Detection And Bioimaging Of T-2 Toxin-induced Apoptosis Markers With Nucleic Acid-functionalized Gold Nano-optical Probes

T-2 toxin is the main source of mycotoxin in grain.It is toxic to the human digestive system,nervous system,and reproduction system because it can induce apoptosis in a variety of ways.Apoptosis plays a crucial role in the growth and development of organisms.Identifying and monitoring toxin-induced apoptotic progression is critical for understanding the toxicity mechanisms and advancements in apoptosis research.Recently,gold nanomaterials（AuNMs）have gained a broad development prospect in the construction of surface-enhanced Raman scattering（SERS）or fluorescent biosensors owing to the excellent optical properties.Multibranched gold nanoparticles（AuNPs）generally exhibited higher SERS activity due to the tip effect.AuNMs are widely employed for intracellular detection and in situ imaging by employing recognition molecules.Among them,functional nucleic acid,as a new type of recognition molecule,has the advantages of simple synthesis,convenient modification and low price.Detection methods based on functional nucleic acids are still in the development stage and have important research significance.In this project,we prepared fluorescence-SERS dual-mode nano-sensing probes and fluorescent multi-color sensing probes by combining the dual advantages of AuNMs and functional nucleic acids,which were used for quantitative and selective detection and imaging of cell apoptosis markers induced by T-2 toxin,respectively.It opens a new avenue to provide a new and effective platform for studying the apoptosis mechanism of toxin-induced cells.The specific research contents are as follows:1.DNAzyme-gold nanostars（AuNSs）probe was used for SERS-fluorescence dual-mode detection and imaging of calcium ions(Ca²⁺)in living cells.Ca²⁺,an important second messenger,plays a key role in T-2 toxin-induced apoptosis.Herein,a novel dual-mode nanoprobe,including"turn-on"fluorescence and"turn-off"SERS,was developed for sensitive and selective Ca²⁺determination.The nanoprobe combines AuNSs and DNAzyme（recognition element）.The Cy5-modified substrate chain was hybridized with the AuNSs surface enzyme chain to establish Ca²⁺-specific DNAzyme and led to the quenching of fluorescence and enhancement of the SERS signal.With the existence of Ca²⁺in the detection system,the Cy5-labeled substrate chain was cleaved and released from the AuNSs surface,leading to the reduction of the SERS signal and recovery of the fluorescence signal.The linear range is 0.4μM-50μM with a minimum detection limit of 0.021μM.Ca²⁺overload events during T-2 toxin-induced apoptosis were successfully monitored using two signal changes.The proposed nanoprobe combined the advantages of SERS and fluorescence and had good universality in various cell lines,which was helpful for better understanding the role of Ca²⁺in the cell pathway.2.DNA modified gold nanoflowers（AuNFs）probe was used for fluorescence and Raman dual-mode quantitative detection and imaging of small-molecule thiols in apoptotic cells.Oxidative stress-induced by T-2 toxin can lead to the collapse of the intracellular antioxidant system and apoptosis.Small-molecule thiols,especially glutathione,play a key role in maintaining redox homeostasis.In this work,a dual-mode nanosensor（C2-C1-AuNFs）was designed to detect small-molecule thiols.DNA was modified by Cy5 and disulfide bonds（recognition elements）.AuNFs were used as fluorescence quenching and SERS enhanced substrates.However,small-molecule thiols can split disulfide bonds and release short DNA chains modified Cy5,leading to the recovery of the fluorescence signal and reduction of the SERS signal.C2-C1-AuNFs were sensitive to small-molecule thiols and represented a good linear range of 0.01 m M-3 m M for glutathione with a detection limit of 913 n M.Most importantly,C2-C1-AuNFs have been successfully used for in situ imaging and quantitative monitoring of small-molecule thiols concentrations during T-2 toxin-induced apoptosis.The developed sensing probe not only combines the dual advantages of fluorescence and SERS but also has universality in other human cell lines.The experimental results showed that C2-C1-AuNFs are good optical imaging tools for the detection of small-molecule thiols,and are expected to be applied in the in-situ studies of related physiological processes.3.Nucleic acid multicolor fluorescent probes based on symmetrical gold nanostars（S-AuNSs）were used for real-time in situ observation of P53-mediated cascade activation of apoptotic pathways.The P53-mediated apoptosis pathway is a very important collection of signaling molecules,which plays a key role in cell apoptosis.Real-time monitoring of upstream and downstream activation relationships of P53 m RNA,Bax m RNA,and Cyt c in signaling pathways is of great significance for understanding the regulatory mechanisms of these signaling molecules in physiological activities.In this work,silicon dioxide-coated S-AuNSs probes（S-AuNSs@SiO₂-P）were developed for highly sensitive in situ real-time imaging of P53 m RNA,Bax m RNA,and Cyt c during T-2 toxin-induced apoptosis.The P53 m RNA recognition chains,Bax m RNA recognition chains,and Cyt c aptamer complementary chains were modified by amide bonds on the surface of S-AuNSs@SiO₂.The complementary and aptamer chains modified with fluorescein groups（FAM,TAMRA,and Cy5）were connected by the complementary matching criterion to construct the multi-color fluorescent probes.In the absence of targets,the fluorescence of the three fluorophores was effectively quenched.In the presence of targets,the DNA double-strands were forced to unbind and the fluorescent strands formed duplexes or complexes with the targets,leading to fluorescence recovery.The probes based on S-AuNSs exhibited excellent optical performance due to the presence of 20 symmetric"hot spots".In the real-time fluorescence imaging of T-2 toxin-induced apoptosis in Hela cells,the fluorescence signals corresponding to P53 m RNA,Bax m RNA,and Cyt c were turned on sequentially.Therefore,S-AuNSs@SiO₂-P is an effective tool for real-time in situ monitoring of P53-mediated cascade activation of apoptotic pathways.It can accurately analyze positively regulated signaling pathways and is easily applicable to in situ detection of other signaling molecules.