-mercapto-1-hexanol (MCH) is dropped to block the non-specific recognition sites.

[0037] Specifically:

[0038] Step 1, before opening the purchased aptamer, vortex for 5 min, then centrifuge at 4000 rpm for 15 min;

[0039] Step 2, open the cap, according to the label on the tube, add 721 μL of 0.05 M Tris-HCl buffer solution containing 0.2 mol / L KCl, 0.1 mol / L NaCl, 5.0 mmol / L MgCl2 and 1.0 mmol / L ethylenediaminetetraacetic acid to the aptamer apt;

[0040] Step 3, shake well, dilute the aptamer to a concentration of 10 μM and store in a 4 ℃ refrigerator for later use.

[0041] In the following embodiments, the preparation method of MDPV standard solution with different concentrations is as follows: MDPV solution is prepared to obtain a series of MDPV standard solutions with different concentrations, and the concentrations of MDPV in the MDPV standard solutions in this embodiment are (a) 1.0×10 -6 g / L; (b) 1.0×10 -7 g / L; (c) 1.0×10 -8 g / L; (d) 1.0×10 -9 g / L; (e) 1.0×10 -10 g / L; (f) 1.0×10 -11 g / L; (g) 1.0×10 -12 g / L; (h) 1.0×10 -13 g / L; (i) 1.0×10 -14 g / L; Embodiment 1

[0042] (1) Preparation of P-COF and RuHPCN-222 materials:

[0043] (1) Preparation of P-COF and RuHPCN-222 materials:

[0044] Accurately weigh 14.058 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 6.486 mg of 1,3,5-benzene tricarboxaldehyde (BTCA) dissolved in 5 mL of acetonitrile and ultrasonic for one minute, then 1.2 mL of acetic acid (AcOH) is poured into the solution. Finally, the mixture is stirred vigorously for 10 s and left to stand at room temperature for 72 h to obtain P-COF.

[0045] Accurately weigh 20 mg of ZrCl4, 500 mg of benzoic acid and 400 μL of H2O dissolved in 4 mL of DMF, dissolved for 5 minutes under magnetic stirring. Next, 20 mg of tetrakis(4-carboxyphenyl)porphyrin (TCPP) is further added to the above solution and kept under magnetic stirring at room temperature for 10 minutes. The resulting homogeneous solution is transferred to a polytetrafluoroethylene-lined stainless steel autoclave, which is then heated at 120 °C for 24 hours. The product is separated by centrifugation at 10000 rpm for 5 minutes and further purified with ethanol several times and dried to obtain a purple solid, which is HPCN-222.

[0046] Accurately weigh 20 mg of HPCN-222 and 200 μL of RuCl3 aqueous solution (20 mg Ru / mL) added to 4 mL of DMF and ultrasonic treated at room temperature for 10 minutes. The mixture is transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 80 °C for 4 hours. Subsequently, RuHPCN-222 is separated by centrifugation at 10000 rpm for 5 minutes, then purified with ethanol several times and dried to obtain a black purple solid, which is RuHPCN-222. RuHPCN-222 is dispersed in DMF, ultrasonic is used to disperse it uniformly, and a RuHPCN-222 dispersion liquid is obtained, the concentration of which is 1 mg / mL.

[0047] (2) Preparation of electrochemiluminescence aptamer sensor for detecting MDPV

[0048] Firstly, the glassy carbon electrode is polished into a mirror surface with polishing powder (Al2O3) on a buffing leather, and then is cleaned with nitric acid solution, ethanol solution and ultrapure water in sequence, and is dried at room temperature to obtain a pretreated glassy carbon electrode. P-COF is self-assembled on the glassy carbon electrode by solvent evaporation method, 4 μL of 1 mg / mL RuHPCN-222 DMF dispersion prepared in step (2) is moved by a microsyringe, and is naturally dried, then 4 μL of prepared Tris-HCl buffer solution containing aptamer apt is modified, and is naturally dried to obtain an apt / RuHPCN-222 / GCE sensor, and 4 μL of 6-mercapto-1-hexanol (MCH) is dropped and coated to block the non-specific recognition sites, and finally the sensor is used as a sensing element for electrochemiluminescence test.

[0049] The concentration of the aptamer solution is 4 μM.

