Real‐Time Digital Micromotor Tracking‐Enabled Ultrasensitive Immunoassay

ABSTRACT Digital bioassays are emerging as a pivotal technology in disease prevention and diagnostics due to their single‐molecule level detection capability. However, elaborate microchamber fabrication, high‐end equipment, and skilled manipulation are required to enable the end ‐ point digital signal readout. Herein, we propose a novel concept of artificial intelligence (AI)‐facilitated real‐time digital micromotor tracking‐enabled immunoassay (AI‐dMIA), which leverages a self‐developed multi‐microparticle tracking system to monitor micromotor motion trajectories in a real‐time manner for digital protein analysis. In this design, even a single molecule‐bridged immunobinding event on the fully‐open microparticle's surface can induce obvious motion behaviors and trajectories, forming a positive micromotor that can be accurately tracked and discriminated from the negative ones in real time by a bright‐field microscope integrated with the AI algorithm. The AI‐dMIA gets rid of the complex process of microchamber fabrication and fluorescence signal yielding/amplification to generate digital counting events. It also exhibits powerful processing capacity to simultaneously track up to thousands of motor trajectories on a large scale, no longer requiring the high‐end equipment that is essential to traditional end‐point digital bioassays. The AI‐dMIA not only demonstrates a robust immunosensing approach but also provides a new alternative for developing next‐generation digital micromotor‐based platforms.