Electrochemiluminescence aptamer sensor based on DNA-Cy7 / MMST / GCE modified electrode

Abstract

The invention relates to the technical field of electrochemical luminescence aptamer sensors, in particular to an electrochemical luminescence aptamer sensor based on a DNA-Cy7 / MMST / GCE modified electrode. When high performance liquid chromatography-tandem mass spectrometry is adopted to detect 4-FMA, although the method is sensitive and efficient, the cost is high, the pretreatment is relatively complicated, and the detection speed is not high enough. In order to solve the technical problems, the invention provides the electrochemical luminescence aptamer sensor based on the DNA-Cy7 / MMST / GCE modified electrode, the DNA-Cy7 / MMST / GCE modified electrode can specifically recognize a detected object 4-FMA, the concentration detection range is 1.0 * 10 <-14 >-1.0 * 10 <-6 > g / L, the minimum detection limit is 1.87 * 10 <-15 > g / L, and the electrochemical luminescence aptamer sensor is simple to manufacture, high in sensitivity, low in cost, high in detection speed and relatively good in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence aptamer sensors, and particularly relates to an electrochemiluminescence aptamer sensor based on a DNA-Cy7 / MMST / GCE modified electrode. Background Art

[0002] An electronic cigarette is an electronic product imitating traditional tobacco, and its market promotion has long focused on shaping the concept of "harmlessness". Some manufacturers conduct marketing by emphasizing that the product does not contain harmful substances such as tar and suspended particulate matter generated by tobacco combustion, and even promote it in the images of "smoking cessation aid" and "lung cleaning alternative". Special studies by the World Health Organization have clearly warned of the risks of electronic cigarettes: Electronic cigarettes are harmful to public health, and their control must be strengthened, especially to avoid health damage to adolescent groups and non-smokers.

[0003] 4-Fluoromethylamphetamine (4-FMA) is a new type of new psychoactive substance of the amphetamine class. It exhibits powerful nerve stimulation and hallucinogenic effects, and has greater activity compared to traditional amphetamine and methamphetamine. However, the toxicological mechanism research and detection of 4-FMA have not been fully carried out. The traditional detection method of 4-FMA is high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), which is sensitive and efficient, but the sample pretreatment cost is high and complex. And the detection method based on electrochemiluminescence (ECL) for 4-FMA is more sensitive, fast, simple and efficient. Currently, there are few reports on the electrochemiluminescence method using a three-electrode system for detecting 4-FMA. Summary of the Invention

[0004] The problems existing in the prior art are as follows: When using high performance liquid chromatography-tandem mass spectrometry to detect 4-FMA, although the method is sensitive and efficient, the cost is high, the pretreatment is relatively complex, and the detection speed is not fast enough. In view of the above technical problems, the present invention provides an electrochemiluminescence aptamer sensor based on a DNA-Cy7 / MMST / GCE modified electrode. The working electrode used in the electrochemiluminescence aptamer sensor is a DNA-Cy7 / MMST / GCE modified electrode, and the DNA-Cy7 / MMST / GCE modified electrode is an electrode obtained by successively modifying MMST and DNA-Cy7 on the surface of GCE.

[0005] Preferably, the preparation method of the MMST includes the following steps:

[0006] Disperse TCPP NPs (porphyrin nanoparticles) evenly in an organic solvent, then inject it into ultrapure water containing MMS, stir and mix evenly, and then obtain MMST through freeze-drying.

[0007] Preferably, the method for preparing the MMS includes the following steps:

[0008] Disperse MIL-101(Fe) uniformly in ultrapure water to obtain a dispersion liquid. Then, add ammonium molybdate and thiourea to the dispersion liquid. After stirring and mixing evenly, transfer it to a high-pressure reaction kettle and heat it at a constant temperature of 180 °C for reaction. After the reaction is completed, the reaction solution is successively subjected to solid-liquid separation and vacuum drying to obtain MMS.

