An immunological detection method of Staphylococcus aureus enterotoxin b based on Raman enhancement effect of gold-silver core-shell structure
An immunological detection method, the technology of staphylococcal gut, applied in the field of immune analysis, can solve the problems of detection sensitivity to be improved, interference, long reaction time, etc.
Active Publication Date: 2018-06-08
JIANGNAN UNIV
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Problems solved by technology
Traditional immunological detection methods for enterotoxins include indirect hemagglutination and immunodiffusion methods. When detecting small molecular weight enterotoxins, the reaction time is long, and positive results can only appear in 16-24 hours, and the detection limit cannot meet the safety limit requirements
At present, some detection kits based on the principle of double-antibody sandwich enzyme-linked adsorbent immunoassay (ELISA) have been used in some areas, and the detection sensitivity can reach the level of ng / L. However, due to the serious interference of food matrices such as meat products and dairy products, It is often necessary to dilute the test sample, so the detection sensitivity of traditional immunological methods needs to be improved
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[0031] (1) Synthesis of gold-silver core-shell nanoparticles with strong Raman enhancement effect, the specific steps are:
[0032] Synthesize 15nm gold nanoparticles Au NPs by citric acid reduction method: first wash the 300 mL Erlenmeyer flask and magnetic stirrer B500 with detergent, then rinse 6 times with ultrapure water; pour 100 mL 0.01% (m / V, Chloroauric acid solution HAuCl in ultrapure water) 4, sealed with 5 layers of plastic wrap, heated and stirred on a heating plate until boiling, and kept for 10 min. Take 200 μL of 10% (m / V, ultrapure water) trisodium citrate solution and quickly add it to the boiling solution, then seal it with 5 layers of plastic wrap and boil for another 15 min until the color of the solution turns wine red and remains unchanged; stop heating, keep stirring until it cools to room temperature, and add ultrapure water until the volume of the solution is 100 mL.
[0033] Coupling of 15nm gold nanoparticles Au NP and beacon molecule 4-NTP via go...
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Abstract
The invention discloses a gold-sliver core-shell structure Raman enhancement effect-based staphylococcus aureus enterotoxin B immunodetection method and belongs to the technical field of immunoassay. The method comprises synthesis of a gold-sliver core-shell structure with strong Raman enhancement effects and building of an enterotoxin B immunodetection method. Silver nitrate on the surface of 4-nitrothiophenol (4-NTP)-modified gold nanoparticle with the size of 15nm is reduced by citric acid so that a sliver shell with the size of 9nm is formed so that the Raman beacon 4-NTP-embedded gold-sliver core-shell structure is obtained. The gold-sliver core-shell structure has strong Raman enhancement effects and realizes uniform and stable Raman signals difficult to lose. The gold-sliver core-shell nanoparticle is labeled with an enterotoxin B monoclonal antibody and a microwell plate is pre-coated with a staphylococcus aureus enterotoxin B capture antibody. The enterotoxin B in a sample is captured by an antibody on the microwell plate and then is bonded to a gold-sliver core-shell nanoparticle-labelled antibody and a Raman signal of the plate is obtained by scanning of a Raman spectrometer. The method has an enterotoxin B detection limit of 0.002ng / mL substantially lower than that of the traditional enzyme-linked immune method.
Description
technical field [0001] The invention relates to an immunological detection method of Staphylococcus aureus enterotoxin B based on the Raman enhancement effect of a gold-silver core-shell structure, and belongs to the technical field of immune analysis. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a common foodborne pathogen, and Staphylococcus aureus enterotoxin is the main hazard factor of Staphylococcus aureus in food. Staphylococcus aureus enterotoxin is a kind of superantigen with a molecular weight of 28-30kDa, acid resistance, heat resistance and protease resistance. The half lethal dose (LD50) of human is only 20ng / kg. Enterotoxin B is a widely studied Staphylococcus aureus enterotoxin and one of the main enterotoxins in food. It has been listed as a Class B biological warfare agent by the US Centers for Disease Control and Prevention. [0003] Meat products, especially deli meat products, are the natural culture medium of Staphyloco...
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IPC IPC(8): G01N33/543G01N21/65
CPCG01N21/658G01N33/54346
Inventor 匡华王文彬胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV