| Reducing substances in organisms to maintain and regulate cellular redox environment has animportant role. Many studies have shown that reducing substances for the body’s antioxidantdefenses, but also has some other physiological functions, such as detoxification, enhanceimmunity, anti-aging and anti-cancer. Since reducing substances with redox regulation ofimportant role, it must come up with some applicable cells reducing species of high selectivity,high-sensitivity detection of small-molecule fluorescent probes, used for intracellular reducingspecies identification, quantitative.Proteins are one of the most important components in the organism, and play an important rolein the life activities. They are extremely numerous, and have a very wide distribution, withdifferent functions. Many physiological and pathological phenomena are associated with lesionsof the protein, such as Huntington’s, Alzheimer’s disease, Parkinson’s disease and cancers. Inorder to treat these diseases effectively, the most important way is to develop new therapeuticdrugs, many of which are target protein. Therefore, the study of protein has a great significancein biomedical research and disease treatment.The protein thiol group is vulnerable to oxidation by reactive oxygen species and reactivenitrogen species, and plays a principal role in maintaining an appropriate oxidation- reductionstate of the protein, regulating signal pathways, and responding to various diseases, such ascancer, diabetes, and neurodegeneration. The protein thiol group mainly refers to the sulfhydrylgroups of cysteine residues which is the most active group in all amino acid residues. Sulfhydrylgroups play an important role in the protein, such as the antioxidant, which can protect cells frombe oxidation. When the redox environment changes, sulfhydryl and disulfide bonds can occurbetween conversions. Therefore, the protein thiol group in living cells is of great significance inbiology and medicine. Fluorescence method, due to its highly sensitivity, good selectivity, shortreaction time, real-time imaging and other advantages, has broad application prospects in thefield of life science and analytical chemistry.At present, a wide range of fluorescent molecular probes are used for the detection of proteinin vivo, but most of them are indirect detection with the help of gene fusion. Fluorescent probesused to directly detect protein in vivo are rarely reported. So, we have designed a newfluorescent probe for the direct detection of double or multiple thiols-proteins in vivo by confocal imaging techniques in real time and in situ. Base on the mechanism of the maleimide onthe fluorescent probes selective binding to thiol substances cause the change of the probefluorescence spectral properties. We have carried out the investigation from two aspects:First, we selected the acridine derivative as a fluorophore and maleimide groups as the bindinggroup to design and synthesize fluorescent probes O-Dm-Ac and M-Dm-Ac for the rapiddetection of GSH and dicysteine peptides. These two fluorescent probes are weak fluorescent,but both of the probes’fluorescence is significantly enhanced when the probes react with GSH.The fluorescence change of fluorescent probes O-Dm-Ac is more obvious than M-Dm-Ac’s. Theexcitation and emission wavelengths of the two fluorescent probes are located at 360nm and460nm, respectively. In simulated physiological conditions, take the fluorescent probe O-Dm-Acfor the study, it is found that the fluorescent probe O-Dm-Ac responses with there small peptidesW-6 peptide, W-7 peptide and W-8 peptide, which containing dithiols. W-7 peptide responsebetter, that is because the distance between the two cysteine residues of W-7 peptide matches thedistance between the two maleimide groups on our probes O-Dm-Ac. There is a good linearrelationship between the fluorescence intensity and W-7 peptide concentration in the range of 0to 5.5μM. In addition, the probe did not respond to other active molecules or metal ions.Second, on the basis of research in the above, we known O-Dm-Ac could react with twosulfhydryl groups separated by an appropriate distance of a series of small peptides. Endogenousmultiple thiols-proteins play a key role in redox homeostasis. The thiol-proteins of organismsoften have multiple thiols, is sensitive to oxidation-reduction state and vulneralble to oxidation.When an oxidative specide exist, two vicinal sulfhydryle groups immediate are oxidated to forma disulfide. For the important function of vicinal-dithiol-containing proteins, there is greatsignificance to directly detection them in proteomics. We have studied the response of thefluorescent probe O-Dm-Ac reacting with reduced bovine serum albumin and have found thatthe probe can detect protein effectively. There is a good linear relationship between thefluorescence intensity and reduced bovine serum albumin concentration in the range of 0 to1.75μM. In addition, the probe did not respond to other active molecules or metal ions. Theprobe has advantages of low toxicity to cells, good light stability and high response rate. Whenscavenged GSH of HepG2 used BSO, the probe can successfully directly detection multiplethiol-proteins with appropriately sized dithiols in living cells using confocal laser scanning microscopy in real-time . |
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