Selection Of Aptamer For The Mycotoxins And The Applications

SELEX （Systematic Evolution of Ligands by Exponential Enrichment）, is a newcombinatorial chemistry technique which was invented successfully in the1990s.We difinedthe selected sequence as aptamer which bind with target in the form of ligands-counterpartfrom the mean of Latin aptus. At present, there are extensive detection methods reported formycotoxins, but each has their own advantages and disadvantages.We got AFB₁-aptamer andAFB₂-aptamer using FluMag-SELEX technique and negative selection and counter selectiontechnique,and set up a new detection method for AFB₁and AFB₂based on Fluorescenceproperties and aptamer identification function. In addition we set up a new naked eyedetection method for FB₁abased on the high identification function of DNA aptamer and thecolor changing effect of AuNPsfirstly,made amine-functionalized magnetic beads as matrix lingked AFB₁andsynthesized a80base ss DNA library containing a central random region of40base flankedby20defined primer binding sequence. After ten selection cycles containing two roundsnegative selection and two rounds counter selection of AFB₂using FluMag-SELEXtechnique,the selected oligonucleotides were cloned and sequenced. Analyzed the first andsecond structure of AFB₁-aptamer utilizing DNAMAN and RNA structure software andsynthesized the representative sequences tagged with biotin after the kin analysis.Affinity andspecificity test was carried out through amine-functionalized magnetic beads wrapped withavidin and set up a new detection method for AFB₁. There was a linear correlation betweenfluorescent intensity and concentration of AFB₁from50ng/L to1500ng/L and a detectionlimit of35ng/L is obtained under the optimial experiment conditions.The standard recoveriesis94.2%-101.2%for peanut oil samples.Secondly,take the AFB₂as molecule taget,using the same fixed progress and librarycontaining two rounds negative selection of magnetic beads and two rounds counter selectionof AFB₁Which structure similar to AFB₂using FluMag-SELEX technique.After10selectioncycles, the selected oligonucleotides were cloned and sequenced.Similarly structure analysis,picking and synthesizing the different kin representative sequences labeled biotion, affinityand specificity analysis of AFB₂-aptamer,and establishing a new detection method of AFB₂using AFB₂-aptamer. There is a linear correlation between fluorescent intensity andconcentration of AFB₂from90ng/L to1800ng/L and a detection limit of55ng/L is obtainedunder the optimal experiment conditions.The standard recoveries is93.1%-97.3%for peanutoil samples.Finally,this work set up a new naked eye detection method for FB₁abased on the highidentification function of DNA aptamer and the color changing effect of AuNPs.This assaymethod taked AuNPs as carrier and linked DNA1to it first and then assembledFB₁-aptamer—DNA1—AuNPs complex throuth the hybridzation process betweenFB₁-aptamer and DNA1-AuNPs.When added different concentrations of FB₁,FB₁-aptamerprefered to connect to FB₁and then subsequently additional DNA2chain that matched with DNA1chain can hybrid with exposed DNA1resulting in AuNPs aggregation.Thus the colorchanges from red to purple and blue could be observed by the naked eye.Optimal conditionsavoided to much salt induced AuNPs aggregation caused error effectively.Furthermore,weadded SDS in DNA1or DNA2chain connected to AuNPs to maintain the color of AuNPs andimproved the concentration of NaCl to500mmol/L and DNA loading enlarge3times.Thisbreaks the traditional boundary of100mmol/L NaCl can caused the color change ofAuNPs.The linear range of the colorimetric aptasensor covers a large variation of FB1concentration from125ng/L to1500ng/L and the detection limit of125ng/L is obtained.