Detection And Cytotoxicity Evaluation Of Vomitoxin Based On Gold Nanoclusters

Deoxynivalenol（DON）,also known as vomitoxin,is a type B trichothecene mycotoxin,which poses a great threat to the health of human and animals by causing acute toxicity,intestinal toxicity,cytotoxicity and other harmful effects at high dose exposure or long-term exposure.Therefore,it holds great importance to establish accurate and sensitive DON detection methods to ensure national food quality and safety and maintain the health of human and animals.In addition,the study of toxic effects and toxic mechanisms of DON is conducive to take effective measures to reduce the risk of exposure and provide targeted intervention and prevention strategies.Fluorescence imaging technology has the advantages of nondestructive,visualization,high specificity and high sensitivity,which can realize real-time in-situ monitoring of the changes of intracellular biomarkers.Gold nanoclusters（Au NCs）are widely applied in the field of sensing detection and fluorescence imaging because of their advantages such as high resistance to photobleaching,easy surface functionalization,luminescence tunability and good biocompatibility.In this work,a dual-mode aptasensor and a dual-color composite probe for the sensitive and specific detection of DON and the cytotoxicity assessment of DON,respectively,were prepared by using Au NCs as signal probes and combining the advantages of easy functionalization,good photobleaching resistance and good biocompatibility of Au NCs with the specific recognition function of aptamers.The main contents are as follows:1.A fluorescent-SERS dual-mode aptasensor based on gold nanoclusters and metal-polydopamine framework was constructed and applied for DON detection.Complementary DNA modified Au NCs（c DNA-Au NCs）was used as fluorescence signals.Silver nanoparticles modified metal-dopamine organic framework（Ag NPs/MPDA）was coupled with TAMRA-labeled DON aptamer and was used as a SERS substrate and a fluorescence quencher.6-carboxyl tetramethylrhodamine（TAMRA）was used as a Raman signal molecule.Au NCs and Ag NPs/MPDA were assembled by nucleic acid hybridization to form the dual-mode aptasensor.The conformation of the aptamer can be altered by the binding of DON to its aptamer,which made c DNA-Au NCs away from Ag NPs/MPDA and TAMRA closer to Ag NPs/MPDA,eventually realizing the recovery of fluorescence signal and the enhancement of SERS signal.Under the optimal conditions,the linear range of this method was 0.1 ng/m L～100 ng/m L in both fluorescence and SERS mode,with the detection limits of 0.08 ng/m L and 0.06 ng/m L,respectively.The dual-mode sensor also showed good specificity and reproducibility,successfully applied for DON detection in wheat flour.The detection results provided by the two signals of this method can be mutually verified,which was conducive to improve the accuracy and reliability of the detection results.2.A DON-controlled dual-color Au NCs composite probe was constructed for simultaneous cell imaging of DON and mi R-34a induced by DON.Hairpin DNA modified dual-color Au NCs were used as energy donors,and Mo S₂ nanosheet was used as energy acceptor.The recognition sequence of mi R-34a in H₁^*was blocked by the aptamer to realize the DON-controlled cell imaging.The spatial distance between the dual-color Au NCs and Mo S₂ nanosheets was increased through the specific binding of DON with its aptamer and the HCR reaction induced by mi R-34a,resulting in the recovery of the fluorescence of the dual-color Au NCs.Under the optimal conditions,the relative fluorescence intensity of red-emitting Au NCs was linearly related to the logarithm of DON concentration(y=137.14 lg C_DON+129.11,R²=0.9807).The relative fluorescence intensity of green-emitting Au NCs was linearly related to the logarithm of mi R-34a concentration(y=287.99 lg C_{mi R-34a}+934.29,R²=0.9894).The expression level of mi R-34a was up-regulated under the stimulation of DON in Hep G2cells.With increasing the concentration（0μg/m L～20μg/m L）and the stimulation time（0 h～12h）of DON,the fluorescence intensities of the dual-color Au NCs were both gradually increased,realizing the simultaneous imaging of DON and mi R-34a induced by DON and providing a new strategy to verify the cytotoxic mechanism of DON.