Adapte sensor for detecting fentanyl based on electrochemiluminescence and application thereof

Abstract

The application belongs to the field of electrochemiluminescence and provides an electrochemiluminescence aptamer sensor for detecting furan fentanyl as well as preparation and application thereof. Based on the resonance energy transfer mechanism between Tg-CNNSs / Co3O4 / NiCo2O4 and Zn-PTC, cDNA is loaded on the Tg-CNNSs / Co3O4 / NiCo2O4 modified electrode, apt-Zn-PTC is drop-coated, apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE is prepared as a working electrode, a three-electrode system is formed, and furan fentanyl is electrochemiluminescence detected. The application realizes specific detection of furan fentanyl by competition of furan fentanyl and cDNA, shedding of apt / Zn-PTC from the electrode surface, and then restoring of the ECL signal. The application is simple in operation, high in sensitivity and wide in linear range.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of electrochemiluminescence, and particularly relates to a preparation method of an electrochemiluminescence aptamer sensor for detecting fentanyl with Tg-CNNSs / Co3O4 / NiCo2O4 as a donor and Zn-PTC as an acceptor and application thereof. BACKGROUND

[0002] Opioids, especially fentanyl and its derivatives, have become an important part of new psychoactive substances due to their strong analgesic effect and low-cost production method. Although fentanyl was originally developed for clinical analgesia, due to its 50-100 times more potent than morphine, only a small amount can produce serious side effects and even lead to death. Therefore, the abuse of fentanyl is increasingly serious, becoming a new street drug and a doping agent for other drugs, which greatly threatens social stability and public health. In order to meet this challenge, there is an urgent need for efficient and accurate detection methods to identify and quantify fentanyl and its analogues. At present, a variety of methods have been used for the detection of fentanyl, including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), ultra-high performance liquid chromatography (UHPLC), and high performance liquid chromatography-mass spectrometry (HPLC-MS). However, these methods usually require the joint testing of multiple instruments and skilled operation skills, consuming a lot of manpower and material resources. Therefore, it is particularly important to develop a detection method with high sensitivity and simple operation.

[0003] Electrochemiluminescence (ECL) technology combines the advantages of spectroscopy and electrochemistry, and has the advantages of low background signal, high sensitivity and wide detection range, and is widely used in the detection of reagent samples. ECL sensors have shown great potential in improving detection sensitivity through signal amplification strategies, and many signal amplification methods have been developed, such as signal amplification through quenching signal and signal amplification using nanomaterials. These methods provide new ideas and means for the detection of fentanyl and its derivatives, and are expected to solve the shortcomings of existing detection methods and provide more efficient and convenient detection schemes. SUMMARY

[0004] The present application provides a method for detecting fentanyl by an electrochemiluminescence sensor, based on the resonance energy transfer mechanism between Tg-CNNSs / Co3O4 / NiCo2O4 and Zn-PTC, and taking Tg-CNNSs as a luminophore and Co3O4 / NiCo2O4 as a catalyst to improve the ECL performance and stability of Tg-CNNSs. Figure 2 ) Based on the different concentrations of the detected fentanyl and the different intensities of the recovered electrochemiluminescence, the detection of fentanyl is realized. Due to the specific binding between the detected fentanyl and cDNA, the sensor has good selectivity.

[0005] The present application adopts the scheme of taking apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 as a working electrode, platinum as an auxiliary electrode, and Ag / AgCl as a reference electrode to construct a three-electrode system, and the specific steps are as follows:

[0006] (1) Preparation method of Tg-CNNSs / Co3O4 / NiCo2O4 composite material:

[0007] Melamine and water are added to a beaker, and after ultrasonic treatment for 30-60 min, they are transferred to a polytetrafluoroethylene reaction kettle. Heat in a 200℃ oven for 12-14 hours to obtain white tubular carbon nitride (T-CN) precursor; wash the precursor with deionized water and dry in a 60℃ vacuum drying oven. The white powder is transferred to an alumina crucible and heated in a muffle furnace at a heating rate of 10℃ / min for 4-5h to obtain light yellow T-CN. Place T-CN in a quartz boat in a 650℃ tube furnace under nitrogen atmosphere at a heating rate of 2℃ / min for 2-3h, and then cool naturally. Porous carbon nitride nanosheets (Tg-CNNSs) are obtained.

