Electrochemical aptamer sensor for detecting kanamycin as well as preparation method and application of electrochemical aptamer sensor

Abstract

The invention discloses an electrochemical aptamer sensor for detecting kanamycin as well as a preparation method and application of the electrochemical aptamer sensor, and belongs to the technical field of electrochemical biosensing analysis. According to the invention, a gold electrode assembled with a kanamycin aptamer with sulfydryl is taken as a sensing interface, a target molecule kanamycin is combined to the sensing interface, and a silver nanoparticle-zirconium-based metal organic framework material signal probe is combined with kanamycin on the sensing interface, so that the sandwich type kanamycin electrochemical aptamer sensor is constructed. The kanamycin electrochemical aptamer sensor prepared by the invention has relatively high sensitivity, relatively low detection limit and relatively wide detection range, the detection limit of the sensor is 0.085 aM, and the detection range is 0.1 aM <-1 > nM. The electrochemical aptamer sensor prepared by the invention is used for quantitative detection of kanamycin, and has the advantages of good stability, strong specificity, good reproducibility and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrochemical biosensing analysis, and particularly relates to an electrochemical aptamer sensor for detecting kanamycin and a preparation method and application thereof. BACKGROUND

[0002] Kanamycin is an aminoglycoside antibiotic, which is used as a feed additive and veterinary drug due to its low price and good antibacterial activity. However, the abuse of kanamycin leads to its residues in animal-derived food, which then enters the human body through the food chain, and harms public health through the accumulation effect. Long-term intake of food containing kanamycin residues can increase bacterial drug resistance and other side effects, such as ototoxicity, nephrotoxicity, and blocking of neuromuscular junctions, causing allergic reactions, and even death in severe cases. Therefore, quantitative detection of kanamycin is of great significance in food analysis.

[0003] Most of the detection technologies for detecting kanamycin adopt high-performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry online detection methods based on laboratory analysis. The sample pretreatment process is complex, time-consuming, and requires professional technical personnel to operate, and the detection cost is high. SUMMARY

[0004] In view of the above problems, the application provides an electrochemical aptamer sensor for detecting kanamycin and a preparation method and application thereof.

[0005] To achieve the above technical purposes and effects, the application is implemented by the following technical solutions. A preparation method of an electrochemical aptamer sensor for detecting kanamycin, comprising the following steps: Step one: kanamycin aptamer with thiol groups is assembled on the surface of a pretreated gold electrode by Au-S interaction to obtain a kanamycin sensing interface. Specifically, the following steps are included: A1.1): Pretreatment of gold electrode: including soaking in piranha solution, polishing, and ultrasonic cleaning steps to achieve the purpose of cleanliness; A1.2): Assembly of kanamycin aptamer with thiol groups on the gold electrode: drop the kanamycin aptamer with thiol groups on the pretreated gold electrode, and incubate at 310 K for 2 h, then rinse with phosphate buffer solution, drop 6-mercapto-1-hexanol solution on the modified electrode surface, and incubate at 310 K for 1 h, and then rinse with phosphate buffer solution.

[0006] Preferably, the concentration of the phosphate buffer solution is 0.01 mol / L.

[0007] As preferred, the sequence of the kanamycin aptamer with thiol group is 5'-SH-(CH2)6-AGA TGG GGGTTG AGG CTA AGC CGA-3'.

[0008] Step two: incubation of kanamycin in the target analyte to bind to the kanamycin sensing interface. Specifically, the following steps are included: drop the target analyte on the electrode surface obtained in step one, incubate, and then wash with phosphate buffer solution.

[0009] As preferred: the incubation time is 1 h, and the incubation temperature is 310 K.

[0010] Step three: preparation of silver nanoparticle-zirconium metal organic framework signal probe.

[0011] As preferred: the zirconium metal organic framework is a double cone structure with a size of about 300 nm; the mass ratio of silver nanoparticles to silver and zirconium in the zirconium metal organic framework is 3.6:1; the silver nanoparticle-zirconium metal organic framework and the kanamycin aptamer with amino group constitute the signal probe of the sensor, and the sequence of the kanamycin aptamer with amino group is 5'-NH2-(CH2)6-AGA TGG GGGTTG AGG CTA AGC CGA-3'.

