Imidazole-enhanced luminol/H₂O₂ chemiluminescent biosensing for SARS-CoV-2 nucleocapsid protein with enzymatic regulation of hemin switch

Owing to the continuous mutations of the coronavirus, the sensitive and accurate detection of the SARS-CoV-2 nucleocapsid protein is still necessary in clinical diagnosis. Aim to the challenge of sensitivity in chemiluminescence (CL) imaging detection and the need for high-throughput clinical screening, this work used imidazole (Im) as an enhancer for hemin-DNA/luminol/H₂O₂ CL system to propose a CL imaging method for SARS-CoV-2 nucleocapsid protein detection. In this system Im could reduce the hemin-DNA catalyzed oxidation energy of luminol by H₂O₂ to promote the CL emission through the accelerated breaking of O-O bond of dianionic cyclic peroxide. The enhancement mechanism was demonstrated with theoretical calculations and several characterization techniques. By combining the proximity-induced initiator release with immunological recognition, an initiator-triggered DNA nanomachine was designed to achieve the release of abundant primers for activating hemin-DNA switch. This signal amplification strategy led to strong CL emission for highly sensitive imaging detection of the target. The Im-enhanced hemin-DNA/luminol/H₂O₂ CL imaging assay exhibited a linear detection range over 4 orders of magnitude with a detection limit down to 2.75 pg/mL for SARS-CoV-2 nucleocapsid protein, exhibiting promising potential of both Im sensitization and the proposed DNA nanomachine to improve the sensitivity of high-throughput CL biosensing for the early diagnosis and clinical screening of the coronavirus and other diseases.