Monitoring Of DON-induced Apoptosis Markers With Construction Of Gold Nano-optical Probes

Deoxynivalenol(DON),also known as vomitoxin,is one of the common trichothecene mycotoxins in nature and widely exists in grains and cereals.It can disrupt the synthesis of DNA,RNA and protein and damage mitochondrial function.The mitochondrial pathway is an essential pathway for DON induced apoptosis,and the identification and monitoring of molecular targets in this pathway plays an important role in studying the relationship between food safety and the homeostasis mechanism of intracellular environment.Gold nanomaterials(Au NMs)exhibit excellent optical properties.Based on their strong localized surface plasma resonance(LSPR)effect and Fluorescence resonance energy transfer(FRET)effect,it can be combined with functional nucleic acids,functional peptides and other novel recognition molecules to construct surface-enhanced Raman scattering(SERS)or fluorescence sensors,which are widely used in the field of food safety analysis and detection.At the same time,Au NMs have good biocompatibility,enabling the gold nano optical probe to be used as a carrier for cell analysis to monitor the dynamic changes of intracellular materials.In this study,we first chose to track the changes of biological information in the nucleus during the process of cell apoptosis.As an important organelle that controls the metabolic cycle of cells,the damage of the nucleus will affect the transcription and translation of genetic material,as well as the synthesis of relevant m RNA and protein.Currently,the analysis of targets in apoptotic events focuses on the Bcl-2 family and Caspase family.Therefore,the key factors Bax m RNA and Caspase-9 were selected as apoptotic markers in subsequent experiments to monitor their dynamic changes in the apoptotic process induced by DON.The dual-mode gold nanoprobe constructed has fluorescence and SERS double imaging capability,which can improve detection accuracy and stability,and has broad development prospects in the recognition and sensing of intracellular substances.The specific research contents are as follows:1.Gold nanoprobes were constructed for real-time in-situ analysis of the changes in the structure of related substances in the nucleus during apoptosis of Hep G2 cells.Nuclear targeting peptides(NLS and RME)were fixed on the surface of gold nanoparticles(Au NPs)by Au-S bond,and NLS-RME@Au NPs was obtained.It could enter Hep G2 cells and achieve nuclear localization,and directly enhance the Raman signal in the nuclear region.Under different stimulation time of DON,the components in the nuclear region changed,and differential spectral Analysis and Principal Component Analysis(PCA)were combined to identify and distinguish cells in different states and summarize typical biomolecular events.The SERS probes had high biocompatibility and realized non-destructive monitoring at the single-cell level,which had advantages in molecular mechanism research.2.The SERS-fluorescence dual-mode nanoprobes(DNA2-DNA1-Au NTs)were constructed for dynamic detection of Bax m RNA in living cells.DON could cause mitochondrial damage and induce cell apoptosis by up-regulating key pro-apoptotic factors such as Bax m RNA in Bcl-2 family.The dual-mode nanoprobes were mainly composed of Gold nanoplates(Au NTs),Bax SH-DNA(DNA1)and Bax-r DNA(DNA2).Au NTs could be used to enhance Raman signal and quench fluorescence.The 5’ terminal of DNA1 was fixed to Au NTs by sulfhydryl functionalization,and the 3’ terminal of DNA2 was labeled by Cyanine 5(Cy5)fluorescence groups with Raman characteristic peaks.After DNA1 was connected to Au NTs surface,DNA2 hybridized with DNA1 based on base complementary pairing principle.In this case,the distance between Cy5 and Au NTs was shortened,Raman signal was enhanced,and fluorescence signal was quenched.In the presence of target chains,the double chains were forced to unravel,and DNA2 tended to bind with fully complementary target chains,making Cy5 far away from Au NTs,resulting in reduced SERS intensity and the quenched fluorescence recovery.The results showed that the dual-mode nanoprobe was highly sensitive,with a linear range of 0.5 nm-10 n M and a minimum detection limit of 0.26 n M,and could be used for the quantitative detection of Bax m RNA in vitro and intracellular real-time in-situ imaging.3.The polypeptide functionalized gold nanoprobe was developed for real-time in situ detection of Caspase-9 in living cells.The LEHD(Leu-Glu-His-Asp)polypeptide was linked to gold nanostars(Au NSs)by Au-S bond.Rhodamine B(Rb)acts as both a Raman beacon molecule and a fluorescent group on the peptide chain modification.In the process of cell apoptosis induced by DON,Caspase-9 was activated,and the activated Caspase-9could specifically shear the recognition site LEHD in the polypeptide substrate,resulting in the drop of the peptide chain modified with Rb group at the end,which was far away from Au NSs,and finally realized the reduction of SERS signal and the enhancement of fluorescence signal in the system.Experimental results showed that the probe was highly sensitive,with a linear range of 5 ng/ ml-400 ng/m L and a detection limit of 0.38 ng/m L,and realized fluorescence and SERS double visualization imaging in different human cell lines.