CN114527113A - Electrochemical luminescence aptamer sensor for specifically detecting enrofloxacin, preparation method and application - Google Patents

Abstract

The invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin, a preparation method and application thereof₄-PEI-PTCA nano composite material modified glassy carbon electrode surface, NaYF₄-the PEI-PTCA nanocomposite is a product of the combination of the up-converted nanoparticles and the perylene tetracarboxylic acid with amide bonds; 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 can realize sensitive detection of enrofloxacin and has the lowest detection limitIs 3.0X 10^‑15mol/L。

Description

Electrochemical luminescence aptamer sensor for specifically detecting enrofloxacin, preparation method and application

Technical Field

The invention belongs to the field of electrochemical luminescence detection, and relates to an electrochemical luminescence aptamer sensor for specifically detecting enrofloxacin, and a preparation method and application thereof. In particular to a perylene derivative (NaYF) based on up-conversion nano particles loaded with aptamer molecules₄-PEI-PTCA) modified glassy carbon electrode surface, i.e. with apt/NaYF₄An electrochemical luminescence analysis method for quantitatively detecting enrofloxacin in river water by taking a PEI-PTCA/GCE electrode as a sensing element.

Background

Enrofloxacin (Enrofloxacin, ENR), a second generation fluoroquinolone antibiotic. The antibacterial agent has the characteristics of wide antibacterial spectrum, strong bactericidal property, less adverse reaction, no cross drug resistance with other antibiotics and the like, and is further widely applied to the fields of animal and aquaculture industry and the like. However, the long-term abuse causes enrofloxacin to be enriched in animal bodies and enter the environment along with excrement of animals, thereby causing environmental pollution. In addition, in the aquaculture industry, a large amount of enrofloxacin remains in the water body, and the water environment is seriously polluted in the discharge process. Once the environment is polluted, factors which seriously affect human health, such as anaphylactic reaction, unbalanced intestinal flora and the like can be generated. At present, the maximum residual quantity of ENR in animal-derived food is regulated in a plurality of countries and regions, for example, the maximum residual quantity of ENR regulated in European Union is 0.3 mug/g, and the maximum residual quantity of muscle and fat regulated in all food animals in China is 100 mug/kg. Based on the method, the establishment of the efficient and sensitive method for detecting enrofloxacin residues is of great significance.

At present, methods for detecting enrofloxacin, which are reported, comprise high performance liquid chromatography, liquid chromatography-mass spectrometry, enzyme linked immunosorbent assay, photoelectric method and the like. However, these analytical methods have many limitations in practical application, for example, high performance liquid chromatography, although having high accuracy, is expensive and has low sensitivity; although the liquid chromatography-mass spectrometry can be used for qualitative and quantitative detection, the instrument operation speciality is high, and the method is not suitable for processing large-batch samples and rapidly detecting on site; the enzyme-linked immunosorbent assay is only suitable for rapidly screening a large number of samples and is not suitable for being used as a single detection method; although the photoelectric method can achieve high sensitivity, the method has disadvantages of being greatly affected by a light source and having poor stability. Therefore, it is necessary and extremely important to develop a simple and rapid electrochemiluminescence specific detection method of enrofloxacin with good selectivity and without introducing an external power source.

Electrochemical luminescence (ECL), also known as electrochemiluminescence, is a combination and extension of an electrochemical method and a chemiluminescence method, and has the advantages of high sensitivity, wide linear range, convenience in observation, simplicity in instrument, good reproducibility of the chemiluminescence method, stability in reagent, easiness in control and the like. And an external light source is not required to be introduced, the luminous intensity spectrum is collected under the assistance of optical instruments such as a photomultiplier and the like, and the relation between the luminous intensity spectrum and the object to be detected is established, so that the microanalysis is realized.

Disclosure of Invention

The invention aims to provide an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin, a preparation method and application aiming at the defects of enrofloxacin detection in the prior art. The invention is based on up-converting nanoparticles (NaYF)₄-PEI) and perylenetetracarboxylic acid (PTCA) to obtain NaYF₄The PEI-PTCA nano composite material is modified on the surface of a glassy carbon electrode, so that the sensitivity and stability of electrochemiluminescence are obviously improved, and then an aptamer is loaded through electrostatic action to obtain an electrochemiluminescence aptamer sensor (apt/NaYF for short)₄A PEI-PTCA/GCE sensor) which can specifically identify the target molecule enrofloxacin and improve the selectivity of enrofloxacin.