[0050] (II) Method for detecting MDPV based on the electrochemiluminescence aptamer sensor

[0051] (1) Drawing of standard curve

[0052] An equal amount of 4 μL of MDPV standard solution with different concentrations is modified on the electrochemiluminescence aptamer sensor as described in (I) and is reacted for the same time of 80 min, so that the electrochemiluminescence aptamer sensor is combined with MDPV, then MDPV / apt / RuHPCN-222 / P-COF / GCE is used as a working electrode, Ag / AgCl is used as a reference electrode, and platinum electrode is used as a counter electrode to form a three-electrode system, a PBS buffer solution containing 0.05 mol / L K2S2O8 with pH of 7.4 is used as an electrolyte, a photomultiplier high voltage is 800 V, a scanning speed is 0.1 V / s, a cyclic voltammetry scanning is performed in an electrochemical window range of -1.0~0 V, a luminescence intensity-time curve is recorded, a linear relationship between the luminescence intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after being combined with MDPV and the logarithmic value of the MDPV concentration in the MDPV standard solution is established, and a corresponding linear regression equation is obtained; ∆ECL = 6527.28 + 1031.92lg C (ng / L), the detection range is 1.0×10 -14 ~1.0×10 -6 g / L, and the detection limit is 4.79×10 -15 g / L.

[0053] (2) Detection of sample

[0054] Take 100 μL of alcoholic beverage from a certain bar in 100 μL of Tris-HCl buffer solution to obtain a dilution, and divide the dilution into several equal parts. Add MDPV standard solution of different concentrations to the above dilution, centrifuge at 3000 rpm for 5 min after sufficient reaction, and collect the supernatant. Take 4 μL of the supernatant to modify the surface of the prepared electrochemiluminescence aptamer sensor, and calculate the concentration of MDPV in the sample to be detected according to the linear regression equation obtained in step (1), and the results are shown in Table 1.

[0055] In this embodiment, P-COF is used as a base material (morphology as shown in Figure 5 ), and a composite material is obtained by secondary drop coating of RuHPCN-222 (morphology as shown in Figure 6 ), which can be stably combined through electrostatic interaction, thereby improving the electrochemiluminescence stability of P-COF monomers. In addition, P-COF can greatly improve the electrochemiluminescence intensity of the single material, has good conductivity and stability, and has good sensor selectivity. Comparative Example 1

[0056] Preparation of apt / RuHPCN-222 / GCE sensor

[0057] 4 μL of 1 mg / mL RuHPCN-222 DMF dispersion was removed with a micro-syringe and drop-coated on the surface of the pretreated glassy carbon electrode (the pretreatment method is the same as in Example 1) to obtain a RuHPCN-222 / GCE chemical modification electrode. After natural air drying, a RuHPCN-222 / GCE sensor was obtained, and 4 μL of 6-mercapto-1-hexanol (MCH) was drop-coated to block the non-specific recognition sites. Finally, the sensor was used as an electrochemiluminescence test sensing element. (The modification amount of the monomer test was kept as a single variable with the sample concentration.)

[0058] (2) Preparation of standard curve and MDPV detection

[0059] The apt / RuHPCN-222 / GCE sensor prepared in step (1) was used as a sensing element, and the standard curve was drawn according to the method of Example 1, and the concentration of MDPV in the sample was detected, and the results are shown in Table 1. Comparative Example 2

[0060] Preparation of apt / HPCN-222 / GCE sensor

[0061] A 4 μL of 1 mg / mL HPCN-222 DMF dispersion was removed by a micro-syringe and dropped onto the surface of a pretreated glassy carbon electrode (pretreatment method same as in Example 1) to obtain a HPCN-222 / GCE chemical modified electrode. After natural air-drying, a HPCN-222 / GCE sensor was obtained. Then 4 μL of 6-mercapto-1-hexanol (MCH) was dropped to block the non-specific recognition sites. Finally, the sensor was used as a sensing element for electrochemiluminescence testing. (The modification amount of the monomer test and the sample concentration remained a single variable in the example.)

[0062] (2) Preparation of standard curve and MDPV detection

[0063] The apt / HPCN-222 / GCE sensor prepared in step (1) was used as a sensing element, and the standard curve was drawn according to the method of Example 1, and the concentration of MDPV in the sample was detected, and the results are shown in Table 1. Comparative Example 3

[0064] (1) Preparation of apt / P-COF / GCE sensor

[0065] P-COF was assembled on the surface of a glassy carbon electrode (pretreatment method same as in Example 1) by solvent evaporation to obtain a P-COF / GCE chemical modified electrode. Then 4 μL of 4 μM aptamer apt was dropped and naturally air-dried to obtain an apt / P-COF / GCE sensor. Then 4 μL of 6-mercapto-1-hexanol (MCH) was dropped to block the non-specific recognition sites. Finally, the sensor was used as a sensing element for electrochemiluminescence testing. (The modification amount of the monomer test and the sample concentration remained a single variable in the example.)

