Aptamers Functionalized Time-resolved Fluorescence Nano-probe Based Detection Methods For Mycotoxins

Food safety is a major challenge both for governing bodies and scientists.Mycotoxins are toxic secondary metabolites produced from fungi and capable of causing death in both humans and animals.The search of mycotoxins has proceeded seriously from agriculture,environmental science immunology,pharmacology and medicine.The insufficiency of chromatographic methods and traditional immunoassays calls for innovative and consistent methods to enable suitable,sensitive and rapid analysis of mycotoxins.Recently,an increasing number of fluorescent aptasensor against various mycotoxins have been selected however,the application of time-resolved fluorescence technique combined with aptamers has just emerged.Time-resolved fluorescence technique?TRF?can eliminate the background fluorescence and offers a signal with remarkably high signal to noise ratio.By setting a certain delay time and gate time,TRF can distinguish the fluorescence lifetime of the fluorescent substance contained in the sample,and thus increased the sensitivity in detection.As a consequence,in this study,several time-resolved fluorescence nanoparticles were synthesized and characterized.Then,combining with various nanomaterials?magnetic nanoparticles,tungsten disulfide and graphitic carbon nitride nanosheet?and techniques?time-resolved fluorescence technique,magnetic separation technique and rolling circle amplification technique?,a series of fast,simple,sensitive and efficient aptasensor for several mycotoxins were developed.Firstly,a novel,highly sensitive and selective time-resolved fluorescence aptasensor was developed for zearalenone?ZEN?detection using reported aptamer of ZEN.The lanthanide(Ln³⁺)doped time-resolved fluorescence nanoparticles?TRFNPs??NaYF₄:Ce/Tb?and amine-functionalized Fe₃O₄ magnetic nanoparticle?MNPs?were synthesized and characterized.The novel strategy utilized ZEN aptamer labeled with MNPs as a capture probe and TRFL nanoparticles labeled with complementary DNA?cDNA?as a signal probe.Under the optimized conditions,TRFL intensity at 544 nm was used to measure ZEN?R²=0.9920?in the range of0.001-10 ng mL^-11 and limits of detection?LOD?for proposed method was 0.21 pg mL^-1.The specificity of aptasensor was also determined by using other mycotoxins and results showed that the aptamer are specific to recognize only ZEN.The analytical applications of the present aptasensor were also examined in maize and wheat samples and results were compared with existing methods.Based on these findings,it is suggested to use current rapid and simple bioassay for the determination of ZEN in food and agricultural products.Secondly,due to variety and serious harm of multiple mycotoxins,simultaneous recognition of two mycotoxins OTA and FB₁ were achieved.For this purpose,another Ln³⁺doped TRFNPs?NH₂-Eu/DPA@SiO₂?was prepared based on preliminary studies.Both nanoparticles?NaYF₄:Ce/Tb and NH₂-Eu/DPA@SiO₂?were excited at same wavelength?273 nm?and their emission spectra did not overlap.Based on these findings both TRFNPs were used for simultaneous recognition of two mycotoxins OTA and FB₁ combined with reported aptamers of OTA and FB₁.NaYF₄:Ce,Tb and NH₂-Eu/DPA@SiO₂ NPs coupled with cDNA of FB₁ aptamer and OTA aptamer respectively,were used as signal probe and aptamers conjugated MNPs act as a capture probe.Under the optimized conditions,the time-resolved fluorescence intensities at 544 and 618nm corresponded with Tb³⁺and Eu³⁺,respectively were used to measure FB₁?R²=0.9917?and OTA?R²=0.9924?,respectively.The LODs for FB₁ and OTA were 0.019 pg mL^-1 and 0.015 pg mL^-1,respectively which were much lower than previously described methods for simultaneous recognition of OTA and FB₁ while detection range varied from 0.0001-0.5 ng mL^-1.This aptasensor was effectively applied to quantity FB₁ and OTA in maize samples and results were compared with ELISA method.This is the first reported TRF based aptasensor to detect two agriculturally