In order to realize the purpose of the invention, the adopted technical scheme is as follows:

an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin is characterized in that an aptamer containing 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3' base sequence is loaded on NaYF₄-PEI-PTCA nanocomposite modified glassy carbon electrode surface, the NaYF₄-PEI-PTCA nanocompositeThe material is a product combined by amide bonds between up-conversion nanoparticles (NaYF4-PEI) and perylene tetracarboxylic acid (PTCA).

The invention also provides a preparation method of the electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin, which comprises the following steps:

(1) preparation of PTCA:

dissolving perylene-3, 4,9, 10-tetracarboxylic dianhydride (PTCDA) in an aqueous solution of NaOH, slowly dropwise adding an HCl solution into the solution to obtain a deep red flocculent precipitate, and centrifuging, washing and drying to obtain a red solid, namely PTCA.

(2) Hollow rod shaped NaYF₄Preparation of PEI:

specifically, the method comprises the following steps: mixing YCl₃、YbCl₃And ErCl₃NaCl, adding into ethanol containing PEI, and adding NH₄Dissolving F in water, adding the mixed solution, stirring, placing in a polytetrafluoroethylene reaction kettle for heating reaction (preferably at 200 deg.C for 24 hr), centrifuging, washing, and drying to obtain hollow rod-shaped white solid, i.e. NaYF₄-PEI。

(3)NaYF₄-preparation of PEI-PTCA:

dissolving the PTCA prepared in the step (1) in DMF to form a PTCA-DMF solution of 1mg/mL, stirring the solution to be uniform, adding EDC and NHS (m/m is 4: 1), stirring the solution in an ice-water bath (aiming at enabling the reaction to be carried out at a constant temperature), and adding NaYF into the solution₄-PEI (m (ptca)/m (NaYF4-PEI) ═ 1: 5), stirring (time generally 16h), allowing both to react sufficiently, then centrifuging, washing, and drying to obtain the product, i.e. NaYF₄-PEI-PTCA。

(4)apt/NaYF₄-preparation of PEI-PTCA/GCE sensor:

polishing a glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, naturally drying the polished glassy carbon electrode for later use, and transferring NaYF₄Dropping the ultrapure water dispersion (1mg/mL) of PEI-PTCA on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain NaYF₄-a PEI-PTCA modified glassy carbon electrode; next, the aptamer was added dropwise to the NaYF₄Surface of PEI-PTCA/GCE, aptamer and NaYF₄-PEI-PTCA by electrostatic binding followed by apt/NaYF₄And (4) placing the PEI-PTCA/GCE modified electrode in a refrigerator for 6-8h to obtain the ECL aptamer sensor. (the refrigerator standing time is the key influence factor of the sensor performance, the temperature is not, 4 ℃ is used in the application.)

Finally, the present invention provides a method for the use of an electrochemiluminescent aptamer sensor for the specific detection of enrofloxacin, comprising the steps of:

firstly preparing a series of enrofloxacin standard solutions with different concentrations, preparing enrofloxacin into a solution by using water, and then adding a K-containing solution₂S₂O₈In the PBS buffer solution to obtain a series of enrofloxacin standard solutions with different concentrations, wherein the concentration range is 1.0 multiplied by 10^-14mol/L～1.0×10^-6mol/L。

Then establishing a linear regression equation, and adding apt/NaYF₄A PEI-PTCA/GCE sensor is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and the three-electrode system is placed in the enrofloxacin standard solutions with different concentrations and the K-containing solution₂S₂O₈Soaking in PBS buffer solution, performing cyclic voltammetry scanning at a scanning speed of 0.1V/s and a photomultiplier tube high voltage of 800V within an electrochemical window range of-1.8-0V, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between a luminous intensity difference before and after adding enrofloxacin and an enrofloxacin concentration logarithm value to obtain a corresponding linear regression equation;

and finally, carrying out sample detection: the sample to be tested is pretreated (the pretreatment is mainly to filter impurities, such as taking 200mL of river water sample, naturally standing for a period of time, centrifuging at 6000rpm for 10min, absorbing supernatant, filtering with 0.22 μm filter membrane), and testing according to the same electrochemiluminescence test conditions (the filtrate is prepared to contain 0.05mol/L K)₂S₂O₈Taking 25mL of the obtained solution in a detection pool, recording the luminous intensity, and calculating the concentration of the enrofloxacin in the sample to be detected by using a linear regression equation corresponding to the obtained standard curve.

