Fluorogenic response of thiamine triggered by Cu2+ for sensitive and economical determination of alkaline phosphatase

Although fluorescent probes have been used extensively for alkaline phosphatase (ALP) sensing, most involve a tedious, prolonged, or high-cost synthesis procedure. Thus, it is still necessary to develop convenient and economical fluorescent probes for sensitive determination of ALP. Herein, a facile fluorescent strategy was designed for ALP detection utilizing the fluorogenic response of thiamine (TH) triggered by Cu²⁺ (Cu²⁺-TH system). It was found that Cu²⁺ facilitated the oxidation of non-fluorescent TH to fluorescent thiochrome (TC) in an alkaline environment, while pyrophosphoric acid (PPi) interacted with Cu²⁺ to form PPi-Cu²⁺ chelates, preventing the oxidation of TH to fluorescent TC. Consequently, a faint fluorescence output was detected in the system. After adding ALP into this system, PPi was catalyzed to phosphate (Pi), which combined weakly with Cu²⁺. A large amount of TH was oxidized into fluorescent TC by free Cu²⁺, leading to high fluorescence. Therefore, an ALP sensing platform was constructed by utilizing the fluorogenic reaction of the Cu²⁺-TH system as the signal reporter. The assay exhibited favorable analytical performance for ALP measurement. A linear correlation was obtained between the ALP concentration (0.01–1 mU/mL) and the fluorescence intensity of the Cu²⁺-TH system, with a limit of detection as low as 0.0038 mU/mL. Additionally, the assay was further employed for analyzing ALP content in complex biological samples. This work thus advances the science of in situ fluorogenic reaction in the field of biosensing.

Graphical Abstract