[0009] Preferably, the method for preparing the MIL-101(Fe) includes the following steps:

[0010] Dissolve trivalent water-soluble iron salt in an organic solvent to form a homogeneous solution. Then, add terephthalic acid to the homogeneous solution. After stirring evenly, transfer it to a high-pressure reaction kettle and react at a high temperature. After the reaction is completed, the reaction solution is subjected to solid-liquid separation and vacuum drying to obtain MIL-100(Fe).

[0011] Preferably, the DNA-Cy7 is a compound formed by adding DNA and Cy7 to a 0.05 mol / L Tris-HCl buffer solution containing KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid to form a homogeneous aptamer dispersion liquid, and then coating it on the electrode surface and drying.

[0012] Preferably, the DNA is single-stranded DNA, and the DNA sequence is 5'-AGG AAT TCA GAT CTC CCT GCA GGTGGT GTT TTT TTG TGT GCT GTG TTT TTG TGA TGC ATG CTC GAG GAG CTC AGG ATC CCG-NH2-3'.

[0013] When performing electrochemical detection, the electrochemiluminescence aptamer sensor is a three-electrode system. The reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum electrode. And a PBS buffer solution containing K2S2O8 with pH = 7.4 is used as the electrolyte, and the concentration of K2S2O8 in the PBS buffer solution is 0.05 mol / L. The electrochemical window for cyclic voltammetry scanning during electrochemical detection is -1.0 to 0 V, the high voltage of the photomultiplier tube is 800 V, and the scanning rate is 0.1 V / s.

[0014] The present invention has the following beneficial effects:

[0015] (1) The present invention designs an electrode modification material with iron-based MOF (MIL-101(Fe)) loaded with molybdenum disulfide as a multivalent carrier to immobilize the luminescent porphyrin supramolecular TCPP NPs. The present invention makes full use of the unique advantages of aptamers and electrochemical luminescence sensors, and utilizes the quenching effect of 4-FMA on the ECL signal intensity of this electrochemical luminescence aptasensor to successfully achieve sensitive detection of 4-FMA. Experiments prove that the electrochemical luminescence aptasensor based on the DNA-Cy7 / MMST / GCE modified electrode obtained in the present invention can specifically recognize and detect the analyte 4-FMA, and the concentration detection range is 1.0×10 -12 ~1.0×10 -6 g / L, and the lowest detection limit is 1.7×10 -13 g / L;

[0016] (2) The detection of 4-FMA in the present invention is simple in operation, good in selectivity, high in sensitivity, and wide in detection range, which has important guiding significance for promoting the application of aptasensors in the trace detection of 4-FMA. Brief Description of the Drawings

[0017] Figure 1 : It is a schematic process flow diagram for the preparation of the electrochemical luminescence aptasensor and the detection of 4-FMA in Example 1 of the present invention.

[0018] Figure 2 : It is the ECL response diagram of the electrochemical luminescence aptasensor constructed in Example 1 after binding with 4-FMA solutions of different concentrations. Among them, the concentrations of 4-FMA from a to g are successively: (a) 1.0×10-14 (b) 1.0×10-13; (c) 1.0×10-12 g / L; (d) 1.0×10-11 g / L; (e) 1.0×10-10 g / L; (f) 1.0×10-9 g / L; (g) 1.0×10-8 g / L; (h) 1.0×10-7 g / L; (i) 1.0×10-6 g / L.

[0019] Figure 3 : It is the standard curve of the difference in luminescence intensity (ΔECL) before and after adding 4-FMA in Example 1 and the logarithm of the 4-FMA concentration.

[0020] Figure 4 : It is the scanning electron microscope image of MMST obtained in Example 1.

[0021] Figure 5 : They are the diagrams of the change of ECL response with time for the MMST modified electrode (a), the DNA-Cy7 / MMST modified electrode (c), and the 4-FMA / DNA-Cy7 / MMST modified electrode (c), respectively. Detailed Embodiments

[0022] The present invention will be described in detail below in conjunction with embodiments. It should be understood that the following embodiments are only illustrative examples of the implementation modes of the present invention, rather than limiting the scope of the present invention.

