WO2022062100A1

WO2022062100A1 - Electrochemical luminescent aptamer sensor for detecting kanamycin and preparation method therefor

Info

Publication number: WO2022062100A1
Application number: PCT/CN2020/127647
Authority: WO
Inventors: 陈智栋; 周利君; 单学凌; 蒋鼎; 王文昌
Original assignee: 常州大学
Priority date: 2020-09-25
Filing date: 2020-11-10
Publication date: 2022-03-31
Also published as: CN112147200B; CN112147200A

Abstract

An electrochemical luminescent aptamer sensor for detecting kanamycin (KAN) and a preparation method therefor. An aptamer is loaded on the surface of a composite Au@HKUST-1/PTC-Cys modified glassy carbon electrode, and an Au-S bond between Au@HKUST-1 and PTC-Cys is jointly modified on the surface of the glassy carbon electrode, so that the sensitivity and stability of electrochemical luminescence are significantly improved, and then the aptamer is loaded to obtain an electrochemical luminescent aptamer sensor, which can specifically recognize a target molecule of KAN, thereby improving the selectivity of KAN detection. The detection range of the electrochemical luminescent aptamer sensor is 1.0 × 10 ^-13 to 1.0 × 10 ^-8 M, and the lowest detection limit is 4.2 × 10 ^-14 M. The preparation method for a modified electrode is simple, and the present invention has high sensitivity of KAN detection, good selectivity, and a wide linear range.

Description

Electrochemiluminescence aptamer sensor for detecting kanamycin and preparation method thereof

technical field

The invention belongs to the field of electrochemiluminescence detection, in particular to an electrochemiluminescence aptamer sensor for detecting kanamycin and a preparation method thereof.

Background technique

Food safety issues have seriously affected people's lives. The monitoring of food safety is very important. Kanamycin (KAN), an aminoglycoside antibiotic, is potentially toxic, and overuse of KAN can cause serious side effects in humans and animals, including ototoxicity, nephrotoxicity, and anaphylactic shock, affecting consumers of health. In order to raise people's awareness of the hazards of antibiotic residues and effectively protect human health, the EU clearly stipulates that the maximum residue limit (MRL) of kanamycin in milk is 150μg·kg ^-1 . Residues in meat, dairy products, and other foods of animal origin that enter the human body through the biological circulatory system, endanger human health, and may endanger public health. Therefore, it is of great significance to strengthen the detection of kanamycin residues.

In the prior art, the commonly used methods for detecting kanamycin mainly include high performance liquid chromatography, enzyme-linked immunosorbent assay, liquid chromatography, gas chromatography, electrochemical method, etc., but these methods generally have complicated experimental operations. And the disadvantages of long time and so on, it is difficult to promote to the market for on-site testing.

The existing aptamer sensor has high detection sensitivity, but the detection cost is high, the measurement time is long, and the result error is large. Therefore, it is necessary to establish a kanamycin detection technology with low cost, high sensitivity, high selectivity and can be used for rapid detection of food safety on-site.

SUMMARY OF THE INVENTION

In view of the defects in the detection of existing kanamycin pointed out in the background art, the present invention develops an electrochemiluminescence aptamer sensor for detecting kanamycin, so as to improve the detection efficiency of kanamycin and improve the Its sensitivity and selectivity make it more practical.

The first object of the present invention is to provide an electrochemiluminescence aptamer sensor for detecting kanamycin, which has the advantages of high sensitivity, good reproducibility, good selectivity and wide linear range.

The above-mentioned technical purpose of the present invention is achieved through the following technical solutions:

The electrochemiluminescence aptamer sensor for detecting kanamycin proposed in the present invention is prepared by loading the aptamer on the surface of the composite material Au@HKUST-1/PTC-Cys modified glassy carbon electrode. The invention makes full use of the Au-S bond between PTC-Cys and Au@HKUST-1 to co-modify the surface of the glassy carbon electrode, so that the sensitivity and stability of electrochemiluminescence are significantly improved. Chemiluminescent aptamer sensor (referred to as Aptamer/Au@HKUST-1/PTC-Cys/GCE sensor) can specifically recognize the target molecule kanamycin and improve the selectivity of kanamycin detection.

Wherein, the aptamer is an aptamer containing 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequence.

