Preparation And Performance Study Of PH Ratiometric Fluorescent Nanosensor Based On Fluorescein And Rhodamine B

Intracellular pH plays a pivotal role in various physiological processes,such as cell proliferation,apoptosis,ion transport,endocytosis and multi-drug resistance.Dysregulated cell pH is often associated with abnormal cell function,proliferation and growth.It is conducive to the discovery of new therapeutic targets and design pH-related disease treatment strategy that the maladjustme of pH in cancer cells and their microenvironment in promoting the rapid development of cancer cell survival,proliferation,invasion,metastasis,drug resistance and apoptosis were studied.Monitoring the pH value in tumor cells can guide the design of targeted drugs and enable loaded small molecule drugs to be released in the tumor under the stimulation of acidic pH environment.Beccause of their good temporal and spatial resolution.,fluorescent nanosensors are widely used as effective tools in the measurement of intracellular pH value.Compared with single fluorescent emission sensor,sensors prepared by ratiometric fluorescent method can effectively eliminate the interference caused by excitation intensity,fluorophore concentration and light scattering wave,then improve the reliability of analysis results.However,the ratio of the two fluorescent component on conventional ratiometric fluorescent nanosensors cannot be fixed precisely,the calibration curves of fluorescent sensors between different batches need to be measured respectively.It takes a lot of time and energy to calibrate the standard curves of nanosensors between different batches frequently,which greatly reduces the detection efficiency.Therefore,the measurement of intracellular metabolite ion concentration would be limited when using nanosensor as a systematic and long-term research method.To solve the above problems,we designed and prepared a new pH ratiometric fluorescent nanosensor with fixed fluorophor ratio.The effective measurement range of this sensor for pH value is 5-7.6.Different batches of sensors can share the same standard curve to avoid repeated calibration,thus greatly improving the research efficiency.In this study,the amino fluorescein and rhodamine B-ethylenediamine molecules were synthesized respectively,and the two molecules were combined in a 1:1 ratio to prepare a dual-emission composite fluorescence molecule.The structures of the synthesized fluorescent molecules were confirmed by MS,NMR and FT-IR.The optical properties of the synthesized molecules were measured by UV-vis and fluorescence spectra.The dual fluorescent molecules were coupled to polyacrylamide nanoparticles,and the pH ratiometric fluorescent nanosensors with fixed fluorophore ratios were prepared.The influences of different fluorescence molecular concentrations on the performance of different batches of nanosensors were investigated,and the standard curves of different batches of fluorescent nanosensors to determine the pH value of buffer solution were drawn.The results show that the consistency of standard curves of different batches of sensors can be achieved by preparing nanosensors with fixed fluorophore ratio.rigin software was used for logistic fitting of the curves,and the fitting equation of the relationship between fluorescence intensity ratio R and pH was obtained,with the fitting degree reaching 0.9994.he experimental results show that the new ratiometric fluorescence pH nanosensor prepared in this study had good biocompatibility and can be used to accurately measure the pH value in HeLa cells,and the performance of different sensors were stable.