[0066] (2) Preparation of standard curve and MDPV detection

[0067] The apt / P-COF / GCE sensor prepared in step (1) was used as a sensing element, and the standard curve was drawn according to the method of Example 1, and the concentration of MDPV in the sample was detected, and the results are shown in Table 1. Comparative Example 4

[0068] (1) Preparation of apt / RuHPCN-222 / P-COF / GCE sensor

[0069] The apt / RuHPCN-222 / P-COF / GCE sensor was prepared according to the method of Example 1.

[0070] (2) Preparation of standard curve

[0071] The apt / RuHPCN-222 / P-COF / GCE sensor prepared in step (1) is used as a sensing element, and an equal amount of different concentrations of the synthesized cannabinoid RCS-4 standard solution is modified on the surface of the sensor and reacts for 80 min, after which it is used as a working electrode, Ag / AgCl is used as a reference electrode, and platinum is used as a counter electrode to form a three-electrode system, and the luminescence intensity is measured in a 0.1 mol / L PBS buffer solution containing 0.05 mol / L K2S2O8 and having a pH of 7.4, in an electrochemical window range of -1.0-0 V, a photomultiplier high voltage of 800 V, and a scan rate of 0.1 V / s, cyclic voltammetry scanning is performed, the luminescence intensity-time curve is recorded, the linear relationship between the luminescence intensity difference before and after the electrochemiluminescence aptamer sensor is combined with RCS-4 and the RCS-4 concentration in the RCS-4 standard solution is established, and the corresponding linear regression equation is obtained.

[0072] (3) Detection of samples

[0073] 100 μL of alcoholic beverages from a certain bar is taken in 100 μL of Tris-HCl buffer solution to obtain a diluent, and the diluent is equally divided into several parts. Different concentrations of RCS-4 standard solution are added to the above diluent, and after sufficient reaction, centrifugation is performed at a speed of 3000 rpm for 5 min, and the supernatant is collected. 4 μL of the supernatant is modified on the surface of the prepared electrochemiluminescence aptamer sensor, and the concentration of RCS-4 in the sample to be detected is calculated according to the linear regression equation obtained in step (1), and the results are shown in Table 1.

[0074] Table 1 Determination results of a certain alcoholic beverage sample

[0075]

[0076] Note: a The average value of three determinations

[0077] As shown in Table 1, the sample is determined in parallel for three times, the standard addition recovery is between 97% and 103%, and the relative standard deviation is less than 5%, indicating that the recovery effect is good. The above experimental results show that the sensing element cannot detect MDPV when the RuHPCN-222 / P-CPF composite material is not used to modify the glassy carbon electrode, and the RuHPCN-222, HPCN-222 and P-COF are used alone to modify the glassy carbon electrode, and has specificity to MDPV, so the sensor of the application can be used to detect MDPV in alcoholic beverages.

[0078] Based on the above verification, it can be known that the application is based on the electrochemiluminescence quenching effect of MDPV on the apt / RuHPCN-222 / P-COF / GCE system, and a new method for rapidly and sensitively detecting MDPV is constructed. Since RuHPCN-222 and P-COF have good electrostatic interaction, RuHPCN-222 / P-COF composite material with high electrochemiluminescence intensity and good stability can be formed. When there is a small amount of MDPV, the aptamer apt can specifically recognize MDPV and fix it on the electrochemiluminescence aptamer sensor, so that the ECL signal of apt / RuHPCN-222 / P-COF / GCE is significantly reduced. It is found through research that the enhanced value (ΔECL) of the ECL signal of the apt / RuHPCN-222 / P-COF / GCE sensor system and the concentration of MDPV present a good linear relationship. The electrochemiluminescence method used in the application not only has the advantages of high sensitivity, fast detection speed, good selectivity and wide linear range, but also has great application potential for the quantitative analysis of MDPV in alcoholic beverages.

[0079] The above examples are only used for the description of the application and are not limited to the application. Those skilled in the art can make corresponding changes without departing from the scope of the application, and therefore all equivalent replacements or equivalent variations form technical solutions within the protection scope of the application.