When PTC-Cys is modified alone on the electrode surface, the electrochemiluminescence response intensity is not high and unstable. When Au@HKUST-1 is loaded, the composite is bound by the Au-S bond, which makes the performance of the composite more stable. Soaked in potassium persulfate solution, the composite material on the electrode will not fall off, so the stability of the sensor can be improved.

The preparation method of the composite Au@HKUST-1/PTC-Cys modified glassy carbon electrode is as follows:

S1. Preparation of PTC-Cys: Dissolve 0.1 g perylene tetracarboxylic dianhydride (PTCDA) in 20 mL aqueous solution containing 0.04 g NaOH, then slowly add 1 M HCl solution to the above solution until there is bright red precipitate To generate, wash the precipitate with deionized water several times until the pH value of the suspension is approximately equal to 7, and dry the washed precipitate (PTCA) for later use.

Accurately weigh 5 mg of the PTCA prepared above, and disperse it in 10 mL of deionized water. After ultrasonically dispersing evenly, stir in an ice-water bath for 30 min, and add 1-ethyl-(3-dimethylaminopropyl) carbodiimide. Hydrochloride (EDC) and N-hydroxysuccinimide (NHS), continue to stir for 2 h to activate the carboxyl group, then, add 100 mg of cysteine, stir overnight, and finally, collect the product PTC-Cys by centrifugation. Disperse PTC-Cys in DMF to make it evenly dispersed to obtain a DMF dispersion of PTC-Cys;

Among them, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (m/m=1:1-4 :1).

The concentration of the obtained DMF dispersion of PTC-Cys was 1 mg/mL.

Preparation of S2.Au@HKUST-1:

Synthesis of Cu ₂ O: 0.2 g of polyvinylpyrrolidone (PVP(K30)) was added to 100 mL of 0.01 M copper nitrate trihydrate (Cu(NO ₃ ) ₂ ·3H ₂ O), followed by 25 mL of 1.5 M sodium hydroxide (NaOH) solution, a blue precipitate was formed immediately, stirred, added 25mL of 0.1M ascorbic acid, after 15min, Cu ₂ O precipitate was formed and dispersed in ethanol;

Au@Cu ₂ O heterostructure: Pipette 10 mL of the 3 mg/mL Cu ₂ O alcoholic solution prepared above, add 10 mL of 3 mM chloroauric acid (HAuCl ₄ ), let it stand, collect the product by centrifugation (8000 rpm, 5 min), and wash to obtain Au @Cu ₂ O heterostructure, dispersed in ethanol.

Mix 5 mL of 0.16M trimesic acid (H ₃ BTC), 5 mL of N,N-dimethylformamide (DMF) with 10 mL of the 10 mg/mL Au@Cu ₂ O heterojunction ethanol solution prepared above, The mixture was stirred overnight, centrifuged, washed, and dried to obtain green crystal Au@HKUST-1; Au@HKUST-1 was dispersed in DMF, and ultrasonically dispersed to obtain a DMF dispersion of Au@HKUST-1;

The concentration of the DMF dispersion of Au@HKUST-1 was 1 mg/mL.

S3. Polish the glassy carbon electrode, ultrasonically clean it with nitric acid solution, ethanol solution and ultrapure water in turn, and dry it at room temperature to obtain the pre-treated glassy carbon electrode for use; use a micro syringe to sequentially remove the PTC- The DMF dispersion of Cys and the DMF dispersion of Au@HKUST-1 in step S2 were drop-coated on the surface of the glassy carbon electrode after pretreatment, and air-dried to obtain the Au@HKUST-1/PTC- Cys composite modified glassy carbon electrode.

Among them, the volume ratio of the composite modification of PTC-Cys and Au@HKUST-1 is: 4:1-4:5.

The second object of the present invention is to provide a preparation method of an electrochemiluminescence aptamer sensor for detecting kanamycin.

The present invention provides a method for preparing an electrochemiluminescence aptamer sensor for detecting kanamycin. The aptamer is loaded on the glassy carbon modified by the Au@HKUST-1/PTC-Cys composite material through gold-sulfur bond binding. The electrode surface was dried naturally to prepare an electrochemiluminescence aptamer sensor.

