A Compartmentalization-Free Digital Immunoassay Based on Plasmonic-Fluorescence Nanoparticles

Ultrasensitive protein detection is crucial for early disease diagnosis, precision medicine, and life science research. Digital immunoassays, especially compartmentalization-free formats, hold significant promise in this field due to their operational simplicity and independence from large-scale instrumentation. However, the performance of such compartmentalization-free digital immunoassays critically depends on the effective single-molecule labels. In this study, we developed a novel compartmentalization-free digital immunoassay utilizing plasmonic-fluorescent nanoparticles (SA-Cy3@BSA-Bio@Au NPs) as labels for individual immunocomplexes. These nanoparticles were synthesized by first modifying gold nanoparticles (Au NPs) with biotinylated bovine serum albumin (BSA-Bio) using a freeze-driven conjugation method, followed by the attachment of streptavidin-conjugated Cy3 (SA-Cy3). The synthesized SA-Cy3@BSA-Bio@Au NPs possess a small size yet exhibit exceptional single-particle brightness and dispersibility. Leveraging these properties, we established a compartmentalization-free digital immunoassay platform and evaluated its performance using interleukin-6 (IL-6) as a model analyte. The developed assay achieved a remarkable detection limit of 95 fg/mL, comparable to existing digital immunoassays. Furthermore, owing to the relatively small dimensions of SA-Cy3@BSA-Bio@Au NPs, this method significantly reduces the assay time to 75 min. The potential practical utility was verified through spiked and recovery experiments using human serum samples. This work provides a new design strategy for labels in digital immunoassays, facilitating sensitive and rapid detection.