HRP-induced wide wavelength-tunable in situ fluorogenic system and its immunoassay for cTnI in clinical samples

The development of wavelength-tunable fluorescence immunoassays for sensitive biomarker detection remains a significant challenge in clinical diagnostics. In this work, we present an HRP-induced in situ fluorogenic immunoassay platform based on the rapid reaction between p-phenylenediamine (PPD) analogues and 6-hydroxyquinoline (6-Hy). The proposed mechanism involves HRP-catalyzed generation of hydroxyl radicals from H₂O₂, which subsequently oxidize PPD to form fluorescent copolymer nanoparticles (FCNPs) with 6-Hy. By strategically modifying the PPD structure with various substituents, we achieved discrete and tunable emission of FCNPs, with maxima spanning from 380 to 600 nm (Δλ ≈ 220 nm). Using cardiac troponin I (cTnI) as a model antigen and zinc-coordination nanoparticles (ZnNPs) as carriers, the immunoassay demonstrated exceptional performance, with a wide dynamic range (0.5-125 ng/mL) and an ultralow detection limit (0.17 ng/mL) for cTnI. The platform was successfully validated in human serum samples, showing excellent correlation with clinical standards. This work not only establishes a novel strategy for wavelength-programmable immunoassays but also provides a versatile platform for advancing biomarker detection in clinical diagnostics.