The specific method for loading the aptamer on the surface of the glassy carbon electrode modified by the Au@HKUST-1/PTC-Cys composite material is as follows: firstly add _KCl , NaCl, MgCl and EDTA into Tris-HCl buffer solution Add aptamer to prepare an aptamer solution with an aptamer concentration of 2-10 μM, then pipette the aptamer solution and drop it on the Au@HKUST-1/PTC-Cys nanocomposite modified material glassy carbon electrode surface.

Further, the concentration of aptamer in the aptamer solution was 8 μM.

The third object of the present invention is to provide an application method of an electrochemiluminescence aptamer sensor for detecting kanamycin.

The specific method of the electrochemiluminescence aptamer sensor for detecting kanamycin provided by the present invention uses the electrochemiluminescence aptamer sensor as the working electrode, Ag/AgCl as the reference electrode, and platinum wire electrode as the counter electrode to form three electrodes In the system, kanamycin in the sample is quantitatively captured on the surface of the sensor, and the detection of kanamycin is realized by the generated luminescence signal.

Further, the specific steps are:

A1. Preparation of PBS buffer solution containing K ₂ S ₂ O ₈ : PBS buffer solution containing 0.05M K ₂ S ₂ O ₈ was prepared with 0.1M PBS buffer solution with a pH of 7.4;

A2. Preparation of kanamycin standard solutions of different concentrations: firstly prepare 1×10 ^-4 M kanamycin solution, dissolve in ultrapure water, and then dilute with water to obtain several kanamycin standard solutions of different concentrations. The concentration range of kanamycin standard solution is 1.0×10 ^-13 ~ 1.0×10 ^-8 M;

A3. Drawing of the standard curve: The electrochemiluminescence aptamer sensor is placed in the kanamycin standard solution of different concentrations prepared in step A2 and soaked for the same time, so that the electrochemiluminescence aptamer sensor binds to kanamycin , then take out and rinse, as the working electrode, Ag/AgCl is the reference electrode, and the platinum electrode is the counter electrode, forming a three-electrode system, using the PBS buffer solution containing K ₂ S ₂ O ₈ in step A1 as the electrolyte, In the electrochemical window range of -1.7 to 0 V, the photomultiplier tube was operated at a high voltage of 800 V and a scan rate of 0.1 V/s, cyclic voltammetry was performed, and the luminescence intensity-time curve was recorded to establish an electrochemiluminescence aptamer sensor combined with kanamycin The linear relationship between the difference in luminescence intensity before and after exposure to kanamycin and the logarithmic value of kanamycin concentration in kanamycin standard solution was obtained, and the corresponding linear regression equation was obtained;

A4. Detection of kanamycin in the sample: the sample is pretreated and then adjusted to pH with the PBS buffer solution containing K ₂ S ₂ O ₈ in step A1, and then added to the prepared kanamycin of different concentrations Put the electrochemiluminescence aptamer sensor into it and soak it for the same time to make the electrochemiluminescence aptamer sensor bind to kanamycin, then take it out and rinse it as the working electrode, and then use the method of step A3 to detect the luminescence intensity, and then use the linear regression method to detect the luminescence intensity. The equation calculates the concentration of kanamycin in the sample.

Further, the soaking time in step A3 is 25min.

To sum up, the present invention has the following beneficial effects:

The present invention designs an electrochemiluminescence aptamer sensor based on Per derivative PTC-Cys and copper-based metal-organic framework Au@HKUST-1 composite material doped with gold nanoparticles. The two materials pass through the Au-S bond. Combined, stable electrochemiluminescence performance can be obtained. The invention makes full use of the advantages of aptamers and electrochemiluminescence sensors. Through the effect of kanamycin on the enhancement of the ECL signal intensity of the system, the successful realization of kanamycin The sensitive detection of this sensing platform can specifically recognize the detection substance kanamycin with high selectivity. The detection range of the present invention is 1.0×10 ^-13 to 1.0×10 ^-8 M, and the minimum detection limit is 4.2×10 ^-14 M. The invention has the advantages of simple operation, good selectivity, low detection cost and high sensitivity for detecting kanamycin. The invention has important significance for promoting the practical application of the aptamer sensor in food safety.

Description of drawings

FIG. 1 is a brief flow chart of the preparation of the electrochemiluminescence aptamer sensor in the present invention and the detection of kanamycin.

