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Biological sensing method for cholera toxin based on agglomeration of phospholipid bilayer membrane covered nanoparticles

A phospholipid bilayer, nanoparticle technology, applied in analytical materials, material excitation analysis, measurement devices, etc., can solve the problems of poor stability, high cost, low sensitivity, etc., and achieve high sensitivity, low cost, and specificity. Good results

Active Publication Date: 2012-07-25
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection techniques include animal experimentation, cell culture, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). These methods require labeling, complex operations, time-consuming, low sensitivity, and poor stability; Flow cytometry method, real-time quantitative PCR method, etc., the operation process requires high technical personnel and expensive instruments

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: content analysis of cholera toxin standard substance

[0023] (1) Preparation of Raman dye-modified phospholipid bilayer membrane-wrapped nanoparticle probes

[0024] Weigh 1.58mg 5,5'-dithiobis(2-nitrobenzoic acid) and dissolve in 4mL 50mM Na 2 HPO4 solution, diluted with sterilized water to 500 μM (protect from light, spare). Take 2mL of 0.3nM nano-gold solution, centrifuge at 10000r / min for 10min, dilute to 2mL with sterilized water, add 500μM DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) 300μL while stirring, Leave for 2 hours. Weigh 2mg 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 0.5mg 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol and 0.1mg GM 1 Add 2 mL of chloroform / methanol (v / v 6:1) to a 5 mL round-bottomed flask, evaporate in a rotary evaporator, set the temperature at 45°C, evaporate to dryness and continue to evaporate for 20 min. Preheat 2.5mL DTNB / AuNPs solution in a constant temperature water bath at 50°C, add it to a round bottom flask,...

Embodiment 2

[0032] Embodiment 2: Content detection of cholera toxin in the cholera vaccine sample for injection

[0033] (1) Preparation of Raman dye-modified phospholipid bilayer membrane-wrapped nanoparticle probes

[0034] Weigh 1.58mg 5,5'-dithiobis(2-nitrobenzoic acid) and dissolve in 4mL 50mM Na 2 HPO4 solution, diluted with sterilized water to 500 μM (protect from light, spare). Take 2mL of 0.3nM nano-gold solution, centrifuge at 10000r / min for 10min, dilute to 2mL with sterilized water, add 500μM DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) 300μL while stirring, Leave for 2 hours. Weigh 2mg 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 0.5mg 1,2-Dipalmitoyl-sn-Glycero-3-Phospho-thioethanol and 0.1mg GM 1 Add 2 mL of chloroform / methanol (v / v 6:1) to a 5 mL round-bottomed flask, evaporate in a rotary evaporator, set the temperature at 45°C, and continue to evaporate for 20 min after evaporating to dryness. After preheating 2.5mL DTNB / AuNPs solution in a constant temperature water b...

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Abstract

The invention discloses a biological sensing method for quantitatively analyzing cholera toxin, wherein a phospholipid bilayer membrane covered Au nanoparticle probe modified by Raman dye is prepared; functional phospholipid, namely phospholipid with a modified sulfydryl at the head end thereof, is selected; and the functional phospholipid can be covalently bound to the surface of Au nanoparticles so that the stability of the Au nanoparticle probe is improved. Cholera toxin subunit B (CTB) is specifically bound with a plurality of acceptor GM1 molecules, thus resulting in the agglomeration of Au nanoparticles covered with GM1 molecules; as a result, surface plasmas are coupled, thereby realizing high-sensitivity SERS (Surface Enhanced Raman Scattering) detection. Simultaneously, the method is capable of causing the change of the light absorption property and also capable of performing detection by a colorimetric method. The method is simple and quick in operation steps, free of applying any mark to the substrate, low in cost, high in sensitivity and good in specificity; the method is hopeful to become a useful technical way for detecting or identifying the cholera toxin; and the method plays an important role in monitoring food security, and controlling and preventing food-borne diseases.

Description

technical field [0001] The invention belongs to a biosensing method for quantitative analysis of cholera toxin, specifically refers to the unique structure and function of phospholipid bilayer membrane, functional modification of nanoparticle surface, surface-enhanced Raman resonance (SERS) and ultraviolet-visible light absorption analysis technology . Background technique [0002] Cholera toxin is a heat-labile enterotoxin secreted by Vibrio cholerae, which is the pathogenic bacteria that causes severe diarrhea in infected persons. It is a common source of contamination for water and food. Traditional detection techniques include animal experimentation, cell culture, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). These methods require labeling, complex operations, time-consuming, low sensitivity, and poor stability; Flow cytometry method, real-time quantitative PCR method, etc. are used, and the operation process requires high technical personnel a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/544G01N21/65
Inventor 王玉蒋健晖楚霞唐丽娟俞汝勤
Owner HUNAN UNIV
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