Spatially differential gold nanocluster amplification of LSPR for ultrasensitive, label-free optical immunoassays

The global impact of COVID-19 highlights the critical need for technologies enabling pre-symptomatic pathogen detection to curb pandemics. Addressing this need requires diagnostic sensors with significantly enhanced sensitivity for reliable low-abundance target detection. Here, we report a planar synergistic Cluster-Pore biosensing microchip (CP-Chip) that couples gold nanoclusters (AuNCs) fluorescence with gold nanopore localized surface plasmon resonance (LSPR). We demonstrate that this plasmonic coupling critically depends on the spatial distribution of AuNCs relative to the nanopores. Exploiting spatially differential AuNCs amplification, we designed the CP-Chip so specific target binding modulates AuNCs distribution. This label-free, wash-free approach amplifies optical signals under target-positive conditions while suppressing background in target-negative states, enhancing differential output. The CP-Chip achieves ultrasensitive SARS-CoV-2 nucleocapsid (N) protein detection with a limit of detection of 150 fg/mL—marking a sensitivity gain exceeding three orders of magnitude over previous LSPR methods and proving suitable for low-abundance analysis. Integration with a mobile health platform enables rapid on-site testing. Validated via simulation and targeted antigen-antibody experiments, the CP-Chip emerges as a promising tool for early-stage infectious disease screening.