[0008] Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to form a pink solution; 2-methylimidazole was dissolved in methanol solution. The two solutions were mixed, and the product was vacuum dried at 60℃ after standing at room temperature for 22h-26h, to obtain ZIF-67. ZIF-67 and Ni(NO3)2·6H2O were dispersed in ethanol at a mass ratio of 1-2:2-3, and ZIF-67 / LDH particles were formed after stirring for 1h-2h, and were collected by centrifugation and dried at 60℃. The ZIF-67 / LDH precursor was annealed in air at 350℃ for 2-2.5h, with a heating rate of 1℃ / min, to form Co3O4 / NiCo2O4 double-shell hollow nanocages.

[0009] The mass ratio of cobalt nitrate hexahydrate to 2-methylimidazole was 1-2:2-3.

[0010] Tg-CNNSs and Co3O4 / NiCo2O4 were ultrasonically mixed at a mass ratio of 1:5-1:7 to form Tg-CNNSs / Co3O4 / NiCo2O4.

[0011] (2) Preparation method of apt / Zn-PTC

[0012] Perylene-3,4,9,10-tetracarboxylic dianhydride and KOH were dissolved in H2O at a molar ratio of 1:5-1:6 and stirred for 12-14h. Next, ethanol was added to obtain K4PTC, and the volume ratio of H2O to ethanol was 4-5:30-40. Then, K4PTC and Zn(CH3COO)2 were dissolved in 15mL ultrapure water at a molar ratio of 0.04-0.06:0.09-0.12 and stirred for 30-60min. Then, the mixture was heated at 100℃ for 12-14h. After washing with H2O, Zn-PTC was collected by centrifugation.

[0013] The volume ratio of H2O to ethanol was 4-5:30-40.

[0014] The molar ratio of K4PTC to Zn(CH3COO)2 was 0.04-0.06:0.09-0.12.

[0015] Zn-PTC was incubated with 1-5μM aptamer (apt) on a shaker for 7-8h to obtain apt / Zn-PTC.

[0016] (3) Preparation of apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE

[0017] The surface of the glassy carbon electrode (the inner core of the glassy carbon electrode is 3 mm) was polished with 0.3 μm alumina polishing powder, then ultrasonically cleaned with ethanol and ultrapure water, and then dried under an incandescent lamp after the polishing powder remaining on the surface of the electrode was removed. 5-7 μL of Tg-CNNSs / Co3O4 / NiCo2O4 was dropped on the surface of the treated electrode, then dried under an incandescent lamp, and then 3-5 μL of 1-5 μM cDNA was incubated on the surface of the electrode for 6-10 h, the DNA sequence of the cDNA being 5'-GTCGTAAGTTCTGCC-3', and then 3-5 μL of apt / Zn-PTC was dropped on the surface of the electrode to combine with the cDNA, thus obtaining an apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCEECL sensor, the DNA sequence of the apt adapter being 5'-TGGCAGAACTTACGACACTGGCAGGAGGGTCGGGTGTGGGGGAACGTGGTCGTAAG-3'.

[0018] In the Tg-CNNSs / Co3O4 / NiCo2O4 composite dispersion liquid, Tg-CNNSs and Co3O4 / NiCo2O4 were mixed in a mass ratio of 1:5-1:7 to prepare 1 mL of a solution (the amount of Tg-CNNSs added was 0.05 mg), and the modification amount was 3-5 μL; the concentrations of cDNA and apt were both 1-5 μM, the modification amount of cDNA was 3-5 μL, and 1-5 μM of apt was used to prepare a 1 mg / mL dispersion liquid of Zn-PTC. The modification amount of Tg-CNNSs / Co3O4 / NiCo2O4 and apt / Zn-PTC was 3-5 μL.

