Cuvette-Integrated Dual-Layer Gold Nanoparticle LSPR Chip with Enhanced Optical Response for Ultrasensitive Detection of SARS-CoV-2 Nucleocapsid Protein

초록

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for rapid, sensitive, and field-deployable diagnostic platforms capable of detecting viral antigens prior to clinical intervention. Although localized surface plasmon resonance (LSPR) biosensors based on gold nanoparticles (AuNPs) offer label-free detection and simple optical readout, their sensitivity remains limited for detecting low-abundance viral proteins at early stages of infection. This study aimed to enhance the detection sensitivity of cuvette-based LSPR biosensors by introducing a dual-layer AuNP architecture that leverages the cumulative optical response of two plasmonically active layers for the ultrasensitive detection of SARS-CoV-2 nucleocapsid protein (NP). A cuvette-integrated dual-layer AuNP LSPR sensor chip was fabricated by vertically assembling two plasmonically active AuNP-coated substrates with a fixed separation distance. Antibody–antigen interactions were monitored through LSPR plasmonic peak shifts using absorbance-based spectrophotometric measurements. Sensor performance was evaluated in phosphate buffer and artificial human saliva, and analytical metrics including limit of detection (LOD), selectivity, coefficient of variation (CV), and recovery were system-atically assessed. The dual-layer AuNP LSPR sensor exhibited a 2.5-fold improvement in detection sensitivity compared with a conventional single-layer configuration, achieving an LOD of 9.7 pM (0.78 ng·mL⁻¹) for SARS-CoV-2 NP with excellent linearity (R² > 0.99, n = 5). The sensor demonstrated high selectivity against non-target viral proteins and com-mon interfering biomolecules. In artificial human saliva, the assay showed excellent analytical precision and accuracy, with CV values of 5.07–7.29% and recovery rates of 100.8–116.2% (n = 5). The enhanced performance of the dual-layer sensor demonstrates that the use of a dual-layer plasmonic architecture provides an effective design strategy for over-coming intrinsic sensitivity limitations of conventional cuvette-based LSPR platforms. Owing to its simple fabrication, label-free operation, and robust analytical performance in complex matrices, the developed dual-layer AuNP LSPR chip represents a promising platform for next-generation point-of-care diagnostics for respiratory viral infections.

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