Electrochemical immunosensing method for detecting Listeria monocytogenes

A Listeria monocytogenes and immune sensor technology, applied in the field of biotechnology detection, can solve the problems of time-consuming, labor-intensive, prone to false positives, cumbersome steps, etc.

Inactive Publication Date: 2015-05-20
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among these methods, the results of conventional culture methods are accurate and reliable, but time-consuming and laborious, and it usually takes 5-7 days to obtain the results; the ELISA method is simple to operate, has strong specificity, and high reaction sensitivity, but it ne

Method used

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  • Electrochemical immunosensing method for detecting Listeria monocytogenes
  • Electrochemical immunosensing method for detecting Listeria monocytogenes
  • Electrochemical immunosensing method for detecting Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0026] Embodiment 1: Fabrication of Immunosensors on the Electrode Surface of Electrochemical Sensors

[0027] (1) Polish the gold electrode in circles on 2000 mesh, 3000 mesh, 5000 mesh sandpaper or in Piranha solution (H 2 o 2 / H 2 SO 4 , 1:3, v / v) for 5 minutes, washed with ultrapure water, and then 1.0μm, 0.3μm, 0.05μm A1 2 o 3 The powder is adjusted into a suspension and polished on the suede, washed with ultra-pure water; the clean gold electrode is placed in 0.1M H 2 SO 4 Carry out cyclic voltammetry scanning (voltage range -0.2V~1.2V, scanning rate 50mV / s) cleaning in the solution until a stable cyclic voltammetry diagram is obtained; then in 0.05M H 2 SO 4 Activate in the solution (voltage range -0.2V~1.6V, scan rate 50mV / s), until a smooth reduction peak is obtained at about 0.94V, wash with ultrapure water, and blow dry with nitrogen;

[0028] (2) Place the treated gold electrode in 2 mL of 1% 3-mercaptopropionic acid ethanol solution for self-assembly, over...

Embodiment approach 2

[0032] Embodiment 2: Detection of Listeria monocytogenes by sandwich electrochemical immunosensor based on enzyme amplification

[0033] (1) React the prepared immunosensor with blank solution PBS at 37°C, then react with HRP-labeled polyclonal antibody at 37°C for 30min, and finally detect with chronoamperometry, and the obtained value is negative;

[0034] (2) Listeria monocytogenes solution (10 7 CFU / ml, 10 6 CFU / ml...10 1 CFU / ml), respectively incubated with the prepared immunosensor at 37°C for 1h, then reacted with HRP-labeled polyclonal antibody, incubated at 37°C for 30min, and finally detected the current response value by chronoamperometry on the electrochemical sensor , use 0.01M glycine-HCl solution to regenerate for 5min, remove the combined LM, and cycle for 10 1 CFU / ml-10 7 LM within the concentration range of CFU / ml was detected.

[0035](3) The difference between the current response and the logarithmic value of the LM concentration and the standard curve...

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Abstract

The invention discloses an electrochemical immunosensing method for detecting Listeria monocytogenes, belonging to the field of the biotechnological detection. The surface of a gold electrode is activated electrochemically and then is immersed in 1% of 3-mercaptopropionic acid ethanol solution to be self-assembled to form a monomolecular film, a capture antibody is combined to the surface of the electrode after activation, an enzyme amplification signal is introduced by a sandwich method, and an enzymatic electroactive substance is subjected to oxidation reduction to obtain a current response signal, therefore, a method for detecting the Listeria monocytogenes by use of the electrochemical immunosensor adopting three electrodes as a system is established; the method has good sensitivity, specificity and stability; the detection system is small, the reagent is saved, the detection cost is reduced remarkably, the method has great potential in application and practical sample detection due to no influence from the turbidity of the sample.

Description

technical field [0001] The invention relates to a method for detecting Listeria monocytogenes by a chronoamperometry based on sandwich method electrochemical immunosensing, and belongs to the technical field of biotechnology detection. Background technique [0002] Listeria monocytogenes is an important pathogen of zoonotic and food-borne diseases. It can cause listeriosis in humans and animals. After infection, the main manifestations are meningitis, sepsis, abortion, etc., and the fatality rate is as high as 30% to 70%, this bacterium has been listed as one of the four major food pathogens in the 1990s in the world. FDA, WHO and other organizations have successively established corresponding institutions or stipulated corresponding strategies to prevent and control the contamination and spread of the bacteria. In 1988, WHO issued an advisory book on "Foodborne Listeriosis", guiding countries around the world on how to prevent LM pollution and poisoning, and advising publi...

Claims

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Application Information

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IPC IPC(8): G01N27/416G01N33/569G01N33/531
Inventor 高志贤彭媛程超男宁保安白家磊
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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