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Electrochemical biosensor for detecting Salmonella typhimurium and preparation method thereof

A biosensor, Salmonella technology, applied in the field of biosensors, can solve the problems of complex instrument operation and long time-consuming, and achieve the effects of good repeatability, improved sensitivity, and sensitive detection

Active Publication Date: 2017-08-11
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The present invention aims at the defects of complicated instrument operation, long time-consuming and professional operators in the existing detection method, and provides a cascading signal based on the assistance of exonuclease III with strong specificity, high sensitivity, low cost and fast detection speed Amplified electrochemical biosensor for the detection of Salmonella typhimurium

Method used

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  • Electrochemical biosensor for detecting Salmonella typhimurium and preparation method thereof
  • Electrochemical biosensor for detecting Salmonella typhimurium and preparation method thereof
  • Electrochemical biosensor for detecting Salmonella typhimurium and preparation method thereof

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preparation example Construction

[0045] The preparation method of described biosensor comprises the following steps:

[0046] (1) Pretreatment of the electrodes;

[0047] (2) Modification of HAP2 to the electrode surface;

[0048] (3) Modification of homogeneous reaction products onto the electrode surface.

[0049] In the preparation method, the operation steps of modifying HAP2 onto the electrode surface are preferably as follows: 10 μL of HAP2 (10 μM) is added dropwise to the pretreated electrode surface, and incubated at 37 °C for 2 h.

[0050] In the preparation method, the preferred steps of modifying the homogeneous reaction product onto the electrode surface are as follows:

[0051] (1) Mix 8 μL sterile water, 2 μL 10× NEBuffer buffer (10 mM Bis Tris Propane-HCl, 10 mM MgCl 2 ,1 mM DTT, pH 7.0), 2 μL aptamer (1 mM), 2 μL primer (1 mM), 2 μL HAP1 (10mM), 2 μL Exonuclease Ⅲ (20 U / μL) and 2 μL of the target substance to be tested were added Place in a centrifuge tube, shake for 30 s, and incubate in ...

Embodiment 1

[0058] The main steps of the electrode modification process are as follows:

[0059] 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;

[0060] b. Add 10 μL of HAP2 (10 μM) dropwise to the electrode surface and incubate at 37 °C for 2 h. Fix the sulfhydryl chains to the electrode surface through Au-S bonds;

[0061] 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:

[0062] a. Mix 8 μL sterile water, 2 μL 10× NEBuffer buffer (10 mM Bis Tris Propane-HCl, 10 mMMgCl 2 ,1 mM DTT, pH 7.0), 2 μL aptamer (1 mM), 2 μL primer (1 mM), 2 μL HAP1 (1 μM, 3 μM, 6 μM, 10 μM, 15 μM, 20 μM), 2 μL Add Exonuclease III (20 U / μL) and 2 μL of the target substance to be tested into a centrifuge tube, shake for 30 s, and incubate in a 37 °C incubator fo...

Embodiment 2

[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 HAP2 (1 μM, 3 μM, 6 μM, 10 μM, 15 μM, 20 μM) onto the electrode surface respectively, and incubate at 37°C for 2 h. 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. Mix 8 μL sterile water, 2 μL 10× NEBuffer buffer (10 mM Bis Tris Propane-HCl, 10 mMMgCl 2 ,1 mM DTT, pH 7.0), 2 μL aptamer (1 mM), 2 μL primer (1 mM), 2 μL HAP1 (10 μM), 2 μL Exonuclease III (20 U / μL) and 2 μL target Add to a centrifuge tube, shake for 30 s, and incubate in a 37°C incubator for 2 h;

[0077] b. Add the...

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Abstract

The invention relates to the technical field of biosensors, in particular to an electrochemical biosensor for detecting Salmonella typhimurium and provides a preparation method of the electrochemical biosensor. The electrochemical biosensor is prepared by modifying an electrode with a HAP2 layer, and homogeneously reacting a mixed liquid. An aptamer of Salmonella typhimurium is used as a recognizing matter to highly specifically detect Salmonella typhimurium in a target by making use of specific recognizing property of the aptamer; the target is recycled through the use of nucleic instrumental enzymes, and signal amplification is achieved.

Description

technical field [0001] The invention relates to the technical field of biosensors, in particular to an electrochemical biosensor for detecting Salmonella typhimurium, and also relates to a preparation method for the electrochemical biosensor for detecting Salmonella typhimurium. Background technique [0002] Salmonella is a Gram-negative bacterium with blunt ends, no spores, and generally no capsule. Except for Salmonella pullorum and Salmonella gallinarum typhi, others have flagella all over the body. Salmonella is a common zoonotic pathogen that can not only cause gastroenteritis, but also various syndromes such as sepsis, typhoid fever, and extraintestinal focal infection. In particular, pathogenic bacteria represented by Salmonella typhimurium have caused a huge threat in food safety, environmental monitoring, disease prevention, etc., and have caused great harm to human health, so they have received strong attention from people. In recent years, the detection techn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3277
Inventor 黄加栋裴倩倩刘素王玉王虹智郭玉娜
Owner UNIV OF JINAN
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