[0023] The DNA used in the following embodiments of the present invention is single-stranded DNA, and the DNA sequence is 5'-AGG AAT TCA GAT CTCCCT GCA GGT GGT GTT TTT TTG TGT GCT GTG TTT TTG TGA TGC ATG CTC GAG GAG CTCAGG ATC CCG-NH2-3'. It is purchased from Sangon Biotech (Shanghai) Co., Ltd., and the product is a DNA solution.

[0024] The 3,3'-diethylthiotricarbocyanine iodide (Cy7) used in the following embodiments of the present invention is a cyanine dye, with the CAS number 3071-70-3, and it is purchased from Aladdin Reagent Company.

[0025] The aptamer solution used in the following embodiments of the present invention is a dispersion formed by DNA and Cy7 in a Tris-HCl buffer solution. The preparation method of the aptamer solution is as follows:

[0026] (1) Before opening the lid of the purchased DNA solution, vortex it for 5 min first, and then centrifuge it at 4000 rpm for 15 min. The solid centrifugate collected is the DNA.

[0027] (2) Add DNA and Cy7 to 106 μL of Tris-HCl buffer solution containing KCl, NaCl, MgCl2 and ethylenediaminetetraacetic acid. The Tris-HCl buffer solution is an aqueous solution containing 0.05 mol / L of tris(hydroxymethyl)aminomethane hydrochloride. After shaking evenly, an aptamer solution is obtained. The concentrations of KCl, NaCl, MgCl2 and ethylenediaminetetraacetic acid in the Tris-HCl buffer solution are 0.2 mol / L, 0.1 mol / L, 5.0 mmol / L and 1.0 mmol / L respectively, and the concentrations of DNA and Cy7 in the Tris-HCl buffer solution are both 2 μmol / L.

[0028] Continue to dilute the aptamer solution with Tris-HCl buffer solution to a concentration of 5 μmol / L, and store it in a refrigerator at 4°C for later use.

[0029] Prepare an aqueous solution of 4-FMA (the solvent is ultrapure water), and then dilute it with Tris-HCl buffer solution to obtain a series of 4-FMA standard solutions with different concentrations. The concentrations of 4-FMA in the standard solutions are respectively (a) 1.0×10 -14 (b) 1.0×10 -13; (c) 1.0×10 -12 g / L; (d) 1.0×10 -11 g / L; (e) 1.0×10 -10 g / L; (f) 1.0×10 -9 g / L; (g) 1.0×10 -8 g / L; (h) 1.0×10 -7 g / L; (i) 1.0×10 -6 g / L.

[0030] Unless otherwise specified, the Tris-HCl buffer solution in the present invention refers to an aqueous solution containing 0.05 mol / L of tris(hydroxymethyl)aminomethane hydrochloride.

[0031] Example 1

[0032] A preparation method of a DNA-Cy7 / MMST / GCE modified electrode is as follows:

[0033] (1) After polishing the glassy carbon electrode (GCE) into a mirror surface with polishing powder (Al2O3) on suede, it is ultrasonically cleaned once with a nitric acid solution with a mass concentration of 69%, an ethanol solution with a mass concentration of 95%, and ultrapure water in sequence, and then naturally dried at room temperature to obtain a clean GCE;

[0034] (2) 4 μL of the MMST dispersion is evenly dropped on the surface of the clean GCE, and after natural drying, an MMST / GCE modified electrode is obtained;

[0035] (3) 4 μL of an aptamer solution with a concentration of 5 μmol / L is evenly dropped on the MMST / GCE modified electrode, and after natural drying, a DNA-Cy7 / MMST / GCE modified electrode is obtained.

