Real‐Time Digital Micromotor Tracking‐Enabled Ultrasensitive Immunoassay

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.