Further, containing K₂S₂O₈The preparation method of the PBS buffer solution comprises the following steps: 0.05mol/L K in 0.1mol/L PBS buffer solution with pH 7.4₂S₂O₈PBS buffer solution of (1).

Further, in an electrochemiluminescence test, the photomultiplier has the high voltage of 800V, the sweeping speed of 0.1V/s and the soaking time of 30min (the detection value is stable when 30 min).

Compared with the prior art, the invention has the following beneficial effects: the invention designs an electrochemiluminescence aptamer sensor of perylene derivatives based on up-conversion nanoparticles, which takes PEI as a bridge to combine PTCA and NaYF₄The invention fully utilizes the advantages of the aptamer and the electrochemical luminescence sensor, realizes the sensitive detection of enrofloxacin by the mechanism of the enhancement of the ECL signal intensity of the enrofloxacin to the system, and has the detection range of 1.0 multiplied by 10^-14～1.0×10^-6mol/L, the lowest detection limit is 3.0 multiplied by 10^-15mol/L. The method for detecting enrofloxacin has the advantages of simple operation, good selectivity, low detection cost, high sensitivity and good stability.

Drawings

FIG. 1 is a NaYF₄-PEI(A)、PTCA(B)、NaYF₄-scanning electron micrographs of PEI-PTCA (C).

FIG. 2 is a graph of ECL-potential curves for different concentrations of enrofloxacin.

Wherein the concentration of the enrofloxacin is as follows according to the peak value of the curve from front to back: 1.0X 10^-14mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^-9mol/L、1.0×10^-8mol/L、1.0×10^-7mol/L、1.0×10^-6mol/L。

FIG. 3 is a standard curve of the difference in luminous intensity before and after the addition of enrofloxacin and the logarithm of the concentration of enrofloxacin.

FIG. 4 is the specific detection of enrofloxacin by the sensor of example 1.

Detailed Description

The present invention is not limited to the following embodiments, and those skilled in the art can implement the present invention in other embodiments according to the disclosure of the present invention, or make simple changes or modifications on the design structure and idea of the present invention, and fall into the protection scope of the present invention. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The invention is described in more detail below with reference to the following examples:

the embodiment provides an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin, which is characterized in that an aptamer containing 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3' base sequence is loaded on NaYF₄-PEI-PTCA nanocomposite modified glassy carbon electrode surface, the NaYF₄-PEI-PTCA nanocomposite, which is the product of the amide bond bonding between upconverted nanoparticles (NaYF4-PEI) and perylenetetracarboxylic acid (PTCA), can be obtained by the method described in example 1, in particular as follows:

example 1:

(1) preparation of PTCA:

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

(2)NaYF₄Preparation of PEI:

add 8mL YCl₃、1.8mL YbCl₃、0.2mL ErCl₃And Tmcl₃10mL of NaCl, added to 60mL of ethanol containing 20mL of PEI, and mixed with 0.12g of NH₄Dissolving the F in 10mL of water, adding the mixed solution, uniformly stirring, and heating in a polytetrafluoroethylene reaction kettle at 200 ℃ for 24 hours. The white solid, i.e., UCPNs-PEI, was obtained by centrifugation, washing and drying.

