Electrochemiluminescent aptamer sensor for specific detection of enrofloxacin and preparation method and application thereof

Abstract

The present invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for specific detection of enrofloxacin, as well as a preparation method and application. The sensor is formed by loading an enrofloxacin aptamer on the surface of a glassy carbon electrode modified with a NaYF4-PEI-PTCA nanocomposite material, wherein the NaYF4-PEI-PTCA nanocomposite material is a product of upconversion nanoparticles and perylene tetracarboxylic acid bound by an amide bond; the nucleotide sequence of the enrofloxacin aptamer is apt:5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3'. The sensor of the present invention can realize sensitive detection of enrofloxacin, and the minimum detection limit is 3.0×10 ‑15 mol / L.

Description

Technical Field

[0001] The present invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for specific detection of enrofloxacin, as well as a preparation method and application thereof. In particular, it relates to an electrochemiluminescence analysis method for quantitatively detecting enrofloxacin in river water, in which aptamer molecules are loaded on the surface of a glassy carbon electrode modified by a perylene derivative (NaYF4-PEI-PTCA) based on upconversion nanoparticles, i.e., using an apt / NaYF4-PEI-PTCA / GCE electrode as a sensing element. Background Art

[0002] Enrofloxacin (ENR) is a second-generation fluoroquinolone antibiotic. It is widely used in animals and aquaculture due to its broad antibacterial spectrum, strong bactericidal activity, few adverse reactions, and no cross-resistance with other antibiotics. However, long-term abuse leads to the accumulation of enrofloxacin in animals and enters the environment with animal excrement, causing environmental pollution. In addition, in aquaculture, a large amount of enrofloxacin remains in the water, and the discharge process also causes serious pollution to the water environment. Once the environment is polluted, allergic reactions and intestinal flora imbalance will occur, which seriously affect human health. At present, many countries and regions have stipulated the maximum residue of ENR in animal-derived foods. For example, the European Union stipulates that the maximum residue of ENR is 0.3μg / g, and my country stipulates that the maximum residue limit in the muscle and fat of all food animals is 100μg / kg. Based on this, it is of great significance to establish an efficient and sensitive method for detecting enrofloxacin residues.

[0003] At present, the methods reported for detecting enrofloxacin include high performance liquid chromatography, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay and photoelectric method. However, these analytical methods have many limitations in practical application. For example, although high performance liquid chromatography has high accuracy, the instrument is expensive and has low sensitivity; although liquid chromatography-mass spectrometry can be used for qualitative and quantitative detection, the instrument operation is highly professional and is not suitable for the processing of large quantities of samples and rapid on-site detection; enzyme-linked immunosorbent assay is only suitable for rapid screening of a large number of samples and is not suitable as a single detection method; although the photoelectric method can achieve high sensitivity, the disadvantage of this method is that it is greatly affected by the light source and has poor stability. Therefore, it is necessary and extremely important to develop a simple, rapid, selective, and external power-free electrochemiluminescence specific detection method for enrofloxacin.

[0004] Electrochemiluminescence (ECL), also known as electrochemiluminescence, is a combination and extension of electrochemical methods and chemiluminescence methods. It has the advantages of high sensitivity, wide linear range, convenient observation and simple instruments of electrochemical methods and good reproducibility, stable reagents and easy control of chemiluminescence methods. In addition, it is a method that does not require the introduction of external light sources, but collects luminescence intensity spectra with the assistance of optical instruments such as photomultiplier tubes, and establishes its relationship with the analyte to achieve microanalysis. Summary of the invention

[0005] The purpose of the present invention is to provide an electrochemiluminescent aptamer sensor for specific detection of enrofloxacin, as well as a preparation method and application thereof, in view of the deficiencies in the prior art for detection of enrofloxacin. The present invention is based on the amide bond binding between upconversion nanoparticles (NaYF4-PEI) and perylenetetracarboxylic acid (PTCA), thereby obtaining a NaYF4-PEI-PTCA nanocomposite material, and modifying it to the surface of a glassy carbon electrode, so that the sensitivity and stability of electrochemiluminescence are significantly improved, and then the aptamer is loaded through electrostatic action to obtain an electrochemiluminescent aptamer sensor (abbreviated as apt / NaYF4-PEI-PTCA / GCE sensor), which can specifically identify the target molecule enrofloxacin and improve the selectivity for enrofloxacin.