[0019] (4) Preparation of a PBS buffer solution containing K2S2O8

[0020] A PBS (0.1 mol / L) solution with pH = 8.0 was used to prepare a buffer solution containing 0.1 mol / L K2S2O8.

[0021] (5) Preparation of fentanyl standard solutions with different concentrations

[0022] Using the solution of step (4), a fentanyl solution with a concentration of 0.1 mg / ml was diluted to prepare a series of fentanyl standard solutions with different concentrations. The concentration range was: 1.0 x 10 -7 g / L-1.0 x 10 -14 g / L.

[0023] (6) Drawing of a standard curve

[0024] The apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE is used as a working electrode, Ag / AgCl is used as a reference electrode, and a platinum electrode is used as a counter electrode to form a three-electrode system, 5-7 muL of furanylfentanyl with different concentrations (1.0*10 -14 g / L-1.0*10 -7 g / L) is added on the surface of the electrode for specific recognition, the PBS buffer solution in step (4) is used as an electrolyte, cyclic voltammetry scanning is carried out in an electrochemical window range of-1.2-0V, the high voltage of a photomultiplier tube is 710V, the amplification order is 3, and the scanning speed is 0.1V / s, and the linear regression equation for detecting furanylfentanyl can be obtained by recording the logarithmic value (logC) of the standard solution concentration and the difference (DeltaECL) between the luminescence intensities before and after the furanylfentanyl is added.

[0025] (7) Actual sample detection: 5-7 muL of a reagent sample containing furanylfentanyl is added on the ECL aptamer sensor apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE constructed, the apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE is used as a working electrode, and the luminescence intensity is detected by using the method in step (6). The concentration of furanylfentanyl in the actual sample can be obtained according to the difference (DeltaECL) between the luminescence intensities before and after the sample is added from the linear regression equation curve corresponding to step (6).

[0026] The working electrode of the present application is apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, when FUF is tested, the FUF needs to be quantitatively added on the apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, put into a solution, and electrochemiluminescence measurement is carried out, and the difference between the two ECLs is DeltaECL.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application relates to an electrochemiluminescence sensor based on ultrathin nanosheet Tg-CNNSs and Zn-PTC quenching. Co3O4 / NiCo2O4 is used as a stabilizer and a promoter to obtain high-efficiency and stable electrochemiluminescence performance. Furanylfentanyl and cDNA compete to combine with apt, and the sensitive detection of furanylfentanyl is realized. The detection range of the present application is 1.0*10 -14 -1.0*10 -7 mg / mL, and the minimum detection limit is 5.7*10 -15 mg / mL. The aptamer sensor shows very good performance in the detection of furanylfentanyl, and also provides a new method for the detection of new psychoactive substances. Attached Figure Description

[0029] Figure 1 A simplified flowchart of the sensor fabrication process and its application in the detection of furanylfentanyl in this invention.

[0030] Figure 2 The electrochemiluminescence response diagrams are shown in 0.1 M K₂S₂O₈ and 0.1 M PBS (pH = 8): a. cDNA / Tg-CNNSs / Co₃O₄ / NiCo₂O₄ / GCE; b. apt-Zn-PTC / cDNA / Tg-CNNSs / Co₃O₄ / NiCo₂O₄ / GCE; c. FUF / apt-Zn-PTC / cDNA / Tg-CNNSs / Co₃O₄ / NiCo₂O₄ / GCE.

[0031] Figure 3 Standard operating curves for the electrochemiluminescence sensor apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE in different concentrations of furanylfentanyl. The furanylfentanyl concentrations were 1.0 × 10⁻⁶. -14 g / L~1.0×10 -7 g / L.

[0032] Figure 4 CV curves and linear relationships for Tg-CNNSs and Tg-CNNSs / Co3O4 / NiCo2O4 modified electrodes; (A) Tg-CNNSs modified electrode and (B) Tg-CNNSs / Co3O4 / NiCo2O4 modified electrode, 5.0 mmol·L -1 [Fe(CN)6] 4- / 3- The scan rate is 25-250 mV·s -1 .