[0012] Specifically, the preparation method of the silver nanoparticle-zirconium metal organic framework signal probe includes the following steps: A3.1) Synthesis of zirconium metal organic framework: 2,2'-dipyridyl 5,5'-dicarboxylic acid and zirconium chloride are respectively dispersed in N,N-dimethylformamide, then mixed together, and acetic acid is added; after ultrasonic treatment, the reaction is refluxed at 393 K for 18 h, centrifuged and collected, then washed with N,N-dimethylformamide and ethanol for several times to obtain the zirconium metal organic framework; A3.2) Synthesis of silver nanoparticle-zirconium metal organic framework composite material: the zirconium metal organic framework material obtained in step is dispersed in ultrapure water, AgNO3 is added, and stirred uniformly; ascorbic acid is added, the mixed solution changes from colorless to gray, and continues to be stirred until the color does not change to obtain the silver nanoparticle-zirconium metal organic framework composite material; A3.3) Synthesis of silver nanoparticle-zirconium-based metal organic framework composite probe: silver nanoparticle-zirconium-based metal organic framework composite was added into ultrapure water to prepare a dispersion solution, and a kanamycin aptamer solution with amino group was added, and the sequence of the kanamycin aptamer with amino group was 5'-NH2-(CH2)6-AGA TGG GGG TTG AGG CTA AGC CGA-3'; NaCl was added for multiple times at 277 K within 8 h, so that the final concentration of NaCl was 0.1 M, the mixture was incubated at 277 K for 40 h, and then centrifuged and washed to obtain the silver nanoparticle-zirconium-based metal organic framework composite probe.

[0013] Step four: binding of silver nanoparticle-zirconium-based metal organic framework composite signal probe to kanamycin on the electrode surface. Specifically comprising the following steps: dispersing the silver nanoparticle-zirconium-based metal organic framework composite probe in a phosphate buffer solution, and then dropping and coating on the electrode surface obtained in step two, and then washing after incubation.

[0014] As preferred: the binding of silver nanoparticle-zirconium-based metal organic framework composite signal probe to kanamycin on the electrode surface is through an incubation process, and the incubation time is 1 h and the incubation temperature is 310 K.

[0015] The above-mentioned electrochemical aptamer sensor for detecting kanamycin is constructed by a sandwich-type sandwich reaction mode among the aptamer on the electrode, kanamycin and the aptamer on the probe.

[0016] The application also provides a method for detecting kanamycin by using the electrochemical aptamer sensor for detecting kanamycin, comprising the following steps: Step 1: the target analyte is prepared into the electrochemical aptamer sensor for detecting kanamycin by the above preparation method, and the electrochemical aptamer sensor for detecting kanamycin is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to construct a three-electrode system; Step 2: different concentrations of kanamycin standard are prepared into the electrochemical aptamer sensor for detecting kanamycin by the above preparation method, and the electrochemical aptamer sensor for detecting kanamycin is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to construct a three-electrode system, electrochemical detection is carried out, and a standard concentration curve is drawn to obtain a linear relationship formula corresponding to the standard concentration curve; Step 3: the aptamer sensor of step 1 is subjected to electrochemical detection to obtain an electrochemical signal value, and the concentration of kanamycin contained in the test sample solution is calculated based on the linear relationship formula obtained in step 2.

[0017] As a preferred method, the electrolyte in the electrochemical detection system is 20 mL of 1 M KCl solution, and the electrochemical detection linear scan voltammetry is used with a scan rate of 0.05 V / s and a sampling interval of 0.001 V.

[0018] The beneficial effects of this invention are as follows: This invention combines electrochemical analysis technology, which has advantages such as high sensitivity, low detection cost, and ease of use, with aptamer sensors that have specific intermolecular binding, to construct a sandwich-type kanamycin electrochemical aptamer sensor. A silver nanoparticle-zirconium-based metal-organic framework composite material is used as a signal marker to amplify the detection signal. The electrochemical aptamer sensor constructed in this invention has a detection limit of 0.085 aM for kanamycin, and has a wide detection range, fast detection speed, and good reproducibility. Attached Figure Description

[0019] Figure 1 Transmission electron microscopy image of a zirconium-based metal-organic framework material; Figure 2 Linear scan voltammetry of the aptamer sensor for different kanamycin concentrations (0.1 aM, 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM); Figure 3 The graph shows the linear relationship between the peak current value of the aptamer sensor and the logarithmic value of the kanamycin concentration. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] In this invention, all water used in the experiments was deionized water, all reagents used in the experiments were analytical grade, and all experiments were conducted at room temperature.