[0006] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0007] An electrochemiluminescent aptamer sensor for specific detection of enrofloxacin is prepared by loading an aptamer containing a 5'-CCC ATCAGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3' base sequence on the surface of a glassy carbon electrode modified with a NaYF4-PEI-PTCA nanocomposite material, wherein the NaYF4-PEI-PTCA nanocomposite material is a product of upconversion nanoparticles (NaYF4-PEI) and perylenetetracarboxylic acid (PTCA) bonded by amide bonds.

[0008] The present invention also provides a method for preparing the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin, comprising the following steps:

[0009] (1) Preparation of PTCA:

[0010] Perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) is dissolved in an aqueous solution of NaOH, and HCl solution is slowly added dropwise to the above solution to obtain a dark red flocculent precipitate. After centrifugation, washing and drying, a red solid, namely PTCA, is obtained.

[0011] (2) Preparation of hollow rod-shaped NaYF4-PEI:

[0012] Specifically: YCl3, YbCl3, ErCl3 and NaCl are mixed evenly, added to ethanol containing PEI, NH4F is dissolved in water and added to the mixed solution, stirred evenly, placed in a polytetrafluoroethylene reactor for heating reaction (preferably at 200°C for 24 hours), and a hollow rod-shaped white solid, namely NaYF4-PEI, is obtained by centrifugation, washing and drying.

[0013] (3) Preparation of NaYF4-PEI-PTCA:

[0014] The PTCA prepared in step (1) is dissolved in DMF to form a 1 mg / mL PTCA-DMF solution, which is stirred until uniform. EDC and NHS (m / m=4:1) are then added and stirred in an ice-water bath (the purpose is to allow the reaction to proceed at a constant temperature). NaYF4-PEI (m(PTCA) / m(NaYF4-PEI)=1:5) is then added and stirred (generally for 16 hours). After the two are fully reacted, the product, namely NaYF4-PEI-PTCA, is obtained by centrifugation, washing and drying.

[0015] (4) Preparation of apt / NaYF4-PEI-PTCA / GCE sensor:

[0016] Polish the glassy carbon electrode, ultrasonicate it with nitric acid, anhydrous ethanol and deionized water respectively, dry it naturally for use, transfer the ultrapure water dispersion of NaYF4-PEI-PTCA (1 mg / mL) and drop it on the surface of the clean glassy carbon electrode, dry it at room temperature, and obtain the glassy carbon electrode modified with NaYF4-PEI-PTCA; then, drop the aptamer onto the surface of NaYF4-PEI-PTCA / GCE, the aptamer and NaYF4-PEI-PTCA are electrostatically bonded, and then place the apt / NaYF4-PEI-PTCA / GCE modified electrode in the refrigerator for 6h-8h to obtain the ECL aptamer sensor. (The refrigerator placement time is the key influencing factor of the sensor performance, the temperature is not, and this application uses 4°C)

[0017] Finally, the present invention provides an application method of an electrochemiluminescent aptamer sensor for specific detection of enrofloxacin, comprising the following steps:

[0018] First, a series of enrofloxacin standard solutions with different concentrations were prepared. Enrofloxacin was prepared into a solution with water, and then added into a PBS buffer solution containing K2S2O8 to obtain a series of enrofloxacin standard solutions with different concentrations ranging from 1.0×10 -14 mol / L~1.0×10 -6 mol / L.