Fig. 2 is the ECL response diagram of the electrochemiluminescence aptamer sensor constructed in Example 1 after binding with different concentrations of kanamycin, wherein the concentrations of kanamycin from left to right are: (a ) 1.0×10 ^-13 M; (b) 1.0×10 ^-12 M; (c) 1.0×10 ^-11 M; (d) 1.0×10 ^-10 M; (e) 1.0×10 ^-9 M; (f) ) 1.0×10 ⁻⁸ M.

Fig. 3 is the standard curve of the difference value of luminescence intensity before and after adding kanamycin in Example 1 and the logarithmic value of kanamycin concentration;

4 is a scanning electron microscope image of the Au@HKUST-1/PTC-Cys composite prepared in Example 1;

Fig. 5 is the electrochemiluminescence stability characterization diagram of the sensor Aptamer/Au@HKUST-1/PTC-Cys/GCE prepared in Example 1 with continuous cycle scanning for 15 cycles;

Figure 6 is a preferred characterization diagram of Example 3 in the amidation reaction, the ratio of EDC to NHS is 4:1 (m/m); in the figure, curve a represents EDC:NHS=1:1 (m/m), Curve b needs to represent EDC:NHS=4:1(m/m);

FIG. 7 is a graph of optimized characterization of aptamer concentrations in Example 4. FIG.

detailed description

The present invention will be further described below in conjunction with the embodiments, but not limited thereto.

In the following examples, the aptamer containing the 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequence was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and the aptamer was loaded on the Au@HKUST-1/PTC-Cys composite material The method for modifying the surface of the glassy carbon electrode is as follows: firstly adding the aptamer to a Tris-HCl buffer solution containing KCl, NaCl, MgCl ₂ and EDTA to prepare an aptamer solution, and then pipetting the aptamer The aptamer solution was drop-coated on the surface of the glassy carbon electrode modified by the Au@HKUST-1/PTC-Cys composite material. specific:

A1. Before opening the cap of the purchased aptamer, vortex for 10 minutes, and then centrifuge at 4000 rpm for 20 minutes;

A2. Slowly open the cap of the tube, according to the label on the tube, add 1020 microliters of 0.05M containing 0.2mol/L KCl, 0.1mol/L NaCl, 5.0mmol/L MgCl ₂ and 1.0mmol/L EDTA Stock solutions of Tris-HCl buffer solutions;

A3. Shake well and evenly, dilute to a concentration of 10 μM, and refrigerate at 4°C for later use.

The preparation methods of kanamycin standard solutions of different concentrations in the following examples are as follows: prepare kanamycin solution, and then dilute it with ultrapure water to obtain a series of kanamycin standard solutions of different concentrations. The concentrations of kanamycin in the standard solution of kanamycin were (a) 1.0×10 ^-13 M; (b) 1.0×10 ^-12 M; (c) 1.0×10 ^-11 M; (d) 1.0× 10 ⁻¹⁰ M; (e) 1.0×10 ⁻⁹ M; (f) 1.0×10 ⁻⁸ M.

Example 1

(1) Assembling an electrochemiluminescent aptasensor for detecting kanamycin

(1) Preparation of PTC-Cys and Au@HKUST-1 materials:

S1. Preparation of precursor PTCA, dissolve 0.1g PTCDA in 20mL aqueous solution containing 0.04g NaOH, then slowly add 1M HCl solution to the above solution until a bright red precipitate is formed, wash the precipitate with deionized water Several times, until the pH value of the suspension is approximately equal to 7, and dry for use. Accurately weigh 5 mg of the PTCA prepared above, disperse it in 10 mL of deionized water, and after ultrasonic dispersion is uniform, stir in an ice-water bath for 30 min, add 40 mg of EDC and 10 mg of NHS (m/m=4:1), and continue to stir for 2 h. The carboxyl group was activated, then, 100 mg of cysteine was added, stirred overnight, and finally, the product was collected by centrifugation as PTC-Cys; PTC-Cys was dispersed in DMF to make it evenly dispersed to obtain PTC-Cys with a concentration of 1 mg/mL of DMF dispersion.