[0022] The application principle of the present invention will be described in detail below with reference to the accompanying drawings. Example

[0023] Fabrication of an electrochemical aptamer sensor: A1 constructs a kanamycin sensing interface by assembling a kanamycin aptamer with a thiol group on a gold electrode:

[0024] A1.1) Pretreatment of gold electrodes: a. Immerse a 3 mm diameter gold electrode (GE) in a piranha solution for 30 min; b. Polishing the GE with 0.3 μm, 0.05 μm Al2O3 polishing powder, and then cleaning with anhydrous ethanol and ultrapure water to obtain the clean gold electrode.

[0025] A1.2) Immobilization of kanamycin aptamer with thiol group on the gold electrode to obtain a kanamycin sensing interface: 5 μL of 1 μmol / L kanamycin aptamer with thiol group was dropped on the GE treated in step A1.1) using a pipette, and the sequence of the kanamycin aptamer was 5'-SH-(CH2)6-AGA TGG GGG TTG AGG CTA AGC CGA-3'. The electrode was incubated at 310 K for 2 h, and then rinsed with 0.01 mol / L phosphate buffer solution. 5 μL of 5 mmol / L 6-mercapto-1-hexanol solution was dropped on the modified electrode surface, and incubated at 310 K for 1 h to reduce non-specific adsorption and block unbound sites. Then the electrode was rinsed with 0.01 mol / L phosphate buffer solution and stored at 277 K for later use.

[0026] A2. Target molecule kanamycin was incubated and combined with the kanamycin sensing interface: 5 μL of a solution of the target analyte was dropped on the electrode surface obtained in step A1, and incubated at 310 K for 1 h to allow the kanamycin in the target analyte to bind to the kanamycin aptamer on the electrode. Then the electrode was rinsed with a buffer solution and stored at 277 K for later use.

[0027] A3. Combination of silver nanoparticle-zirconium metal organic framework composite signal probe with kanamycin on the electrode surface: A3.1) Synthesis and morphology characterization of zirconium metal organic framework material: 40 mg of 2,2'-dipyridyl 5,5'-dicarboxylic acid and 12.06 mg of zirconium chloride were dispersed in 3 mL and 6 mL of N,N-dimethylformamide, respectively, and then the two reactants were mixed together. 200 μL of acetic acid was added to the reaction mixture. After ultrasonic treatment, the mixture solution was refluxed at 393 K for 18 h. After centrifugal collection, the zirconium metal organic framework material was washed several times with N,N-dimethylformamide and ethanol.

[0028] The prepared zirconium metal organic framework material was subjected to morphology characterization, such as Figure 1 As shown in the transmission electron microscopy image, the zirconium metal organic framework material was a bipyramidal structure of about 300 nm.

[0029] A3.2) Synthesis of silver nanoparticle-zirconium metal organic framework composite: The 2.5 mg zirconium-based metal organic framework material prepared in step A3.1 was dispersed in ultrapure water, 200 μΐ of 1 M AgN03 was added to the dispersion, and stirred uniformly; 200 μΐ of 1 M ascorbic acid was slowly added to the mixture, the mixture changed from colorless to gray, and continued to stir until the color did not change, to obtain a silver nanoparticle-zirconium-based metal organic framework composite material.

[0030] A3.3) Synthesis of silver nanoparticle-zirconium-based metal organic framework composite material probe: The 0.2 mg silver nanoparticle-zirconium-based metal organic framework composite material prepared in step A3.2 was added to 0.1 mL of ultrapure water to prepare a 2 mg / mL silver nanoparticle-zirconium-based metal organic framework composite material dispersion, and 2 μΐ of 100 μιηοΙ / L amino-containing kanamycin aptamer solution was added. The sequence of the amino-containing kanamycin aptamer was 5'-NH2-(CH2)6-AGA TGG GGG TTG AGG CTA AGC CGA-3'. The dispersion was added with NaCl multiple times within 8 h at 277 K, so that the final concentration of NaCl was 0.1 M. The mixture was placed in a refrigerator at 277 K for about 40 h, then centrifuged and washed to obtain a silver nanoparticle-zirconium-based metal organic framework composite material probe; finally, the obtained product was redispersed in 0.1 mL of 0.01 mol / L pH 7.4 phosphate buffer solution and stored at 277 K for standby.

[0031] A3.4) Binding of silver nanoparticle-zirconium-based metal organic framework composite material probe to kanamycin on the surface of the electrode: The kanamycin sensing interface prepared in step A2 which combined kanamycin in the target analyte was added with 5 μΐ of the silver nanoparticle-zirconium-based metal organic framework composite material signal probe prepared in step A3.3, and incubated at 310 K for 1 h, and then eluted with 0.01 mol / L phosphate buffer solution to obtain an electrochemical aptamer sensor for detecting kanamycin. The sensor was a sandwich-type kanamycin electrochemical aptamer sensor. The kanamycin aptamer on the electrode in step A1 specifically bound to the target molecule kanamycin, and the kanamycin in the target analyte was combined to the surface of the electrode, and then the silver nanoparticle-zirconium-based metal organic framework material signal probe was combined to the target molecule kanamycin, and the signal probe was combined to the surface of the electrode. Through this sandwich-type sandwich reaction mode, a new type of electrochemical aptamer sensor for kanamycin was constructed.

[0032] B Electrochemical kanamycin aptamer sensor for detection of kanamycin: B1 Detection method

[0033] The electrochemical kanamycin detection system adopts a classic three-electrode working system: working electrode, reference electrode and counter electrode. The working electrode is the electrochemical aptamer sensor for detecting kanamycin prepared in step A, the counter electrode is a platinum wire electrode, the reference electrode is a saturated calomel electrode, the determination solution is 20 mL of 1 M KCl solution, the linear sweep voltammetry method is adopted, the scanning speed is 0.05 V / s, and the sampling interval is 0.001 V.

[0034] B2 Draw standard concentration working curve Referring to step A, different known concentrations of kanamycin incubation solution (0.1 aM, 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM) are added dropwise to the kanamycin sensing interface prepared in step A1 as target analytes. The kanamycin in the incubation solution can specifically bind to the aptamer on the surface of the electrode, and then the signal probe silver nanoparticle-zirconium-based metal organic framework is added, which can specifically bind to the aptamer and kanamycin, thereby fixing the probe to the surface of the sensor, catalyzing the oxidation of silver, and generating an electrochemical signal. The oxidation peak current value increases with the increase of the concentration of kanamycin in the incubation solution Figure 2 ). Then a relationship is constructed according to the obtained data, as shown in Figure 3 , the increase of the oxidation peak current value is proportional to the logarithm of the concentration of the kanamycin to be detected in the incubation solution in the range of 0.1 aM-1 nM, and the linear correlation coefficient is 0.9975. The detection limit of the electrochemical aptamer sensor is 0.085 aM. It can be seen that the sensor manufactured by the method has good stability and low detection limit for detecting kanamycin.

[0035] B3 Detection of actual samples The actual sample is a milk product, 1 mL of sample is taken into a sample tube, 4 mL of phosphate buffer is added, mixed, ultrasonic for 20 min, centrifuged at 10000 rpm for 10 min, and the upper whey is taken for standby. Add phosphate buffer solution to 5 mL in the whey. 5 μL of the treated sample as the target analyte is dropped on the kanamycin aptamer sensing interface prepared in step A1, and then 5 μL of the silver nanoparticle-zirconium-based metal organic framework composite signal probe prepared in step A3 is dropped on the electrode surface. The electrochemical linear sweep voltammetry method in step B1 is used to detect the concentration of kanamycin on the sensor, and each sample is measured 3 times. The response peak current obtained can be used to calculate the concentration of kanamycin in the test solution according to the standard curve obtained in step B2. The test results are shown in Table 1. The results show that 6 of the 8 milk samples are not detected for kanamycin, and the concentrations of kanamycin detected in the other 2 milk samples are 1.1 fM and 0.8 fM, respectively, both of which are lower than the maximum residual limit of 150 μg / kg (0.3 μM) in milk specified in the national standard. The accuracy of the method is verified by the standard addition recovery method, and the results show that the recovery rate of kanamycin in the 8 actual samples is 96.0% to 100.4%. It shows that this method has good accuracy, and the method can be used for the analysis of kanamycin in milk samples.

[0036] Table 1 Detection results of kanamycin in actual samples

[0037] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an electrochemical aptamer sensor for detecting kanamycin, characterized in that, It comprises the following steps: Step one: assembling kanamycin aptamer with thiol on the surface of gold electrode to obtain kanamycin sensing interface, Step two: binding kanamycin in target analyte to the kanamycin sensing interface; Step three: combining silver nanoparticle-zirconium metal organic framework signal probe with the electrode obtained in step two to obtain an electrochemical aptamer sensor for detecting kanamycin, wherein the silver nanoparticle-zirconium metal organic framework signal probe comprises kanamycin aptamer with amino.

