Preparation method and application of electrochemical immunosensor based on enzyme induction

An immunosensor and electrochemical technology, applied in the field of electrochemistry, can solve the problems that the sensitivity of molecularly imprinted electrochemical sensors cannot meet the detection requirements, and achieve the effects of low detection cost, simple operation, high sensitivity and selectivity

Pending Publication Date: 2021-02-12
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Obviously, the sensitivity of molecularly imprinted elect

Method used

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  • Preparation method and application of electrochemical immunosensor based on enzyme induction
  • Preparation method and application of electrochemical immunosensor based on enzyme induction
  • Preparation method and application of electrochemical immunosensor based on enzyme induction

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Experimental program
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Embodiment 1

[0043] 1. Electrode cleaning

[0044] The glassy carbon electrode was polished sequentially with polishing powders with particle sizes of 1.0 μm, 0.3 μm, and 0.05 μm, then cleaned with ultrapure water, ultrasonicated for 10 s, and finally washed again with ultrapure water.

[0045] 2. Probe construction

[0046] Add 6 μL of urethane-coated original (10 μg / mL Ag) to the glassy carbon electrode to be used, incubate for 1 hour, wash with pH7.4 0.1MPBS buffer three times, and wash off the urethane that is not immobilized on the electrode Coating original; soak the glassy carbon electrode in 1% BSA solution to seal for 0.5h, wash with pH7.4 0.1M PBS buffer three times to wash off excess BSA; then soak the glassy carbon electrode in pH7.4 0.1M PBS Incubate in urethane indirect rabbit polyclonal antibody (10μg / mL Ab1) prepared in buffer for 0.5h; wash with pH 7.4 0.1M PBS buffer three times to wash off unbound urethane indirect rabbit polyclonal antibody; finally, glass Soak the carb...

Embodiment 2

[0055] 1. Electrode cleaning

[0056] Cleaning of the electrode: Polish with 1.0 μm, 0.3 μm, and 0.05 μm polishing powder in sequence, then clean with ultrapure water, then ultrasonic for 10 s, and finally wash with ultrapure water to obtain a glassy carbon electrode to be used.

[0057] 2. Probe construction

[0058] Add 20 μL of urethane-coated original (40 μg / mL Ag) to the glassy carbon electrode to be used, incubate for 1 hour, wash with pH7.4 0.1M PBS buffer three times, and wash off the urethane that is not fixed on the electrode Coating original; Soak the glassy carbon electrode in 1% BSA solution to seal for 1h, wash with pH7.4 0.1M PBS buffer three times to wash off excess BSA; then soak the glassy carbon electrode in pH7.4 0.1M PBS Incubate in urethane indirect rabbit polyclonal antibody (40μg / mL Ab1) prepared in buffer for 1h; wash with pH7.4 0.1M PBS buffer three times to wash off unbound urethane indirect rabbit polyclonal antibody; finally, glass Soak the carbo...

Embodiment 3

[0067] 1. Electrode cleaning

[0068] Cleaning of the electrode: Polish with 1.0 μm, 0.3 μm, and 0.05 μm polishing powder in sequence, then clean with ultrapure water, then ultrasonic for 10 s, and finally wash with ultrapure water to obtain a glassy carbon electrode to be used.

[0069] 2. Probe construction

[0070] Drop 10 μL of urethane-coated original (20 μg / mL Ag) to the glassy carbon electrode to be used, incubate for 1 h and wash with pH7.4 0.1MPBS buffer three times to wash off the urethane coating not fixed on the electrode. Cover the original; soak the glassy carbon electrode in 1% BSA solution for 1 hour, wash with pH7.4 0.1M PBS buffer three times to wash off excess BSA; then soak the glassy carbon electrode in pH7.4 0.1M PBS buffer Incubate in the prepared urethane indirect rabbit polyclonal antibody (15 μg / mL Ab1) for 1 h; wash with pH7.4 0.1M PBS buffer three times to wash off the unbound urethane indirect rabbit polyclonal antibody; finally, the glassy carbon...

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Abstract

The invention discloses a preparation method and application of an electrochemical immunosensor based on enzyme induction. The preparation method comprises the following steps of: (1) cleaning a glassy carbon electrode; and (2) constructing a probe; The construction of the probe specifically comprises the following steps of: dropwise adding an ethyl carbamate coating antigen (Ag) to the glassy carbon electrode for incubation; soaking the glassy carbon electrode in a BSA solution; soaking the glassy carbon electrode in an ethyl carbamate indirect rabbit polyclonal antibody (Ab1) for incubation;and finally, soaking the glassy carbon electrode in mouse polyclonal antibody-alkaline phosphatase (Ab2-ALP) for incubation. With the preparation method of the invention adopted, a competitive electrochemical immunosensor based on basic phosphatase (ALP) induced Cu <2+>/Cu<+> conversion is developed. The competitive electrochemical immunosensor can be used for sensitively detecting ethyl carbamate. Compared with the detection sensitivity of a traditional enzyme-linked immunosorbent assay, the detection sensitivity of the competitive electrochemical immunosensor is improved through three rounds of signal amplification, namely catalytic reaction of ALP enzyme, Cu<2+>/Cu<+> conversion and square wave voltammetry signal output.

Description

technical field [0001] The invention belongs to the field of electrochemical technology, in particular to a preparation method and application of an enzyme-induced electrochemical immunosensor. Background technique [0002] Urethane (urethane) is listed as a Class 2A carcinogen and is a chemical pollutant widely present in fermented foods, especially fermented alcoholic beverages. For the sake of human health, many countries have stipulated the legal limit of ethyl carbamate, such as Canada, the United States, France, Japan, etc., have set the legal limit of ethyl carbamate in alcoholic beverages as 15-1000g / L. At present, the commonly used methods for the detection of ethyl carbamate at home and abroad mainly include gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography-fluorescence detection (HPLC-FLD), etc. However, these methods mainly rely on large-scale instruments, requir...

Claims

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Application Information

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IPC IPC(8): G01N27/30G01N27/327G01N33/543
CPCG01N27/308G01N27/3271G01N33/5438
Inventor 吴龙王亚生
Owner HUBEI UNIV OF TECH
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