Design Of Hydrogen-bond Tuning Fluorescent Probe For PH And Its Application In Daphnia Magna Imaging

pH is an important indicator of water environmental monitoring.Compared with traditional pH measurement methods,pH fluorescent molecular probes have excellent sensitivity and specificity,and can highly selectively identify the required pH range.Rhodamine-based substances have the advantages of long emission wavelength,high fluorescence quantum yield,high extinction coefficient,etc.,especially their unique spiro switch characteristics,which make them the first choice for the design of fluorescent probes.There are many researches on rhodamine-based fluorescent probes,but there are few fluorescent probes for pH detection of microbial lipid membranes.In view of the important role of proton channels on lipid membranes in microorganisms and environmental toxicology,this paper designs and two types of fluorescent probes were synthesized,and the corresponding properties were tested.The first part uses rhodamine B as the parent to synthesize rhodamine B acid chloride,then react with 2-amino-1,3-propanediol to generate intermediates,and then react with decanoyl chloride to obtain RK-1 and RK-2 fluorescence Probe.According to the test results of the RK-1 and RK-2 fluorescent probes,the RK-1 fluorescent probe exhibits a more sensitive spectral response in the range of pH 3.0-6.0,and its p Ka is3.51(±0.02)(Intensity)and 3.19(±0.07)(Abs);RK-2 exhibits a more sensitive spectral response in the range of pH 3.5-7.0,and its p Ka is 4.68(±0.05)(Intensity)and 4.26(±0.001)(Abs).It is verified by Gaussian fitting that RK-2 has a wider pH response range under the action of intramolecular hydrogen bonds.In addition,RK-1 and RK-2are not interfered by metal ions and show good acid-base reversibility;the solvent effect test further proves that the spectra of RK-1 and RK-2 fluorescent probes will not be affected.Other solvents and polarity interference;the large flea staining experiment shows that the amphiphilic chain in the RK-1 structure can perform fluorescence imaging of the pH of the large flea surface,which will provide a visual tool for monitoring the pH fluctuation of the large flea in the environmental water.RK-2 can perform fluorescence imaging of the digestive tract of large fleas,which will provide a visualization tool for the pH changes of the lower digestive tract stimulated by pollutants.In the second part,pyrrole was used as the parent to react with thiophosgene and then Mc Murry was coupled to synthesize the TPE probe,and a series of tests were performed to determine the properties of the TPE probe;TPE probes with different water contents were set The solution was screened to have strong fluorescence conditions,which indicates that the substance has AIE properties,which may enable the probe to accumulate on the surface of the cell membrane to indicate changes in pH.The TPE probe has been tested for pH spectrum response,metal ion interference test,ATP balance time test,and phosphate interference test.The results show that the probe is sensitive to changes in pH within the range of pH 2.0-12.0;and is not subject to metal Interference of ions and phosphate;can produce fluorescence response to ATP,fluorescence equilibrium time is 15 min.In addition to indicating changes in pH,the probe can also perform fluorescence detection of changes in ATP.