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Fluorescent biosensor for detecting kanamycin, preparation method and application thereof

A biosensor, kanamycin technology, applied in the field of biosensors, can solve the problems of cumbersome sample pretreatment or concentration process, difficult to popularize, expensive instruments, etc., and achieve the effect of fast detection speed, low detection limit and easy operation

Active Publication Date: 2021-03-02
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 cumbersome operation of the method for detecting kanamycin in the prior art, cumbersome sample pretreatment or concentration process, and expensive instruments that are difficult to popularize, the present invention provides a target substance with strong specificity, high affinity, and binding A biosensor for detecting kanamycin by hybridization chain reaction amplification fluorescence method with multiple, smaller molecular weight and simple synthesis process, and also provides its preparation method

Method used

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  • Fluorescent biosensor for detecting kanamycin, preparation method and application thereof
  • Fluorescent biosensor for detecting kanamycin, preparation method and application thereof
  • Fluorescent biosensor for detecting kanamycin, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A method for preparing a fluorescent biosensor for detecting kanamycin, comprising the following steps:

[0061] (1) Preparation of gold nanoparticles;

[0062] (2) Preparation of modified gold nanoparticles solution: modify Aptamer, Walker and Track on the surface of gold nanoparticles;

[0063] (3) Hybrid chain reaction: Mix the modified gold nanometer solution with the homogeneous reaction solution;

[0064] (4) Fluorescence detection.

[0065] In the step (1), the preferred preparation steps of gold nanoparticles are as follows:

[0066] (1) Install the required instruments and add 200mL ultrapure water into the three-necked flask (be careful not to let dust fall into the three-necked flask).

[0067] (2) Take 500uL (0.04g / mL) HAuCl4 in a single-package centrifuge tube, use a pipette gun to take 500uL and 200mL ultrapure water, stir and heat at a stirring speed of about 450 rpm, until boiled.

[0068] (3) Under the condition of stirring, take 3mL of 1% trisodium...

Embodiment 2

[0084] A method for preparing a fluorescent biosensor for detecting kanamycin, comprising the following steps:

[0085] (1) Preparation of gold nanoparticles;

[0086] (2) Preparation of modified gold nanoparticles solution: modify Aptamer, Walker and Track on the surface of gold nanoparticles;

[0087] (3) Hybrid chain reaction: Mix the modified gold nanometer solution with the homogeneous reaction solution;

[0088] (4) Fluorescence detection.

[0089] Steps (1), (2), and (4) are the same as in Example 1.

[0090] The main steps of the reaction process of step (3) are as follows: sterilized water, target substance (4 μL), modified nano-gold solution (6 μL), HAP1 (2 μL) (final concentrations were 0.1 nM, 0.2 nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM), 10× buffer (buffer) (4 μL), restriction endonuclease IV (3 μL), signal probe (6 μL), added to In pre-prepared sterile EP tubes. Shake for 30 s and incubate for 2 h in a 37°C incubator.

[0091] The excitation wavelength of the fluore...

Embodiment 3

[0094] A method for preparing a fluorescent biosensor for detecting kanamycin, comprising the following steps:

[0095] (1) Preparation of gold nanoparticles;

[0096] (2) Preparation of modified gold nanoparticles solution: modify Aptamer, Walker and Track on the surface of gold nanoparticles;

[0097] (3) Hybrid chain reaction: Mix the modified gold nanometer solution with the homogeneous reaction solution;

[0098] (4) Fluorescence detection.

[0099] Steps (1), (2), and (4) are the same as in Example 1.

[0100] The main steps of the reaction process in step (3) are as follows: sterilized water, target substance (4 μL), modified nano-gold solution (6 μL), HAP1 (2 μL), HAP2 (2 μL), 10× buffer (3 μL), restriction endonuclease IV (3 μL), signal probe (6 μL) (final concentrations were 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2μM, 1.4μM), added to the pre-prepared sterilized EP tube. Shake for 30 s and incubate for 2 h in a 37°C incubator.

[0101] The excitation wavelen...

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Abstract

The invention relates to the technical field of biosensors, in particular to fluorescent biosensors based on hybridization chain reaction amplification. 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. In the invention, the combination of the endonuclease IV and the chain hybridization chain reaction realizes the circular amplification effect, the fluorescence resonance energy transfer of the fluorescent group and the quenching group, and the homogeneous reaction mixed solution. Preparation method: prepare gold nanoparticles; modify Walker and Track on the surface of gold nanoparticles; mix the labeled nano-gold solution with a homogeneous reaction solution; hybridization chain reaction, fluorescence detection; Aptamer's highly specific detection of the target kanamycin; the use of hybridization chain reaction amplification to achieve signal amplification.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to a fluorescent biosensor for detecting kanamycin by a hybridization chain reaction amplification fluorescence method, and also relates to a preparation method and application thereof. Background technique [0002] Kanamycin is a broad-spectrum aminoglycoside antibiotic, used as a water-soluble sulfate, effective against many Gram-negative bacteria, including gonorrhea, salmonella, and tuberculosis infections, and widely used as a form of second-line antibiotics Shared medicines for humans and animals. Kanamycin overdose can cause ototoxicity and nephrotoxicity. Kanamycin can accumulate in animals and pass through the food chain. Therefore, kanamycin in foods of animal origin is potentially dangerous to human health. [0003] At present, in the analytical methods of kanamycin, commonly used are high performance liquid chromatography, capillary electrophoresis, immunoassay and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 刘素瞿晓楠姜磊王玉黄加栋王海旺王敬锋张雪张儒峰赵一菡李莎莎
Owner UNIV OF JINAN
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