| Mushroom poisoning due to accidental consumption occurs every year around the world.The most important and lethal toxin causing mushroom poisoning isα-amanitin.As a cyclic octapeptide,α-amanitin has special structural properties and application value,and the lethal dose to human is about 0.1 mg/kg.Toxic wild mushroom poisoning has not yet found a special antidote,and rapid detection of wild mushrooms before consumption or rapid identification of toxins after poisoning can effectively reduce the occurrence of fatal cases.The purpose of this study is to use a novel recognition factor,aptamer,to investigate its recognition mechanism withα-amanitin,to evaluate the effect of different tailoring and modification of aptamer on the toxicity ofα-amanitin,and to establish two rapid detection methods forα-amanitin-fluorescence assay and carbon dots gold nanoassay.Firstly,the sequence ofα-amanitin aptamer was optimized based on a series of cuts based on the secondary structure of the aptamer,truncating the primer region of the removed aptamer in base pairs until its stem-loop structure was truncated.The affinity and specificity of the trimmed aptamer sequences were characterized by colloidal gold spectrophotometry,and an Apt-20 with comparable affinity to the original chain but with significantly higher specificity was obtained.thermodynamic changes,conformational changes and binding sites of different aptamer sequences toα-amanitin were investigated by isothermal titration calorimetry,circular dichroism and molecular docking techniques.Secondly,a rapid,label-free fluorescence assay forα-amanitin was developed.The nucleic acid dye SYBR GreenⅠwas introduced into the detection system,and its binding to the small groove of ds DNA increased the fluorescence intensity by 1000-fold.Using the optimal aptamer Apt-20 obtained by tailoring optimization,the experimental system was designed using the principle that the complementary strands ofα-amanitin and the aptamer competitively bind to the aptamer resulting in a change in fluorescence signal value.α-Amanitin concentration showed a linear relationship with fluorescence signal in the range of 50-800 ng/m L,Y=1.243x+351.6,R~2=0.991,and the detection limit was The specificity of the assay system was good,and the recoveries in urine were 93.88%-95.01%.urine samples only required simple centrifugation,and the detection time was 1 h,which can realize the rapid detection of urine samples.Thirdly,a method based on carbon dots gold nanoenzyme was established for the detection ofα-amanitin.Using glucose carbon dots and sodium citrate as reducing and stabilizing agents,a carbon dots gold nanase with strong peroxidase-like activity was prepared,which could oxidize the substrate TMB to blue o-TMB,which could cover the active site of the nanase after specific binding toα-amanitin and lead to a decrease in its catalytic activity and absorbance at 652 nm value decreased.The regression equation Y=-0.06083x+0.9643,R~2=0.996,the limit of detection was 48.03 ng/m L,and the recoveries in urine were 91.2%-97.6%.The method enables the visualization ofα-amanitin whole detection process was completed within 20 min,which can be subsequently applied to the quantitative and qualitative determination ofα-amanitin samples.Finally,in order to enhance the in vivo stability and and blood circulation time of the aptamer,different types of modifications were performed on the aptamer prochain:3’odification of inverted thymine(id T),2’OH modification by 2’fluorine and 2’oxo-methyl,5’binding to PEG,mirror image treatment,etc.The effects of aptamers onα-amanitin in the cellular environment were comprehensively evaluated by cytotoxicological analysis,intracellular oxidative stress damage markers(MDA,SOD,GSH/GSSG)and hepatocyte damage markers(AST,ALT)activity studies,which provided a solid theoretical basis for subsequent aptamer design optimization and targeted drug therapy based on aptamers. |
PDF Full Text Request