Study On Mitochondria-and Lysosomes-targeted Carbazole-based Dual-response Two-photon Fluorescent Probes For Autophagy Detection

Small molecule two-photon fluorescent probes have helped us understand complex biological systems more comprehensively through their high sensitivity to specific analytes.However,the current design of fluorescence sensors tends to be selective probes for a single analyte rather than monitoring multiple analytes at the same time,and many biological processes involve more than one chemical parameter.Therefore,it is meaningful to design a single probe reasonably so that it can respond to two analytes at the same time,named dual-response probes.Mitochondria have several main functions such as energy supply,signal transmission,cell differentiation,and maintenance of cell cycle and cell growth control.The lysosome is a double-membrane organelle,which is the main digestion chamber for degrading cellular components such as proteins,nucleic acids,carbohydrates,and lipids.Abnormal functions of mitochondria and lysosomes often lead to a variety of diseases.Autophagy is of great significance to the circulation of biomolecules under physiological conditions,and is essential for cleaning damaged organelles and useless macromolecules.Besides,it also plays an indispensable role in a variety of important biological events such as programmed cell death,cell growth and circulation,nutrient signal transduction,and tumor suppression.Therefore,in order to deepen the understanding of autophagy and promote the development of related biopathological areas,the method of real-time and in-situ observation of autophagy can be used.Combining recent literature and the work of our group,two dual-response two-photon fluorescent probes with carbazole as the fluorescent matrix were designed and synthesized in this paper.(1)A new fluorescent probe(Mito-FG)was designed and synthesized.According to the test of the optical experiment,the probe Mito-FG responds to polarity at short-wave emission(412 nm),and responds to G-quadruplex at long-wave emission(530 nm).Moreover,the change of fluorescence intensity and polarity or the concentration of G-quadruplex has a satisfied linear relationship.In addition,the probe Mito-FG also has better two-photon properties and low cytotoxicity.And it also can be stably located in the mitochondria without being affected by the mitochondrial membrane potential.Therefore,the probe Mito-FG can be used for long-term detection of changes in polarity and G-quadruplex to monitor mitochondrial autophagy.(2)A new fluorescent probe(Lyso-FNN)was designed and synthesized.Optical experiments showed that the probe Lyso-FNN can simultaneously respond to both polarity(short-wave emission,434 nm)and p H(long-wave emission,521 nm).Cytotoxicity experiments also displayed that the probe Lyso-FNN has lower cytotoxicity and can be safely in the cell body.Co-localization experiments proved that the probe Lyso-FNN can be localized in the lysosome and has good photostability.Therefore,this probe is suitable for long-term detection of lysosomal polarity and p H fluctuations to monitor autophagy.