[0033] Figure 5 The selectivity of the ECL aptamer sensor for RCS-4, 2-chlorodiazepam, fluazolomide, isobutylfentanyl and furanylfentanyl.

[0034] Figure 6 ECL values ​​for Tg-CNNSs and Co3O4 / NiCo2O4 at different mass ratios. Detailed Implementation

[0035] To further illustrate the technical methods and detection effects adopted in this invention, a series of embodiments are provided in detail. In this embodiment, the concentration of the furanylfentanyl standard solution is (a) 1.0 × 10⁻⁶. -14 g / L; (b) 1.0×10 -13 g / L; (c) 1.0×10 -12g / L; (d) 1.0 x 10 -11 g / L; (e) 1.0 x 10 -10 g / L; (f) 1.0 x 10 -9 g / L; (g) 1.0 x 10 - 8 g / L; (h) 1.0 x 10 -7 g / L.cDNA and apt were purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and the DNA sequence of cDNA was 5'-GTCGTAAGTTCTGCC-3'; the DNA sequence of apt was 5'-TGGCAGAACTTACGACACTGGCAGGAGGGTCGGGTGTGGGGGAACGTGGTCGTAAG-3'.

[0036] Example 1: apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE for detection of fentanyl

[0037] (1) Preparation of Tg-CNNSs / Co3O4 / NiCo2O4 / GCE

[0038] 5 g of melamine and 20 mL of water were added to a beaker, and after ultrasonic treatment for 30-60 min, they were transferred to a 50 mL polytetrafluoroethylene reaction kettle. They were heated in an oven at 200°C for 12-14 h to obtain a white tubular carbon nitride (T-CN) precursor, which was washed thoroughly with deionized water and then dried in a vacuum drying box at 60°C. Finally, the white powder was transferred to an alumina crucible and heated in a muffle furnace at a temperature increasing rate of 10°C / min for 4-5 h at 500°C to obtain a light yellow T-CN. The T-CN was placed in a quartz boat and placed in a tube furnace at 650°C under the protection of a nitrogen atmosphere at a temperature increasing rate of 2°C / min for 2-3 h, and then naturally cooled. Carbon nitride nanosheets (Tg-CNNSs) were obtained.

[0039] 0.182 g of cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in 10 mL of methanol to form a pink solution; 0.205 g of 2-methylimidazole was dissolved in 10 mL of methanol solution. The above two solutions were mixed, and after standing at room temperature for 22-26 h, the product was dried at 60°C under vacuum to obtain ZIF-67. ZIF-67 and Ni(NO3)2·6H2O were dispersed in ethanol at a mass ratio of 1:2. After stirring for 1-2 h, ZIF-67 / LDH particles were formed, which were collected by centrifugation and dried at 60°C. The ZIF-67 / LDH precursor was annealed in air at 350°C for 2-2.5 h at a temperature increasing rate of 1°C / min to form Co3O4 / NiCo2O4 double-shell hollow nanocages.

[0040] Tg-CNNSs were mixed with Co3O4 / NiCo2O4 at a mass ratio of 1:5 by ultrasonic mixing to form Tg-CNNSs / Co3O4 / NiCo2O4

[0041] (2) Preparation of apt / Zn-PTC

[0042] 392.32 mg of perylene-3,4,9,10-tetracarboxylic dianhydride and 56.11 mg of KOH were dissolved in 4-5 mL of H2O and stirred for 12-14 hours. Next, 30-40 mL of ethanol was added to obtain K4PTC. Then, 87 mg of K4PTC was dissolved in a Zn(CH3COO)2 solution (15 mL, 0.007 mol / L) and stirred for 30-60 minutes. Then, the mixture was heated at 100°C for 12-14 hours. After washing with H2O, the Zn-PTC was collected by centrifugation.

[0043] The apt / Zn-PTC was obtained by incubating the Zn-PTC with 3 μM of aptamer for 7-8 hours.

