Beads‐on‐a‐Tip testing for ultrasensitive antigen detection across a large dynamic range

Abstract Lateral flow immunoassays (LFIAs) are low‐cost, rapid, and easy to use for point‐of‐care testing (POCT), but the majority of the available LFIA tests are indicative, rather than quantitative, and their sensitivity in antigen tests are usually limited at the nanogram range, which is primarily due to the passive capillary fluidics through nitrocellulose membranes, often associated with non‐specific bindings and high background noise. To overcome this challenge, we report a Beads‐on‐a‐Tip design by replacing nitrocellulose membranes with a pipette tip loaded with magnetic beads. The beads are pre‐conjugated with capture antibodies that support a typical sandwich immunoassay. This design enriches the low‐abundant antigen proteins and allows an active washing process to significantly reduce non‐specific bindings. To further improve the detection sensitivity, we employed upconversion nanoparticles (UCNPs) as luminescent reporters and SARS‐CoV‐2 spike (S) antigen as a model analyte to benchmark the performance of this design against our previously reported methods. We found that the key to enhance the immunocomplex formation and signal‐to‐noise ratio lay in optimizing incubation time and the UCNP‐to‐bead ratio. We therefore successfully demonstrated that the new method can achieve a very large dynamic range from 500 fg/mL to 10 μg/mL, across over 7 digits, and a limit of detection of 706 fg/mL, nearly another order of magnitude lower than the best reported LFIA using UCNPs in COVID‐19 spike antigen detection. Our system offers a promising solution for ultra‐sensitive and quantitative POCT diagnostics.