Raman Spectroscopic Analysis Of Phosphorylation-functional Biomolecules

Phosphorylated functionalization is one of the most common biochemical processes in living organisms,with the chemical link of the phosphate group to biomolecules.Some small biomolecules,such as riboflavin（RF）,bind to enzymes through the phosphate group after phosphorylation as coenzyme molecules to participate in enzymatic reactions.This kind of flavin biological functional molecule contains excellent chromophore,which has the application potential as the reporter molecule of SERS nanosensors.Phosphorylated proteins play important role in physiological and pathological processes such as cell signal transduction,neural activity and immune protection.Analysis of phosphorylated proteins based on Raman spectroscopy is of great significance for the identification of phosphorylation sites,early diagnosis of diseases and targeted therapy.Resonance Raman scattering（RRS）spectroscopy and Surface-enhanced Raman scattering（SERS）,capable of providing rich molecular fingerprint information,can realize rapid,highly sensitive and highly selective detection in physiological environment,which makes Raman spectroscopy have excellent performance in the detection of small biomolecules,analysis of the structure and function of protein and observation of life dynamic process.Several phosphorylation-functional biomolecules were studied mainly by RRS and SERS spectroscopy in this thesis.The major research contents are as follows:1.SERS Spectroscopic Analysis of Flavin MoleculesRiboflavin exists as two phosphate functionalized derivative forms of flavin mononucleotide（FMN）and flavin adenine dinucleotide（FAD）in living organisms.Their molecular structures contain the isoalloxazine group,which has good Raman activity.We prepared Ag nanoparticles self-assembled films,and conducted SERS characterization on riboflavin（RF）,FMN amd FAD,also lumichrome（LC）and lumiflavin（LF）which contained the isoalloxazine group as well,respectively.Combined with density functional theory（DFT）,their Raman characteristic peaks were calculated and assigned in detail.It was found that the Raman signals of these molecules are almost identical except LC,arising from the isoalloxazine group.In order to explore the adsorption mode of flavin molecules on Ag nanoparticles,Ag atoms were introduced to design three possible adsorption models with the aid of DFT calculation.It was determined that flavin molecules were more likely to be adsorbed by bonding with Ag at N₃ site.Subsequently,through the substitution experiments among FAD,adenine and adenosine triphosphate（ATP）on Ag nano-films,it was proved that the adsorption capacity of the isoalloxazine group was stronger than that of the adenine group.Finally,the optimal p H range and lowest detectable concentration of flavin molecules on Ag nanoparticles were investigated.The content of this chapter has theoretical guiding significance for the study of flavoprotein research and SERS nanosensors with flavin molecules as reporter molecules.2.Dual Detection of GOx Enzyme Activity and Glucose Based on RRS SpectroscopyGlucose oxidase（GOx）is a common FAD-coenzyme oxidoreductase,which oxidizes glucose,with the subsequence electron delivery to oxygen receptors through the FAD-redox active center to produce H₂O₂.The enzyme activity of GOx is of great significance in the food,pharmaceutical and clinical medicine fields.Reduced cytochrome c（Cyt c）can be oxidized to oxidized type by H₂O₂,accompanied by the change of intrinsic Raman signal of Cyt c,capable of being used to monitor the enzymatic reaction of GOx.In this chapter,reduced Cyt c was obtained by rapid reduction and separation with prepared magnet nickel nanowires. RRS spectroscopy was utilized to determine the linear relationship of the concentration proportion between the oxidized and reduced Cyt c at a given total concentration.The change of RRS spectra of reduced Cyt c was used to quantitatively monitor the catalytic oxidation of glucose by GOx.Finally,under the condition of fixed GOx enzyme activity or initial concentration of glucose,the dual linear detection of glucose and GOx enzyme activity was realized.This method provides a new pathway for the determination of enzyme activity or substrate concentration in the enzymatic reaction system with H₂O₂ as product3.Re-titration of the Reduction Potential of Erv1 and Raman Spectroscopic Analysis of FAD-binding DomainErv1 is a member of the FAD-coenzyme sulfhydryl oxidase family,which widely exists in the mitochondrial intermembrane space of eukaryotic organisms.It participates in the folding process of the disulfide bond of the mitochondrial protein peptide chain and maintains normal mitochondrial function.Erv1 transfers electrons from upstream Mia40 to downstream Cyt c receptors through its three internal redox centers.The previously reported reduction potential of the Mia40-Erv1 disulfide bond transfer system is thermodynamically adverse.Therefore,redox titration of the GSH/GSSG and DTT/DTT_ox redox buffer systems and SDS-PAGE technique were used to re-evaluate the reduction potential of the shuttle disulphide bond in Erv1,getting a more favorable result in thermodynamics.Also,we prepared magnetic Ti O₂ nanomaterial,and realized a rapid separation of reduced FAD from reductant under anaerobic condition,subsequently investigating the direct reduction of Cyt c by the free FADH₂.We concluded that the FAD-binding domain plays an important role in electron transport in the redox center of Erv1 and promotes the contact between FAD and Cyt c,giving a further understanding of the significance of the phosphate-functionalization of riboflavin to flavoproteins.4.In Situ SERS Determination of Phosphorylated ProteinsPhosphorylated proteins exist widely in biological cells and tissues,maintaining various life activities of cells.Abnormal dynamic regulation of phosphorylated proteins is closely related to the occurrence of cancer and neurodegenerative diseases.The highly sensitive and site-specific identification and detection of phosphorylated proteins are of great help in revealing many important signal transduction pathways and tumor targeted therapy.In this chapter,we designed a kind of SERS nanochip mediated by metal ions,specifically capturing and SERS recognizing four phosphorylated amino acids.We interpreted and assigned their intrinsic Raman vibration information with the aid of DFT calculation.Subsequently,we achieved specific identification of various phosphorylation site of peptides with the contrast of the SERS feature peaks of four amine acids above.This method has also been successfully applied in the non-immunospecific identification of Tau protein phosphorylated at serine site,which has far-reaching significance in the detection of biomarkers for Alzheimer’s disease.