[0036] The MMST dispersion is a solution with a concentration of 0.5 mg / mL formed by uniformly dispersing MMST in DMF. The preparation method of the MMST is as follows:

[0037] (1) 10 mg of 5,10,15,20-tetrakis(4-carboxymethoxyphenyl)-21H,23H-porphine (TCPP) is dispersed in 20 mL of tetrahydrofuran, ultrasonically dissolved until completely dissolved, then the dissolved solution is injected into 20 mL of ultrapure water, and after mixing evenly, porphyrin nanoparticles (TCPP NPs) are obtained by freeze-drying;

[0038] (2) Accurately weigh 0.675 g of FeCl3·6H2O and dissolve it in 30 mL of N,N-dimethylformamide (DMF) to form a homogeneous solution. Next, after stirring for 10 minutes and ultrasonic dispersion for 10 min in sequence, add 0.206 g of terephthalic acid to the above solution. After stirring evenly, transfer the obtained mixture to a high-pressure reaction kettle and react at a constant temperature of 110 °C for 20 h. After the reaction is completed, cool it naturally to room temperature. Finally, after centrifugal washing of the reaction solution and vacuum drying at 60 °C, the MOF material MIL-100(Fe) is obtained;

[0039] (3) Add 0.1 g of MIL-101(Fe) to 70 mL of ultrapure water and ultrasonic disperse for 30 min to obtain a homogeneous solution. Add 1.24 g of ammonium molybdate tetrahydrate and 1.06 g of thiourea to the above homogeneous solution and stir for 30 min. After stirring evenly, transfer it to a high-pressure reaction kettle and react at a constant temperature of 180 °C for 12 h. After centrifugal washing 3 times, vacuum dry at 60 °C for 12 h to obtain MIL-101(Fe)@MoS2 (MMS);

[0040] (4) Disperse 10 mg of TCPP NPs in 20 mL of tetrahydrofuran and ultrasonic disperse evenly. Then add the solution to 20 mL of ultrapure water containing 15 mg of MMS. After stirring evenly, obtain MMST after freeze-drying.

[0041] The method for detecting 4-FMA by an electrochemiluminescence aptasensor is as follows:

[0042] (1) Drawing of the standard curve

[0043] Take 4 μL of a series of 4-FMA standard solutions with different concentrations and evenly drop them on the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After natural drying at room temperature, a series of different 4-FMA / DNA-Cy7 / MMST / GCE electrodes are obtained. Respectively, use different 4-FMA / DNA-Cy7 / MMST / GCE electrodes as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system of the electrochemiluminescence aptasensor. Use a PBS buffer solution with pH = 7.4 (containing K2S2O8, and the concentration of K2S2O8 in the PBS buffer solution is 0.05 mol / L) as the electrolyte, and perform electrochemical cyclic voltammetry scanning in the electrochemical window range of -1.0 to 0 V. The high voltage of the photomultiplier tube is 800 V, and the scanning rate is 0.1 V / s. Record the luminescence intensity-time curve of the corresponding 4-FMA / DNA-Cy7 / MMST / GCE electrode, establish the linear relationship between the stable luminescence intensity difference (ΔECL) before and after the DNA-Cy7 / MMST / GCE modified electrode binds 4-FMA and the logarithm of the concentration of the 4-FMA standard solution, and obtain the corresponding linear regression equation; ΔI ECL = 15433.83 + 856.41LgC 4-FMA , the detection range is 1.0×10 -14 ~1.0×10 -6 g / L, and the detection limit is 1.87×10 -15 g / L.