[0019] Then, a linear regression equation was established, and the apt / NaYF4-PEI-PTCA / GCE sensor was used as a working electrode, a platinum electrode was used as an auxiliary electrode, and Ag / AgCl was used as a reference electrode to form a three-electrode system. The three-electrode system was immersed in the above-mentioned series of enrofloxacin standard solutions with different concentrations and the PBS buffer solution containing K2S2O8. Within the electrochemical window range of -1.8 to 0V, a photomultiplier tube high voltage of 800V and a scan rate of 0.1V / s were used to perform cyclic voltammetry scanning, and a potential-luminescence intensity curve (E-ECL) was recorded. A linear relationship between the luminescence intensity difference before and after the addition of enrofloxacin and the logarithm of the enrofloxacin concentration was established to obtain a corresponding linear regression equation;

[0020] Finally, sample testing is carried out: the sample to be tested is first pre-treated (the pre-treatment mainly involves filtering impurities, for example, taking 200 mL of river water sample, letting it stand naturally for a period of time, centrifuging it at 6000 rpm for 10 min, aspirating the upper clear liquid, and filtering it through a 0.22 μm filter membrane), and then tested under the same electrochemiluminescence test conditions as mentioned above (the filtrate is prepared into a 0.1 mol / L PBS buffer solution (pH 7.4) containing 0.05 mol / L K2S2O8, and 25 mL of the resulting solution is placed in the detection cell), the luminescence intensity is recorded, and the obtained luminescence intensity is used to calculate the concentration of enrofloxacin in the sample to be tested using the linear regression equation corresponding to the obtained standard curve.

[0021] Furthermore, the preparation method of the PBS buffer solution containing K2S2O8 is as follows: using 0.1 mol / L PBS buffer solution with pH 7.4 to prepare a PBS buffer solution containing 0.05 mol / L K2S2O8.

[0022] Furthermore, in the electrochemiluminescence test, the photomultiplier tube high voltage was 800 V, the scanning rate was 0.1 V / s, and the immersion time was 30 min (the detection value was stable at 30 min).

[0023] Compared with the prior art, the present invention has achieved the following beneficial effects: the present invention designs an electrochemiluminescent aptamer sensor based on perylene derivatives of upconversion nanoparticles, and combines PTCA and NaYF4 with PEI as a bridge to obtain an electrochemiluminescent body with high intensity and stable performance. The present invention makes full use of the advantages of aptamers and electrochemiluminescent sensors, and realizes the sensitive detection of enrofloxacin through the mechanism of enrofloxacin enhancing the ECL signal intensity of the system. The detection range of the present invention is 1.0×10 -14 ~1.0×10 -6 mol / L, the minimum detection limit is 3.0×10 -15mol / L. The method for detecting enrofloxacin has the advantages of simple operation, good selectivity, low detection cost, high sensitivity and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are scanning electron micrographs of NaYF4-PEI (A), PTCA (B), and NaYF4-PEI-PTCA (C).

[0025] Figure 2 It is the ECL-potential curve of different concentrations of enrofloxacin.

[0026] The concentration of enrofloxacin is as follows from the peak of the curve from front to back: 1.0×10 -14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 -9 mol / L、1.0×10 -8 mol / L、1.0×10 -7 mol / L、1.0×10 -6 mol / L.

[0027] Figure 3 It is the standard curve of the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithm of the enrofloxacin concentration.

[0028] Figure 4 This is the specific detection of enrofloxacin by the sensor of Example 1. DETAILED DESCRIPTION

[0029] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any simple changes or modifications made to the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0030] The present invention is further described in detail below in conjunction with embodiments:

[0031] The embodiment provides an electrochemiluminescent aptamer sensor for specific detection of enrofloxacin, which is composed of an aptamer containing a 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATTTCA GGG GGA-3' base sequence loaded on the surface of a glassy carbon electrode modified with a NaYF4-PEI-PTCA nanocomposite material, wherein the NaYF4-PEI-PTCA nanocomposite material is a product of an upconversion nanoparticle (NaYF4-PEI) and perylenetetracarboxylic acid (PTCA) bonded by an amide bond, and can be obtained by the method described in Example 1, as follows:

[0032] Embodiment 1:

[0033] (1) Preparation of PTCA:

[0034] 0.1009 g of perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) was dissolved in an aqueous solution of NaOH (0.05 M), and a 1 M HCl solution was slowly added dropwise to the above solution until a dark red flocculent precipitate was observed. The red solid, i.e., PTCA, was obtained by centrifugation, washing, and drying.