(3)NaYF₄-preparation of PEI-PTCA:

10mg of PTCA was dissolvedDissolving in 10mL DMF, stirring for 1h, adding 40mg EDC and 10mg NHS, stirring in ice-water bath for 4h, adding 50mg NaYF₄PEI, stirred overnight. Centrifuging, washing and drying to obtain the product NaYF₄-PEI-PTCA。

(4) Modified electrode apt/NaYF₄-preparation of PEI-PTCA/GCE:

polishing glassy carbon electrode, sequentially performing ultrasonic treatment with nitric acid, anhydrous ethanol and deionized water, naturally drying, and transferring 4 μ L NaYF with 5 μ L microsyringe₄Dripping the solution of-PEI-PTCA on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain NaYF₄-PEI-PTCA modified glassy carbon electrode. Next, 4. mu.L of 3. mu. mol/L aptamer was added dropwise to the NaYF₄Surface of PEI-PTCA/GCE, aptamer and NaYF₄Through electrostatic combination of-PEI-PTCA, apt/NaYF is obtained₄-PEI-PTCA/GCE. Finally, apt/NaYF₄And (3) placing the-PEI-PTCA/GCE modified electrode in a refrigerator at 4 ℃ for 6 hours to obtain the ECL aptamer sensor.

The recognition molecule apt sequence is: apt:5'-HOOC-AGA TGG GGG TTG AGG CTA AGC CGA-3' (manufacturer is Industrial bioengineering (Shanghai) GmbH)

(5) Drawing of standard curve

Will modify the electrode apt/NaYF₄-PEI-PTCA/GCE is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and the three-electrode system is placed in a series of enrofloxacin concentrations (1.0 multiplied by 10)^{- 14}mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^{- 9}mol/L、1.0×10^-8mol/L、1.0×10^-7mol/L and 1.0X 10^-6mol/L) contains 0.05mol/L of K₂S₂O₈Soaking the mixture in 0.1mol/L PBS buffer solution with the pH of 7.4 for 30min, performing cyclic voltammetry scanning at the high voltage of 800V and the sweeping speed of 0.1V/s of a photomultiplier within the electrochemical window range of-1.8-0V, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between the luminous intensity difference before and after the enrofloxacin is added and the concentration logarithm of the enrofloxacinThe corresponding linear regression equation is obtained as: delta I_ECL5149.83162+1001.84055LogC (mol/L), the correlation coefficient (R) is 0.9977. The detection range of the linear regression equation is 1.0 multiplied by 10^-14～1.0×10^-6mol/L, the lowest detection limit is 3.0 multiplied by 10^-15mol/L。

(6) Detection of samples

Taking a river water sample, naturally standing for a period of time, centrifugally separating and absorbing an upper layer solution, filtering through a 0.22 mu m filter membrane to collect filtrate, and adding a solution containing 0.05mol/LK₂S₂O₈Adjusting the pH value of the 0.1mol/L PBS buffer solution to 7.4, taking 25mL of the obtained solution for electrochemical luminescence analysis, testing according to the electrochemical luminescence test conditions in the step (4), recording the luminescence intensity, calculating the concentration of enrofloxacin in the sample to be detected according to the linear regression equation obtained in the step (5), and obtaining the results shown in Table 1.

Compared with the common electrochemiluminescence sensor, the electrochemiluminescence sensor has the following two remarkable advantages: NaYF₄-PEI is tightly bound to PTCA via amide bond and exhibits synergy of both in ECL; the perylene substance has good electrochemical activity and is an up-conversion nano particle (NaYF)₄) Has excellent optical characteristics, such as high intensity and stable cathode signal, and is a promising new ECL luminophore. The invention fully utilizes the advantages of the aptamer and the electrochemical luminescence sensor, successfully realizes the sensitive detection of enrofloxacin through the mechanism of enhancing the ECL signal intensity of the system by enrofloxacin, and the sensing platform can specifically identify the enrofloxacin as a detection object and has high selectivity. The invention has important significance for popularizing the practical application of the aptamer sensor in the aspects of water environment monitoring and the like.

And the electrochemical luminescence sensor for detecting enrofloxacin prepared in the embodiment 1 is further processed by anti-interference detection, wherein working electrodes after the incubation of the aptamer are respectively arranged at 10^-6M Norfloxacin (NFX), Ciprofloxacin (CIP), Chloramphenicol (CAP), Kanamycin (KAN), chlortetracycline (CTC), Streptomycin (STP) interferents, andat 10^-8M Enrofloxacin (ENR) standard solution, and detecting the working electrode in the mixed solution of the substances, wherein the detection result is shown in figure 4.

As can be seen from FIG. 4, the modified electrode with excellent electrochemical properties has a selective recognition effect on enrofloxacin after incubation of the aptamer, and the interference substance with 100 times concentration after mixing has a slight effect on detection of enrofloxacin. Therefore, the working electrode can realize anti-interference selective detection of enrofloxacin.

