Application Of Surface-enhanced Raman Spectroscopy In Detection Of Reactive Oxygen And Related Substances

When the living body is in a normal state,the generation and disappearance of oxides and antioxidants in the body are in a dynamic balance.At this point,the generated reactive oxygen species?ROS?with an appropriate amount has important physiological significances for intracellular immune regulation and signal transduction.However,when the body is subjected to some exogenous or endogenous stimuli,the normal metabolic process of ROS is interrupted,causing an outbreak of ROS,resulting in oxidative stress.The occurrence of oxidative stress usually leads to a series of serious consequences:acceleration of the body's aging,the disorder of acid-base balance in the body and the occurrence of various diseases.Therefore,it is of great importance to develop a series of detection probes and analytical methods for the analysis and detection of intracellular ROS and related substances.It is of profound significance for us to analyze the process of oxidative stress and its related processes.At the same time,it can further elaborate the role of ROS and related substances in the physiological and pathological processes,which is of great significance for the early detection,treatment and prevention of certain diseases.As the commander of the nervous system,the brain controls a variety of physiological activities,and most of these activities are achieved by neurons through the regulation of signal transduction.At the same time,under some stimulating conditions,the signal changes produced by neurons will map to the brain,causing some changes in brain functions,further causing some changes in certain physiological functions,which may lead to diseases such as cerebral ischemia injury,cerebral infarction,and Parkinson's disease.Therefore,on the basis of cell analysis and detection,the construction of a probe which can be applied to the detection of brain level and cell level at the same time is very important.It can provide further insights into the role of ROS and related substances in the physiological processes of these diseases and it is very practical significance for guiding the diagnosis and treatment of diseases.This thesis provides new research methods for studying the role and association of ROS and related substances in oxidative stress,and completes the progression from cell detection to brain detection,and further analyzes the changes of ROS and related substances in the relevant stimulation models.The details are as follows:?1?.A single nanoprobe for ratiometric imaging and biosensing of hypochlorite and glutathione in live cells using surface-enhanced Raman scattering.In this work,we first chosed a specific response molecule 4-Mercaptophenol?4-MP?that can detect hypochlorite?ClO^-?and glutathione?GSH?,and then modified 4-MP to the surface of gold flower?AuFs?to construct a surface-enhanced Raman scattering?SERS?probe?AuF/MP?.The hydroxyl group of 4-MP changed in the presence of ClO^-and GSH which induced the variation of SERS spectra of the probe.The peak located at 635 cm^-1 in SERS spectra gradually increased with the increasing concentration of ClO^-,while that observed at 1077 cm^-1 decreased.The SERS spectra recovered after addition of GSH,demonstrating that the SERS probe can also be employed for determination of GSH followed by ClO^-.The peak obtained at 822 cm^-1 stayed almost constant with the changes of both ClO^-and GSH,which can play as an inner-reference element to provide the ratiometric determination of ClO^-and GSH with high accuracy.The developed probe with high selectivity,accuracy and sensitivity was successfully applied for monitoring of ClO^-and GSH in live cells upon oxidative stress.?2?.A ratio-type SERS probe for intracellular pH imaging and biosensing.In this work,we designed and developed a ratiometric SERS probe for pH detection and imaging in live cells.We used gold flowers?AuFs?as the Raman-enhanced substrate material and selected 4-Mercaptobenzoic acid?MBA?as a pH-specific recognition component.Changes in the carboxyl groups of the MBA in different pH values caused the variation of SERS spectra of the probe.The peak located at 1391 cm^-1 in SERS spectra gradually increased with the increasing of pH value,while that observed at 1700cm^-1 decreased.Vice versa.Therefore,the pH detection was achieved according to the relationship of I₁₃₉₁/I₁₇₀₀ and pH.The probe with extremely high sensitivity,stability and accuracy was successfully applied to study the changes of intracellular pH under different stimuli.?3?.A SERS optophysiological probe for the real-time mapping and simultaneous determination of the carbonate concentration and pH value in a live mouse brain.Different tip sizes of quartz tapers?QTs?from nanometer to micron size were pulled and coated with a layer of rough gold film?Au-QT?as the Raman-enhanced substrate material to meet different detection needs.The specific recognition molecules of 1-?4-aminophenyl?-2,2,2-trifluoroethanone?AT?for CO₃^2-,4-mercaptobenzoic acid?MBA?for pH and reference molecule 4-mercaptobenzonitrile?MBN?were modified into the surface of Au-QT to form the SERS optophysiological probe and realized the detection of CO₃^2-and pH simultaneously.The present SERS technique can be combined with electrophysiology without cross-talk to record both electrical and chemical signals in brains.A SERS microarray of eight microprobes with high selectivity,good stability,high accuracy and good biocompatibility was used for real-time mapping and simultaneous quantification of CO₃^2-and pH in cortex of live mouse brain under ischemic conditions.To deepen our understanding of the mechanism of ischemia on the single-cell level,a SERS nanoprobe with a tip size of 200 nm was developed for use in a single neuron.