Claims

1. An electrochemiluminescence aptamer sensor for detecting MDPV, characterized in that: The electrochemiluminescence aptamer sensor is formed by loading aptamer apt on the surface of a composite RuHPCN-222 / TAPB-BTCA-COF modified glassy carbon electrode through a Ru-S bond, and is denoted as apt / RuHPCN-222 / P-COF / GCE; wherein the composite RuHPCN-222 / TAPB-BTCA-COF is a photonic crystal assembled by Ru-anchored Zr-based porphyrin MOF and TAPB-BTCA-COF and is formed through electrostatic interaction; the base sequence of the aptamer apt is 5'-SH-(CH2)6-ACCTTAAGTGGGGTTGGGTGGAGTTTATGGGGT-Fc-3'; The preparation method of the TAPB-BTCA-COF is as follows: 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-benzene tricarboxaldehyde are weighed and dissolved in acetonitrile and are ultrasonically mixed uniformly, the molar ratio of the 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-benzene tricarboxaldehyde is 1:1; then acetic acid is added to obtain a mixture; the mixture is stirred vigorously for 10 s and is left to stand at room temperature for 72 h, and the TAPB-BTCA-COF is obtained; The preparation method of the RuHPCN-222 is as follows: HPCN-222 and a RuCl3 aqueous solution are added into DMF and are ultrasonically dispersed uniformly at room temperature to obtain a mixture; the mixture is transferred into a polytetrafluoroethylene-lined stainless steel autoclave, is heated at 80 DEG C for 4 hours, and the product is collected by centrifugation, washed and dried to obtain the RuHPCN-222; wherein the mass ratio of HPCN-222 to Ru in the RuCl3 aqueous solution is 5:1; The preparation method of the HPCN-222 is as follows: ZrCl4, benzoic acid and H2O are added into DMF and are stirred and dissolved; tetra(4-carboxyphenyl)porphyrin is added and is continuously stirred and dissolved and mixed uniformly at room temperature to obtain a uniform solution; the obtained uniform solution is transferred into a polytetrafluoroethylene-lined stainless steel autoclave, is heated at 120 DEG C for 24 hours, and the product is collected by centrifugation, washed and dried to obtain the HPCN-222; wherein the mass ratio of ZrCl4, tetra(4-carboxyphenyl)porphyrin and benzoic acid is 1:1:25; the volume ratio of H2O to DMF is 1:

10.

2. A method for preparing the electrochemiluminescence aptamer sensor for detecting MDPV according to claim 1, characterized in that: The method comprises the following specific steps: (1) TAPB-BTCA-COF is self-assembled on GCE through evaporation induction to obtain TAPB-BTCA-COF / GCE, denoted as P-COF / GCE; (2) RuHPCN-222 is dispersed in N,N-dimethylformamide, is ultrasonically dispersed uniformly to obtain a RuHPCN-222 dispersion, and is then drop-coated on the surface of P-COF / GCE and is naturally air-dried to obtain the modified electrode RuHPCN-222 / P-COF / GCE; (3) An aptamer solution is prepared, is drop-coated on the surface of the modified electrode RuHPCN-222 / P-COF / GCE, is naturally air-dried, and then 6-mercapto-1-hexanol is drop-coated, and the electrochemiluminescence aptamer sensor is obtained.

3. The method for preparing an electrochemiluminescence aptamer sensor for detecting MDPV according to claim 2, characterized in that: The concentration of the RuHPCN-222 dispersion liquid is 1 mg / mL, and the drop coating amount is 4 μL.

4. The method for preparing an electrochemiluminescence aptamer sensor for detecting MDPV according to claim 2, characterized in that: The concentration of the aptamer apt in the aptamer solution is 0.5-5 μmol / L, and the drop coating amount is 4 μL; the drop coating amount of 6-mercapto-1-hexanol is 4 μL.

5. A method for electrochemiluminescence detection of MDPV, characterized in that: The detection method uses the apt / RuHPCN-222 / P-COF / GCE as claimed in claim 1 as a working electrode, Ag / AgCl as a reference electrode, and a platinum wire electrode as a counter electrode to form a three-electrode system, so that MDPV in a sample is quantitatively captured on the surface of the sensor, and the detection of MDPV is realized through the generated luminescence signal.

6. The method of electrochemiluminescence detection of MDPV according to claim 5, wherein: The electrochemiluminescence detection of MDPV uses a PBS buffer solution containing K2S2O8 as an electrolyte, and the electrochemical window range is -1.0-0 V, the high voltage of the photomultiplier tube is 800 V, the scanning speed is 0.1 V / s, and cyclic voltammetry scanning is performed; and the luminescence intensity before and after the combination of MDPV is recorded.

7. The method of electrochemiluminescence detection of MDPV according to claim 6, wherein: The reaction time of MDPV and the aptamer apt is 80 min.

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