Design,Synthesis And Bioimaging Applications Of Fluorescence Probes For Discriminating Live And Dead Cells

Discrimination of live and dead cells and monitoring cell viability are crucial and necessary in the research of biology and medicine.In biology,discrimination of live and dead cells is an important task for studying the process of apoptosis.In the field of medicine,discrimination of live and dead cells and monitoring cell viability are the most direct method to confirm drug efficacy and cytotoxicity.Therefore,reagents that can distinguish and detect dead and living cells are important research tools,and the development of such tools is of great significance in the field of life science and medicine,which have broad commercial prospects.Up to now,various methods have been applied for the detection of cell apoptosis,including morphological analysis,colorimetric detection,and fluorometric analysis.Among them,the fluorescence detection method displays superior advantages,such as high sensitivity,good selectivity,in situ and real time imaging.Therefore,it has been widely used in the field of life science and medicine.Moreover,compared with the“turn-on/off”fluorescent probes with only one emission color,the ratiometric fluorescent probes can effectively improve the accuracy of the detection results by self-calibration of two emission bands.However,there are few reports on the construction of ratiometric fluorescent probes for detecting cell apoptosis.Therefore,the development of ratiometric fluorescent probes for real-time and in situ visualization of cell apoptosis with high accuracy is still in urgent need.In this paper,we designed and synthesized three ratiometric fluorescent probes for the detection of cell apoptosis using pH response,excited state intramolecular proton transfer?ESIPT?process,and fluorescence resonance energy transfer?FRET?mechanism.Firstly,a pH-responsive fluorescent probe PVMR that can target mitochondria and RNA was constructed for ratiometric detection of cell apoptosis.Then,a two-photon fluorescent probe DACA can discriminating live and dead cells in dual-color mode based on ESIPT mechanism was developed.Finally using the FRET mechanism and the different re-localization behaviors before and after the??_m change,the ratiometric imaging of cell apoptosis was achieved by FRET pairs HVQ-MTDR and MTR-1-G-1.In chapter 2,we have rationally designed and synthesized a pH-responsive and dual-targets probe PVMR for real-time and ratiometric monitor of the cells apoptosis process.The probe consists of pH-responsive and blue-emitting 7-hydroxylcoumarin,and red-emitting quinoline salt fluorophores.In live cells,the probe targets mitochondria with basic matrixes and high membrane potential???_m?,displays intense emission in the blue and red channels.During apoptosis,??_m decreases,so the probe is released from mitochondria,binds to basophilic RNA.At the same time,the probe only shows strong emission in the single red channel.Based on the above process,PVMR enables the ratiometric observation of the oxidative damage of live cells by hydrogen peroxide?H₂O₂?,and the monitor of drug-induced apoptosis by paclitaxel and rotenone.In chapter 3,we constructed the probe DACA by acetylating the fluorescent dye 3-hydroxyflavone with ESIPT properties.In live cells,the acetyl part of the probe is hydrolyzed by active esterase in living cells,and the recovery of the ESIPT process causes the cells to display orange fluorescence peaked at 570 nm.In dead cells,only the blue fluorescence of the probe itself peaked at 440 nm was detected.Consequently,probe DACA can discriminate dead and living cells in a dual-color manner,and the emission wavelength separation in dead and live cells is up to 130 nm,which could be easily discriminated under fluorescence microscope.In addition,the probe has good biocompatibility,modest two-photon properties,and high photostability.With this probe,we successfully detected the apoptosis process of cervical cancer cells induced by H₂O₂ and ultraviolet?UV?radiation.Besides,the live and dead zebrafishes were successfully distinguished under a two-photon microscope.In chapter 4,by means of the FRET mechanism and the??_m-dependent subcellular migration properties of probes,FRET probe pairs HVQ-MTDR and MTR-1-G-1 for imaging the apoptosis process were developed.These probes have excellent optical properties and cell membrane permeability.Bioimaging experiments show that when HVQ and MTDR are used in combination,FRET signals can be produced in living cells,which gives the deep red fluorescence of MTDR under short-wave excitation.In dead cells,FRET process is blocked,under short-wave excitation,the fluorescence signal in the deep-red channel gradually disappears,and the emission in the red channel becomes stronger.Similarly,when MTR-1 and G-1 are used together,through the presence or absence of the FRET effect,under short-wave excitation,the change of the fluorescent signal in the red and green channels can be used to identify dead and living cells.The combination of HVQ-MTDR,and MTR-1-G-1 both successfully monitored the apoptosis process induced by hydrogen peroxide and CCCP in a ratiometric manner.