[0044] (3) Preparation of apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE

[0045] After the electrode surface was polished with 0.3 μm alumina polishing powder and ultrasonically cleaned with ethanol and ultrapure water, the polishing powder remaining on the electrode surface was removed, and the treated electrode was dried under an incandescent lamp for standby. 5 μL of Tg-CNNSs / Co3O4 / NiCo2O4 was dropped on the treated electrode surface, and the electrode was dried under an incandescent lamp. Then, 3 μL of 2 μM cDNA was incubated on the electrode surface for 6-10 hours, and then 5 μL of apt / Zn-PTC was dropped on the electrode surface to bind with the cDNA, thereby obtaining an apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE ECL sensor.

[0046] (4) Preparation of standard curve

[0047] 7 μL of fentanyl (FUF) with different concentrations (concentration range: 1.0 x 10 -7 g / L-1.0 x 10 -14After 7 μL of the FUF / apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE electrode was dropped on the above electrochemiluminescence sensor and incubated for 2 h, a three-electrode system was formed with the FUF / apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE electrode as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode, and a PBS buffer solution containing 0.1 mol / L K2S2O8 and having a pH of 8 was used as the electrolyte. In an electrochemical window range of -1.2-0 V, a photomultiplier high voltage of 710 V, and a scanning speed of 0.1 V / s, cyclic voltammetry scanning was performed, and a linear relationship between the difference in luminescence intensity (ΔECL) of the electrochemiluminescence aptamer sensor before and after the combination of furan fentanyl and the logarithmic value of the furan fentanyl concentration in the furan fentanyl standard solution was established, to obtain a corresponding linear regression equation; ΔECL = 16871.7 + 1123.58 log C (g / L). The detection linear range was 1.0 x 10 -14 ~1.0 x 10 -7 g / L, and the detection limit was 5.7 x 10 -15 g / L. Figure 3

[0048] (5) Detection of samples

[0049] The constructed sensor was used for the detection of furan fentanyl in mineral water and fruit wine. The mineral water purchased from a supermarket was directly used without purification. The fruit wine was a commercial product purchased from a supermarket and stored in a refrigerator below -4°C before use. After the possible solid substances were removed by centrifugation at 10000 rpm, the supernatant was used. 7 μL of the actual sample was dropped on the apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, and after incubation for 2-3 h, the electrode was used as the working electrode. The difference in luminescence intensity (ΔECL) of the electrochemiluminescence aptamer sensor before and after the combination of furan fentanyl was determined, and the concentration of furan fentanyl in the actual sample corresponding to ΔECL was obtained according to the linear regression equation. The analysis results are shown in Table 1.

[0050] Figure 2 Electrochemiluminescence response diagrams of a.cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE; b.apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE; c.FUF / apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE in 0.1 M K2S2O8 and 0.1 M PBS (pH = 8) Figure 2 ​As can be seen from the curve, the ECL value of cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE is approximately 13500 a.u. (curve a). When apt-Zn-PTC is added in layers to the surface of cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE to form apt-Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, the ECL value is severely quenched due to the quenching effect of Zn-PTC, dropping to approximately 580 a.u. (curve b). When the analyte FUF is added to the electrode surface, FUF competes with cDNA and binds more strongly to apt, causing apt-Zn-PTC to detach from the electrode surface. The resulting electrochemiluminescence quenching is partially recovered (curve c), thus demonstrating the successful construction of the aptamer sensor.

[0051] Figure 4 For (A) Tg-CNNSs modified electrode and (B) Tg-CNNSs / Co3O4 / NiCo2O4 modified electrode, 5.0 mmol·L -1 [Fe(CN)6] 4- / 3- The scan rate is 25-250 mV·s -1 The electrochemically active area of ​​Tg-CNNSs is 0.064 cm². 2 ( Figure 4 A) The electrochemical active area of ​​Tg-CNNSs / Co3O4 / NiCo2O4 increased to 0.074 cm². 2 ( Figure 4 B) indicates that Co3O4 / NiCo2O4 can act as a catalyst for Tg-CNNSs, providing more active sites and improving the stability of Tg-CNNSs. Simultaneously, it can act as a co-reaction promoter, accelerating the decomposition of K2S2O8 and increasing the luminescence intensity.

