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Construction method of fluorescent biosensor for rapid detection of Listeria monocytogenes

A biosensor and sensor technology, applied in fluorescence/phosphorescence, instruments, measuring devices, etc., can solve the problems of easy false positive results, high design requirements, complicated operation, etc., and achieve easy on-site instant detection and strong anti-interference ability , to overcome the effect of low sensitivity

Pending Publication Date: 2022-01-28
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Common nucleic acid amplification methods such as qPCR, mPCR, loop-mediated isothermal amplification, etc., but the operation is complex, requiring professionals and special instruments, and the design of primers and probes is demanding and difficult, and false positive results are prone to occur

Method used

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  • Construction method of fluorescent biosensor for rapid detection of Listeria monocytogenes
  • Construction method of fluorescent biosensor for rapid detection of Listeria monocytogenes
  • Construction method of fluorescent biosensor for rapid detection of Listeria monocytogenes

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Embodiment 1

[0020] 1. Fabrication of Fluorescent Biosensors

[0021] (1) Preparation of UCNPs

[0022] 1) Weigh 0.1164g YCl respectively 3 ·6H 2 O, 0.0892g GdCl 3 ·6H 2 O, 0.062g YbCl 3 ·6H 2 O and 0.006g ErCl 3 ·6H 2 O was dissolved in 4mL of methanol solution, sonicated until completely dissolved, and then poured into a three-necked flask rinsed with a little methanol (the three-necked flask must be kept clean, if necessary, it can be soaked overnight in aqua regia); then poured into 6mL of oleic acid and 14 mL of 1-octadecene.

[0023] 2) Under continuous magnetic stirring, the above mixture was placed under nitrogen protection condition, heated to 160°C, kept for 30min, and then cooled to about 50°C.

[0024] 3) Weigh 0.2964g NH respectively 4 F and 0.2g NaOH were dissolved in 20mL methanol solution, and ultrasonicated for about 15min until completely dissolved; then, under continuous stirring, the solution was added dropwise to the above flask solution. The mixture was the...

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Abstract

The invention relates to a fluorescent biosensor constructed based on a double-site recognition strategy and a fluorescent inner filter effect. An up-conversion nanoparticle (UCNPs) mediated fluorescent biosensor is constructed, and is used for rapid and sensitive detection of Listeria monocytogenes. The method comprises the following steps: preparing an MNPs-Van magnetic nano probe by taking vancomycin (Van) as a first recognition molecule; and taking a biotinylated aptamer as a second recognition molecule and combining thesecond recognition molecule with internalized protein A on the cell wall of the listeria monocytogenes. Two recognition molecules are used for respectively targeting different sites on target bacteria, so that the target bacteria are specifically captured, and a sandwich type compound (MNPs-Van / Listeria monocytogenes / aptamer) is formed. A HRP-TMB enzyme catalysis system is introduced through a horse radish peroxidase marker (HRP-SA) to generate a blue substance, and the fluorescence intensity of the UCNPs is reduced by combining the fluorescence inner filter effect of the UCNPs, so that the quantitative detection of the listeria monocytogenes is realized.

Description

technical field [0001] The invention belongs to the technical field of food safety detection and analysis. Specifically, it involves the construction of a fluorescent biosensor based on a two-site recognition strategy and a fluorescent inner filter effect, thereby realizing a new method for rapid detection of Listeria monocytogenes in food. Background technique [0002] "Food is the most important thing for the people, and food safety is the first priority." Food safety is one of the livelihood issues that ordinary people are most concerned about. Food-borne diseases caused by food-borne pathogenic microorganisms are important public health problems, and they are also the key monitoring objects of food safety in various countries. There are reports of food poisoning and food hygiene incidents every year, especially pathogenic microorganisms, which are extremely unfavorable for the healthy and orderly development of the country. From the data analysis of foodborne disease o...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 潘磊庆李悦屠康陈敏彭菁王振杰
Owner NANJING AGRICULTURAL UNIVERSITY
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