[0044] (2) Detection of samples

[0045] Take 100 μL of the e-liquid of a certain brand of e-cigarette and place it in 100 μL of Tris-HCl buffer solution with a concentration of 0.01 mol / L to obtain a dilution. Use the dilution containing the e-cigarette liquid to dilute the 4-FMA sample to a concentration of 1 ng / L, react overnight, centrifuge at 3000 rpm for 5 min, and collect the supernatant. Take 4 μL of the e-cigarette liquid dilution without 4-FMA (0 ng / L) and the supernatant containing 1 ng / L 4-FMA sample and modify and drop them onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, use it as the working electrode, Ag / AgCl as the reference electrode, and the platinum electrode as the counter electrode to respectively form a three-electrode system of the electrochemiluminescence aptasensor. Use PBS buffer solution with pH = 7.4 (containing K2S2O8, and the concentration of K2S2O8 in the PBS buffer solution is 0.05 mol / L) as the electrolyte, perform cyclic voltammetry scanning in the electrochemical window range of -1.0 to 0 V, with the high voltage of the photomultiplier tube being 800 V and the scanning rate being 0.1 V / s. Record the luminescence intensity-time curve corresponding to the 4-FMA / DNA-Cy7 / MMST / GCE electrode, establish the ΔECL before and after the electrochemiluminescence aptasensor binds to 4-FMA, and substitute it into the linear regression equation obtained in step (1) to calculate the concentration and actual amount of 4-FMA in the sample to be detected. a The test results of the recovery rate and RSD are listed in Table 1. Each data is tested in parallel three times. The specific calculation results of the three groups of parallel tests are processed as follows:

[0046] (1) Drop the supernatant containing 1 ng / L of e-cigarette liquid onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, use it as the working electrode. The measured ΔECL difference is approximately 7315, and the difference corresponding to 10-9 in the linear equation obtained in step (1) is approximately 7700. 7315 / 7700 = 0.95, and the actual amount in Table 1 is obtained. a Is 0.95.

[0047] (2) Drop the supernatant containing 1 ng / L of e-cigarette liquid onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, use it as the working electrode. The measured ΔECL difference is approximately 7854. 7854 / 7700 = 1.02, and the actual amount in Table 1 is obtained. a Is 1.02.

[0048] (3) Drop the supernatant containing 1 ng / L of e-cigarette liquid onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, use it as the working electrode. The measured ΔECL difference is approximately 7623. 7623 / 7700 = 0.99, and the actual amount in Table 1 is obtained.a is 0.99.

[0049] The working electrode of the electrochemiluminescence aptamer sensor of the present invention uses MMST as the substrate material (the morphology is as Figure 4 ), and MMS is used as the multivalent carrier. This variable valence process can increase the redox activity of the ECL emitter, greatly improve the electrochemiluminescence intensity of the electrode active material, has good conductivity, good stability, and good selectivity of the sensor.

[0050] The process flow diagram of the preparation of the electrochemiluminescence aptamer sensor constructed in Example 1 and the detection of 4-FMA is shown in the attached Figure 1 description.

[0051] The ECL response diagram of the electrochemiluminescence aptamer sensor constructed in Example 1 after binding with 4-FMA solutions of different concentrations is shown in the attached Figure 2 description, where the concentrations of 4-FMA from a to g are in turn: (a) 1.0×10-14 (b) 1.0×

[0052] 10-13; (c) 1.0×10-12 g / L; (d) 1.0×10-11 g / L; (e) 1.0×10-10 g / L; (f) 1.0×10-9 g / L; (g) 1.0×10-8 g / L; (h) 1.0×10-7 g / L; (i) 1.0×10-6 g / L.

[0053] The standard curve diagram of the difference in luminescence intensity (ΔECL) before and after adding 4-FMA in Example 1 and the logarithm of the 4-FMA concentration is shown in the attached Figure 3 description.

[0054] The scanning electron microscope image of the MMST obtained in Example 1 is shown in the attached Figure 4 description.

[0055] The diagram of the change of ECL response of MMST / GCE (a) and DNA-Cy7 / MMST / GCE (b) 4-FMA / DNA-Cy7 / MMST / GCE (c) prepared in Example 1 with time is shown in the attached Figure 5 description. The detailed test steps are as follows: The above three electrodes are the working electrode, Ag / AgCl is the reference electrode, and the platinum electrode is the counter electrode. All use PBS buffer solution with pH = 7.4 (containing K2S2O8, and the concentration of K2S2O8 in the PBS buffer solution is 0.05 mol / L) as the electrolyte, and perform electrochemical cyclic voltammetry scanning in the electrochemical window range of -1.0 to 0 V. The high voltage of the photomultiplier tube is 800 V, the scanning speed is 0.1 V / s, and the corresponding luminescence intensity-time curve is recorded.

