A Self-Unlocking Biocatalytic Cascade Enabled by a Laccase-Entrapping Metal–Organic Framework for Point-of-Care Detection of Alkaline Phosphatase

Abstract The point-of-care testing (POCT) for alkaline phosphatase (ALP), a critical biomarker for hepatobiliary diseases, bone disorders, and specific malignancies, remains analytically challenging due to signal interference from complex biological matrices. Herein, we report the development of a laccase-entrapping zeolitic imidazolate framework-8 nanoprobe (LAC@ZIF-8) that enables a self-unlocking biocatalytic cascade (SUBC) for the selective colorimetric detection of ALP. The SUBC mechanism proceeds via three discrete steps: (1) ALP-catalyzed hydrolysis of disodium phenyl phosphate yields phenol and inorganic phosphate (Pi); (2) Pi induces the structural collapse of the ZIF-8 scaffold via competitive coordination, thereby, releasing the encapsulated LAC which is otherwise sterically locked by intact ZIF-8 with a narrow microporous aperture (ca. 3.4 Å) and inaccessible to consequent bioreaction; (3) the cogenerated phenol is subsequently oxidized by the released LAC in the presence of 4-aminoantipyrine to generate a quantifiable red quinoneimine chromophore. By requiring the concerted presence of both Pi and phenol, the dual products of ALP catalysis, this LAC@ZIF-8-mediated SUBC design effectively suppresses false-positive signals originating from endogenous phosphate-containing compounds or phenolic interferents. This biosensor exhibits exceptional specificity for ALP activity, demonstrating negligible cross-reactivity with common ionic species (K+, Na+, Ca2+, CO32–), glucose, amino acids, proteins, phosphate-containing compounds, and endogenous phenols. The assay achieves a linear dynamic range of 0.5–50 U/L with a detection limit of 0.43 U/L. Furthermore, a smartphone-coupled POCT hydrogel kit was developed, facilitating on-site, instrument-free visual detection of ALP activity and its inhibitor Na3VO4. This work presents a generalized strategy for the design of highly specific biosensors through the integration of metal–organic framework (MOF)-gated selectivity with biocatalytic signal amplification.