Iron Sequestration–Modulated Dual‐Readout Sensing of Pyrophosphate and Alkaline Phosphatase via Fenton Oxidation and Fluorescence Recovery

A dual‐mode sensing platform integrating colorimetric and fluorometric detection has been developed for the simultaneous determination of pyrophosphate (PPi) and alkaline phosphatase (ALP) activity. Accurate monitoring of both analytes is clinically significant, as elevated PPi activity and aberrant ALP activity serve as early indicators of disrupted phosphate homeostasis and bone turnover across CKD stages. The platform exploits the Fenton reaction between Fe 2+ and H 2 O 2 to generate Fe 3+ and hydroxyl radicals (•OH); the latter oxidizes TMB to produce a colorimetric signal while Fe 3+ simultaneously quenches carbon dot fluorescence, establishing a coupled dual‐channel baseline. PPi sequesters both Fe 2+ and Fe 3+ , suppressing Fenton chemistry and producing concentration‐dependent attenuation of both signals, while ALP hydrolyzes PPi to release iron ions and restore both channels proportionally to enzyme activity. The colorimetric channel showed a linearity over 1.0–25.0 μM for PPi and over 3.0–18.0 U/L for ALP, while the fluorometric channel demonstrated linear responses across 0.05–7.0 μM for PPi and 0.5–12.0 U/L for ALP, with detection limits of 210 and 14 nM for PPi and 0.91 and 0.15 U/L for ALP in the colorimetric and fluorometric modes, respectively. Validation in spiked serum yielded recoveries of 96.0%–100.9% with RSDs of 1.96%–3.85%, indicating satisfactory accuracy and negligible interference.