Sensitive And Homogenous Immunoassay Of Fumonisin In Foods Using Single Molecule Methods

Fumonisins are a class of mycotoxins primarily produced by Fusarium moniliforme.More than 11 structurally ralated fumonisins found in several types of foods worldwide.Fumonisin B₁（FB₁）is the most prevalent and also the most toxic of the Fumonisins,and it accounts for 70%-80%of the total amount of Fumonisins.FB₁ is considered to be the most prevalent mycotoxin in naturally contaminated cereals throughout the world and is potentially hazardous to humans and animals.Therefore,it is necessary to develop sensitive,fast and reliable detection methods of FB₁.In this study,we combined single molecule methods with homogeneous immunoassays for sensitive detection of FB₁ in maize.The main work is included in the following parts.1.We reported a homogeneous immunoassay for sensitive detection of FB₁ in maize using single molecular fluorescence correlation spectroscopy（FCS）.FB₁ was labeled with fluorescent dye as a fluorescent tracer.The principle of immunoassay is based on sensitively distinguishing the fluorescent tracer and tracer-antibody complex by FCS technique due to significant difference of the characteristic diffusion times between the tracer and tracer-antibody complex.We firstly synthesized the fluorescent FB₁ tracer using Alexa 488 as labeling probes,and then optimized the experimental conditions for immunoassay.We observed good linear relations between the fraction of Alexa 488-FB₁ immune complex in reaction solution and the FB₁ concentration in sample.Under optimal conditions,the linear range is from 1.0μg/L to 25.0μg/L,and the detection limit is 1.0μg/L for FB₁.This method was successfully used for determination of FB₁ in spiked and natural samples.The results obtained by FCS are in good agreement with that by ELISA method.2.We demonstrated a homogeneous immunoassay for sensitive detection of FB₁ in maize using single particle resonance light scattering correlation spectroscopy（RLSCS）.FB₁ was attached to the surface of GNPs.The combination of GNPs-FB₁ and antibody will casue to the increase of particle size of GNPs,which leads to the significant increase in the characteristic diffusion time of GNPs in the detection volume.The RLSCS method can sensitively detect the light scattering signal of gold nanoparticles and the changes in the characteristic diffusion time of GNPs before and after immune reactions.Firstly,FB₁ was attached to the surface of GNPs,and then the experimental conditions were optimized for immunoassay.We observed good linear relations between characteristic diffusion time of GNPs and the FB₁ concentration in samples.Under optimal conditions,the linear range of FB₁ is from 10.0μg/L to 200.0μg/L,and the detection limit is 10.0μg/L for FB₁.This method was successfully used for determination of FB₁ in spiked and natural samples.The results obtained by RLSCS are in good agreement with that by ELISA method.Our results demonstrate that the quantitative FCS and RLSCS methods are rapid,simple and highly sensitive,and it can easily be extended to detect other chemical contaminants for food safety.