[0035] (2) Preparation of NaYF4-PEI:

[0036] 8 mL YCl3, 1.8 mL YbCl3, 0.2 mL ErCl3 and TmCl3, 10 mL NaCl were mixed evenly, added to 60 mL ethanol containing 20 mL PEI, 0.12 g NH4F was dissolved in 10 mL water, added to the above mixed solution, stirred evenly, placed in a polytetrafluoroethylene reactor and heated at 200 ° C for 24 h. After centrifugation, washing and drying, a white solid, namely UCPNs-PEI, was obtained.

[0037] (3) Preparation of NaYF4-PEI-PTCA:

[0038] 10 mg PTCA was dissolved in 10 mL DMF, stirred for 1 h, then 40 mg EDC and 10 mg NHS were added, stirred in an ice-water bath for 4 h, then 50 mg NaYF4-PEI was added, stirred overnight, and the product, NaYF4-PEI-PTCA, was obtained by centrifugation, washing, and drying.

[0039] (4) Preparation of modified electrode apt / NaYF4-PEI-PTCA / GCE:

[0040] The glassy carbon electrode was polished, ultrasonicated with nitric acid, anhydrous ethanol and deionized water respectively, and dried naturally for use. A 5μL microinjector was used to transfer 4μL of NaYF4-PEI-PTCA solution and drop it on the surface of the clean glassy carbon electrode. It was dried at room temperature to obtain a glassy carbon electrode modified with NaYF4-PEI-PTCA. Next, 4μL of 3μmol / L aptamer was added to the surface of NaYF4-PEI-PTCA / GCE. The aptamer and NaYF4-PEI-PTCA were electrostatically bonded to obtain apt / NaYF4-PEI-PTCA / GCE. Finally, the apt / NaYF4-PEI-PTCA / GCE modified electrode was placed in a refrigerator at 4°C for 6h to obtain an ECL aptamer sensor.

[0041] The recognition molecule apt sequence is: apt:5'-HOOC-AGA TGG GGG TTG AGG CTA AGC CGA-3' (manufacturer is Sangon Biotech (Shanghai) Co., Ltd.)

[0042] (5) Drawing of standard curve

[0043] The modified electrode apt / NaYF4-PEI-PTCA / GCE was used as the working electrode, the platinum electrode as the auxiliary electrode, and the Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in a series of enrofloxacin concentrations (1.0×10 - 14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 - 9 mol / L、1.0×10 -8 mol / L、1.0×10 -7 mol / L and 1.0×10 -6 mol / L) in a buffer solution of 0.1 mol / L PBS at pH 7.4 containing 0.05 mol / L K2S2O8 for 30 min. Cyclic voltammetry was performed in the electrochemical window range of -1.8 to 0 V with a photomultiplier tube high voltage of 800 V and a scan rate of 0.1 V / s. The potential-luminescence intensity curve (E-ECL) was recorded. A linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithmic value of the enrofloxacin concentration was established, and the corresponding linear regression equation was obtained as follows: △I ECL=5149.83162+1001.84055LogC(mol / L), the correlation coefficient (R) is 0.9977. The detection range of the linear regression equation is 1.0×10 -14 ~1.0×10 -6 mol / L, the minimum detection limit is 3.0×10 -15 mol / L.

[0044] (6) Sample testing

[0045] A water sample from a certain river was taken, and after being naturally allowed to stand for a period of time, the upper solution was absorbed by centrifugation, and the filtrate was collected by filtration through a 0.22 μm filter membrane. A 0.1 mol / L PBS buffer solution containing 0.05 mol / L K2S2O8 was added to adjust the pH to 7.4, and 25 mL of the resulting solution was used for electrochemiluminescence analysis. The test was performed under the same electrochemiluminescence test conditions as step (4), and the luminescence intensity was recorded. The concentration of enrofloxacin in the sample to be tested was calculated according to the linear regression equation obtained in step (5). The results are listed in Table 1.

[0046] The significant advantage of the present invention is that an electrochemiluminescence sensor for detecting enrofloxacin and its preparation method have been developed. Compared with ordinary electrochemiluminescence sensors, it has the following two significant advantages: NaYF4-PEI and PTCA are tightly combined through amide bonds, and the synergistic effect of the two is reflected in ECL; perylene substances have good electrochemical activity, and upconversion nanoparticles (NaYF4) have excellent optical properties, such as high-intensity and stable cathode signals, and are a very promising new ECL luminophore. The invention fully utilizes the advantages of aptamers and electrochemiluminescence sensors, and successfully realizes sensitive detection of enrofloxacin through the mechanism of enrofloxacin enhancing the ECL signal intensity of the system. The sensing platform can specifically identify the detection object enrofloxacin and has high selectivity. The present invention is of great significance to the promotion of the practical application of aptamer sensors in water environment monitoring and other aspects.

[0047] The electrochemiluminescent sensor for detecting enrofloxacin prepared in Example 1 was used for anti-interference detection, wherein the working electrode after incubation with the aptamer was incubated at 10 -6 M Norfloxacin (NFX), ciprofloxacin (CIP), chloramphenicol (CAP), kanamycin (KAN), chlortetracycline (CTC), streptomycin (STP) interfere with the -8 The working electrode was tested in the mixed solution of the above substances. The test results are shown in Figure 4 .

[0048] from Figure 4It can be seen that the modified electrode with excellent electrochemical performance has a selective recognition effect on enrofloxacin after incubation of the aptamer, and the interference substance with a concentration of 100 times after mixing has little effect on the detection of enrofloxacin. Therefore, the working electrode can achieve the selective detection of enrofloxacin with anti-interference.

[0049] Comparative Example 1:

[0050] (1) Preparation of apt / NaYF4-PEI-PTCA / GCE modified electrode

[0051] The glassy carbon electrode was polished, ultrasonicated with nitric acid, anhydrous ethanol and deionized water respectively, and dried naturally for use. 4.0 μL of 1.0 mg / mL NaYF4-PEI-PTCA aqueous solution was pipetted with a microinjector and dropped on the surface of the clean glassy carbon electrode, dried at room temperature to obtain a NaYF4-PEI-PTCA / GCE modified electrode; 4.0 μL of 3.0 μM aptamer (same as Example 1) was then dropped on the surface of the NaYF4-PEI-PTCA / GCE modified electrode, and dried naturally for 4 hours to obtain an apt / NaYF4-PEI-PTCA / GCE sensor as a working electrode for electrochemiluminescence testing.

[0052] (2) Drawing of standard curve

[0053] The apt / NaYF4-PEI-PTCA / GCE modified electrode was used as the working electrode, the platinum electrode as the auxiliary electrode, and the Ag / AgCl as the reference electrode to form a three-electrode system. The luminescence intensity was detected using a 0.1 mol / L PBS buffer solution at pH 7.4 containing 0.05 mol / L K2S2O8 as the blank solution. The three-electrode system was placed in a series of enrofloxacin concentrations (1.0×10 - 14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 - 9 mol / L、1.0×10 -8 mol / L、1.0×10 -7 mol / L and 1.0×10 -6The cyclic voltammetry was performed in a 0.1 mol / L PBS buffer solution of pH 7.4 containing 0.05 mol / L K2S2O8 in the electrochemical window range of -1.8 to 0 V with a photomultiplier tube high voltage of 800 V and a scan rate of 0.1 V / s. The E-ECL curve was recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithm of the enrofloxacin concentration was established to obtain the corresponding linear regression equation.

[0054] (3) Sample testing

[0055] 25 mL of treated river water was added to a 0.1 mol / L PBS buffer solution at pH 7.4 containing 0.05 mol / L K2S2O8 for electrochemiluminescence detection. The concentration of enrofloxacin in the sample to be tested was calculated according to the linear regression equation corresponding to the above step (2). The results are listed in Table 1.

