Construction And Application Of Fluorescent Dyes And Probes With A Large Stokes Shift

Due to the advantages of simple operation and good reproducibility,biomolecular labeling and molecular fluorescent probes based on organic fluorescent dyes have been widely used for real-time and non-destructive detection of biomolecules and tracking of biological processes.However,most fluorescent dyes?such as fluorescein,rhodamine,oxazine,and cyanine?have very small Stokes shifts?generally<30 nm?,resulting in severe overlap between the excitation and emission spectra,and low signal to noise ratio and severe fluorescence self-quenching,which limit its application in bioimaging.Compared with common dyes,the fluorescence excitation and emission spectra of fluorescent dyes with the large Stokes shift can be well separated,which can effectively eliminate the self-quenching of fluorescence caused by self-absorption of the dyes,and significantly improve the signal-to-noise ratio in bioimaging applications.Therefore,it is highly favored in the imaging applications of cells,tissues and in vivo.In this paper,to solve the problem of small Stokes shifts of the rhodamine-based fluorescent dyes and probes,a series of rhodamine-based fluorescent dyes were developed by rational design method,and based on this,probes with large Stokes shifts for mercury ion and peroxynitrite were synthesized.The main works are shown as follows:?1?We have combined the rhodamine and phenothiazine dyes to develop a series of novel fluorescent dyes with an"on-off"ring structure conjugated with oxonium and phenothiazine dyes.Compared with traditional rhodamine dyes,those dyes have a significantly red-shifted emission spectrum with absorption wavelengths greater than 570 nm and emission wavelengths greater than 730 nm,which all have large Stokes shifts?>130 nm?.Among them,the dye PZ-R6G has an excitation wavelength matched with a conventional laser confocal microscope?575 nm in acetonitrile solution?,a large Stokes shift?159 nm in acetonitrile solution?and a suitable fluorescence quantum yield?9.0%in acetonitrile solution?and is suitable for bioimaging applications.?2?The selected fluorescent dye PZ-R6G has similar structural characteristics to the rhodamine dye,and the near-infrared fluorescent probe with large Stokes shift can be designed by using the"off-on"design of the spiral ring.Thus we synthesized a near-infrared fluorescent probe PZS-R6G containing thio spironolactone by reacting PZ-R6G with Lawson's reagent.The probe fluorescence is significantly enhanced in the presence of mercury ions and other coexisting analytes can not affect their fluorescence properties,indicating that it has a good selectivity for mercury ions.The results of spectral experiments show that the fluorescence intensity of PZS-R6G has a good linear relationship with Hg²⁺from 1 to 20?M,and the detection limit is 0.21?M.The probe has also been successfully applied to imaging studies of mercury ions in cells and zebrafish.?3?Although the above dyes have solved the problem that the small Stokes shift of the rhodamine-based fluorescent dye,the fluorescence quantum yield is low,and it is not an ideal fluorescent dye for bioimaging applications.In response to the above problem,in this chapter,we have combined a fluorescein dye and a reduced diazepine structure and developed a new fluorescent dye DQF-560 with a large Stokes shift?110nm??an appropriate fluorescence quantum yield?0.31 in ethanol?,and an optically tunable hydroxyl group.Then we further constructed a peroxynitrite near-infrared fluorescent probe with large Stokes shift using DQF-560 dye.The probe showed a fluorescence enhancement after selective oxidation with peroxynitrite,and can also be used for imaging intracellular peroxynitrite.