| Mycotoxin contamination is a key problem in the quality and safety control of traditional Chinese medicine(TCM).TCM mainly derives from natural medicines and processed products,which is consequently susceptible to mycotoxins in each of the processes in TCM production,such as planting,growing,harvesting,pre-processing,transportation and storage.Mycotoxins like aflatoxin,zearalenone(ZEN),patulin(PAT),etc.,are regarded as the most common and harmful ones,which have great neurotoxicity and genetic toxicity to human body.And even trace amount of them can bring serious toxic effects to multiple organs of human body.Therefore,much attention should be paid to the detection and control of mycotoxins in the practical production of TCM.However,owing to the relatively complex compositions of TCM,the detection of mycotoxins in the practical production processes of TCM is very difficult.At the same time,chromatography,mass spectrometry,ELISA and other traditional detection methods for mycotoxins detection are limited in the practical application of TCM production due to high cost,complex operation,cumbersome pretreatment process and so on so forth.Therefore,it is in an urgent need to develop a rapid,simple and cost-effective detection methods,which can be flexibly applied into the detection of mycotoxins in various production and development scenarios of TCM.Aptamer is single-stranded DNA(ss DNA)or RNA screened by systematic evolution of ligands by exponential enrichment(SELEX),which performs specific recognition and binding to the targets.Owing to these advantages like good stability,strong specificity,in vitro synthesis,low cost and easy modification,aptamers have been employed as a newly kind of receptor element,and widely used in the field of biochemical sensing detection.Besides,many aptasensors have been developed in the application of small molecular detection,and achieved ultra low detection limit and high sensitivity.In order to provide a practical-oriented mycotoxin detection method as well as new means for the quality and safety control of TCM.Herein,based on aptamer and highly sensitive fluorescence spectrum detection methods,this thesis developed two facile and economical methods with the advantages of excellent efficiency,sensitivity and accuracy for the rapid determination of mycotoxins in the production of TCM.At the same time,this thesis also analyzed the binding mechanism between aptamer and target mycotoxin via simulation software,in order to realize the scientific and reasonable design of aptasensor,and improve the biochemical sensing efficiency of the mycotoxin detection.The specific research contents are as follows:1.Development of "turn-on" fluorescent aptasensor for the simultaneous detection of PAT and ZEN in Coicis Semen.In order to achieve efficient simultaneous detection of two mycotoxins in TCM samples,this aptasensor was established upon the specific recognition of aptamer and the adsorption-induced fluorescence quenching of graphene oxide(GO).This aptasensor exploited FRET effect,used low-cost materials including aptamers and GO,and achieved a simple,cost-effective and sensitive determination of targets.In this study,the aptamers of PAT and ZEN were labeled by FAM and Cy3,respectively,serving as fluorescence probes.Both aptamers could be adsorbed on the surface of graphene oxide(GO)via π-πstacking,which will consequently result in the occurrence of FRET between the fluorophores and GO.In the absence of the targets,the fluorescence would be quenched.In the presence of any of the dual mycotoxins,the corresponding aptamers would interact with the targets and release from GO due to the conformational variation,leading to fluorescence “turn-on” effect.The limit of detection of this difunctional aptasensor was 2.29 n M for PAT and 0.037 n M for ZEN,respectively,which could satisfactorily meet the requirements in Chinese Pharmacopoeia.This aptasensing platform exhibited satisfactory selectivity against interferents and reliability in Coicis Semen sample detection.To our knowledge,it is the first aptasensor based on GO and FRET that realizes simultaneous detection of dual mycotoxin in TCM.Moreover,it is economical for a single assay with the cost equivalent to about 1 RMB yuan,and the measurement takes merely ～60 min,which can realize fast,cost-effective and reliable simultaneous detection of dual mycotoxins in TCM.2.Development of ratiometric fluorescent aptasensor for the detection of PAT in Semen Nelumbinis.In order to improve the sensitivity of PAT detection,this study developed a ratiometric fluorescent aptasensor for PAT detection in Semen Nelumbinis based on FRET and synchronous fluorescence spectrometry detection method.This aptasensor was consisted of an aptamer and its complementary sequence(c DNA),which could spontaneously bind with each other when existing alone in solution system.Meanwhile,in order to construct a FRET system based on the complementary binding between PAT-APT and c DNA,the fluorophores FAM and ROX were modified on PAT-APT and c DNA,respectively,and serving as the fluorescence donor and receptor,in which the fluorescence of FAM would be weakened and ROX would be enhanced.When adding PAT in the FRET system,the c DNA would dissociate from the aptamer due to the competitive binding between PAT and PAT-APT.Then the FRET effect in the aptasensor would consequently be weakened,and the fluorescence of donor FAM would be partially recovered,while the fluorescence of receptor would weaken.According to the ratio of the variation in the fluorescence intensity of these two fluorophores before and after detection,the aptasensor achieved accurate detection of PAT.Besides,this aptasensor achieved a lower detection limit of 0.16 n M for PAT detection,and also performed a satisfactory recovery rate in Semen Nelumbinis samples.3.Mechanism of the interaction between aptamer and mycotoxin.In order to elucidate the mechanism of the interaction between aptamer and mycotoxin,PAT and PAT-APT were selected as research objects in this study,and the mechanism of the binding between PAT and PAT-APT was investigated by circular dichroism and computer-aided simulation software.Firstly,the structures of PAT-PAT and ZEN-APT were characterized by circular dichroism spectroscopy.The result showed that the structures of these two aptamers changed significantly after binding with PAT and ZEN,respectively,which confirmed the specific binding between the aptamers and their target mycotoxins.Then,this study utilized Autodock,Gromacs as well as other molecular docking and molecular dynamics simulation software to investigate the mechanism of the binding between PAT and PAT-APT,including the binding process,binding sites and binding energy,etc.The results showed that the complex of PAT and PAT-APT could reach a stable state during the simulation process,and C-11,C-37 and C-38 were the key base sites for the specific recognition and binding between PAT and PAT-APT.The results of molecular docking and dynamics simulation can provide theoretical guidance for scientific and rational design of aptasensors for the detection of PAT. |
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