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An electrochemical biosensor for detecting kanamycin and its application

A biosensor, kanamycin technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of high cost, low specificity and sensitivity, and achieve the effects of fast speed, improved sensitivity and stable performance

Active Publication Date: 2020-09-29
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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  • An electrochemical biosensor for detecting kanamycin and its application
  • An electrochemical biosensor for detecting kanamycin and its application
  • An electrochemical biosensor for detecting kanamycin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

Embodiment 2

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

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

[0061] (2) 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 After the solution is mixed, incubate at constant temperature;

[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 °C for 2 h;

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

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

[0065] (6) containing H 2 o 2 (10 μM) PBS buffer solution, with Ag / AgCl as the reference electrode and Pt electrode as the counter electrode, the current change was measured by differential pulse voltammetry, the potential was 0 to -0.6 ...

Embodiment 3

[0067] Example 3 Effects 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) Mix the probe in step (1), HAP1 (final concentration is 1 μM, 5 μM, 10 μM, 15 μM, 20 μM), 10 μM HAP2, 10 μM HAP3, 1U / μL Nt.BbvCI endonuclease and 1U / μL exonuclease III, 10× cutsmart The buffer solution and 5nM kanamycin standard solution were mixed and then incubated at a constant temperature of 37°C;

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

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

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

[0073] (6) containing H 2 o 2 (10 μM) PBS buffer solution, with Ag / AgCl as the reference electrode and Pt electrode as the ...

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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 a nucleic acid aptamer, in particular to a target-induced conformational change of a nucleic acid aptamer and Nt.BbvCI The invention relates to an electrochemical biosensor for detecting kanamycin with endonuclease and exonuclease III assisted cycle amplification and detection, belonging to the technical field of electrochemical biosensors. Background technique [0002] Antibiotics have the function of killing and destroying microbial cell structures, and are widely used in the preparation of various antibacterial drugs around the world, but at the same time, the residual problem caused by the abuse of antibiotics has become a problem that needs to be solved urgently worldwide. Overuse of antibiotics can spur infection-causing bacteria to become resistant. [0003] Kanamycin is a protein biosynthesis inhibitor and an antibiotic. It is used orally for the treatment of intestinal infe...

Claims

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Application Information

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Patent Type & Authority Patents(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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