[0056] Comparative Example 2:

[0057] (1) Preparation of apt / NaYF4-PEI / GCE modified electrode

[0058] The glassy carbon electrode was polished, ultrasonicated with nitric acid, anhydrous ethanol and deionized water respectively, and dried naturally for use. 4.0 μL of 1.0 mg / mL NaYF4-PEI material aqueous solution was pipetted with a microinjector and dropped on the surface of the clean glassy carbon electrode, dried at room temperature, and a NaYF4-PEI / GCE modified electrode was obtained; 4.0 μL of 3.0 μM aptamer (same as Example 1) was then dropped on the surface of the NaYF4-PEI / GCE modified electrode, and dried naturally for 10 hours to obtain an apt / NaYF4-PEI / GCE sensor as a working electrode for electrochemiluminescence testing.

[0059] (2) Drawing of standard curve

[0060] The apt / NaYF4-PEI / GCE modified electrode was used as the working electrode, the platinum electrode as the auxiliary electrode, and the Ag / AgCl as the reference electrode to form a three-electrode system. The luminescence intensity was detected using a 0.1 mol / L PBS buffer solution with pH = 7.4 containing 0.05 mol / L K2S2O8 as the blank solution. The three-electrode system was placed in a series of enrofloxacin concentrations (1.0×10 -14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 -9 mol / L、1.0×10 -8mol / L、1.0×10 -7 mol / L and 1.0×10 -6 The luminescent intensity of enrofloxacin was measured in a 0.1 mol / L PBS buffer solution of pH 7.4 containing 0.05 mol / L K2S2O8. Cyclic voltammetry was performed in the electrochemical window range of -1.8 to 0 V with a photomultiplier tube high voltage of 800 V and a scan rate of 0.1 V / s. The E-ECL curve was recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithm of the enrofloxacin concentration was established to obtain the corresponding linear regression equation.

[0061] (3) Sample testing

[0062] 25 mL of treated river water was added to a 0.1 mol / L PBS buffer solution at pH 7.4 containing 0.05 mol / L K2S2O8 for electrochemiluminescence detection. The concentration of enrofloxacin in the sample to be tested was calculated according to the linear regression equation corresponding to the above step (2). The results are listed in Table 1.

[0063] Comparative Example 3:

[0064] (1) Preparation of apt / PTCA / GCE modified electrodes

[0065] The glassy carbon electrode was polished, ultrasonicated with nitric acid, anhydrous ethanol and deionized water respectively, and dried naturally for use. 4.0 μL of PTCA aqueous solution was pipetted with a microinjector and dropped on the surface of the clean glassy carbon electrode, and dried at room temperature to obtain a PTCA / GCE modified electrode; 4.0 μL of 3.0 μM aptamer (same as Example 1) was then dropped on the surface of the PTCA / GCE modified electrode, and dried naturally for 10 hours to obtain an apt / PTCA / GCE sensor as a working electrode for electrochemiluminescence testing.

[0066] (2) Drawing of standard curve

[0067] The apt / PTCA / GCE modified electrode was used as the working electrode, the platinum electrode as the auxiliary electrode, and the Ag / AgCl as the reference electrode to form a three-electrode system. The luminescence intensity was detected using a 0.1 mol / L PBS buffer solution with pH = 7.4 containing 0.05 mol / L K2S2O8 as the blank solution. The three-electrode system was placed in a series of enrofloxacin concentrations (1.0×10 -14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 -9 mol / L、1.0×10- 8 mol / L、1.0×10 -7 mol / L and 1.0×10 -6 mol / L) standard solution for 30 min, taken out and rinsed, used as the working electrode, and performed cyclic voltammetry scanning in the electrochemical window range of -1.8 to 0 V with a photomultiplier tube high voltage of 800 V and a scan rate of 0.1 V / s. The E-ECL curve was recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithmic value of the enrofloxacin concentration was established to obtain the corresponding linear regression equation.

[0068] (3) Sample testing

[0069] 25 mL of treated river water was added to a 0.1 mol / L PBS buffer solution at pH 7.4 containing 0.05 mol / L K2S2O8 for electrochemiluminescence detection. The concentration of enrofloxacin in the sample to be tested was calculated according to the linear regression equation corresponding to the above step (2). The results are listed in Table 1.