S2. Preparation of Au@HKUST-1

Synthesis of Cu ₂ O: 0.2 g of polyvinylpyrrolidone (PVP(K30)) was added to 100 mL of 0.01 M copper nitrate trihydrate (Cu(NO ₃ ) ₂ ·3H ₂ O), followed by 25 mL of 1.5 M sodium hydroxide (NaOH) solution , a blue precipitate was formed immediately, stirred, and 0.4403 g of ascorbic acid was added. After 15 minutes, Cu ₂ O precipitate was formed and dispersed in ethanol;

5mL of 0.16M H ₃ BTC and 5 mL of DMF were mixed with 10 mL of the ethanol solution of 10 mg/mL Au@Cu ₂ O heterojunction prepared above, the mixture was stirred overnight, centrifuged and washed to obtain Au@HKUST-1; Au@HKUST-1 was obtained; -1 was dispersed in DMF, sonicated to make it evenly dispersed, and a DMF dispersion of 1 mg/mL Au@HKUST-1 was obtained.

(2) Preparation of electrochemiluminescence aptamer sensor for detecting kanamycin

Firstly, polishing powder (Al ₂ O ₃ ) for glassy carbon electrode was polished on the chamois into a mirror surface, then ultrasonically cleaned with nitric acid solution, ethanol solution and ultrapure water for 3 minutes, and dried at room temperature to obtain the pretreated glassy carbon electrode . Pipette 4 μL of 1 mg/mL PTC-Cys DMF dispersion prepared in step (1) with a micro syringe in turn, and after natural drying, drop 3 μL of 1 mg/mL Au@HKUST-1 DMF dispersion , after natural drying, the Au@HKUST-1/PTC-Cys/GCE modified electrode was obtained, which was naturally dried for use; on the surface of the Au@HKUST-1/PTC-Cys/GCE modified electrode, 5 μL of the prepared The Tris-HCl buffer solution of the ligand was dried naturally for 10 h to obtain the Aptamer/Au@HKSUT-1/PTC-Cys/GCE sensor, which was used as the sensing element for the electrochemiluminescence test.

Among them, the concentration of aptamer in the aptamer solution was 8 μM.

(2) Application method of electrochemiluminescence aptamer sensor for detecting kanamycin

A1. Preparation of K ₂ S ₂ O ₈ -containing PBS buffer solution:

Prepare a PBS buffer solution containing 0.05M K ₂ S ₂ O ₈ with a 0.1M PBS buffer solution with a pH of 7.4;

A2. Preparation of kanamycin standard solutions of different concentrations: firstly prepare 1×10 ^-4 M kanamycin solution, dissolve with ultrapure water, and then dilute with ultrapure water to obtain a series of kanamycin with different concentrations The concentration range of kanamycin standard solution is 1.0×10 ^-13 ~ 1.0×10 ^-8 M; in this example, the concentration of kanamycin in the kanamycin standard solution is (a) 1.0 ×10 ^-13 M; (b) 1.0×10 ^-12 M; (c) 1.0×10 ^-11 M; (d) 1.0×10 ^-10 M; (e) 1.0×10 ^-9 M; (f) 1.0 × ^10-8M .

A3. Drawing of the standard curve: The electrochemiluminescence aptamer sensor for detecting kanamycin in A1 was used as the sensing element, and it was soaked in kanamycin standard solutions of different concentrations for 25 minutes, and then rinsed after taking it out. , as the working electrode, Ag/AgCl is the reference electrode, and the platinum electrode is the counter electrode, forming a three-electrode system, and using 0.1M PBS buffer containing 0.05MK ₂ S ₂ O ₈ at pH 7.4 as the electrolyte to measure the luminescence intensity, In the electrochemical window range of -1.7 to 0 V, the photomultiplier tube was operated at a high voltage of 800 V and a scan rate of 0.1 V/s, cyclic voltammetry was performed, and the luminescence intensity-time curve was recorded to establish an electrochemiluminescence aptamer sensor combined with kanamycin The linear relationship between the difference in luminescence intensity before and after exposure to kanamycin and the logarithmic value of kanamycin concentration in the standard solution of kanamycin, the corresponding linear regression equation obtained is; ΔECL=9277.5598+2109.2115lgC (nM), the detection range is 1.0 ×10 ^-13 ～1.0×10 ^-8 M, and the detection limit was 4.2×10 ^-14 M.

