Establishment And Application Of Aptamer-based Detection Methods For Biotoxins

Biotoxins are kinds of toxic chemicals produced by organisms,which can be divided into animal toxins,plant toxins and microbial toxins according to different sources.?-amanitin and OTA are the focus of biotoxins,with stable chemical properties and strong toxicity.?-amanitin is the principal lethal toxin in amanita poisoning,and the lethal dose to human is about 0.1 mg/kg.OTA is identified as a class of 2B carcinogen by International Agency for Research on Cancer(IARC),widely exists in food and posing a serious threat to liver and kidney of human and animals.Currently,the common detection methods of?-amanitin and OTA include high performance liquid chromatography(HPLC),liquid chromatography mass spectrometry(LC-MS),enzyme linked immunosorbent assay(ELISA)and Lateral flow immunoassay(LFIA).Most of the existing detection methods are based on the specific recognition between the antigen and antibody.In this paper,a series of detection methods targeting at?-amanitin and OTA were established by utilizing the aptamer as a new recognition factor.Firstly,on the basis of traditional ELISA,a magnetic bead-based enzyme linked immunoassay(MELISA)method for?-amanitin was established using immunomagnetic beads to fix aptamers.The detection limit is 0.1?g/ml and the linear relationship is good in the range of 0.01-6?g/ml.The MELISA method also have the application for the determination of mushroom and urine.Secondly,based on the aggregation phenomenon of colloidal gold in salt solution,and the inhibitory effect of aptamers on colloidal gold aggregation,a label free and unimmobilized colloidal gold spectrophotometric method for?-amanitin was established.The detection limit is 0.312?g/ml,and the A_620nm/A_520nm ratio of Au NPs increases linearly in the concentration range of 0.05-2?g/ml.The strategy can be applied to the determination of mushroom samples.Thirdly,in order to improve the detection sensitivity,fluorescence labeling method and the fluorescent carbon dot method were presented for the determination of OTA.The fluorescence labeling method is based on the principle of fluorescence resonance energy transfer(FRET),with aptamer and complementary strand(c DNA)as the main body.It can achieve the purpose of quantitative detection of OTA with a detection limit of 0.01?g/ml and a good linear relationship in the range of 0.01-0.25?g/ml.Additionally,the method can be carried out for the determination of actual sample and the recovery ranged from 90%to 99%.Fluorescent carbon dot method combines the prepared fluorescent carbon dots with aptamers was established and used as fluorescent probes for detection.The detection limit of the method reached 4 ng/ml,and OTA in the concentration range of 0.004-1?g/ml can be detected quantitatively.The applicability of the assay was evaluated in the determination of OTA in spiked wine samples,and the recovery ranged from 80%to 96%.In order to study the binding mechanism between aptamer and target,the recognition sites and binding pockets of?-amanitin and aptamer in vitro were studied by molecular docking.The main binding bases of?-amanitin and hydrophobic grooves on the surface of aptamer are T3,G4,C5,T6,T7,C67 and A68.