[0056] Comparative Example 1 was the same as Example 1, except that in Comparative Example 1, the MMS dispersion was used to replace the MMST dispersion in Example 1. The MMS dispersion was a solution with a concentration of 0.5 mg / mL formed by uniformly dispersing MMS in DMF.

[0057] The difference in the stable luminescence intensity (ΔECL) before and after the DNA-Cy7 / MMS / GCE modified electrode obtained in Comparative Example 1 bound 4-FMA did not show a linear relationship with the logarithm of the concentration of the 4-FMA standard solution, and no linear regression equation was obtained.

[0058] In the same manner as in Example 1, the DNA-Cy7 / MMS / GCE modified electrode obtained in Comparative Example 1 was used to replace the DNA-Cy7 / MMST / GCE modified electrode in Example 1 to perform electrochemical detection of 4-FMA in the same brand and same volume of e-cigarette liquid. Due to poor ECL performance, test results could not be obtained, and all were not detected, as shown in Table 1.

[0059] Comparative Example 2 was the same as Example 1, except that in Comparative Example 2, the TCPPNPs dispersion was used to replace the MMST dispersion in Example 1. The TCPPNPs dispersion was a solution with a concentration of 0.5 mg / mL formed by uniformly dispersing TCPPNPs in DMF.

[0060] The difference in the stable luminescence intensity (ΔECL) before and after the DNA-Cy7 / TCPPNPs / GCE modified electrode obtained in Comparative Example 2 bound 4-FMA did not show a linear relationship with the logarithm of the concentration of the 4-FMA standard solution, and no linear regression equation was obtained.

[0061] In the same manner as in Example 1, the DNA-Cy7 / TCPPNPs / GCE modified electrode obtained in Comparative Example 2 was used to replace the DNA-Cy7 / MMST / GCE modified electrode in Example 1 to perform electrochemical detection of 4-FMA in the same brand and same volume of e-cigarette liquid. Due to poor ECL performance, test results could not be obtained, and all were not detected, as shown in Table 1.

[0062] Comparative Example 3 was the same as Example 1, except that in Comparative Example 3, a DNA dispersion was used to replace the aptamer solution in Example 1. The DNA dispersion was formed by uniformly dispersing DNA (DNA sequence: 5'-AGG AAT TCA GAT CTC CCT GCA GGT GGT GTT TTT TTG TGT GCT GTG TTT TTG TGA TGC ATG CTC GAG GAG CTC AGG ATC CCG-NH2-3') in a Tris-HCl buffer solution. The DNA dispersion was obtained by adding DNA to 106 μL of a Tris-HCl buffer solution containing KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid at a concentration of 0.05 mol / L, and shaking evenly. The concentrations of KCl, NaCl, MgCl2, and ethylenediaminetetraacetic acid in the Tris-HCl buffer solution were 0.2 mol / L, 0.1 mol / L, 5.0 mmol / L, and 1.0 mmol / L, respectively, and the concentration of DNA in the Tris-HCl buffer solution was 2 μmol / L.

[0063] The difference in the stable luminescence intensity (ΔECL) before and after the DNA / MMST / GCE modified electrode obtained in Comparative Example 3 combined with 4-FMA showed a linear relationship with the logarithm of the concentration of the 4-FMA standard solution. The linear regression equation was: ΔI ECL = 16430.25 + 1176.06LgC 4-FMA , and the detection range was 1.0×10 -13 ~1.0×10 -6 g / L, and the detection limit was 5.50×10 -14 g / L.