important mycotoxins in the maize.The developed aptasensor has potential to be used for detection of toxins in food safety fields.Thirdly,three mycotoxins zearalenone?ZEN?,trichothecenes A?T-2?and aflatoxin B₁?AFB₁?were detected simultaneously.Three multicolor Ln³⁺(Ln³⁺=Dy³⁺,Tb³⁺,Eu³⁺)-doped time-resolved fluorescence nanoparticles(KYF₄:Dy³⁺,KYF₄:Tb³⁺,KYF₄:Eu³⁺)were synthesized and characterized.By combining quenching efficiency of tungsten disulfide?WS₂?nanosheet and the interaction between WS₂ and DNA molecules,the multicolor Ln³⁺-doped TRFNPs?excited at same wavelength?were used to fabricate a new“turn on”aptasensor for the simultaneous detection of three mycotoxins.KYF₄:Dy³⁺,KYF₄:Tb³⁺and KYF₄:Eu³⁺TRFNPs functionalized with ZEN,T-2 and AFB₁ aptamers respectively,were applied as bioprobes and WS₂ nanosheet as a fluorescence quencher.Under the optimum conditions,the time-resolved fluorescence intensities at 488,544 and 619 nm corresponded with Dy³⁺,Tb³⁺and Eu³⁺were used to measure ZEN?R²=0.9947?,T-2?R²=0.9972?and AFB₁?R²=0.9909?,respectively.The developed aptasensor presented high selectivity and sensitivity to ZEN,T-2 and AFB₁ with LODs of 0.51,0.33 and 0.40pg mL^-1,respectively.Moreover,the interference test confirmed that present aptasensor can detect three mycotoxins simultaneously without interfering each other.The developed aptasensor was effectively applied to quantify ZEN,T-2 and AFB₁ in maize samples and results were compared with ELISA method.The results revealed that the developed aptasensor is homogeneous and multiple targets can be detected within one hour.Therefore,the developed aptasensor has the potential to become an alternative to other monotonous heterogeneous methods,e.g.based on enzyme-linked immune-sorbent assay?ELISA?and can also be used for multiplex analysis of various targets with the replacement of aptamer.Fourthly,based on previous methods,rolling circle amplification?RCA?technology was further used to improve the sensitivity of detection method.By using the modified KYF₄:Eu³⁺TRFNPs as signal probe and graphitic carbon nitride?g-C₃N₄?nanosheet as a quencher,the fluorescent detection of aflatoxin M₁?AFM₁?,was achieved.The sensor utilized reported aptamer of AFM₁ as recognition element and aptamer also act as primer for DNA synthesis.TRFNPs attached with cDNA?TRFNPs-cDNA?was act as signal probe.In the absence of target,rolling circle template?RCT?,which was designed to be complementary to both ends of the target aptamer,successfully ligated to target aptamer and RCA reaction initiated.The TRFNPs-cDNA complementary to target aptamer was annealed to multiple sites of amplified RCA product?RCAP?to form RCAP/TRFNPs-cDNA duplex.This RCAP/TRFNPs-cDNA duplex is less adsorbed on g-C₃N₄monolayer,thus fluorescence increases.In the presence of AFM₁,aptamer recognized target and aptamer-target complex was formed.Hence,no aptamer hybridized with RCT,thus no RCA and duplex formation.Consequently,free TRFNPs-cDNA is adsorbed onto g-C₃N₄ monolayer.This“signal-off”assay could avoid adsorption of non-specific DNA in sensing system.The assay provides lower detection limit?0.0194 pg/mL?than previous reported assay for AFM₁ detection.Furthermore,it is highly sensitive,specific and has good recoveries between 92-99.8%,thus can be used to detect AFM₁ in milk.Hence,g-C₃N₄-based fluorescence detection combined with RCA would be a convenient method for quantification of small molecules.In conclusion,using the long life time fluorescence emission of Ln³⁺ion compounds can diminish the background noises efficiently.Thus,as compared to conventional fluorescent assays,time-resolved fluorescent assay offers high signal-to-noise ratio in fluorescence based biodetection.Combined with time-resolved fluorescence detection technique,magnetic separation technique and signal amplification technique,aptasensors have been successfully developed and applied in real food sample.This study provides a novel idea and support to improve the detection and monitoring technologies of mycotoxins.