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Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria

A technology of luminescent immunity and pathogenic bacteria, which is applied in the field of electrochemiluminescence immune sensor, can solve the problems that have not been reported, and achieve the effect of good reproducibility, high sensitivity and high accuracy

Active Publication Date: 2016-02-03
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the sandwich type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria by capturing bacteria with immunomagnetic beads and using multifunctional graphene oxide as a marker

Method used

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  • Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria
  • Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria
  • Preparing method and application of sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria

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specific Embodiment 1

[0035] A method for preparing a sandwich type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria, comprising the following steps:

[0036] (1) Synthesis of immunomagnetic beads

[0037] Add 5-10 mg of aminated magnetic beads to a clean flask, then add 2-5 mL of 0.1 mol / L pH7.4 phosphate buffer solution containing 3 wt % glutaraldehyde, incubate at 37 ° C for 3 h, and then add a magnet for magnetic separation Wash with 0.1mol / L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and dilute to 2~5mL with 0.1mol / LpH7.4 phosphate buffer solution; add 50~150μL of 0.5mg / mL Marine pathogen primary antibody solution, incubate at 37°C for 1 hour, add a magnet for magnetic separation, wash with 0.1mol / L pH7.4 phosphate buffer solution for 3 to 5 times, remove the supernatant, and wash with 0.1mol / LpH7.4 phosphate buffer solution Dilute the phosphate buffer solution to 2-5mL; add 50-150μL of 2wt% bovine serum albumin solution to block th...

specific Embodiment 2

[0046] The sandwich-type electrochemiluminescent immunosensor for detecting marine pathogenic bacteria is used to detect marine pathogenic bacteria. The specific steps are as follows:

[0047] Soak the magnetic glassy carbon electrodes with immunomagnetic beads immobilized on the surface prepared in step (3) of the above-mentioned specific example 1 respectively in solutions containing different concentrations of marine pathogenic bacteria, and incubate at 37° C. for 1 hour, and dilute with secondary water After cleaning, soak in the multifunctional graphene oxide solution prepared in the first step (2) of the above specific example, take it out after incubating at 37° C. for 1 h, and use it as a working electrode after washing with secondary water; use a platinum electrode as a counter electrode, Ag / AgCl electrode or saturated calomel electrode as a reference electrode to form a three-electrode system; put the above three-electrode system into the buffer solution containing c...

specific Embodiment 3

[0051] A preparation method for detecting a sandwich type electrochemiluminescence immunosensor for Vibrio parahaemolyticus (VP), comprising the following steps:

[0052] (1) Synthesis of immunomagnetic beads

[0053] Add 8mg of aminated magnetic beads to a clean flask, then add 3.5mL of 0.1mol / L pH7.4 phosphate buffer solution containing 3wt% glutaraldehyde, incubate at 37°C for 3h, add a magnet for magnetic separation and use 0.1mol / LpH7.4 phosphate buffer solution washed 4 times, remove the supernatant, 0.1mol / LpH7.4 phosphate buffer solution to 2 ~ 5mL; add 100μL 0.5mg / mL Vibrio parahaemolyticus primary antibody solution , incubate at 37°C for 1 hour, add magnetic separation, wash with 0.1mol / L pH7.4 phosphate buffer solution for 4 times, remove the supernatant, and dilute to 3mL with 0.1mol / L pH7.4 phosphate buffer solution; Add 100 μL of 2 wt% bovine serum albumin solution to block non-specific active sites, incubate at 37°C for 1 h, apply magnetic separation with a ma...

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Abstract

The invention discloses a preparing method and application of a sandwich type electrochemical luminescence immunosensor for detecting marine pathogenic bacteria. The method is characterized by comprising the steps of obtaining an immunomagnetic bead solution the surface of which is covered with marine pathogenic bacterium antibodies by immobilizing the marine pathogenic bacterium antibodies onto the surfaces of aminated magnetic beads; synthesizing multifunctional oxidized graphene through chemical bonding of the marine pathogenic bacterium antibodies and an electrochemical illuminant; conducting electrode pretreatment and immobilizing immunomagnetic beads onto the surface of a magnetic glassy carbon electrode; finally adsorbing the marine pathogenic bacteria to be detected and the multifunctional oxidized graphene onto the surface of the magnetic glassy carbon electrode in sequence. The obtained sandwich type electrochemical luminescence immunosensor serves as a working electrode, a platinum electrode is taken as a counter electrode and an Ag / AgCl electrode or saturated calomel electrode is taken as a reference electrode to calculate the accurate concentration of the marine pathogenic bacteria in a sample solution to be detected. Detection speed is high, the sensitivity and accuracy of detection results are high, and specificity is high.

Description

technical field [0001] The invention relates to an electrochemiluminescence immunosensor, in particular to a preparation method and application of a sandwich type electrochemiluminescence immunosensor for detecting marine pathogenic bacteria. Background technique [0002] Marine pathogenic bacteria are prokaryotic single-celled organisms that are spherical, rod-shaped, helical, etc. Marine pathogenic bacteria can cause diseases in marine aquatic animals and plants, for example: Vibrio parahaemolyticus can cause devastating death of juvenile Litopenaeus vannamei. Some marine pathogenic bacteria are important pathogens of zoonotic diseases. Humans are infected with pathogenic bacteria through eating contaminated seafood or contacting seawater with damaged skin, which can cause diseases such as otitis media, urinary system infection, sepsis and even death. Therefore, rapid and accurate detection of marine pathogens is an important way to protect human health and reduce economi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/569
Inventor 郭智勇沙玉红苏秀榕武琳张宁宋信信贾亚茹卢静李温柔
Owner NINGBO UNIV
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