Development Of Mitochondria-targeted Thiophenol Fluorescent Probe And Senescence- Associated ?-Galactosidase Fluorescent Probe And Their Application In Bioimaging

Mitochondria as cellular powerhouses are the preferential targets affected by thiophenols,an important class of highly toxic environmental pollutants,and are linked to the production of pathogenic reactive oxygen species(ROS)induced by trace thiophenol residues.For realtime and accurate sensing,a highly sensitive fluorescent probe for the specific detection of mitochondrial thiophenols was developed in this paper.The determination of senescent cells is conducive to elucidate senescence-associated process as well as age-related disease diagnosis.Senescence-associated ?-gal(SA-?-gal),as a lysosomal hydrolase,is currently the most widely used biomarker for cellular senescence.However,non-senescence-associated ?-gal usually interferes with the detection results of SA-?-gal.Considering that the lysosomal pH of SA-?-gal may differ from that of other nonsenescence-associated ?-gal,a fluorescent ?-gal probe for sensing lysosomal pH was developed in this paper.The specific contents are as follows:(1)A novel hybrid structure of fluorescein and rhodamine(rhodol)dye ROAP and a mitochondria-targeted thiophenol fluorescent probe ROAL,composed of ROAP as fluorescence reporter,the 2,4-dinitrophenyl moiety as the thiophenol-active trigger and pyridinium cations as a mitochondria-targeted moiety,were developed in this paper.ROAL proved to be highly selective to thiophenols among various analytes including aliphatic thiols,and renders an ultrasensitive off-on fluorescence response to thiophenols with a remarkable detection limit(8.1 nM).Colocalization experiments showed that ROAL efficiently stained mitochondria with a high Pearson's co-localization coefficient(0.95),thereby ensuring the specific detection of mitochondrial thiophenols.In addition,the probe ROAL was also applied to the imaging studies of thiophenol-stimulated fixed and living cells.The results showed that the fixed cells showed bright fluorescence,in contrast,the living cells exhibited weak fluorescence,suggesting that mitochondria can produce endogenous reactive oxygen species(ROS)due to the attack of thiophenols and then thiophenols can be eliminated by endogenous ROS in living cells.(2)A fluorescent ?-gal probe CA for sensing lysosomal pH,by selecting pH-sensitive hemicyanine dye CPH as a fluorophore and the ?-galactoside bond as the response site,was designed and synthesized in this paper.Since the pH range of lysosome is 4.0-6.0,A.oryzae ?-galactosidase(A.oryzae ?-gal)is optimally active at pH 4.0-5.0 and E.coli ?-galactosidase(E.coli ?-gal)is optimally active at pH 7.4,A.oryzae ?-gal and E.coil ?-gal were chosen to verify the pH sensitivity of the probe CA in vitro.A strong green fluorescence was observed after the reaction of A.oryzae ?-gal and CA,while the addition of E.coil ?-gal into the buffer solution of CA elicited red fluorescence.The results indicated that CA could detect ?-gal at different pH and showed different fluorescence signals,which proved that CA can simultaneously detect lysosomal pH and ?-gal.At the cellular level,the probe CA was applied to image ?-gal in ovarian cancer cells and human lung fibroblast cells with different degrees of senescence.The results showed that CA could not only distinguish SA-?-gal and SKOV-3 ?-gal,but also successfully monitored the senescent process of cells.