Rapid Quantification of Virus-Like Particles via Gold Nanoparticle Sensors and Dark-Field Differential Dynamic Microscopy

We present a novel diagnostic platform using differential dynamic microscopy (DDM) to quantify viral load in salivary samples. This method leverages gold nanoparticle-based sensors that form heteroaggregates with virus-like particles (VLPs), designed to mimic viruses. The sensors were sequentially functionalized with biotin, streptavidin, and biotinylated angiotensin-converting enzyme 2, while VLPs were functionalized with streptavidin and the spike S1 receptor-binding domain of SARS-CoV-2 as the model virus. Viral load is quantified by tracking changes in the sensor nanoparticle dynamics during their interactions with VLPs. Initially optimized in buffer and subsequently adapted for salivary samples, the assay leverages dark-field DDM to remove possible interference from unbound VLPs. This approach enables the quantification of VLPs that were otherwise undetectable by dark-field DDM alone, by exploiting the slower diffusion of nanosensor-VLP heteroaggregates, achieving a detection limit of 9 × 10³ VLPs/mL (20 pg/mL), within clinically relevant viral loads. The platform requires minimal sample preparation, a 5 min incubation, and no fluorescent labeling or washing steps. With only a conventional microscope and camera, this rapid assay provides quantitative results in 10-15 min. This proof-of-concept offers an accessible tool for rapid and precise viral load quantification in laboratory settings with potential for point-of-care applications through setup miniaturization. With its simplicity, speed, and sensitivity, this platform represents a promising advancement in infectious disease diagnostics.