Design,Synthesis And Application In Cell Of Lysosomal PH Probe

Hydrion plays a significant role in cell proliferation,apoptosis,enzymatic activity and tumor growth.As an important indication of cellular health,minor variations of intracellular pH may cause cellular dysfunction and serious diseases,such as colorectal cancer,breast cancer and cystic fibrosis.Lysosome with acidic environment(pH 4.0-6.0)is identified as the first defence line of cells.Many diseases,such as cancer,stroke and Alzheimer,could be caused when the pH in lysosome changes.Therefore,quantitative detection of pH in biological environments makes good sense in cellular analysis or diagnosis.Many detection methods have been used to measure pH value.Compared with those,fluorescent probe has many advantages including high selectivity and sensitivity,good spatiotemporal resolution,convenient operation and possibility of real time monitoring.Most reported fluorescent pH probes depended on the measurement of fluorescence intensity changes in single-emission windows.However,the signal output tends to be affected by many exterior factors,such as temperature,solvent polarity,probe concentration and instrument sensitivity.A ratiometric probe with two emission peaks can resolve above issues via the self-calibration of two emission bands.Therefore,more precise detection of pH could be obtained by means of ratiometric probes even in complicated biological systems.Constructing an FRET system is a wonderful way to fabricate a ratiometric fluorescent probe.Because FRET-based ratiometric probes with large Stoke shifts can reduce fluorescence detection error and self-quenching.To date,the reported ratiometric pH probes based on FRET are still rare compared with the demand.We designed pH fluorescent probes by use of imidazo[1,5-a]pyridine fluorophore as a fluorescence resonance energy transfer(FRET)donor and rhodamine B fluorophore as a FRET acceptor.We have further explored the optical properties and dedicated to the applied research of them in the water samples and cells.The concrete content includes the two parts.1.Design,synthesis,optical properties and application research of pH probe basedon FRETIn this study,we designed and synthesized a ratiometric pH probe Rh MP with high selectivity based on FRET.The probe was fabricated by integrating the imidazo [1,5-a]pyridine moiety as the FRET donor and the rhodamine moiety as the FRET acceptor.Rhodamine moiety as an FRET acceptor has been widely used because of its excellent biocompatibility and photophysical properties.What's more,the rhodamine moiety is non-fluorescent at basic and neutral pH condition due to its spirocyclic structure.In acidic solution,the spirocyclic form of the rhodamine moiety transformed into the ring-opening form,resulting in strong fluorescence.On the other hand,we had synthesized a imidazo [1,5-a]pyridine-based pH fluorescent probe with fast response,high selectivity and sensitivity.However,this probe was a single-emission probe.And so far,imidazo[1,5-a]-pyridine fluorophore is not used as an FRET donor.Thus,we have been focusing on the design and synthesis of ratiometric fluorescent probe based on FRET applying imidazo[1,5-a]pyridine.Through our research,we know the emission spectrums of imidazo[1,5-a]pyridine and rhodamine are well separated.The probe RhMP has been successfully applied in fluorescence imaging in HeLa cells and could selectively stain lysosome.2.Design,synthesis,optical properties and application research of water-soluble pHprobe based on FRETIn this work,we focused on the design and synthesis of a water-soluble ratiometric fluorescent probe based on FRET platform involving imidazo[1,5-a]pyridine and Rhodamine.To avoid the protonation of 2-nitrogen in imidazo[1,5-a]pyridine in acidic condition and increase water-soluble of the probe,we changed imidazo[1,5-a]pyridine into imidazo-[1,5-a]pyridine quaternary ammonium salt which is also wonderful fluorophore,with good water-solubility and broad emission band.The broad emission band makes it possible to increase the spectral overlap with the absorption spectrum of the rhodamine acceptor.The spectral overlap is beneficial to FRET mechanism.The probe RMPM has been successfully applied in fluorescence imaging in HeLa cells and could selectively stain lysosome.