Rational design of in situ localization solid-state fluorescence probe for bio-imaging of intracellular endogenous...

Fluorescence detection technology has been widely concerned for its advantages of low cost, simple operation, good sensitivity, real-time and non-destructive biological imaging. However, most fluorophores emit bright fluorescence in solution, and the fluorescence decreases significantly in the high concentration or solid/aggregated state, which is called aggregation-caused quenching (ACQ). Cysteine (Cys) is an important kind of amino-acid in the field of bio-medicine, whose main function is to participate in metabolism and protein synthesis, detoxification, but intracellular cysteine concentrations (30-200 μM) are much low, and direct detection of endogenous cysteine is hampered by interference with other thiols. To solve the above problems, based on solid-state fluorophore HPQ, we for the first time prepared a novel solid-state fluorescence probe MA-HPQ, for monitoring of endogenous Cys, operated by the mechanism of excited intramolecular proton transfer (ESIPT). MeO-HPQ is completely insoluble in water, has very strong solid-state fluorescence with the maximum emission wavelength of 510 nm and the maximum excitation wavelength of 365 nm. This special property makes it very suitable for confocal microscopy compared with ordinary water-soluble fluorescent dyes. Due to the large Stokes shift (145 nm), MA-HPQ has very desirable advantages: reduced interference of background fluorescence, increased sensitivity, and enhanced contrast of biological imaging. More importantly, by preventing it from establishing internal hydrogen bonds, which is between imine nitrogen and phenolic hydroxyl groups, it can be made insoluble in water and have strong fluorescence properties, and the process is reversible. The ESIPT process can be blocked by masking phenolic hydroxyl, which can inhibit fluorescence to a large extent. In the presence of Cys, the probe reacts, releasing free MeO-HPQ, and begins to form a precipitated solid. The precipitated solid emitted bright green solid-state fluorescence, which was enhanced 43 times more than MA-HPQ. These results indicate that the probe MA-HPQ can be suitable to real spatiotemporal imaging of endogenous cysteine in HeLa cells. The excellent performance of the probe makes it applying for the visualization detection of endogenous cysteine in living cells and tissues with obtaining satisfactory results.