Ratiometric Fluorescent Strategy for Localizing Alkaline Phosphatase Activity in Mitochondria Based on the ESIPT Process.

Fluorescent probes are powerful tools for detecting and mapping the species of interest in vitro and in vivo. Although the probes always show high selectivity and sensitivity, they are usually affected by some factors, such as detecting conditions and the probe concentrations. Ratiometric fluorescent strategies, possessing advantage of low background noise, would solve the problem effectively and lead to a higher sensing performance. Thus, an ESIPT-based ratiometric probe (HBTP-mito) was developed on the basis of a phosphorylated 2-(2'-hydroxyphenyl)-benzothiazole derivative for the determination of ALP activity. HBTP-mito is water soluble and emits green fluorescence in TBS buffer due to the blockage of ESIPT. Upon the introduction of ALP, the phosphate ester of HBTP-mito was hydrolyzed and the ESIPT process was restored. Accordingly, the fluorescence at 514 nm decreases, while emission at 650 nm shows a "turn-on" response. The ratio of intensity (I514nm/I650nm) decreases linearly with ALP activity increasing from 0 to 60 mU/mL, obtained an LOD of 0.072 mU/mL. The favorable performance of the probe enables its application not only in the detection of ALP activity in biological samples, but also in the localization of the ALP levels in living cells and in vivo.