ON-OFF ¹O₂ photoelectrochemical immunosensing strategy based on enzymatic conversion: interleukin-6 detection as...

Singlet oxygen (¹O₂)-mediated photoelectrochemistry (¹O₂-PEC) biosensing has emerged as a highly sensitive and selective platform for biomolecule detection, benefiting from low background signals due to the light ON-OFF switching mode. However, the compatibility of this technology is limited to the modification of affinity molecules with an adequate Type II photosensitizer, hindering widespread applicability. Here, we introduce a novel ON-OFF ¹O₂-PEC sensing strategy based on the use of alkaline phosphatase (ALP) as enzymatic label for signal generation. In this approach, ALP catalyzes the dephosphorylation of an inactive photosensitizer precursor (OFF), fluorescein diphosphate (FDP), turned ON under enzymatic activation. This ON-OFF strategy enables flexible assay design and straightforward integration with standard immunoassay workflows. As a proof-of-concept, we demonstrate the detection of interleukin-6 (IL-6) as clinically relevant inflammatory biomarker, showing excellent performance with a limit of detection of 85.4 pg/mL and a linear range of 100 - 500 pg/mL and adequate performance in diluted human serum samples. The developed strategy establishes ALP as a general and adaptable label for PEC immunoassays, offering a simple route toward compact and sensitive point-of-care diagnostic platforms.