Development Of Novel Fluorescent Probes For Hypochlorite And Their Application In Environmental Analysis

As an endogenous fungicide,hypochlorous acid/hypochlorite(HClO/ClO~-)is widely used in disinfection and cleaning products because of its strong oxidizing properties.HClO/ClO~-would accumulate in vivo through material cycle and metabolism of ecosystem.An appropriate amount of HClO/ClO~-has a positive effect on tissue repair and disease resistance.But when the concentration is abnormal,it will be accompanied by some diseases.The advent of fluorescent probe technology has made it possible to detect HClO/ClO~-easily and rapidly.For practical application,the residual background fluorescence signal from some present“ON-OFF/OFF-ON”probes tend to decrease the detection sensitivity,and even cause false positive results.To get an accurate and reliable detection,the fluorescent probe for ClO~-with extremely low background fluorescence is highly desirable.In addition,the built-in self-calibration function of the ratio fluorescent probes with two emission bands have a good application prospect.Because they can effectively reduce the interference of the probe concentration and distribution,environmental polarity,pH and photobleaching,making the detection results more precise.In this paper,we chose coumarin as a fluorescent platform and constructed two fluorescent probes(LH-1 and LH-3),which were used to detect hypochlorite in aqueous solution,cells and zebrafish.(1)Coumarin 343 and coumarin 334 were synthesized from m-aminophenol.The sensitive probe LH-1 with very low background fluorescence was obtained by binding 2,4-dinitrophenylhydrazine with coumarin 343.The excitation ratio response probe LH-3 was prepared by dehydration condensation of coumarin 334 and2-mercaptoethanol.The molecular structures were determined by nuclear magnetic resonance and mass spectrometry methods.(2)LH-1 was successfully used for the detection of ClO~-in actual environmental water samples and organisms.We rationally developed a novel fluorescent probe for hypochlorite with extremely low background fluorescence by introducing two types of fluorescence quenching groups(the free rotation of N-N single bond in acetohydrazide group and 2,4-dinitrobenzene group).Notably,due to the combined effect of two fluorescence quenching groups,the probe could keep low background fluorescence even in a solution with high viscosity.The optical experiments indicated that probe LH-1 displayed dramatical fluorescence enhancement response to ClO~-,and the fluorescence intensity at 488 nm was enhanced 739 folds within 20 minutes after adding 40μM ClO~-.At the same time the probe had high sensitivity and selectivity,and the detection limits were determined to be 10.5 nM.Additionally,there was a good linear relationship between fluorescence enhancement and the concentration of ClO~-(0~20μM).The practical application demonstrated that the probe LH-1 was able to quantitatively detect ClO~-in various water samples with good recovery(>93%).Moreover,the probe also showed low extremely background fluorescence and was capable of detecting minor variation of ClO~-in living cells.Significantly,combined with biological imaging technology,the probe was successfully applied to the imaging of different concentrations of hypochlorite in zebrafish.(3)The probe LH-3 with coumarin 334 as the fluorophore and monothioketal as the recognition group has also been successfully applied to the detection of ClO~-in aqueous solutions and living cells.The probe exhibited a red shift of 91 nm,a typical ratio emission mode,after incubation with ClO~-in solution.The reaction between the probe and ClO~-in the aqueous solution could be completed within 10 minutes,which is very advantageous for the detection in vivo.In fluorescent imaging experiments,the probe was also applied to image ClO~-in living cells,which shows its potential application value in the field of biology.