[0070] Comparative Example 4:

[0071] (1) Preparation of NaYF4-PEI-PTCA / GCE modified electrode

[0072] The glassy carbon electrode was polished, ultrasonicated with nitric acid, anhydrous ethanol and deionized water respectively, and dried naturally for use. 4.0 μL of 1.0 mg / mL NaYF4-PEI-PTCA aqueous solution was pipetted with a microinjector and dropped onto the clean glassy carbon electrode surface, and dried at room temperature to obtain a NaYF4-PEI-PTCA / GCE modified electrode.

[0073] (2) Drawing of standard curve

[0074] The three-electrode system was composed of NaYF4-PEI-PTCA / GCE modified electrode as the working electrode, platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. The luminescence intensity was detected using 0.1 mol / L PBS buffer at pH 7.4 containing 0.05 mol / L K2S2O8 as the blank solution. The three-electrode system was placed in a series of enrofloxacin concentrations (1.0×10 -14 mol / L、1.0×10 -13 mol / L、1.0×10 -12 mol / L、1.0×10 -11 mol / L、1.0×10 -10 mol / L、1.0×10 -9 mol / L、1.0×10 -8 mol / L、1.0×10-7 mol / L and 1.0×10 -6 The luminescent intensity of enrofloxacin was measured in a 0.1 mol / L PBS buffer solution of pH 7.4 containing 0.05 mol / L K2S2O8. Cyclic voltammetry was performed in the electrochemical window range of -1.8 to 0 V with a photomultiplier tube high voltage of 800 V and a scan rate of 0.1 V / s. The E-ECL curve was recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithm of the enrofloxacin concentration was established to obtain the corresponding linear regression equation.

[0075] (3) Sample testing

[0076] 25 mL of treated river water was added to a 0.1 mol / L PBS buffer solution at pH 7.4 containing 0.05 mol / L K2S2O8 for electrochemiluminescence detection. The concentration of enrofloxacin in the sample to be tested was calculated according to the linear regression equation corresponding to the above step (2). The results are listed in Table 1.

[0077] Table 1 Determination results of enrofloxacin in river water

[0078]

[0079]

[0080] Remark: a The average value of three measurements

[0081] As shown in Table 1, the sample was tested three times in parallel, the relative standard deviation was less than 5%, and the spike recovery range was 96% to 102%. The above results show that it is difficult to detect enrofloxacin by further assembling a sensor element after the glassy carbon electrode modified with NaYF4-PEI or PTCA alone without modification with the NaYF4-PEI-PTCA composite material, and the composite electrode material of the present invention is feasible for detecting enrofloxacin in river water.

[0082] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention. Sequence Listing <110> Changzhou University <120> Electrochemiluminescent aptamer sensor for specific detection of enrofloxacin and preparation method and application thereof <141> 2022-02-17 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 60 <212> PRT <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <400> 1 Cys Cys Cys Ala Thr Cys Ala Gly Gly Gly Gly Gly Cys Thr Ala Gly 1 5 10 15 Gly Cys Thr Ala Ala Cys Ala Cys Gly Gly Thr Thr Cys Gly Gly Cys 20 25 30 Thr Cys Thr Cys Thr Gly Ala Gly Cys Cys Cys Gly Gly Gly Thr Thr 35 40 45 Ala Thr Thr Thr Cys Ala Gly Gly Gly Gly Gly Ala 50 55 60

Claims

1. An electrochemiluminescent aptamer sensor for specific detection of enrofloxacin, characterized in that: The enrofloxacin aptamer is loaded on the surface of a glassy carbon electrode modified by a NaYF4-PEI-PTCA nanocomposite material, wherein the NaYF4-PEI-PTCA nanocomposite material is a product of upconversion nanoparticles NaYF4-PEI and perylenetetracarboxylic acid PTCA bonded by an amide bond; The enrofloxacin aptamer nucleotide sequence is as follows: apt: 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTTATT TCA GGG GGA-3'.