[0064] According to the method of Example 1, the DNA / MMST / GCE modified electrode obtained in Comparative Example 3 was used to replace the DNA-Cy7 / MMST / GCE modified electrode in Example 1, and 4-FMA in the same brand, same volume, and same concentration of e-cigarette liquid was electrochemically detected. The test results were compared with the values in the linear equation obtained in Example 1 to obtain the actual quantity results, which are listed in Table 1. Each data was tested in parallel three times. The test process and results are as follows:

[0065] (1) The supernatant containing 1 ng / L of e-cigarette liquid was dropped onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Comparative Example 3. After reacting for 30 min, it was used as the working electrode. The measured ΔECL difference was about 6853, while the difference corresponding to 10-9 in the linear equation obtained in step (1) was about 7700. 6853 / 7700 = 0.89, and the actual quantity in Table 1 a was 0.89.

[0066] (2) The supernatant containing 1 ng / L of e-cigarette liquid was dropped onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, it was used as the working electrode. The measured ΔECL difference was approximately 8855, and 8855 / 7700 = 1.15, obtaining the actual amount in Table 1 a to be 1.15.

[0067] (3) The supernatant containing 1 ng / L of e-cigarette liquid was dropped onto the surface of the DNA-Cy7 / MMST / GCE modified electrode obtained in Example 1. After reacting for 30 min, it was used as the working electrode. The measured ΔECL difference was approximately 7007, and 7007 / 7700 = 0.91, obtaining the actual amount in Table 1 a to be 0.91.

[0068] Comparative Example 4 was the same as Example 1, except that in Comparative Example 4, the TCPP NPs dispersion, MMS dispersion, and aptamer solution were sequentially and uniformly drop-coated on the surface of a clean GCE to obtain a DNA-Cy7 / MMS / TCPP NPs / GCE modified electrode. The TCPP NPs dispersion was the same as that in Comparative Example 2, the MMS dispersion was the same as that in Comparative Example 1, and the aptamer solution was the same as that in Example 1.

[0069] There was no linear relationship between the difference in stable luminescence intensity (ΔECL) before and after the DNA-Cy7 / MMS / TCPP NPs / GCE modified electrode obtained in Comparative Example 4 bound 4-FMA and the logarithm of the concentration of the 4-FMA standard solution, and no linear regression equation was obtained.

[0070] In the same manner as in Example 1, the DNA-Cy7 / MMS / TCPP NPs / GCE modified electrode obtained in Comparative Example 4 was used to replace the DNA-Cy7 / MMST / GCE modified electrode in Example 1, and 4-FMA in the same brand, same volume, and same concentration of e-cigarette liquid was electrochemically detected. Due to poor ECL performance, test results could not be obtained, and all were not detected, as shown in Table 1.

[0071] Table 1

[0072]

[0073] Note: a in Table 1 is the average value of three determinations.

[0074] As shown in Table 1, the samples were determined in parallel three times. The spiked recovery rate was between 95% and 102%, and the relative standard deviation was less than 5%, indicating good recovery. The above experimental results show that 4-FMA cannot be detected when the glassy carbon electrode is modified with TCPP NPs, MMS or MMS / TCPP NPs alone without MMST composite material modification. Therefore, the sensor of the present invention can be used to detect 4-FMA in electronic cigarettes. In addition, when the aptamer DNA does not bind to the cyanine dye Cy7, the displacement reaction cannot occur. Although 4-FMA can still be detected, the spiked recovery rate has a significantly larger error compared to Example 1, indicating that the addition of the cyanine dye can effectively improve the recognition performance of the sensor.

[0075] Based on the above verification, a new method for rapidly and sensitively detecting 4-FMA was constructed based on the electrochemiluminescence quenching effect of 4-FMA on the DNA-Cy7 / MMST / GCE system according to the present invention. Since the cyanine dye can undergo a displacement reaction with 4-FMA. When a trace amount of 4-FMA is present, DNA-Cy7 / MMST / GCE specifically recognizes the analyte 4-FMA. When the analyte binds to the aptamer, it is immobilized on the electrode surface and displaces the cyanine dye, resulting in a significant decrease in the ECL signal of DNA-Cy7 / MMST / GCE. It was found through research that the enhanced value (ΔECL) of the ECL signal of the DNA-Cy7 / MMST / GCE sensor system has a good linear relationship with the concentration of 4-FMA. The electrochemiluminescence method used in the present invention 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 trace quantitative analysis of 4-FMA in electronic cigarettes.