2. The method of claim 1, wherein the method is characterized by: The gold electrode in step one is pretreated, and the pretreatment comprises soaking piranha solution, polishing and ultrasonic cleaning steps; The assembling in step one comprises the following specific steps: dropping kanamycin aptamer with thiol on the pretreated gold electrode, standing at 310 K for 2 h, washing with phosphate buffer solution, dropping 6-mercapto-1-hexanol solution on the surface of the modified electrode, incubating at 310 K for 1 h, and washing with phosphate buffer solution.

3. The method of claim 1, wherein the method is characterized by: The sequence of the kanamycin aptamer with thiol is 5'-SH-(CH2)6-AGA TGG GGG TTG AGG CTA AGC CGA-3'.

4. The method of claim 1, wherein the method is characterized by: The sequence of the kanamycin aptamer with amino is 5'-NH2-(CH2)6-AGA TGG GGG TTG AGG CTA AGC CGA-3'.

5. The method of claim 1, wherein the method is characterized by: The combining in step two comprises the following specific steps: dropping target analyte on the electrode surface obtained in step one, washing with phosphate buffer solution after incubation; the incubation time is 1 h, and the incubation temperature is 310 K.

6. The method of claim 1, wherein the method is characterized by: The preparation method of the silver nanoparticle-zirconium metal organic framework signal probe comprises the following steps: A1) Synthesis of zirconium metal organic framework: dispersing 2,2'-dipyridyl 5,5'-dicarboxylic acid and zirconium chloride in N,N-dimethylformamide respectively, then mixing them together, and adding acetic acid; after ultrasonic treatment, refluxing at 393 K for 18 h, centrifuging and collecting, and washing with N,N-dimethylformamide and ethanol for several times to obtain the zirconium metal organic framework; A2) Synthesis of silver nanoparticle-zirconium metal organic framework composite material: dispersing the zirconium metal organic framework material in ultrapure water, adding AgNO3, stirring uniformly; adding ascorbic acid, and the mixed solution changes from colorless to gray; continue stirring until the color does not change to obtain the silver nanoparticle-zirconium metal organic framework composite material; A3) Synthesis of silver nanoparticle-zirconium metal organic framework composite material probe: adding the silver nanoparticle-zirconium metal organic framework composite material to ultrapure water to prepare a dispersion liquid, adding kanamycin aptamer solution with amino, adding NaCl for multiple times within 8 h at 277 K to make the final concentration of NaCl reach 0.1 M, standing the mixture at 277 K for 40 h, then centrifuging and washing to obtain the silver nanoparticle-zirconium metal organic framework composite material probe.

7. The method of claim 6, wherein the method is characterized by, The mass ratio of silver to zirconium in the silver nanoparticle-zirconium metal organic framework composite material is 3.6:

1.

8. The electrochemical aptamer sensor for detecting kanamycin prepared by the method according to any one of claims 1-7.

9. Use of an electrochemical aptamer sensor for detecting kanamycin according to claim 8, characterized in that, The method comprises the following steps: Step 1: The target analyte is subjected to the electrochemical aptamer sensor for detecting kanamycin prepared by the method according to claim 1, and the electrochemical aptamer sensor for detecting kanamycin is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to construct a three-electrode system; Step 2: Different concentrations of kanamycin standard samples are used as target analytes to prepare the electrochemical aptamer sensor for detecting kanamycin by the method according to claim 1, and the electrochemical aptamer sensor for detecting kanamycin is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode to construct a three-electrode system, electrochemical detection is performed, and a standard concentration curve is drawn to obtain a linear relationship corresponding to the standard concentration curve; Step 3: The aptamer sensor of step 1 is subjected to electrochemical detection to obtain an electrochemical signal value, and the concentration of kanamycin contained in the test sample solution is calculated based on the linear relationship obtained in step 2.

10. Use according to claim 9, characterised in that, In the electrochemical detection system, the electrolyte is 20 mL of 1 M KCl solution, the electrochemical detection is linear sweep voltammetry, the scanning speed is 0.05 V / s, and the sampling interval is 0.001 V.

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