A Molecular Fluorescent Probe That Visually Detects Endogenous Hydrogen Peroxide

With the increasing advancement of scientific technology,scientists have invested more and more research interests into the cellular level,especially the research on cell messenger molecules,which has become the froniter of physiological pathology research.Cell messenger molecular theory divides all messenger molecules into two major categories:the first messenger molecules and the second cell molecules.Among them,the second messenger is a kind of endogenous non-protein small molecules,which produced by the first messenger.Its concentration level changed by responding to the cell signal from the first messenger molecule,then regulating the activity of the enzyme and the non-enzymatic protein in the cell.Through this manner,the intracelluar signal is transmitted step by step,and finally the messenger effect is terminated through the degradation.The second messenger molecule works in two ways:one is to act on the target protein directly;the other is to induce a series of protein phosphorylation by activating the corresponding kinase,then produce the cellular effect.Recent research about hydrogen peroxide confirms that endogenous hydrogen perioxide serves an emerging second messenger molecule in signal transduction in cell activities such as cell division,differentiation and metastasis.The level of endogenous hydrogen peroxide is significant related to the rate of cell mitosis,especially during the critical period brain development in mammals.Molecular fluorescence probe technology is mainly used to detect various small molecules in living organisms.It has the advantage of high selectivity,low detection limit and no toxicity to cells.As far as we known,the current fluorescence probes for detecting hydrogen perioxide have not been able to realize imaging in mammals.The main reason is that the probe excitation and emission wavelength is close to the living tissue self-background fluorescence region,the two interfere with each other,therefore it is difficult to achieve imaging in vivo.And the level of endogenous hydrogen perioxide for cell motisis is extremely low,it is easily converted into other active oxygen interfering substances in the cell environment.Therefore,we invested our research interest into the detection and imaging of endogenous hydrogen perioxide in vivo by molecular fluorescence probe.My graduation thesis consists of the following parts:In Chapter 1,there is an introductory chapter about fluorescence probe technology.In this chapter,we focus on the componets of the fluorescence probe,the mechanism type of the probe,and illustrate the various design mechanisms and papers in combination with the excellent work that has been published by the predecessors.Moreover,the existing hydrogen perioxide probes were classfied and compared,we found out the shortcomings of current hydrogen perioxide probe.In Chapter 2,we developed a ratiometric near-infrared fluorescent probe BCy-PFS（BenzohemiCyanine-Pentafluorobenzenesulfonyl）and used in detection endogenous H₂O₂ in normal human cells,human skin cancer cells,mouse hippocampal neuronal cell mitosis and mouse brain development.We used the probe BCy-PFS to detect H₂O₂ high selectively in many ROS within 200 seconds.Rely on the low toxicity of the probe BCy-PFS,we confirmed the role of endogenous H₂O₂ in accelerating the process of cell mitosis.Rely on the excitation and emission wavelengths in the near-infrared（NIR）region of this probe,we realized the detection and imaging endogenous hydrogen peroxide in the living mouse brain.The results of cell experiments showed that EGF stimulates the production of endogenous H₂O₂ in cells,then the endogenous H₂O₂promoted the rate of cell mitosis.The results of hydrogen peroxide imaging in mice brain revealed the changes of H₂O₂ levels affects the development of the mouse brain directly.The results for the first time illustrated the close relationship between endogenous H₂O₂ and brain development through promoting cell mitosis.This work may provide futher information for exploring H₂O₂ as a chemical messenger molecule in cell mitosis.In Chapter 3,we synthesised a two-photon fluorescent probe DCPO-PFS（2-（2-（4-Hydroxystyryl）-4H-chromen-4-ylidene）malononitrile-Pentafluorobenzenesulfonyl）for the two-photon detection of endogenous H₂O₂ in Hela cells produced by PMA stimlulation.The probe DCPO-PFS has a large two-photon absorption cross section of 65 GM at 830 nm,the fluorescence quantum yield isΦ=0.45,and the hydrogen peroxide detection limit is 68 nm.We used the probe DCPO-PFS to detect H₂O₂ high selectively in many ROS in 180 seconds.Due to the two-photon excitation wavelength of the probe DCPO-PFS,we detected the endogenous H₂O₂ in HeLa cells at the two-photon excitation wavelength of 820 nm.Two-photon fluorescent probes have many advantages such as:eliminating background fluorescence,reducing photobleaching,and deep tissue imaging.Therefore,we plan to use the probe DCPO-PFS for imaging H₂O₂ in deep tissues of living organisms.In Chapter 4,summarizes the current work results and makes a futher plan for the research on the multi-functional drug delivery fluorescent probes.