ABSTRACT Respiratory syncytial virus (RSV) is one of the most common causes to pediatric death globally. Rapid RSV diagnostics is important for judicial use of antibiotics, to reduce disease spread in healthcare facilities, and to enable prompt treatment since several RSV antiviral therapies are on the horizon. Current diagnostic methods rely on time-consuming laboratory-based tests including virus culture and polymerase chain reaction (PCR), and rapid diagnostic tests (e.g., lateral flow immunoassay, LFA) are not sufficiently sensitive as standalone diagnosis. Therefore, there is an unmet need for rapid and ultrasensitive diagnostic tests for RSV. The plasmonic coupling assay is a rapid colorimetric diagnostic test that makes use of the optical response of gold nanoparticles (AuNPs) during the process of target recognition to analyze its concentration. Despite its easy operation, the sensitivity of the plasmonic coupling assay is limited. In this proposed work, we aim to substantially improve the limit of detection (LOD) of the plasmonic coupling assay by innovative digital nanobubble detection. Specifically, we propose to directly detect intact RSV particles with antibody-conjugated AuNPs that recognize the RSV surface fusion (F) protein. AuNPs bind to multiple RSV F proteins and lead to plasmonic coupling. Ultrashort laser pulse selectively activates coupled AuNPs due to their enhanced absorption compared with a single AuNP. This greater optical absorption leads to nanoscale cavitation bubbles, i.e. nanobubbles, which can be measured easily from the bubble-induced and transient scattering. Single nanobubble generation leads to a sensitive digital detection with “on” and “off” signals. Our preliminary results suggest ~3 orders of magnitude improvement of LOD in detecting RSV. In this proposed work, we will firstly innovate the AuNP probe by optimizing the AuNP formulation (size, concentration and conjugation), investigating the non-spherical nanoparticle for more efficient virus binding, and exploring the asymmetric antibody-coated Janus nanoparticles for controlled binding. Next, we will design and build a prototype device for automated sample loading, reading, and data processing for the digital nanobubble assay. Lastly, we will test this assay with clinical RSV isolates (from diverse genetic strains) and clinical specimens. By comparing with current PCR and rapid diagnostic tests, we will establish the clinical sensitivity and specificity for the digital nanobubble test. This test is fast (< 30 minutes), low-cost (AuNP reagent cost is similar to lateral flow immunoassay), and highly sensitive and specific for RSV. Furthermore, the direct detection of virus particles eliminates the need of extensive sample preparation such as nucleic acid extraction. Success of our project will meet urgent demands of rapid and sensitive RSV diagnostics and address a major healthcare need for pediatric patients that are affected by RSV.