Optimization Of Gold Nanoparticles Based Lateral Flow Immunoassay For Rapid Detection Of Escherichia Coli O157:H7

Shiga Toxin-Producing Escherichia coli,including 0157 and non-0157 serotype,is one of the top three foodborne pathogenic bacteria in the world.E.coli 0157:H7 can cause diarrhoea,enterorrhagid,haemolytic uraemic syndrome(HUS),and may even cause death.It is important to establish a method with high sensitivity and specificity to control E.coli 0157:H7.There are a great diversity of methods for detection of E.coli 0157:H7.Traditional methods are time consuming and strenuous.Lateral flow immunoassay method attracts more and more attention as its high sensitivity,rapid and easy to handle.In this paper,gold nanoparticles were prepared by refluxing and non-refluxing methods.The prepared gold nanoparticles were used to label anti-E.coli 0157:H7 monoclonal antibody,and prepare E.coli 0157:H7 test strip for the detection of E.coli 0157:H7.Five gold nanoparticles in different sizes were prepared by refluxing method.The result suggested that the sensitivity of the test strip increased first and then decreased when the size of gold nanoparticles increased.Thirty-five nanometer gold nanoparticles with UV-vis maximum apsorption peak of 526 nm owned the highest sensitivity of 2.8×102 CFU/mL.The sensitivity of the test strip with gold nanoparticles prepared by non-refluxing method prepared gold nanoparticles was 2.8×104 CFU/mL.Two kind of gold nanoparticles which had same UV-vis maximum absorption peak of 526 nm and different PDI were mixed by different proportion.Five kind of gold nanoparticles solution with different PDI was prepared by these mixtures.The prepared gold nanoparticles were used to label anti-E.coli 0157:H7 monoclonal antibody,and prepared E.coli 0157:H7 test strip for the detection of E.coli 0157:H7.The result suggested that when PDI increased from 0.2 to 0.6,the sensitivity of the test strip decreased from 2.8×102 CFU/mL to 2.8×105 CFU/mL.Gold nanoparticles were coupled with anti-E.coli 0157:H7 monoclonal antibody by the bridge of CMHG to form the gold nanoparticle-CMHG-antibody complex.The result suggested that this coupling method achieved a sensitivity of 102 CFU/mL.Gold nanoparticles modified by CMHG need no blocking step.NC membrane in test strip need not block.This method of coupling avoided non-specificity reaction by covering whole surfaces of gold nanoparticles by CMHG and preventing the non-specificity reaction between protein on NC membrance and gold nanoparticles.