Study On Mitochondria-targeted Ratiometric Two-photon Fluorescetprobe For Visualization Of Apoptosis

Most of the metabolism of cells are mainly done in the mitochondria,and many processes involved in mitochondria include ATP production,redox sites,and apoptosis.During apoptosis,mitochondria are damaged and affect the balance within the cell,and can even lead to neurodegenerative and neuromuscular diseases.Therefore,monitoring the progression of the apoptotic process is very important for the assessment of disease progression.Traditional methods of apoptosis detection,such as the TUNEL assay and the Annexin V-FITC apoptosis assay,are cumbersome and can not give real-time results.Therefore,methods for monitoring the apoptotic process in real time are needed.Among all developed methods of apoptosis,fluorescent probe-based optical assays have been widely accepted because they facilitate accurate and economical manipulation and can detect target analytes in a biological environment.Therefore,developing a two-photon fluorescent probe to detect apoptosis is a very good idea.Biothiols such as Cys,Hey and GSH are involved in many biological processes.Abnormal levels of biothiol can cause many diseases.And Cys is more susceptible to oxidation than GSH and Hey,and is more sensitive to fluctuations in mitochondrial ROS(reactive oxygen species).Therefore,the reduction of Cys can serve as an early marker of apoptosis.Detection of early apoptosis is very helpful in the treatment of disease.Therefore,monitoring the level of Cys in mitochondria is very important for biomedical research and diagnosis.Up to now,a variety of analytical techniques have been developed to detect biothiols,including voltammetry,etc.These methods provide high precision,but they require skilled workers,expensive analytical instruments and cumbersome pre-processing procedures.Among all the methods developed,fluorescent probe-based optical assays have been widely accepted since they are convenient to operate accurately and economically,and can detect targeted analytes in biological environment.Polarity is an important parameter in chemical research.And some organic and inorganic processes are closely related to the surrounding polarity.In biological systems,especially in cellular systems,polarity determines the interaction of some proteins and enzymes and reflects the permeability of some organelle membranes.In addition,abnormal fluctuations in polarity are closely related to certain diseases.Therefore,new tools are needed to detect polarity in living cells.The process of apoptosis leads to changes in mitochondrial polarity,and a two-photon fluorescent probe for mitochondrial targeted localization can be developed for rapid and accurate monitoring of polarity fluctuations.This has great significance for the exploration of apoptosis.Carbazole is a very good two-photon fluorophore,so our group has done a lot of work with carbazole as the parent.There are many sites on the carbazole group that can be modified,which provides a good basis for the design of fluorescent probes.It has been reported that when designing Cys and polar probes,the ICT mechanism is:first selected,which is not only beneficial for th e realization of two-channel detection of analytes,but also that the ICT mechanism is very sensitive to polarity.Therefore,in this paper,we synthesized two probes for detecting mitochondrial Cys and polarity on the basis of carbazole.More significantly,the two probes are capable of real-time tracking and the ability of dual-channel TPM to visualize apoptosis,which is of great significance for studying apoptosis and provides a good idea.(1)We separately synthesized a pair of carbazole derivatives(Mito-SCHO and Mito-DCHO)in one-dimensional/two-dimensional ICT system.Both of them can be easily synthesized in three steps.As expected,Mito-DCHO with a two-dimensional ICT system can specifically detect Cys with a ratio signal(119 nm emission shift).The donor(D)of carbazole skeleton and the acceptors(A)of the double aldehyde groups in Mito-DCHO constructed a strong ICT system,which also endowed the probe with the desired two-photon character.Besides,triphenylphosphine(TPP)was worked as a mitochondrial-located group.More importantly,we monitored early apoptosis by detecting mitochondrial oxidative stress levels.(2)We synthesized two compounds,Mito-1 and Mito-PF,in which Mito-1 is interfered by viscosity while detecting polarity,while Mito-PF specifically responds to polarity and is not affected by the external environment.1-ethynyl-4-fluorobenzene of Mito-PF as a polar responsive group,and benzothiazole salt acts as a mitochondrial localization group.Mito-PF can be well localized in lysosomes along with the good biocompatibility.More importantly,we monitored apoptosis by detecting mitochondrial polarity fluctuations.