Ratiometric differential sensing of biophosphates using a ligand-diversified Fe-MOF nanozyme array

Discriminating structurally similar biophosphates remains challenging for conventional single-channel sensing systems. Here, we develop a ratiometric differential sensing strategy based on a three-channel array of ligand-diversified Fe-MOF nanozymes, Fe-IPA-NH₂, Fe-TPA-NH₂, and Fe-TPA-OH. Each Fe-MOF combines intrinsic fluorescence with peroxidase-like activity and catalyzes the H₂O₂-mediated oxidation of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Phosphate-dependent modulation of the catalytic process produces coupled changes in the DAP emission and the intrinsic Fe-MOF emission. These outputs were converted into an internally referenced ratio (R = F₅₅₀/F_MOF) and then normalized to the phosphate-free response (R/R₀), generating self-referenced cross-reactive fingerprints. Chemometric analysis enabled discrimination of PPi, Pi, ATP, ADP, and AMP at 5 µM and maintained distinct clustering at 3, 1, and 0.5 µM. The array also differentiated PPi concentration levels and resolved binary PPi/Pi mixtures with varying compositions. By integrating ligand-diversified cross-reactivity, ratiometric normalization, and multivariate decoding, this work provides a proof-of-concept strategy for differential sensing of structurally related analytes.