[0052] The selectivity of ECL aptamer sensors for RCS-4, 2-chlorodiazepam, fluazolam, isobutylfentanyl, and furanylfentanyl, such as Figure 5 As shown, the results indicate that the modified ECL aptamer sensor has good selectivity for furanylfentanyl.

[0053] The effect of the mass ratio of Tg-CNNSs to Co3O4 / NiCo2O4 on ECL values ​​was studied. Results for mass ratios of 1:2, 1:4, 1:5, 1:6, and 1:8 are shown below. Figure 6 As shown, the ECL value reaches its maximum when the mass ratio is 1:5, indicating that the mass ratio of Tg-CNNSs to Co3O4 / NiCo2O4 is optimal at this time.

[0054] Comparison Implementation 1

[0055] (1) Preparation of Tg-CNNSs / Co3O4 / NiCo2O4 / GCE

[0056] Take 3-5 μL of Tg-CNNSs / Co3O4 / NiCo2O4 mass ratio of 1:5 in ultrapure water dispersion solution, drop-coat on the surface of the pretreated glassy carbon electrode (pretreatment method same as example 1), and dry under the incandescent lamp to obtain Tg-CNNSs / Co3O4 / NiCo2O4 / GCE ECL sensor.

[0057] (2) Preparation of standard curve

[0058] After 5-7 μL of furan fentanyl of different concentrations is drop-coated on the above-mentioned electrochemiluminescence sensor and incubated for 2-3 h, a three-electrode system is formed with the electrode as the working electrode, Ag / AgCl as the reference electrode, and platinum electrode as the counter electrode, a PBS buffer solution containing 0.1 mol / L K2S2O8 and having pH of 8 is used as the electrolyte, cyclic voltammetry scanning is performed in the electrochemical window range of -1.2-0 V, the high voltage of the photomultiplier tube is 710 V, and the scanning speed is 0.1 V / s, the linear relationship between the light intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after binding with furan fentanyl and the logarithmic value of the furan fentanyl concentration in the furan fentanyl standard solution is established, and the corresponding linear regression equation is obtained.

[0059] (3) Detection of sample

[0060] The constructed sensor is used for detection of furan fentanyl in mineral water and fruit wine. The mineral water purchased from the supermarket does not need to be purified and can be directly used. The fruit wine is a commercial product purchased from the supermarket and is stored in a refrigerator below -4°C before use. After centrifugation at 10000 rpm to remove possible solid substances, the supernatant is taken for standby use. 5-7 μL of the actual sample is drop-coated on the Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, incubated for 2-3 h, and then the electrode is used as the working electrode. The light intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after binding with furan fentanyl is determined, and the concentration of furan fentanyl in the actual sample corresponding to ΔECL can be obtained according to the linear regression equation. The analysis results are shown in Table 1.

[0061] Comparative Example 2

[0062] In Comparative Example 2, apt is incubated on the surface of the Tg-CNNSs / Co3O4 / NiCo2O4 / GCE electrode for 6-10 h to obtain apt-Tg-CNNSs / Co3O4 / NiCo2O4 / GCE ECL sensor, and other operations are the same as those in Comparative Example 1.

[0063] (1) Preparation of apt-Zn-PTC / GCE

[0064] Take 3-5 μL apt-Zn-PTC and drop it on the surface of the pretreated glassy carbon electrode (the pretreatment method is the same as that in Example 1), and dry it under the incandescent lamp, to obtain the apt-Zn-PTC / GCE ECL sensor.

[0065] (2) Preparation of standard curve

[0066] Take 5-7 μL furanconitine of different concentrations and drop it on the above-mentioned electrochemiluminescence sensor and incubate it for 2-3 h, then take the electrode as the working electrode, Ag / AgCl as the reference electrode, and platinum electrode as the counter electrode to form a three-electrode system, take PBS buffer solution containing 0.1 mol / L K2S2O8 with pH of 8 as the electrolyte, carry out cyclic voltammetry scanning in the electrochemical window range of -1.2-0 V, the high voltage of photomultiplier is 710 V, and the scanning speed is 0.1 V / s, to establish the linear relationship between the light intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after combining with furanconitine and the logarithmic value of the furanconitine concentration in the furanconitine standard solution, and obtain the corresponding linear regression equation.

