Fluorescent Probe Design And Cell Application For Specific Detection Of Cysteine

Cysteine(Cys)is the only amino acid that has a reducing thiol group.It can be converted from methionine(Met,an essential amino acid in vivo),and can also be converted into each other with cystine(Cys2).Cys is involved in many important physiological processes such as detoxification,metabolism and redox regulation of animal cells.The abnormal concentration of Cys in the organism is related to the pathogenesis of cancer,cardiovascular and cerebrovascular diseases and other diseases.So that specific semiquantitative analyses of intracellular Cys concentration change and distribution have guiding significance for the pathogenesis of its related diseases.In this article,a series of functional fluorescent dyes were designed and synthesized,which is based on the unique advantages of fluorescent probe in detecting non-invasive markers of target substances in living cells.They were applied to the specific fluorescence detection of Cys in cytoplasm,mitochondria and cancer cells.Specific research works are as follows:1.We modified the naphthofluorescein with acryl as the specific reaction site of Cys to synthesize a fluorescent probe NFA.The introduction of carboxyl group in NFA effectively improves the problem of poor water solubility commonly existing in Cys fluorescent probes.It can specifically react with Cys in PBS and exhibit an enhanced fluorescence response with an emission wavelength of 545 nm.We are able to achieve Cys fluorescence detection in PBS and cytoplasmic environments.The detection process is based on the specific release of naphthofluorescein after tandem nucleophilic reaction between acryl group and a specific mercaptoethylamine of Cys,resulting in a change in the fluorescence signal.In addition,the introduction of a carboxyl group provides an entry point for further modification of the probe.2.Based on the above work,we prepared an asymmetric fluoresceinderivative,and introduced acryl as the specific reaction site of Cys and biotin as the targeting group of cancer cells to obtain the fluorescent probe AC that can target and label Cys in cancer cells.The probe reacted with Cys in PBS and showed an enhanced fluorescence response(115-fold increase in fluorescence intensity)with an emission wavelength of 550 nm,and its detection limit for Cys was 0.307 ?M.We can selectively stain He La cells in a culture dish incubated with He La and RAW264.7 cells with this probe.This probe furnishes that a powerful tool is studying the concentration changes of Cys in cancer cells and normal cells.3.Mitochondria are energy factories in cells and the starting unit of oxidative stress.In order to investigate the role of Cys in the regulation of cellular redox levels,we synthesized a probe Mito-1 using quaternary phosphine salt as the targeting group of mitochondria to detect Cys in mitochondria.As a reaction site,?,?-unsaturated ketones in Mito-1 can specifically react with Cys in weak alkaline environment(PB,p H 8.0,that is similar with the mitochondrial environment),resulting in the system to increase fluorescence intensity at 498 nm.The probe can present semiquantitative fluorescence analysis of Cys concentration changes in mitochondria of cells,which provides the means to further understand the correlation between Cys levels in mitochondria and disease.