Construction Of Reaction-based Fluorescent Probes For Reactive Oxygen Species Of Water Samples And Living Organisms

Reactive oxygen species?ROS?are generally produced from oxidative metabolism of cellular.On the one hand,ROS are believed to play influential roles in various biological functions.On the other hand,overproduced ROS will cause oxidative stress and subsequently lead to many diseases.It is,therefore,considerable to develop techniques in the detection of ROSs with good selectivity and sensitivity.From many detecting methods,fluorescent methodology is an outstanding method for the simple operation,high sensitivity,low cost and high spatial resolution.In this paper,four fluorescent probes were designed and synthesized to detect ROS in environmental water and living cells.All the structures were confirmed by nuclear magnetic resonance and mass spectrometry.Absorbance spectra and fluorescence emission spectra were determined to study the performance of these fluorescent probes.The main studies are as follows:?1?A novel fluorescent probe,which aimed at qualitatively and quantitatively detecting ClO^- ,was synthesized by introducing 2,4-dinitrophenylhydrazine into the BODIPY fluorophore.Probe 1 was almost no fluorescent due to the C=N isomerization.The green fluorescence was restored after the oxidation of C=N bond by ClO^- ,with a colour change from pink to orange.The specific reaction between Probe 1 and ClO^- gave Probe 1 ability to identify ClO^- from many other ROSs.With the higher concentration of ClO^- ,the fluorescence intensity gradually increased.The fluorescence intensity shows an excellent linearity with ClO^- in the range of 0-340 ?M with a detection limit of 228 nM.Based on the changes of fluorescence intensity and colour,we further applied Probe 1 in test strips for the visual detection of ClO^- .In addition,2,4-dinitrophenylhydrazine was replaced by diaminomaleonitrile to prepare Probe 2,which was more effective to monitor ClO^- .Upon the ClO^- added into Probe solution,a green fluorescence appeared dramatically.The response time is 10 s and the detection limit is as low as 19.8 nM.Probe 2 showed high sensitivity to ClO^- over other ROSs.Further studies of test strips based on Probe 2 were used to monitor ClO visually.Importantly,Probe 2 was successfully employed for quantitative measurement of ClO^- in tap water,Yangtze River and sodium hypochlorite disinfectant.?2?A coumarin derivative?Probe 3?was designed and synthesized based on the specific reaction between hypochlorite and oxime group,which was applied to detect ClO^- in environmental water and biological samples.Probe 3 was found to be non-fluorescent,but displayed strong blue-fluorescence after the reaction promoted by ClO^- .The fluorescence intensity was found to reach a plateau in 20 s and was hardly disturbed by the other ions,showing the high selectivity.On account of the good linear relationship between emission intensity and ClO^- concentration,the detection limit of Probe 3 was found to be 8.3 nM,making the test strips of Probe 3 sensitive enough to monitor ClO^- of the tap water.Standard addition method with a recovery rate of 98.3%-118.9% further demonstrated that Probe 1 was applicable for quantitative detection of ClO^- in tap water,Yangtze River and sodium hypochlorite disinfectant.In addition,Probe 3 had good water-solubility and biocompatibility and had practical application in imaging exogenous ClO^- of macrophage RAW264.7.?3?By introducing boronate esters as a recognition unit,we developed a fluorescent probe?Probe 4?for the qualitative and quantitative determination of hydrogen peroxide.Absorbance spectra and fluorescence emission spectra of Probe 4 and the reaction product were studied.It was found that maximum absorbance peak changed from 321 nm to 372 nm and an emission peak centered at 451 nm appeared.The fluorescence intensity at 451 nm enhanced with the linear increase of the H₂O₂ concentration?0-180 ?M?and the detection limit was calculated to be 118.2 nM.Even in the presence of various ions,the fluorescence intensity remained relatively unchanged.Moreover,Probe 4 was further used to image macrophage RAW264.7 and colon cancer cell SW480 because it worked excellently under leaning alkalinity condition,which indicated the good cell-membrane permeability of Probe 4 and its potential ability to detect the exogenous H₂O₂ in living organisms.