The development of an expandable sensing platform that responds to high-energy phosphate bonds in phosphorus-containing biomolecules is crucial for both basic research and clinical diagnostics. Herein we develop a highly universal enzyme-immobilized sensing platform based on hierarchically mesoporous amine-functionalized UiO-66(Zr) (HMUiO-66-NH2). The platform utilizes a cascade response system where the immobilized dephosphorylating enzymes such as bovine intestinal alkaline phosphatase (ALP) hydrolyze high-energy phosphate compounds to generate inorganic phosphate (Pi). Competitive coordination of Pi and BDC-NH2 weakens the ligand-to-metal charge transfer (LMCT) effect in framework, which triggers fluorescence enhanced responses in the surrounding ALP@HMUiO-66-NH2. Mechanistic studies reveal that the much stronger coordination between degradation products of high-energy phosphate compounds and Zr-OH sites enables the the pronounced fluorescence response of ALP@HMUiO-66-NH2 within a certain reaction time. In contrast, organic phosphate species such as organophosphate pesticides, nerve agent simulants and phospholipids exhibit a much lower fluorescence response than high-energy phosphate compounds. Finally, the platform is integrated with apyrase, which exhibits enhanced specificity for selective detection of ATP and ADP in high energy phosphate compounds. Both ATP and ADP exhibited linear responses within the concentration range of 5-200 μM, with the limit of detection (LOD) of 0.23 μM and 0.24 μM, respectively. The successful integration of apyrase and the superior detection performance underscore the high versatility of this platform, suggesting its broad applicability in biochemical and diagnostic fields.

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