Target-Triggered Microspheres Magnetization for Multiplexed Nucleic Acids Detection Using Vertically Encoded Microspheres and Flow Cytometric Counting.

In conventional multiplex detection using fluorescently encoded microspheres, the reporting signal also originates from fluorescence, which often leads to mutual interference between encoding and detection signals. Furthermore, the reporting signal occupies an independent spectral band, thereby limiting the multiplexing capacity of the fluorescently encoded microspheres. To overcome this limitation, we propose a strategy termed target-triggered microsphere magnetization, which decouples encoding from signaling by employing vertically encoded microspheres for target identification and magnetic nanoparticles as magnetic tags. In the presence of target analytes, sandwich complexes are formed, purified by magnetic separation, and subsequently analyzed by flow cytometry. Target types are identified based on the positions of encoded microspheres in two-dimensional scatter plots, and target concentrations are determined by the event counts at the corresponding positions. Validated using nine gene fragment samples, this method achieves high-specificity multiplex detection with favorable sensitivity. By unlocking the encoding potential of fluorescent microspheres, this strategy offers a novel approach for large-scale, multi-index analysis of complex samples.