Fluoroimmunoassay Based On Quantum Dots For The Determination Of Alfatoxin B₁in Peanuts

With the frequent occurrings of accident related with food safety, there has been much attention tofood safety issures caused by biotoxin contaminates for the last few years. For example, the overstandard cases of aflatoxin B₁in edible oil and aflatoxin M1in milk seriously affected people’s healthand social stability. Aflatoxins, one kind of highly toxic, mutagenic, teratogenetic and carcinogeniccompounds, are so far recognized as one of the most toxic compounds, in which aflatoxin B₁（AFB₁） isthe most abundant aflatoxin that contaminates agricultural products and can have the most acute toxicityand carcinogenicity. Thus, there has been a hotspot to establish a rapid and sensitive analysis method interm of biotoxin research. Quantum dots （QDs）, a new type of fluorescent label, have advantages ofbroad excitation spectra, a narraw emission spectra and high photostability, consequensly promising abroad application prospect, especially in the application field of fluoroimmunoassay. QDs-basedfluorescent probe used in AFB₁deteciton are supposed to lay a foundation for detecion methods ofother mycotoxins.In this thesis, a simple and rapid method has been developed for synthesis of water solubleglutathione-capped CdTe quantum dots by using microwave irradiation heating method （MIRH）. Withan optimized synthesis conditions, the proper QDs were chosen for the next conjugation procedure. Theoptimized condition of MIRH for QDs synthesis was a reflux procedure at a pH at10.5for45min. Theresulting QDs were then characterized by UV/vis absorption spectra, fluorescence spectra and highresolution transmission electron microscopes. Results showed in the optimized conditions, thesynthesized QDs had the strongest fluorescence intensity and highest quantum yields. Moreover, QDshad no affaction of the emission fluorescence of AFB₁. The synthesized QDs had superior luminescenceproperties with a dameter of about2.6nm.The monoclonal against AFB₁with high purity was prepared via cell trawing and the purity ofascites. The CdTe QDs were successfully covalently bound to the monoclonal antibody against AFB₁,suggesting that a successful synthesis of MAb-CdTe probe for AFB₁detecion. This conjugates wereprove to have their fluorescent characteristic of QDs by SDS-PAGE. The bioactivity of QDs-antibodyconjugates were further proved by traditional indirect enzyme linked immunosorbent assay （ELISA）.On the other hand, the secondary antibody-CdTe QDs was further successfully synthezied by covalentlycoupling between CdTe QDs and the rabbit anti-mouse IgG antibody.With the prepared MAb-CdTe QDs conjugates, a novel direct competitive fluorescence-linkedimmunosorbent assay （dcFLISA） for detection of AFB₁in peanuts was developed. In the peanutsmatrix, results showed a hemi-inhibitory concentration （IC50） of0.148ng/mL, a limit of detection（LOD） of0.016ng/mL, a detection linear range of0.041-1.753ng/mL. Moreover, recoveries rangingfrom85.3%to117.3%were obtained by recovery experiment of blank peanut samples, along withcoefficients of variation （CVs） less than10%. These results suggested that the developed method couldmeet the need of AFB₁analysis and then be applied in AFB₁analysis of peanuts.An indirect competitive FLISA （icFLISA） method was as well established by using either the secondary antibody-CdTe QDs conjugates or the commercialized fluoresceinisothiocyanate （FITC）labeled secondary antibody. Results from the former method showed the IC50of0.059ng/mL in thepeanuts matrix, while those from the later method presented the IC50of0.588ng/mL. Both methodssuggested recoveries ranging from80%to120%, with CVs lower than10%.The comparation of these three fluorescence immunosorbent assay method in the thesis suggestesthat all these methods can meet with the prerequisite of AFB₁analysis that can be applied in AFB₁detection in peanuts. Based on MAb-CdTe QDs conjugates, dcFLISA method illustrated the shortest,rapidest and most specific merits. Results from real samples demonstrated a high consistency whencompared with HPLC method.