Electrochemical biosensor for detecting kanamycin as well as preparation method and application

A biosensor, kanamycin technology, applied in electrochemical variables of materials, scientific instruments, instruments, etc., can solve the problems of high cost, low specificity and sensitivity, and achieve high speed, improved sensitivity, and low process cost. Effect

Active Publication Date: 2019-03-22
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of relatively low specificity and sensitivity and high cost of the method for detecting kanamycin in the above prior art, the present invention provides a target-induced detection method with simple operation, high specificity and sensitivity, and low cost. conformational changes of nucleic acid aptamers and Nt.BbvCI An electrochemical biosensor for detection of kanamycin with endo- and exonuclease III-assisted loop amplification

Method used

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  • Electrochemical biosensor for detecting kanamycin as well as preparation method and application
  • Electrochemical biosensor for detecting kanamycin as well as preparation method and application
  • Electrochemical biosensor for detecting kanamycin as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of CP modified gold electrode.

[0057] a) Gold electrode polishing treatment: The gold electrode is polished to the mirror surface in 0.3 µm and 0.05 µm alumina slurry in sequence, and washed with ultrapure water 5 times;

[0058] b) Drop 10 μL of CP solution (final concentration of 1 μM, 5 μM, 10 μM, 15 μM, 20 μM) on the surface of the gold electrode after the above treatment, and incubate at 37 ℃ for 2 hours to obtain a CP-modified gold electrode S1- S6.

Embodiment 2

[0059] Example 2 The influence of different CP concentrations on the detection of kanamycin.

[0060] (1) Hybridize Aptamer and Primer in PBS buffer into Probe (10 μM);

[0061] (2) Combine the probe Probe, 10 μM HAP1, 10 μM HAP2, 10 μM HAP3, 1U / μL Nt.BbvCI endonuclease and 1U / μL exonuclease III, 10×cutsmart buffer, 5nM kanamycin standard Incubate the solution at a constant temperature after mixing;

[0062] (3) Add the mixed solution in step (2) dropwise to gold electrodes S1-S6 modified with different concentrations of CP, and then incubate at 37 ℃ for 2 hours;

[0063] (4) Drop 5 μL of heme solution (10 μM) onto the gold electrode, and then incubate at 37°C for 1 hour;

[0064] (5) Rinse the electrode in step (4) 3 times with PBS buffer solution;

[0065] (6) When containing H 2 O 2 (10 μM) PBS buffer solution, Ag / AgCl as the reference electrode, Pt electrode as the counter electrode, differential pulse voltammetry to measure the current change, potential 0 to -0.6 V, pulse width 0.0...

Embodiment 3

[0067] Example 3 The influence of different HAP1 concentrations on the detection of kanamycin.

[0068] (1) Hybridize Aptamer and Primer in PBS buffer to form Probe (10 μM);

[0069] (2) Combine the probes in step (1), HAP1 (final concentration is 1 μM, 5 μM, 10 μM, 15 μM, 20 μM), 10 μMHAP2, 10 μM HAP3, 1U / μL Nt.BbvCI endonuclease And 1U / μL Exonuclease III, 10× cutsmart Buffer and 5nM kanamycin standard solution were mixed and incubated at 37°C;

[0070] (3) Add the mixed solution in step (2) dropwise to the modified CP gold electrode S4, and then incubate at 37°C for 2h;

[0071] (4) Add 5 μL of heme solution (10 μM) dropwise to the electrode obtained in step (3), and then incubate at 37 ℃ for 1 hour;

[0072] (5) Rinse the electrode in step (4) 3 times with PBS buffer solution;

[0073] (6) When containing H 2 O 2 (10 μM) PBS buffer solution, Ag / AgCl as the reference electrode, Pt electrode as the counter electrode, and differential pulse voltammetry to measure the current change, p...

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Abstract

The invention relates to an electrochemical biosensor for detecting kanamycin based on an aptamer, and belongs to the technical field of electrochemical biosensors. By utilizing an Nt.BbvCI incision enzyme with a function of cutting identification, cyclic utilization of a primer is realized, a detection signal is amplified, and the detection sensitivity is improved; by utilizing the specificity idenfitication and the hydrolytic action of an exonuclease III, the circle amplification at a second step is achieved, so that the detection sensitivity is further improved. According to the electrochemical biosensor disclosed by the invention, the specific detection can be improved; the electrochemical biosensor is mild in reaction conditions and high in reaction speed; a process of making an electrode is low in cost, so that the electrochemical biosensor is suitable for the requirement of low cost in industrialization and is also suitable for the detection of the kanamycin in food safety and the actual industrial application of the electrochemical biosensors.

Description

Technical field [0001] The invention relates to a biosensor for detecting kanamycin based on nucleic acid aptamers, in particular to a target-induced conformational change of nucleic acid aptamers and Nt.BbvCI An electrochemical biosensor with endonuclease and exonuclease III assisted circulation amplification and detection of kanamycin belongs to the technical field of electrochemical biosensors. Background technique [0002] Antibiotics have the function of killing and destroying the cell structure of microorganisms, and are widely used in the preparation of various antibacterial drugs around the world. At the same time, the residual problem caused by the abuse of antibiotics has become an urgent problem in the world. Excessive use of antibiotics can stimulate the bacteria caused by infection to become resistant. [0003] Kanamycin (Kanamycin) is a protein biosynthesis inhibitor and an antibiotic. It is used orally to treat intestinal infections caused by sensitive bacteria and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/30G01N27/327
CPCG01N27/30G01N27/3271
Inventor 刘素张儒峰黄加栋王玉宋晓蕾瞿晓南赵一菡李莎莎江龙张曼茹
Owner UNIV OF JINAN
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