Study On Cyclodextrins-based Fluorescence Enhancement Technology For Aflatoxin B₁ Detection

Aflatoxin B₁ (AFB₁), one of the most natural chemical toxicity carcinogens, commonly contaminated peanuts, corn, feed, other agricultural products and its products and food, seriously threatened human and animal healthy or life safety. The development of detecting AFBB₁ is the hot issue and key problem in the research of agricultural quality safety and food safety. To solve the problem of the serious matrix interferences and low detection sensitivity during pretreatment involved, the aflatoxin immunaffinity column (IAC) combined with fluorescence enhancement technology based detection was developed, and a high sensitivity detection technique to AFB₁B was established. The results had significant theoretical meaning and applications value to guarantee agriculture produce quality and food safety, and to break down agricultural technical trade barriers. The main research results were shown as follow:1. Fluorescence excitation and emission wavelength of AFBB₁β-cyclodextrin (β-CD) and its derivative 2,6-Methyl-β-cyclodextrin were determinated for 365 nm and 440 nm. The results were also showed the best performance with 1 mLβ-cyclodextrin or its derivatives 2,6-Methyl-β-cyclodextrin mixed into 2 mL10.0μg/kg aflatoxin solutions, the enhancement was 5 and 8 times, respectively. The derivatives'fluorescence enhancement inclusion property of AFB₁B and CD was also deduced from experiments, with a 1:1 host: gust complexities, and K=12.237. An excellent correlation (R2>0.99) was observed between before and after the fluorescence enhancement. There was no significient influences between AFBB₁ and CDs with existence of ions Mg2 +,Ca ²⁺,K⁺,Na⁺,Cl^-,(H2PO4) (c<0.01 mol/L) and matrix at room temperature.2. A novel NH2-silica gel microparticle-based IAC was developed with the reported ultrasensitive generic monoclonal antibody. The successful conjugation between NH2-silica gel and AFB₁ monoclonal antibody were verified by near infrared (NIR). And the capacity of each IAC with 0.2 mL NH2-silica gel carrier and the crude AFB₁ monoclonal antibody powder,the capacity of each IAC covered a range of 30-107 ng for AFB₁, showing a average coupling rate of 87%. After optimization of applied conditions, 20% methanol was the best sampling buffer, PBS for washing, and pure methanol for elution buffer. With a validated high performance liquid chromatography method, the results of this IAC was no significant difference compared with commercial IAC and multifunctional column. Finally, compared with commercial AFB₁ IAC, the developed IAC had the advantages of low AFB₁ detect levels of 0.01μg/kg, high efficiency of 107 ng, low cost, simple operation, good stability, strongly suggesting it's suitable to fast detection of samples.3. The pretreatment of AFBB₁ in peanuts was optimized, and a detection method based on novel fluorescence enhanced reagent was developed. After the sample was grinded with sieve of 20 aperture. It was extracted by 80% methanol using the ultrasonic wave assisted method, showing the optimized best recovery of 90%. Thus, a novel method was developed for accurate, rapid, convenient AFB₁B determination. Fluorescence enhancement of CD may be introduced to IAC test and sample pretreatment method in agricultural production and food. The LOD was 0.3μg/kg, and the recovery of AFB₁ in spiked samples was 90%-115%. Compared with national standard HPLC, ELISA and anogold probe-based immunochromatographic assay of peanuts, feeds, corn and oil, no significant differences was detected. The newly developed method in this study with superiority of simple process, rapid detection time, high accuracy, coupled with more safety to environment and testing staff, which afforded a preferential method for aflatoxin detection method.