Sepsis is a critical condition associated with substantial mortality, thus driving a pressing demand for rapid diagnostic approaches ranging from simple, readily deployable point-of-care testing (POCT) to highly sensitive laboratory-based detection methods. Heparin-binding protein (HBP), a neutrophil-derived protein, rises earlier and correlates more closely with disease severity than conventional biomarkers including procalcitonin and C-reactive protein, making it a promising biomarker for timely sepsis detection. To simultaneously meet these needs, we established a dual-mode biosensor that combines electrochemiluminescence (ECL) and multicolor for highly sensitive analysis of HBP. The sensing platform is constructed from an aluminum-copper layered double hydroxide (AlCu-LDH) scaffold conjugated with an HBP-specific aptamer and complementary DNA immobilized on magnetic beads (MBs). In the absence of HBP, the assembled AlCu-LDH-MBs probe suppresses ECL emission through ferrocene-mediated quenching while catalyzing efficient TMB oxidation to produce a strong multicolor response. Upon HBP binding, conformational switching induces probe disassembly, releasing AlCu-LDH and ferrocene from the duplex, thereby restoring ECL output while simultaneously attenuating the multicolor signal. Under optimized conditions, the biosensor achieved an ultralow detection limit of 0.023 pM in ECL mode with a linear range of 0.10 pM–10 nM, whereas the multicolor mode showed a detection limit of 0.43 nM and exhibited linearity across 0.50–10 nM, accompanied by an intuitive multicolor transition from gray to purple. Notably, this strategy avoids tedious electrode surface modification and relies on magnetic separation to regulate signal generation, thereby simplifying operation while offering a sensitive and practical tool for early sepsis diagnosis that bridges laboratory-grade analysis with instrument-free POCT.
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