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Electrochemical sensor for detection of kanamycin based on nucleic acid aptamer and preparation method thereof

A nucleic acid aptamer and kanamycin technology, applied in the field of its preparation, can solve the problems of low specificity, sensitivity, and high cost, and achieve the effects of improved sensitivity, low detection limit, and simple electrodes

Inactive Publication Date: 2017-12-26
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 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-based induction method with high specificity and sensitivity, low cost and fast detection speed. A biosensor for the detection of kanamycin by conformational changes of nucleic acid aptamers

Method used

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  • Electrochemical sensor for detection of kanamycin based on nucleic acid aptamer and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The main steps of the electrode modification process are as follows:

[0043] a. The gold electrode is first polished in 0.3 and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed repeatedly with PBS and secondary water;

[0044] b. Add 10 μL of the mixture of HAP2 and Helper (10 μM) onto the electrode surface, and incubate at 37°C for 2 hours. Fix the sulfhydryl chains to the electrode surface through Au-S bonds;

[0045] So far, the modification process of the electrode has come to an end. The following describes the reaction in the homogeneous solution and the main steps in the homogeneous reaction:

[0046] a. Sterilized water, 10× buffer buffer, different concentrations of HAP1 (1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM), Primer (10 μM), phi29 DNA polymerase (2 μL ), dNTPs (2 μL), Nt.AlwI Endonuclease (1 μL) and the target substance to be tested were added to the centrifuge tube, shaken for 30s, and incubated in a 37°C incubator for 2h.

[0...

Embodiment 2

[0059] The main steps of the electrode modification process are as follows:

[0060] a. The gold electrode is first polished in 0.3 and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed repeatedly with PBS and secondary water;

[0061] b. Add 10 μL of the mixture of HAP2 and Helper (10 μM) onto the electrode surface, and incubate at 37°C for 2 hours. Fix the sulfhydryl chains to the electrode surface through Au-S bonds;

[0062] So far, the modification process of the electrode has come to an end. The following describes the reaction in the homogeneous solution and the main steps in the homogeneous reaction:

[0063] a. Sterilized water, 10× buffer buffer, HAP1 (10 μM), different concentrations of Primer (1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM), phi29 DNA polymerase (2 μL), dNTPs (2 μL), Nt.AlwI Endonuclease (1 μL) and the target substance to be tested were added to the centrifuge tube, shaken for 30 seconds, and incubated in a 37°C incubator for 2 h...

Embodiment 3

[0072] The main steps of the electrode modification process are as follows:

[0073] a. The gold electrode is first polished in 0.3 and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed repeatedly with PBS and secondary water;

[0074] b. Add 10 μL of the mixture of HAP2 and Helper at different concentrations (1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM) onto the electrode surface and incubate at 37°C for 2 hours. Fix the sulfhydryl chains to the electrode surface through Au-S bonds;

[0075] So far, the modification process of the electrode has come to an end. The following describes the reaction in the homogeneous solution and the main steps in the homogeneous reaction:

[0076] a. Sterilized water, 10× buffer buffer, HAP1 (10 μM), Primer (10 μM), phi29 DNA polymerase (2 μL), dNTPs (2 μL), Nt.AlwI Endonuclease (1 μL) and the target substance to be tested were added to the centrifuge tube, shaken for 30s, and incubated in a 37°C incubator for 2h.

[0077...

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Abstract

The invention relates to a biosensor for detecting kanamycin based on target-induced conformational changes of nucleic acid aptamers. The invention is prepared by sequentially modifying an electrode with a HAP2 layer, a Helper layer, and a homogeneous reaction mixture. The present invention utilizes the specific type recognition of nucleic acid aptamer, uses the aptamer of kanamycin as the recognition substance to realize the highly specific detection of the target kanamycin; uses the nucleic acid tool enzyme to realize the recycling of the target , played the role of signal amplification, thereby improving the detection sensitivity.

Description

technical field [0001] The invention relates to the technical field of electrochemical sensors, in particular to an electrochemical biosensor for detecting kanamycin based on target-induced conformational changes of nucleic acid aptamers, and to a preparation method thereof. Background technique [0002] Kanamycin (Ka) is an aminoglycoside antibiotic produced by the fermentation of Streptomyces sp. and is widely used to treat mistranslation-induced infections and indirectly inhibit translocation during protein synthesis. Like other aminoglycosides, Ka can accumulate in animals and transfer to the food chain, which may cause potential hazards to human health, such as hearing loss, nephrotoxicity and drug allergic reactions. EU organizations have established maximum residues of Ka in edible tissues and milk. The currently reported methods for detecting Ka include high performance liquid chromatography, capillary electrophoresis, surface plasmon resonance and fluorescence reso...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王玉王虹智黄加栋刘素郭玉娜许颖邱婷婷崔洁崔雪君冷雪琪韩聪裴倩倩
Owner UNIV OF JINAN
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