A Prolonged Multimodal Lateral Flow Immunoassay Enabled by Synergistic Peroxidase–Heme Nanohybrids

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Horseradish peroxidase (HRP)-catalyzed lateral flow immunoassays (LFIAs) are powerful platforms for sensitive biosensing. However, the limited stability and short-lived catalytic activity of HRP significantly constrain their broader applications. Conventional HRP-based chemiluminescence (CL)-LFIAs typically emit for about 1 min before rapid decay, restricting signal capture. Herein, we report for the first time a prolonged CL/colorimetric LFIA enabled by β-cyclodextrin (β-CD)-functionalized polydopamine (PDA) nanospheres coloaded with HRP and heme, denoted β-CD-PDA/Heme/HRP (CPHH). This hybrid architecture integrates the complementary properties of both catalytic components: heme provides long-lasting but moderate CL, while HRP contributes strong yet transient luminescence. This synergistic interplay yields clearly enhanced and sustained signal output lasting up to 5 min on paper-based substrates, allowing sensitive and stable detection of cardiac troponin I (cTnI). When applied in an LFIA device, the CPHH probe can offer three readout modes, colorimetric (CM), enhanced colorimetric (ECM), and chemiluminescent (CL), covering a wide detection range of 0.1-500 ng/mL with limit of detection values of 10 ng/mL (CM), 0.47 ng/mL (ECM), and 0.95 ng/mL (CL). Further clinical research confirms its robustness and accuracy in real human samples. This work provides a new design paradigm for durable chemiluminescent nanoprobes and multifunctional LFIA systems for rapid and reliable cTnI detection.