Studies On Benzothiazole Fluorescent Probes For Detection Of Formaldehyde,pH And Glutathione

Formaldehyde(FA)and glutathione(GSH)can regulate a variety of life processes.As a key parameter in intracellular environment,p H plays an essential role in many physiological processes such as cell proliferation and differentiation,ion transport and protein degradation.Canceration of cells may lead to abnormal levels of FA,GSH or lower p H values.The levels of FA/GSH and p H can be used as important parameters to reflect the growth status of cells.Traditional methods for detection FA,p H and GSH include high-performance liquid chromatography,flow cytometry and so on.These methods have the disadvantages of expensive instruments and complex operation procedures,which limit the applications of them in the detection of analytes.Fluorescent probes have the advantages of high sensitivity,good selectivity,low cost and little toxicity to cells.They have been widely used in the imaging of biomolecules.Fluorescent probes with excited-state intramolecular proton transfer(ESIPT)have many advantages such as dual wavelength ratio fluorescence detection,large stokes shifts and high fluorescence quantum yield.2-(2?-Hydroxyphenyl)benzothiazole,a typical ESIPT molecule,has been widely used to design novel fluorescence probes.In this thesis,three fluorescent probes were synthesized by introducing the substituents at different sites of their parent fluorophores and they were respectively used for the detection of FA,p H and GSH.In this paper,by introducing the amino group at position 5 as the recognition site for FA,two FA fluorescent probes(BT-1/BT-2)with large stokes shifts were synthesized.The existence of photo-induced electron transfer(PET)process between the amino group and the fluorophore of probe led to the fluorescence quenching of probe.In the presence of FA,the PET process was inhibited and the fluorescence of probe recovered.Based on the different fluorescence signals before and after addition of FA,probes were used to specifically detect FA.In the detection on test strip,BT-1 showed different fluorescence colors to FA and other common gases.It was successfully used to distinguish FA from other common gases.In addition,based on the difference in the fluorescence signal of BT-1 emitted in different concentrations of FA solution,it could be used to detect different concentrations of FA solution.In live cell experiments,the fluorescence signal of BT-1 under confocal microscope was significantly enhanced after addition of FA,indicating that BT-1 could be successfully used for imaging of FA in living cells.In addition,BT-1 could image exogenous and endogenous FA in Arabidopsis.By introducing the succinimide group on the amino site of BT-1,a fluorescent probe BTSA with the properties of ESIPT and aggregation-induced emission(AIE)was designed.By observing the aggregation and dispersion state of BTSA in different p H solutions under scanning electron microscope,we found that the acidic solution is more conducive to the aggregation of BTSA.In spectral experiments,BTSA mainly emitted aggregated fluorescence signals under acidic solution.The hydroxyl group of BTSA was deprotonated by basic solution,therefore,BTSA showed deprotonated fluorescence signals under basic solution.Adjusting the protonation process of BTSA by acid and alkali to change its aggregation and dispersion state,BTSA was successfully used to ratiometrically and reversibly detected p H of different solutions in the pure water.In addition,due to the lower p H of cancer cells to normal cells,BTSA had been successfully used to distinguish cancer cells from normal cells.Probe(BTHA)was designed and synthesized by introducing the acryloyl group,the specific recognition group of the thiol amino acid,into the ortho position of the hydroxyl group on parent fluorophore.In the fluorescence titration experiment,the fluorescence emission peak of BTHA was at 510 nm.Upon addition of GSH,the fluorescence signal at 510 nm gradually disappeared,at the same time,the fluorescence signal at 460 nm was continuously enhanced.Moreover,there is a good linear relationship between the ratio of fluorescence intensity at two wavelengths and the concentrations of GSH.Therefore,BTHA was successfully used for ratio detection of GSH in vitro.In addition,it may be due to the difference in the hydrogen bonding between the hydroxyl groups and the carboxyl groups of the three thiol amino acids.After respective addition of GSH,Cys,and Hcy,BTHA exhibited different fluorescence signals.In cell experiments,BTHA emitted different fluorescent colors after the addition of GSH or Cys.In addition,BTHA could image endogenous biothiols.