2. The method for preparing the electrochemiluminescent aptasensor for specific detection of enrofloxacin according to claim 1, characterized in that: The steps include: After polishing and cleaning the glassy carbon electrode GCE, an aqueous dispersion of NaYF4-PEI-PTCA is added to the surface of GCE and dried naturally to obtain a NaYF4-PEI-PTCA / GCE modified electrode. Then, the aptamer is applied to obtain an apt / NaYF4-PEI-PTCA / GCE modified electrode, which is stored at low temperature for 6-8 hours for use to obtain an ECL aptamer sensor; NaYF4-PEI-PTCA is obtained by the following method: NaYF4-PEI, NHS and EDC with a hollow rod structure are dispersed in water, PTCA is added thereto, stirred at room temperature, and NaYF4-PEI-PTCA is obtained by centrifugation and drying.

3. The method for preparing the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin according to claim 2, characterized in that: The preparation method of hollow rod-shaped NaYF4-PEI includes: dissolving YCl3, YbCl3, ErCl3, TmCl3, and NaCl in ethanol containing PEI, adding NH4F under stirring, placing the mixture in a polytetrafluoroethylene reactor for heating reaction, and obtaining a white solid, namely NaYF4-PEI, by centrifugation, washing, and drying.

4. The method for preparing the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin according to claim 2, characterized in that: The preparation method of PTCA includes: dissolving perylene-3,4,9,10-tetracarboxylic dianhydride PTCDA in an aqueous solution of NaOH, slowly adding a dilute hydrochloric acid solution to the above solution until a deep red flocculent precipitate can be observed in the solution, and obtaining a red solid, namely PTCA, by centrifugation, washing, and drying.

5. Use of the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin according to claim 1 in detection of enrofloxacin in water, characterized in that: The steps include: (1) Prepare a phosphate PBS buffer solution containing potassium persulfate K2S2O8; (2) Preparation of PBS buffer solutions containing different concentrations of enrofloxacin and 0.05 mol / L K2S2O8; Prepare enrofloxacin aqueous solution, then add a certain amount of enrofloxacin aqueous solution into 0.1 mol / L PBS buffer solution with pH=7.4 containing 0.05 mol / L K2S2O8 to obtain a series of enrofloxacin standard solutions with different concentrations ranging from 1.0×10 -14 ~1.0×10 -6 mol / L; (3) Drawing of standard curve The modified electrode apt / NaYF4-PEI-PTCA / GCE was used as the working electrode, the platinum electrode was used as the auxiliary electrode, and the Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was immersed in the above-mentioned PBS buffer solution containing a series of different concentrations of enrofloxacin and 0.05 mol / L K2S2O8 for a certain period of time, and the luminescence intensity was detected using a 0.1 mol / L PBS buffer solution with pH=7.4 containing 0.05 mol / L K2S2O8 as a blank solution; within the electrochemical window range of -1.8~0V, the photomultiplier tube was used at a high voltage of 800V and a scan rate of 0.1V / s to perform cyclic voltammetry scanning, and the potential-luminescence intensity curve E-ECL was recorded, and a linear relationship between the difference in luminescence intensity before and after the addition of enrofloxacin and the logarithmic value of the enrofloxacin concentration was established, and the corresponding linear regression equation was obtained; (4) Actual sample testing The actual sample test is first pre-treated and then the pH is adjusted. The test is carried out according to the same electrochemiluminescence test conditions as in step (3) above, and the luminescence intensity is recorded. The concentration of enrofloxacin in the sample to be tested is calculated using the linear regression equation corresponding to the obtained standard curve using the obtained luminescence intensity.

6. Use of the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin according to claim 5 in detection of enrofloxacin in water, characterized in that: In step (3), the immersion time of the modified electrode apt / NaYF4-PEI-PTCA / GCE is 30 min.

7. Use of the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin according to claim 5 in detection of enrofloxacin in water, characterized in that: The pretreatment method in step (4) includes: allowing the sample to be tested to stand naturally for a period of time, centrifuging and absorbing the supernatant, and then filtering it through a 0.22 μm filter membrane.

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