[0067] (3) Detection of samples

[0068] The constructed sensor is used for the detection of furanconitine in mineral water and fruit wine. The mineral water purchased from the supermarket does not need to be purified and can be directly used. The fruit wine is a commercial product purchased from the supermarket and is stored in a refrigerator below -4°C before use. After centrifugation at 10000 rpm to remove the possible solid substances, the supernatant is taken for standby use. Take 5-7 μL of the actual sample and drop it on the apt / Zn-PTC / GCE, incubate it for 2-3 h, then take the electrode as the working electrode, and determine the light intensity difference (ΔECL) of the electrochemiluminescence aptamer sensor before and after combining with furanconitine. According to the linear regression equation, the concentration of furanconitine contained in the actual sample corresponding to ΔECL can be obtained. The analysis results are shown in Table 1.

[0069] Table 1 Determination results of furanconitine in real samples

[0070]

[0071]

[0072] Note: a The average value of three determinations

[0073] As shown in Table 1, the sample is determined in parallel for three times. In the example, the standard addition recovery rate is between 94%-102%, and the relative standard deviation is less than 3%, indicating that the recovery effect is good. The above experimental results show that the constructed electrochemiluminescence aptamer sensor can be used for the detection of furanconitine in real samples without the need of labeling and separation.

[0074] apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4, while the sensor system of Tg-CNNSs / Co3O4 / NiCo2O4 or apt / Zn-PTC alone cannot detect fentanyl, so the sensor of the application can be used to detect fentanyl in water environment.

[0075] Based on the above verification, it is known that the application is based on the electrochemiluminescence quenching effect between Tg-CNNSs / Co3O4 / NiCo2O4 and Zn-PTC, and a new method for rapid and sensitive detection of fentanyl is constructed. Since cDNA and fentanyl will competitively bind to apt, when there is a trace amount of fentanyl, fentanyl will bind to apt, causing apt-Zn-PTC to fall off from the electrode surface, and thus the electrochemiluminescence quenching caused by the apt-Zn-PTC modified on the surface of Tg-CNNSs / Co3O4 / NiCo2O4 electrode will be partially restored. Based on the different concentrations of the detected fentanyl and the intensity of electrochemiluminescence recovery, the detection of fentanyl is realized. Through research, it is found that

[0076] The enhanced value (ΔECL) of the ECL signal of the apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 sensor system and the concentration of fentanyl show a good linear relationship. The electrochemiluminescence method used in the application not only has the advantages of high sensitivity, fast detection speed, good selectivity (ΔECL) and wide linear range, but also has great application potential for quantitative analysis of fentanyl. Figure 5 ) and linear range, and great application potential for quantitative analysis of fentanyl.

[0077] The above examples are only used to illustrate the use of the application and are not a limitation of the application. Those skilled in the art can make corresponding changes without departing from the scope of the application, so all equivalent replacements or equivalent variations formed by technical solutions belong to the protection scope of the application.

