Construction And Properties Of A Photoactivated Internal Standard Fluorescent Probe Based On The Synergistic Mechanism Of FRET And IFE

Fluorescence imaging technology is an important method to monitor living cells,biomolecules and life activities in vivo.It is highly sensitive,can be spatially resolved imaging,and has little light damage to living cells,less background interference.As a new imaging technology,it can not only be used for the detection of biothiols and ions,cancer diagnosis,but also can be used in pharmaceutical research and development,environmental monitoring,etc.Fluorescence imaging technology is a kind of low-light imaging technology,and its imaging quality depends on the photophysical properties of the dyes used in fluorescence dyeing.Fluorescent probes constructed by photoactivation technology have the advantages of high spatial and temporal resolution and good biocompatibility,they are widely used in the biomedical field.At present,the traditional photoactivated fluorescent probes are still subject to many environmental interference factors,and the test error is large.The development of this work improves the accurate detection of photoactivated fluorescent probes in biomedical imaging and avoids the influence of biological microenvironment.The specific research is as follows:In this paper,coumarin was used as a functional internal standard to design and synthesize a group of photoactivated fluorescence probes and study their properties.Based on the synergistic cancellation strategy of fluorescence resonance energy transfer(FRET)and internal filter effect(IFE),a novel single-molecule photoactivated fluorescence probe consisting of functional internal standard(FIS),fluorescence reporter(reporter)and controlled filter(filter)was constructed.The structures were characterized by hydrogen NMR spectroscopy,carbon spectroscopy and high-resolution mass spectrometry.The spectral results showed that the fluorescence of FIS(469 nm)was almost unchanged before and after irradiation under physiological conditions,while the fluorescence of semi-Rhodamine semi-fluorescein fluorophore(540 nm)was significantly enhanced by FRET at 400 nm.To confirm the synergistic counteracting mechanism of FRET and IFE effects,it was found that the fluorescence intensity of FIS remained stable before and after illumination by adjusting the intensity of FRET and IFE effects separately.In addition,confocal cell experiments showed that the photoluminescence platform exhibited stable internal standard signals in cells,which confirmed that the photoluminescence platform could eliminate the effects of biological microenvironment through intramolecular synergistic cancellation,and could be an effective tool to accurately monitor the fluorescence release of living cells in the field of photoluminescence.Based on the synthesis of coumarin derivatives,a synergistic mechanism of intra-molecular fluorescence resonance energy transfer and internal filtering effect was established,and a photoactivated internal standard coumarin fluorescence probe was synthesized successfully.This kind of probe has a new improvement in the precision detection,so that it has a broad prospect in medicine,analysis and other applications.