Aptamers Functionalized Metal Nanoclusters As Novel Fluorescence Nanoprobes For Selective And Sensitive Detection Of Mycotoxins

Food safety is the foremost and major subject of community health concerns.Mycotoxins are the primary challenger in the field of food safety.Mycotoxins,structurally diverse fungal secondary metabolites,stimulate an extensive range of toxicological effects.Some mycotoxins can alter the normal immune system although exist in food at levels below discernible overt toxicity.The search of mycotoxins has been progressed extremely from agriculture,pharmacology,immunology,environment,and medicine.Various conventional methods have been employed for the determination of mycotoxins,however,the limitations and inadequacy of conventional and immunoassays demands consistent and innovative methods to permit rapid,sensitive and suitable determination of mycotoxins.Recently,several luminous aptasensors have been developed for mycotoxin quantification,though,the applications of aptamer functionalized metal nanoclusters?MNCs?have just emerged in the field of bioimaging and biosensing.MNCs can be easily synthesized,functionalized with aptamer,and have excellent biocompatibility and photostability with ultra-small size.They have more stable nature,easy binding nature with DNA,and offer more effective energy transfer to other fluorophores and quenchers.Enchanting features of fluorescent MNCs have open new pave for the exciting construction and developments of new analytical methods for fluorescent biosensing.In the present study,we synthesized several fluorescent MNCs and characterized them.Afterward,aptamer stabilized MNCs were employed with numerous nanomaterials such as MoS₂,AuNPs,and WS₂ nanosheet.Thus,a series of sensitive,simple,efficient,and fast bioassays are developed for quantification of several mycotoxins.Firstly,we prepared apt-AgNCs for fluorescent recognition of T-2,which is dependent on the FRET mechanism.Apt-AgNCs?fluorescent donor?was employed as a fluorescent sensing probe whose fluorescence was quenched by MoS₂?energy acceptor?.The introduction of T-2 in designed bioassay leads to the desorption of fluorescence probe from MoS₂ which results in recovered fluorescence intensity?F.I?in T-2 concentration dependent-manner ranged from 0.005-500 ng mL^-1 with LOD of 0.93 pg mL^-1.Spiking investigation discloses the strength and efficiency of the established bioassay with recovery percentages,ranged from 90.4 to 107.8%and 89.5 to104.9%in wheat and maize specimens,respectively.The outcomes were cross-verified using standard commercial T-2 ELISA kit.In conclusion,the established detection assembly may expose the excellent utility in analysis for risk evaluation of T-2 toxin.Secondly,T-2 toxin was again quantified and photo-visualized using AuNCs.For this purpose,we synthesized a simple,rapid,and sensitive method for quantification and photo-visualization of T-2 toxin,which offers much lower LOD than the previous one.Therefore,in view of numerous encouraging requisitions,we synthesized a high luminous green-emitting Arg@ATT-AuNCs by host-guest assemblies.The Arg@ATT-AuNCs were capped with PAA shell for the construction of PAA@Arg@ATT-AuNCs nanoparticles?NPs?to prevent the AuNCs aggregation from PDDA which may influence the F.I for the detection of T-2 toxin.The fluorescent and visual detection of T-2 agglomerated is based on soluble PAA@Arg@ATT-AuNCs NPs and PDDA-aggregated-AuNPs.The scattered AuNPs quench the F.I of PAA@Arg@ATT-AuNCs NPs,while the PDDA-agglomerated AuNPs may have little influence.The electrostatic interaction of the aptamer-PDDA complex prevents the aggregation of the AuNPs?absorbance at 520 nm?and the F.I of PAA@Arg@ATT-AuNCs NPs?emission at 530 nm?is quenched following the FRET mechanism.Upon the addition of T-2 toxin,toxin–aptamer assemblies are formed,and the succeeding PDDA is free to aggregate the AuNPs?absorbance 665nm?,resulted in the recovered F.I of PAA@Arg@ATT-AuNCs NPs.This label-free fluorescent and colorimetric method sensitively used for quantification of T-2 toxin with a smaller LOD of0.57 pg m L^-1(range 0.001-100 ng mL^-1).The assay is selective for T-2 over other interfering compounds.We envision that the present study will substantively contribute to synthesize new core-shell multifunctional NCs and can be considered as a new method for the onsite detection of the environmental and agricultural samples.Thirdly,the combined toxic effect of more than one mycotoxins posit severe toxic threats to animal and human health.For this reason,it is necessary to recognize the multiple toxins by a single diagnostic methodology.To best of our knowledge,for the first time,we synthesized an aptamer tempted“turn on”FRET biosensor using dual-color AuNCs?Lp-AuNCs&BSA-AuNCs?with WS₂ nanosheet for concurrent quantification of aflatoxin B₁?AFB₁?and zearalenone?ZEN?by single excitation.Here,AFB₁-apt-Lp-AuNCs?at 442 nm?and ZEN-apt-BSA-AuNCs?at 650nm?were engaged as the fluorescent donor and WS₂ nanosheet?fluorescence quencher?.With the addition of AFB₁ and ZEN,the variation in F.I peaks was documented at 442 nm and 650 nm for concurrent quantification with LOD of 0.34 pg mL^-1?R²=0.9931?and 0.53 pg mL^-1?R²=0.9934?,respectively.Predominantly,the semi-quantitative recognition of AFB₁ and ZEN can also be performed through photo-visualization.In conclusion,ultra-small size,facile synthesis,high photostability,and F.I make MNCs a promising candidate in the field of biosensing.Therefore,as compared to conventional fluorescent assays,aptamer functionalized MNCs offer a new route in fluorescence-based biodetection.A combination of aptamer functionalized MNCs with other nanomaterials has been successfully established and applied in real food samples.The present research work affords a supportive and novel approach to advance the recognition and determination methodologies of mycotoxins.The present approach opens a new avenue to develop economic,sensitive,convenient,and selective MNCs fluorescent“switch-on”probes with potential applications in multipurpose biosensing.