Ce⁴⁺-triggered cascade reaction for ratiometric fluorescence detection of alendronate

A ratiometric fluorescence assay for alendronate (ALDS) has been designed with Ce⁴⁺-triggered cascade chromogenic reaction. This strategy involves three processes: (1) Ce⁴⁺ oxidizes ascorbic acid (AA) into dehydroascorbic acid (DHAA), which then condenses with o-phenlenediamine (OPD) to generate fluorescent 3-(dihydroxyethyl)furo[3,4-b] quinoxaline-1-one (DFQ), presenting the maximum emission at 434 nm; (2) As oxidase-mimics, Ce⁴⁺ can oxidize OPD into fluorescent 2,3-diaminophenazine (DAP) which shows a strong emission at 568 nm; (3) ALDS inhibits the oxidation ability of Ce⁴⁺ towards OPD, thus inhibiting the generation of DAP. Accordingly, a homogeneous ratiometric fluorescence system with dual emission comes into being and the presence of ALDS can change the fluorescence intensity ratio obviously. With F₄₃₄/F₅₆₈ as readout, ALDS can be detected sensitively with the detection limit of 30 nM. Moreover, this ratiometric method was used to analyze ALDS in both human serum and pharmaceutical samples.