Comparative example 1:

(1)apt/NaYF₄preparation of-PEI-PTCA/GCE modified electrode

Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. 4.0 μ L of 1.0mg/mL NaYF was pipetted with a microsyringe₄Dripping an aqueous solution of the-PEI-PTCA material on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain NaYF₄-PEI-PTCA/GCE modified electrode; in NaYF₄The surface of the-PEI-PTCA/GCE modified electrode is dripped with 4.0 mu L of 3.0 mu M aptamer (same as the example 1), and the mixture is naturally aired for 4 hours to obtain apt/NaYF₄PEI-PTCA/GCE sensor as working electrode for electrochemiluminescence test.

(2) Drawing of standard curve

By apt/NaYF₄-PEI-PTCA/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L₂S₂O₈The pH of the solution (2) is 7.4, 0.1mol/L PBS buffer solution is used as a blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of enrofloxacin concentrations (1.0 multiplied by 10)^{- 14}mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^{- 9}mol/L、1.0×10^-8mol/L、1.0×10^-7mol/L and 1.0X 10^-6mol/L) contains 0.05mol/L of K₂S₂O₈In 0.1mol/L PBS buffer solution with pH of 7.4, in the electrochemical window range of-1.8-0V, the high voltage of a photomultiplier is 800V, the sweep rate is 0.1V/s, and cyclic voltammetry sweep is carried outAnd (3) describing, recording an E-ECL curve, and establishing a linear relation between the luminous intensity difference value before and after the enrofloxacin is added and the logarithm value of the enrofloxacin concentration to obtain a corresponding linear regression equation.

(3) Detection of samples

25mL of the treated river water was added to a solution containing 0.05mol/L of K₂S₂O₈The buffer solution of 0.1mol/L PBS with the pH value of 7.4 is used for electrochemiluminescence detection, the concentration of enrofloxacin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2), and the result is shown in Table 1.

Comparative example 2:

(1) preparation of apt/NaYF4-PEI/GCE modified electrode

Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. 4.0 mu L of 1.0mg/mL NaYF4-PEI material aqueous solution is transferred by a microsyringe and dripped on the surface of a clean glassy carbon electrode, and the mixture is dried at room temperature to obtain a NaYF4-PEI/GCE modified electrode; and dripping 4.0 mu L of 3.0 mu M aptamer (the same as the example 1) on the surface of the NaYF4-PEI/GCE modified electrode, and naturally airing for 10h to obtain the apt/NaYF4-PEI/GCE sensor serving as a working electrode for electrochemiluminescence test.

(2) Drawing of standard curve

An apt/NaYF4-PEI/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L₂S₂O₈0.1mol/L PBS buffer solution with pH of 7.4 as blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of enrofloxacin concentrations (1.0 × 10)^-14mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^-9mol/L、1.0×10^-8mol/L、1.0×10^-7mol/L and 1.0X 10^-6mol/L) contains 0.05mol/L of K₂S₂O₈In 0.1mol/L PBS buffer solution with pH of 7.4, in the electrochemical window range of-1.8-0V, the photomultiplier high voltage is 800V, the sweep rate is 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded,and establishing a linear relation between the luminous intensity difference before and after the enrofloxacin addition and the enrofloxacin concentration logarithm value to obtain a corresponding linear regression equation.

(3) Detection of samples

25mL of the treated river water was added to a solution containing 0.05mol/L of K₂S₂O₈The buffer solution of 0.1mol/L PBS with the pH value of 7.4 is used for electrochemiluminescence detection, the concentration of enrofloxacin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2), and the result is shown in Table 1.

Comparative example 3:

(1) preparation of apt/PTCA/GCE modified electrode

Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. Transferring 4.0 mu L of PTCA aqueous solution onto the surface of a clean glassy carbon electrode by using a microsyringe, and drying at room temperature to obtain a PTCA/GCE modified electrode; and (3) dripping 4.0 muL of 3.0 muM aptamer (the same as the example 1) on the surface of the PTCA/GCE modified electrode, and naturally airing for 10h to obtain the apt/PTCA/GCE sensor serving as a working electrode for electrochemiluminescence test.