A4. Detection of samples: Briefly, 10 mL of milk samples were diluted with 10 mL of PBS solution (pH 7.4, 0.01 μM). It was then centrifuged at 12,000 rpm for about 20 minutes and left at room temperature for 20 minutes. The supernatant of milk was extracted, filtered with a 0.22 mm microporous membrane, and then KAN standard solution was spiked into five-fold diluted milk to prepare kanamycin sample solutions of different concentrations for analytical determination. And add 0.1M PBS buffer solution containing 0.05M K ₂ S ₂ O ₈ to adjust the pH to 7.4, take 25 mL of the obtained solution for electrochemiluminescence analysis, and calculate the kanamycin in the sample to be detected according to the linear regression equation obtained in step A1. The concentration of the element is listed in Table 1.

In this example, PTC-Cys is used as the base material, and the composite material is obtained by secondary drop coating of Au@HKUST-1. The morphology is shown in Figure 4. The two can be stably combined through the Au-S bond. The material combination is novel and can greatly improve the The electrochemiluminescence intensity of the individual material is good, the sensitivity is good, the stability is good, and the sensor selectivity is good, and the stability is good.

Example 2

Example 1 (1) Assembling an electrochemiluminescent aptamer sensor for detecting kanamycin

Step (1) During the preparation of the PTC-Cys material, in the amidation reaction of S1, the ratio of EDC to NHS was changed to 1:1 (m/m), and the others were the same as in Example 1.

Example 3

In Example 1 (1) assembling an electrochemiluminescence aptamer sensor for detecting kanamycin, in step (2), the concentration of aptamer in the aptamer solution is 2-10 μM.

Comparative Example 1

(1) Preparation of Aptamer/Au@HKUST-1/GCE sensor

Pipette 3 μL of 1 mg/mL DMF dispersion of Au@HKUST-1 with a pipette and apply it to the surface of the pre-treated glassy carbon electrode (the pre-treatment method is the same as in Example 1) to obtain Au@HKUST-1/GCE The chemically modified electrode was left to dry naturally; 5 μL of 8 μM aptamer was then dropped on the surface of the Au@HKUST-1/GCE chemically modified electrode, and it was naturally dried for 10 h to obtain the Aptamer/Au@HKUST-1/GCE sensor, which was used as an electro- Sensing element for chemiluminescence testing.

(2) Drawing of standard curve

The Aptamer/Au@HKUST-1/GCE sensor obtained in step (1) was used as a sensing element, and it was soaked in standard solutions of kanamycin with different concentrations for 25 min, taken out and rinsed, and used as a working electrode, Ag /AgCl as the reference electrode, platinum electrode as the counter electrode, constitute a three-electrode system, and use 0.1M PBS buffer containing 0.05MK ₂ S ₂ O ₈ pH 7.4 as the electrolyte to measure the luminescence intensity, at -1.7 ~ 0V Within the range of the electrochemical window, the photomultiplier tube was operated at a high voltage of 800V and a scan rate of 0.1V/s. Cyclic voltammetry was performed to record the luminescence intensity-time curve to establish the luminescence intensity difference between the electrochemiluminescence aptamer sensor and kanamycin The linear relationship between the value and the logarithm of the kanamycin concentration in the kanamycin standard solution was obtained, and the corresponding linear regression equation was obtained.

(3) Detection of samples

A4. Detection of samples: Briefly, 10 mL of milk samples were diluted with 10 mL of PBS solution (pH 7.4, 0.01 μM). It was then centrifuged at 12,000 rpm for about 20 minutes and left at room temperature for 20 minutes. The supernatant of the milk was extracted, filtered with a 0.22mm microporous membrane, and then the KAN standard solution was spiked into the five-fold diluted milk to prepare kanamycin sample solutions of different concentrations for analytical determination. The pH of the 0.1M PBS buffer solution of MK ₂ S ₂ O ₈ was adjusted to 7.4, and 25 mL of the obtained solution was taken for electrochemiluminescence analysis. concentration, the results are listed in Table 1.

Comparative Example 2

(1) Preparation of Aptamer/PTC-Cys/GCE sensor

Pipette 4 μL of 1 mg/mL PTC-Cys DMF dispersion with a micro-syringe and drop it onto the surface of the pre-treated glassy carbon electrode (the pre-treatment method is the same as in Example 1) to obtain a PTC-Cys/GCE chemically modified electrode. Dry for use; apply 5 μL of 8 μM on the surface of the PTC-Cys/GCE chemically modified electrode, and let it dry naturally for 10 h to obtain an Aptamer/PTC-Cys/GCE sensor, which is used as a sensing element for electrochemiluminescence testing.

