Time-resolved and wash-free digital immunoassay based on dynamic tracking of single binding events

Here we introduce a novel digital immunoassay that offers both time-resolved analysis and wash-free convenience. This assay enables dynamic tracking of individual binding events, facilitating a one-step and ultrasensitive detection of biomarkers. The proposed digital immunoassay utilizes functionalized plasmonic gold nanoparticles (AuNPs) as molecular labels to enhance signal readout. To further enhance detection capabilities, we incorporate a microlens chip as a superlens layer, which efficiently boosts the scattering intensity of AuNPs, allowing for precise enumeration of individual AuNPs through a low-cost conventional optical setup. By continuously tracking individual binding events over time, we can significantly minimize non-specific binding, eliminating the need for washing steps altogether. This streamlined approach reduces the time-to-result to less than 20 minutes. As a validation of this technology platform, we demonstrate the immunoassay's efficacy in detecting cardiac troponin I with an extensive linear range (0.1-10,000 pg/mL) and a low detection limit (0.013 pg/mL), underscoring its potential for rapid, accurate, and on-site diagnosis of acute diseases.