Claims

1. An aptamer sensor for detecting furan fentanyl based on electrochemiluminescence method, characterized in that: The sensor is modified on the surface of a glassy carbon electrode by apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4, to obtain a modified electrode, which is used as a working electrode for electrochemiluminescence test, to form a three-electrode system for detecting fentanyl by electrochemiluminescence method; wherein the DNA sequence of cDNA is: 5'-GTCGTAAGTTCTGCC-3'; and the DNA sequence of apt is: 5'-TGGCAGAACTTACGACACTGGCAGGAGGGTCGGGTGTGGGGGAACGTGGTCGTAAG-3'; The preparation method of the aptamer sensor comprises the following steps: (1) preparing Tg-CNNSs / Co3O4 / NiCo2O4 composite material; (2) preparing apt / Zn-PTC, incubating Zn-PTC with aptamer on a shaker to obtain apt / Zn-PTC; (3) dropping Tg-CNNSs / Co3O4 / NiCo2O4 on the surface of a treated glassy carbon electrode, drying, incubating cDNA on the surface of the dried electrode, then dropping apt / Zn-PTC on the surface of the electrode to combine with cDNA, to obtain the aptamer sensor apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE; The preparation method of the Tg-CNNSs / Co3O4 / NiCo2O4 composite material is as follows: (1) mixing melamine and water, ultrasonic treatment, and then transferring into a reaction kettle, heating at 200 DEG C for 12-14 hours to obtain tubular carbon nitride precursor; after washing and drying, heating at 500 DEG C in a muffle furnace for 4-5 hours to obtain T-CN; placing T-CN in a 650 DEG C tube furnace, heating for 2-3 hours under the protection of nitrogen atmosphere to obtain Tg-CNNSs; (2) dispersing ZIF-67 and Ni(NO3)2·6H2O in ethanol at a mass ratio of 1-2:2-3, stirring for 1-2 hours to form ZIF-67 / LDH particles, centrifuging, drying, annealing the ZIF-67 / LDH precursor in air at 350 DEG C for 2-2.5 hours to generate Co3O4 / NiCo2O4 double-shell hollow nanocage; (3) ultrasonic mixing Tg-CNNSs and Co3O4 / NiCo2O4 at a mass ratio of 1:5-1:7 to form Tg-CNNSs / Co3O4 / NiCo2O4 composite material; The preparation method of the apt / Zn-PTC is as follows: perylene-3,4,9,10-tetracarboxylic dianhydride and KOH are dissolved in water at a molar ratio of 1:5-1:6, and after stirring and mixing, ethanol is added to obtain K4PTC; K4PTC and Zn(CH3COO)2 are dissolved in ultrapure water at a molar ratio of 0.04-0.06:0.09-0.12 and stirred and mixed, and the mixture is heated at 100 ℃ for 12-14 h to obtain Zn-PTC; the Zn-PTC is incubated with 1-5 μM of aptamer on a shaking bed for 7-8 h to obtain apt / Zn-PTC.

2. Application of the aptamer sensor of claim 1 in electrochemiluminescence detection of furan fentanyl.

3. Use according to claim 2, characterized in that, The specific detection steps are as follows: (1) Preparation of PBS buffer solution containing K2S2O8: a PBS buffer solution containing 0.1 mol / L K2S2O8 is prepared by using pH=8.0 PBS; (2) Preparation of different concentrations of fentanyl standard solution: using the buffer solution of step (1), dilute the fentanyl solution with a concentration of 0.1 mg / ml into a series of different concentrations of fentanyl standard solution, the concentration range is 1.0 x 10 -7 g / L ~ 1.0 x 10 -14 g / L; (3) Preparation of standard curve: apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / 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, different concentrations of furan fentanyl are added to the surface of the electrode for specific recognition, the buffer solution in step (1) is used as an electrolyte, cyclic voltammetry scanning is performed, and the logarithmic value log C of the standard solution concentration and the difference ΔECL of the luminescence intensity before and after the addition of furan fentanyl are recorded to obtain a linear regression equation for detecting furan fentanyl; (4) Actual sample detection: a reagent sample containing furan fentanyl is added to the constructed ECL aptamer sensor apt / Zn-PTC / cDNA / Tg-CNNSs / Co3O4 / NiCo2O4 / GCE, which is used as a working electrode, and the luminescence intensity is detected by the method in step (3); the concentration of furan fentanyl in the actual sample is obtained according to the difference in luminescence intensity before and after the addition of the sample from the linear regression equation curve in step (3).

4. Use according to claim 3, characterized in that: The conditions for cyclic voltammetry scanning are as follows: in the electrochemical window range of-1.2-0 V, the high voltage of the photomultiplier tube is 710 V, the amplification factor is 3, and the scanning speed is 0.1 V / s.

Images