[0076] Taking the above ideal embodiments of the present invention as inspiration, through the above description, relevant workers can make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An electrochemical luminescence aptasensor based on a DNA-Cy7 / MMST / GCE modified electrode, characterized in that, The DNA-Cy7 / MMST / GCE modified electrode is a modified electrode obtained by sequentially modifying MMST and DNA-Cy7 on the surface of GCE.

2. The electrochemical luminescence aptamer sensor based on a DNA-Cy7 / MMST / GCE modified electrode according to claim 1, wherein The preparation method of the MMST includes the following steps: Disperse TCPP NPs evenly in an organic solvent, then inject it into ultrapure water containing MMS, stir and mix evenly, and then obtain MMST by freeze-drying.

3. An electrochemiluminescence aptamer sensor based on a DNA-Cy7 / MMST / GCE modified electrode according to claim 2, wherein The preparation method of the MMS includes the following steps: Disperse MIL-101(Fe) evenly in ultrapure water to obtain a dispersion, then add ammonium molybdate and thiourea to the dispersion, stir and mix evenly, transfer it to a high-pressure reaction kettle, and heat it at a constant temperature of 180 °C for reaction. After the reaction is completed, the reaction solution is subjected to solid-liquid separation and vacuum drying in sequence to obtain MMS.

4. An electrochemical luminescence aptasensor based on a DNA-Cy7 / MMST / GCE modified electrode according to claim 3, characterized in that, The preparation method of the MIL-101(Fe) includes the following steps: Dissolve trivalent water-soluble iron salt in an organic solvent to form a homogeneous solution, then add terephthalic acid to the homogeneous solution, stir evenly, transfer it to a high-pressure reaction kettle, and then react at a high temperature. After the reaction is completed, the reaction solution is subjected to solid-liquid separation and vacuum drying to obtain MIL-100(Fe).

5. An electrochemiluminescence aptasensor based on a DNA-Cy7 / MMST / GCE modified electrode according to claim 1, characterized in that, The DNA-Cy7 is a compound formed by adding DNA and Cy7 to a 0.05 mol / L Tris-HCl buffer solution containing KCl, NaCl, MgCl2 and ethylenediaminetetraacetic acid to form a homogeneous aptamer dispersion, and then coating it on the electrode surface and drying.

6. An electrochemiluminescence aptasensor based on a DNA-Cy7 / MMST / GCE modified electrode according to claim 5, wherein, The DNA is single-stranded DNA, and the DNA sequence is 5'-AGG AAT TCA GAT CTC CCT GCA GGTGGT GTT TTT TTG TGT GCT GTG TTT TTG TGA TGC ATG CTC GAG GAG CTC AGG ATC CCG-NH2-3'.

7. An electrochemiluminescence aptamer sensor, characterized in that, Use the DNA-Cy7 / MMST / GCE modified electrode described in any one of claims 1-6 as its working electrode.

8. An electrochemiluminescent aptamer sensor according to claim 7, characterized in that, During electrochemical detection, it is a three-electrode system. The reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum electrode.

9. The electrochemiluminescent aptamer sensor according to claim 7, characterized in that During electrochemical detection, use a PBS buffer solution containing K2S2O8 with a pH of 7.4 as the electrolyte, and the concentration of K2S2O8 in the PBS buffer solution is 0.05 mol / L.

10. An electrochemiluminescent aptamer sensor according to claim 7, characterized in that, During electrochemical detection, the electrochemical window for cyclic voltammetry scanning is -1.0 to 0 V, the high voltage of the photomultiplier tube is 800 V, and the scanning rate is 0.1 V / s.

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