Detection Of Hydrogen Peroxide In Cancer Cells Based On Bifunctional Nanozyme Fe₃O₄@Ag And In Situ SERS Imaging Analysis Of Single Cells

Cells play an important role in most life activities.Single-cell analysis is helpful to understand their characteristics and heterogeneity,while single-cell imaging will further achieve sensitive and precise determination of intracellular component content and location in single cells,which is crucial for explaining some important physiological and pathological processes.Hydrogen peroxide（H₂O₂）is a major reactive oxygen species in cellular environment,high concentration of H₂O₂ will cause cell damage,leading to cancer,pancreatic dysfunction and inflammatory diseases and many other diseases related to oxidative stress.However,the content of H₂O₂ in cancer cells is low and the diffusion is fast,which makes the in situ detection of H₂O₂ in single cells difficult.Therefore,developing a rapid,simple and highly sensitive method for in situ analysis of H₂O₂ in single cells is conducive to understand the role of H₂O₂ in oxidative stress-related diseases,and has vital significance for the diagnosis and treatment of these diseases.Surface-enhanced Raman scattering（SERS）technology is widely used in biological samples analysis,especially in single-cell analysis with the advantages of its high sensitivity and no damage to biological samples.In this thesis,we proposed an in situ SERS imaging for discrimination of H₂O₂ in single cells using a dualfunctional nanomaterial Fe₃O₄@Ag with peroxidase-like activity and SERS activity.Based on the principle that the substrate can catalyze H₂O₂ to oxidize 3,3’,5,5’-tetramethylbenzidine（TMB）and lead to the changes of the SERS patterns,on the light of the linear relationship between the concentration of H₂O₂ and the SERS intensity,we have achieved the simple and high sensitivity quantitative detection of H₂O₂ released from living cancer cells,and obtained the average content of H₂O₂ released from each cell.Meanwhile,we have also achieved in situ imaging of H₂O₂ in single cells.In chapter 1,the significance of determination of H₂O₂ in cancer cells and detection methods were summarized firstly;Secondly,we introduced the methods of single cell imaging.Then,the SERS principle,the types of SERS substrates,and the advantages of SERS technology in cell analysis,especially in imaging analysis were reviewed.Finally,the design ideas and significance of this work were proposed.In chapter 2,we prepared the Fe₃O₄@Ag magnetic nanoparticles with peroxidase-like activity.Scanning electron microscopy,transmission electron microscopy,X-ray diffraction spectroscopy and hysteresis loop curve were used to characterize the prepared nanoparticles.TMB was selected as the chromogenic substrate,and the peroxidase-like activity of the prepared Fe₃O₄@Ag was investigated by using ultraviolet spectroscopy and SERS.Then we optimized the enzymatic reaction conditions.Through the study of catalytic reaction kinetics and mechanism,it was proved that the prepared Fe₃O₄@Ag nanomaterials have good catalytic activity,and the content of H₂O₂ can be detected based on the enzymatic reaction of the nanomaterials.In chapter 3,we investigated the SERS spectral activity and reproducibility of Fe₃O₄@Ag nanomaterials.The oxidation reaction of TMB was monitored in real time by SERS spectroscopy,and the reaction time of catalytic oxidation of TMB was determined to be 16 min.Under the optimized catalytic reaction conditions,the content of H₂O₂ in solution was detected by SERS,and the detection concentration could be as low as 10-15 mol L^-1.According to the linear relationship between the intensity value of the SERS characteristic peak and the concentration of H₂O₂,a fast and ultra-sensitive method was established to detect the content of H₂O₂ in living cells,and the H₂O₂ in single cells was further analyzed by in situ imaging.In chapter 4,we summarized the proposed method of in situ SERS imaging of H₂O₂ in single cells,and its application was prospected.