(2) Drawing of standard curve

An apt/PTCA/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L₂S₂O₈The pH of the solution (1.0 × 10) is 7.4, 0.1mol/L PBS buffer solution is used as blank solution for detecting the luminous intensity, and the three-electrode system is placed in a series of enrofloxacin concentrations (1.0 × 10)^-14mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^-9mol/L、1.0×10^{- 8}mol/L、1.0×10^-7mol/L and 1.0X 10^-6mol/L) for 30min, taking out and rinsing, taking the solution as a working electrode, performing cyclic voltammetry scanning on the solution within an electrochemical window range of-1.8-0V at a photomultiplier high voltage of 800V and a scanning speed of 0.1V/s, recording an E-ECL curve, establishing a linear relation between a luminous intensity difference before and after the enrofloxacin is added and an enrofloxacin concentration logarithm value,and obtaining a corresponding linear regression equation.

(3) Detection of samples

25mL of the treated river water was added to a solution containing 0.05mol/L of K₂S₂O₈The buffer solution of 0.1mol/L PBS with the pH value of 7.4 is used for electrochemiluminescence detection, the concentration of enrofloxacin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2), and the result is shown in Table 1.

Comparative example 4:

(1)NaYF₄preparation of-PEI-PTCA/GCE modified electrode

Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. Transfer 4.0 μ L of 1.0mg/mL NaYF with a microsyringe₄Dripping an aqueous solution of the-PEI-PTCA material on the surface of a clean glassy carbon electrode, and drying at room temperature to obtain NaYF₄-PEI-PTCA/GCE modified electrode.

(2) Drawing of standard curve

With NaYF₄-PEI-PTCA/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L₂S₂O₈The pH of the solution (1.0X 10) is 0.1mol/L PBS buffer solution as blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of enrofloxacin concentrations (1.0X 10)^-14mol/L、1.0×10^-13mol/L、1.0×10^-12mol/L、1.0×10^-11mol/L、1.0×10^-10mol/L、1.0×10^-9mol/L、1.0×10^-8mol/L、1.0×10^-7mol/L and 1.0X 10^-6mol/L) contains 0.05mol/L of K₂S₂O₈In the 0.1mol/L PBS buffer solution with the pH of 7.4, in the electrochemical window range of-1.8-0V, the photomultiplier has the high voltage of 800V and the sweep rate of 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded, a linear relation between the luminous intensity difference before and after the enrofloxacin is added and the logarithm value of the enrofloxacin concentration is established, and a corresponding linear regression equation is obtained.

(3) Detection of samples

Taking 25mL of the treated riverWater was added to the solution containing 0.05mol/L of K₂S₂O₈The buffer solution of 0.1mol/L PBS with the pH value of 7.4 is used for electrochemiluminescence detection, the concentration of enrofloxacin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2), and the result is shown in Table 1.

TABLE 1 determination of enrofloxacin in river Water

Remarking:^ais the average of three determinations

As shown in Table 1, the samples were tested in parallel for 3 times, the relative standard deviation was less than 5%, and the recovery rate of spiking ranged from 96% to 102%. The above results show that NaYF is not used₄-PEI-PTCA composite modification with NaYF alone₄And the PEI or PTCA modified glassy carbon electrode is further assembled with a sensing element to be difficult to detect enrofloxacin, and the composite electrode material is feasible to detect enrofloxacin in river water.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Changzhou university

<120> electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin, preparation method and application

<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 (7)

1. An electrochemiluminescent aptamer sensor for specifically detecting enrofloxacin, characterized by: is loaded by enrofloxacin aptamer on NaYF₄-PEI-PTCA nanocomposite modified glassy carbon electrode surface, the NaYF₄-PEI-PTCA nanocomposite is a product of the coupling between up-converted nanoparticles (NaYF4-PEI) and perylene tetracarboxylic acid (PTCA) with amide bonds;

the nucleotide sequence of the enrofloxacin aptamer is shown as follows:

apt:5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3'。

2. the method for preparing an electrochemiluminescence aptamer sensor for specific detection of enrofloxacin according to claim 1, wherein the electrochemiluminescence aptamer sensor comprises: the method comprises the following steps:

polishing and cleaning a Glassy Carbon Electrode (GCE), and then dropwise adding NaYF₄Putting the aqueous dispersion of-PEI-PTCA on the surface of GCE, and naturally airing to obtain NaYF₄-PEI-PTCA/GCE modified electrode, then, dropping aptamer to obtain apt/NaYF₄The PEI-PTCA/GCE modified electrode is stored at low temperature for 6-8h for later use, and the ECL aptamer sensor is obtained; NaYF₄-PEI-PTCA was obtained by the following method: mixing hollow rod-shaped NaYF₄Dispersing PEI, NHS and EDC in water, adding PTCA, stirring at room temperature, centrifuging, and drying to obtain NaYF₄-PEI-PTCA。

3. The method for preparing an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin according to claim 2, wherein the electrochemiluminescence aptamer sensor comprises: hollow rod shaped NaYF₄-the method of preparation of PEI comprises: mixing YCl₃、YbCl₃、ErCl₃、TmCl₃NaCl was dissolved in ethanol containing PEI, and NH was added with stirring₄F, placing the mixture in a polytetrafluoroethylene reaction kettle for heating reaction, centrifuging, washing and drying to obtain a white solid, namely NaYF₄-PEI。

4. The method for preparing an electrochemiluminescence aptamer sensor for specifically detecting enrofloxacin according to claim 2, wherein the electrochemiluminescence aptamer sensor comprises: the preparation method of PTCA comprises the following steps: dissolving perylene-3, 4,9, 10-tetracarboxylic dianhydride (PTCDA) in an aqueous solution of NaOH, slowly dropwise adding a dilute hydrochloric acid solution into the solution until a dark red flocculent precipitate can be observed in the solution, and centrifuging, washing and drying to obtain a red solid, namely PTCA.

5. The use of the electrochemiluminescent aptamer sensor for specific detection of enrofloxacin in water according to claim 1, characterized by comprising the following steps:

(1) preparing potassium persulfate (K)₂S₂O₈) Phosphate (PBS) buffer solution of (a);

(2) contains enrofloxacin with different concentrations and 0.05mol/L K₂S₂O₈Preparing a PBS buffer solution;

preparing enrofloxacin aqueous solution, adding a certain amount of enrofloxacin aqueous solution into the solution containing 0.05mol/L K₂S₂O₈In PBS buffer solution with pH value of 7.4 of 0.1mol/L to obtain a series of enrofloxacin standard solutions with different concentrations, wherein the concentration range is 1.0 multiplied by 10^-14～1.0×10^-6mol/L；

(3) Drawing of Standard Curve

Modified electrode apt/NaYF₄-PEI-PTCA/GCE is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and the three-electrode system is placed in a container containing a series of enrofloxacin with different concentrations and 0.05mol/L K₂S₂O₈The PBS buffer solution is soaked for a certain time and K with the concentration of 0.05mol/L₂S₂O₈The luminescence intensity was measured using 0.1mol/L PBS buffer solution with pH of 7.4 as a blank solution; in the electrochemical window range of-1.8-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 800V and a scanning speed of 0.1V/s, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between a luminous intensity difference value before and after adding enrofloxacin and an enrofloxacin concentration logarithm value to obtain a corresponding linear regression equation;

(4) actual sample detection

And (3) carrying out pretreatment and pH adjustment on the actual sample detection, testing according to the same electrochemical luminescence test conditions in the step (3), recording the luminescence intensity, and calculating the concentration of enrofloxacin in the sample to be detected by using a linear regression equation corresponding to the obtained standard curve of the obtained luminescence intensity.

6. The use of the electrochemiluminescent aptamer sensor for the specific detection of enrofloxacin according to claim 5 in the detection of enrofloxacin in water, wherein: modifying the electrode apt/NaYF in the step (3)₄-PEI-PTCA/GCE soak time 30 min.

7. The use of the electrochemiluminescent aptamer sensor for the specific detection of enrofloxacin according to claim 5 in the detection of enrofloxacin in water, wherein: the pretreatment method in the step (4) comprises the following steps: after the sample to be tested is naturally stood for a period of time, the supernatant is centrifugally absorbed, and then the supernatant is filtered by a 0.22 mu m filter membrane.

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