Enhanced Raman Scattering Spectroscopic Detection Of S-nitrosated Proteins

Resonance Raman scattering(RRS)and surface enhanced Raman scattering(SERS)have wide applications in life science because of their high sensitivity and selectivity,Protein S-nitrosylation is an important post-translational modification,in which the thiol of cysteine residue is converted to nitrosothiol.Increasing evidences have shown that S-nitrosylation has important effect on protein functions like enzyme activity,protein localization and metabolic pathway.This thesis is focusing on developing SERS and RRS based detection methods for S-nitrosated proteins.S-nitrosylated proteins play an important role in modulating human functions and are applied as a disease biomarker.The detection methods established in our study demonstrated their great potential in early diagnosis and prevention of relevant diseases.The major contributions of this thesis are as follows:(1)Detection of S-nitrosated proteins based on RRSWe developed a label-free detection method for S-nitrosated proteins based on RRS.Indirect quantification of S-nitrosothiols(RSNO)was achieved by chemical properties of photo-induced nitric oxide(NO)release from RSNO and the redox reaction of ferrous cytochrome c(Fe²⁺Cyt c)with NO.UV-Vis and laser at the excitation wavelength of 532 nm were used to explore the reaction mechanism and to optimize experimental conditions.The results showed that NO released by the photo-induced decomposition of RSNO can fast oxidize Fe²⁺Cyt c and replace the Met ligation as a new ligand to form a new coordination bond Fe³⁺-NO with the central porphyrin.Typical Raman band intensities of RRS present a linear relationship with the concentration of RSNO.This detection method has good reproducibility and high accuracy,and the limit of detection(LOD)was as low as 10 n M.This study suggests a reliable assessment of total RSNO in the clinical applications.(2)Detection of S-nitrosated proteins based on SERSWe established a quantification method of RSNO based on Raman frequency shifts and developed a SERS chip which can specifically recognize RSNO.In the first step of the study,4-Aminothiophenol(PATP)was assembled on silver substrate via Ag-S bond,and then 4-(Diphenylphosphino)benzoic acid(LCOOH)was introduced to connect with PATP on the silver substrate.The-SNO group of RSNO can oxidize LCOOH,causing SERS frequency shifts.The results showed that SERS frequency shifts presented an exponential linear relationship with the concentration of RSNO.Compared with the conventional immunoreaction method,this method is sensitive,specific,simple and effective.It has great potential in RSNO biomarkers detection in linical diagnosis.