Design And Synthesis Novel Fluorescence Ratio Probes For The Detection Of Intracellular Active Small Molecules

Fluorescence analysis has obvious advantages: good selectivity, high sensitivity,easy to operate. In field of biology, chemistry and medicine, we can find that moreand more fluorescent probes have been used to detect the small active molecules incells. Fluorescence analysis is indispensable in the analytical methods. Sometimeusing a single emission peak increase or decrease as a signal to detect small activemolecules is inaccurate, which is easy to affect by environment. In contrast,ratiometric fluorescent probes use ratio of emission intensities at two differentwavelengths as singles, which can overcome the drawbacks of probe concentration、light intensity、sensitivity of the instrument、environmental effects and increase theselectivity， sensitivity. Ratiometric fluorescent probes with a same exitationwavelength can further reduce the error.Generation two emission wavelengths for fluorescent ratio probes are usuallyrequired. There are two methods for generating two emission wavelengths: doublechromophores and a single chromophore. Dual chromophores refers to doublechromophores and each of them emits their corresponding fluorescence wavelengths,single chromophore means the chromophore connects with analyte before and after,the fluorescence emission wavelength can blue shift or red shift. Currently, based onthe the single chromophore fluorescence ratio probes had been reported a lot, whilefluorescence ratio probes use dual chromophores research little. Fluorescence ratioprobes with an excitation wavelength and different emission wavelengths are morerarely.For fluorescence ratio probes, the linker is very important. Different linker has adifferent influence on the properties of probes. Therefor, linker is one of the mostimportant parts of the probe. So we must select a suitable linker when we design andsynthesize probes. On the basis of the research status of ratiometric fluorescent probes and theimportant of linker, this paper carried out the work of the following two aspects:First, linkers which affect the optical properties of the probe have an importantrole for dual chromophores fluorescence ratio probes. In this paper,6-methoxyquinoline was used as a chloride ion-sensitive group while Dansylamide as achloride ion-insensitive group. Then we connected the two fluorophores with threedifferent linkers respectively, as three methylenes, N-methylene-piperazine,phenylmethyl bromide. So we designed and synthesised three kinds of fluorescenceratio probes which have a same excitation wavelength and different emissionwavelengths for the detection of chloride ion. We find that the probe connected bythree methylenes almost has no response to chloride ion. While the probe whichconnected by N-methylene-piperazine can respond to chloride ion. But thefluorescence intensity of quinoline part and dansylamide part simultaneously changeswhen the chloride ion concentration changed. The probe using phenylmethyl bromideas linker shows a better selectivity and high sensitivity to chloride ion and instantlyresponds to different concentrations of chloride ion. The fluorescence intensity ofquinoline part is reduced linearly with the increased concentration of chloride ionwhile the fluorescence intensity of dansylamide part is constant. At the same time, theratio of the fluorescence intensity of dansylamide and quinoline showed a good linearrelationship with the concentration of chlorine ions. The fluorescence ratio probeprovides a potential method for the detection of changes in the chloride ionconcentration within the cells. Our experimental results show that linkers indeedaffect the optical properties of the probe, which provides a guiding direction forchoosing a good linker when designing fluorescence ratio probes.Second, we designed and synthesised a new probe Cyb-Rhodamine-Tpy with asame excitation wavelength and different emission wavelengths to detect ferrous ionsin living cells. There are two distinctly different emission wavelengths whenCyb-Rhodamine was excited by a same excitation wavelength, which rarely influorescence ratio probes. The intensity of Rhodamine-Tpy group of probe is changedby addition of Fe²⁺, while the Dye6group is unchanged.