Research On The Construction Of Nanobody-based Fluorescence Immunoassays For The Detection Of Ochratoxin A

As one of the most common and most endangered mycotoxins.ochratoxin A(OTA)is a secondary metabolite produced by Aspergillus and Penicillium and is toxic to humans or animals.Many researches have revealed the diverse toxicities of OTA,such as nephrotoxic and immunosuppressive effects.The detection process of mycotoxins.especially OTA,is usually time-consuming and laborious.So it is of great significance to establish a rapid detection method to achieve simple,rapid and efficient determination of OTA.Fluorescence immunoassay is a simple,sensitive,and reproducible immunoassay method.The development of highly sensitive immunoassay technology has always been an important research direction in the field of food analysis.It is significant to establish a detection method of mycotoxins,trace elements and other food contaminants.Nanobody(Nb)is the smallest functional units with intact antigen-binding properties.Due to their large surface area,high affinity,and good water solubility,Nb is superior to traditional antibodies in many fields.Nb has strong fluorescent properties and can generate effective FRET reactions with OTA molecules in homogeneous systems.Therefore,it is possible to establish a labele-free fluorescent immunoassay for OTA.This study is based on the Nb28 that already developed in the laboratory previously.OTA and OTB were used as the detection targets,and the fluorescent immunoassay system was constructed by using the Nb with the advantages of small molecular weight and high fluorescence quenching efficiency.The results show that compared with the traditional method,the method achieves the advantages of fast detection speed(10 minutes),greatly improved sensitivity(0.06 ng/mL),and convenient operation.Thus achieves ultrasensitive homogeneous immunoassay for OTA and OTB.Furthermore,it provides experimental basis of the establishment of ultrasensitive fluorescence immunoassay for small molecule compounds in the field of food safety.The quantum dots(QDs)has the characteristics of stable optical properties and easy surface modification.The assay was consisted of amino water-soluble QDs coupled with OTA as energy donor and carboxyl water-soluble QDs coupled with Nb28 as energy acceptor.The FRET homogeneous competition detection system was constructed based on the principle of immunology.According to the properties of the conjugates,DCC/NHS and EDC/NHSS methods were used to prepare QDs conjugates with different labeling ratios.Various parameters including labeling ratio,donor-receptor reaction ratio and other buffer conditions such as immunoreaction time.pH value,methanol content and ionic strength were systematically investigated.Furthermore,the effects of matrix effects of grain extracts were further explored.The QDs-FRET detection system was successfully applied to the quantitative analysis of OTA in cereals with a detection limit of 5 pg/mL and a linear range of 5-1000 pg/mL.The Nb28 gene was cloned into the green fluorescent protein(sfGFP)prokaryotic expression vector and the sfGFP-Nb28 fusion protein was obtained after induction,purification and identification.The OTA was coupled to the amino water-soluble QDs as previous work.Then the sfGFP-Nb28 fusion was acted as the energy donor and the OTA-RQD was used as the energy acceptor to estalish the FRET system.Furthermore,the FRET assay method eliminated the matrix effect of the grain extract.Eventually,the FRET method was successfully applied to the quantitative analysis of OTA in cereals with a detection limit of 5 pg/mL and a linear range of 5-5000 pg/mL.