(2) Drawing of standard curve

The Aptamer/PTC-Cys/GCE sensor obtained in step (1) was used as a sensing element, and it was soaked in standard solutions of kanamycin with different concentrations for 25 minutes, taken out and rinsed, as a working electrode, Ag/AgCl As the reference electrode, the platinum electrode is the counter electrode, forming a three-electrode system, and using 0.1M PBS buffer with pH 7.4 containing 0.05MK ₂ S ₂ O ₈ as the electrolyte to measure the luminescence intensity, the electrochemical measurement at -1.7 ~ 0V Within the window range, the high voltage of the photomultiplier tube was 800V, the scanning speed was 0.1V/s, cyclic voltammetry was performed, and the luminescence intensity-time curve was recorded. The linear relationship between the kanamycin concentration in the kanamycin standard solution and the logarithm value is obtained, and the corresponding linear regression equation is obtained.

(3) Detection of samples

Briefly, 10 mL of milk samples were diluted with 10 mL of PBS solution (pH 7.4, 0.01 μM). It was then centrifuged at 12,000 rpm for about 20 minutes and left at room temperature for 20 minutes. The supernatant of milk was extracted, filtered with a 0.22mm microporous membrane, and then KAN standard solution was spiked into five-fold diluted milk to prepare kanamycin sample solutions of different concentrations for analytical determination. The pH of the 0.1M PBS buffer solution of MK ₂ S ₂ O ₈ was adjusted to 7.4, and 25 mL of the obtained solution was taken for electrochemiluminescence analysis. concentration, the results are listed in Table 1.

Table 1 Determination results of a milk sample

Among them: the actual amount ^a is the average value of three measurements

As shown in Table 1, the samples were tested in parallel for 3 times, the recovery rate of standard addition was between 96% and 103%, the relative standard deviation was less than 5%, and the recovery effect was good. The above experimental results show that kanamycin cannot be detected by further assembling the sensing element without modification of Au@HKUST-1/PTC-Cys composite material and the glassy carbon electrode modified with Au@HKUST-1 or PTC-Cys alone. The invented sensor can be used to detect kanamycin in milk.

The above are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Technical personnel, within the scope of the technical solution of the present invention, can make some changes or modifications to equivalent examples of equivalent changes by using the technical content disclosed above, but any content that does not depart from the technical solution of the present invention, according to the present invention. The technical essence of the invention Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solutions of the present invention.

Claims

An electrochemiluminescence aptamer sensor is characterized in that, the electrochemiluminescence aptamer sensor is prepared by loading the aptamer on the surface of the composite material Au@HKUST-1/PTC-Cys modified glassy carbon electrode.
The electrochemiluminescence aptamer sensor according to claim 1, wherein the aptamer is an aptamer containing 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequence.
The electrochemiluminescence aptamer sensor according to claim 1, wherein the preparation method of the composite material Au@HKUST-1/PTC-Cys modified glassy carbon electrode is as follows:

S1. Preparation of PTC-Cys: perylene tetracarboxylic dianhydride (PTCDA) was dissolved in an aqueous solution of sodium hydroxide, and then HCl was added to it to obtain a red precipitate, which was washed with deionized water to remove excess The reactant is then dispersed to obtain a PTCA solution, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinyl are successively added to the PTCA solution. imine (NHS), add L-cysteine, continue stirring, and then centrifuge and dry to obtain a pink powdery product PTC-Cys; disperse PTC-Cys in DMF to make it evenly dispersed to obtain DMF dispersion of PTC-Cys;

Preparation of S2.Au@HKUST-1: trimellitic acid (H 3 BTC), N,N-dimethylformamide (DMF) and Au@Cu 2 O heterojunction in ethanol were mixed, and the mixture was stirred overnight , centrifugal separation and washing to obtain Au@HKUST-1, disperse Au@HKUST-1 in N,N-dimethylformamide, and ultrasonically disperse it uniformly to obtain the DMF dispersion of Au@HKUST-1;

S3. Polish the glassy carbon electrode, ultrasonically clean it with nitric acid solution, ethanol solution and ultrapure water in turn, and dry it at room temperature to obtain a pre-treated glassy carbon electrode for later use; sequentially remove the DMF dispersion of PTC-Cys in step S1 solution and the DMF dispersion of Au@HKUST-1 in step S2, and drop-coated it on the surface of the glassy carbon electrode after pre-treatment, and air-dried naturally to obtain the modified Au@HKUST-1/PTC-Cys composite material glassy carbon electrode.
The electrochemiluminescence aptamer sensor according to claim 3, wherein in the step S1, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) The molar ratio to N-hydroxysuccinimide (NHS) is 1:1-4:1; the concentration of the obtained DMF dispersion of PTC-Cys is 1 mg/mL.
The electrochemiluminescence aptamer sensor according to claim 3, wherein the concentration of the DMF dispersion liquid of Au@HKUST-1 obtained in the step S2 is 1 mg/mL.
The electrochemiluminescence aptamer sensor according to claim 3, wherein the volume ratio of the composite modification of PTC-Cys and Au@HKUST-1 in the step S3 is: 4:1-4:5.
A method for preparing an electrochemiluminescence aptamer sensor according to any one of claims 1-6, wherein the method loads the aptamer on Au@HKUST-1 through Au-S bond binding The surface of the glassy carbon electrode modified by the /PTC-Cys composite material was air-dried to prepare an electrochemiluminescence aptamer sensor.
The method for preparing an electrochemiluminescence aptamer sensor according to claim 7, wherein the specific method for loading the aptamer on the surface of the glassy carbon electrode modified by the Au@HKUST-1/PTC-Cys nanocomposite material is as follows: : First, add the aptamer to the Tris-HCl buffer solution containing KCl, NaCl, MgCl 2 and EDTA to prepare an aptamer solution with an aptamer concentration of 2-10 μM, and then pipette the aptamer The bulk solution was drop-coated on the surface of the glassy carbon electrode modified by the Au@HKUST-1/PTC-Cys composite material.
An electrochemiluminescence aptamer sensor according to any one of claims 1-6 for detecting kanamycin, wherein the application method is: using the electrochemiluminescence aptamer sensor as a working electrode , Ag/AgCl is the reference electrode, and the platinum wire electrode is the counter electrode to form a three-electrode system. The kanamycin in the sample is quantitatively captured on the surface of the sensor, and the detection of kanamycin is realized by the generated luminescence signal.
The application of the electrochemiluminescence aptamer sensor according to claim 9, wherein the specific steps of the application method are:

A1. Preparation of PBS buffer solution containing K 2 S 2 O 8 : PBS buffer solution containing 0.05M K 2 S 2 O 8 was prepared with 0.1M PBS buffer solution with a pH of 7.4;

A2. Preparation of kanamycin standard solutions of different concentrations: firstly prepare 1×10 -4 M kanamycin solution, dissolve it in ultrapure water, and then dilute it with ultrapure water to obtain kanamycin standards of different concentrations solution, the concentration range of kanamycin standard solution is 1.0×10 -13 ~ 1.0×10 -8 M;

A3. Drawing of the standard curve: The electrochemiluminescence aptamer sensor is placed in the kanamycin standard solution of different concentrations prepared in step A2 and soaked for the same time, so that the electrochemiluminescence aptamer sensor binds to kanamycin , then take out and rinse, as the working electrode, Ag/AgCl is the reference electrode, and the platinum electrode is the counter electrode, forming a three-electrode system, using the PBS buffer solution containing K 2 S 2 O 8 in step A1 as the electrolyte, In the electrochemical window range of -1.7 to 0 V, the photomultiplier tube was operated at a high voltage of 800 V and a scan rate of 0.1 V/s, cyclic voltammetry was performed, and the luminescence intensity-time curve was recorded to establish an electrochemiluminescence aptamer sensor combined with kanamycin The linear relationship between the difference in luminescence intensity before and after exposure to kanamycin and the logarithmic value of kanamycin concentration in kanamycin standard solution was obtained, and the corresponding linear regression equation was obtained;

A4. Detection of kanamycin in the sample: the sample is pretreated first, then the pH is adjusted with the PBS buffer solution containing K 2 S 2 O 8 in step A1, and then placed in the electrochemiluminescence aptamer sensor to soak the same time, make the electrochemiluminescence aptamer sensor bind to kanamycin, then take it out and rinse it as a working electrode, then use the method of step A3 to detect the luminescence intensity, and then calculate the concentration of kanamycin in the sample according to the linear regression equation .