Acoustofluidic Focusing for High-Throughput and Sensitive Analysis of Shape-Encoded Hydrogel Microparticles in Multiplex Immunoassay.

Shape-encoded hydrogel microparticles (SHMPs) offer robust sensitivity and multiplexing capacity in immunoassays. However, their analysis has conventionally required labor-intensive imaging-based methods. Although flow-through analysis has been explored as an alternative approach, its throughput and sensitivity remain limited. In this study, we developed a high-throughput and highly sensitive multiplex immunoassay platform by integrating SHMPs with acoustofluidic focusing. Acoustofluidic focusing is a powerful technique that enables high-throughput analysis owing to its capability to densely align micro-objects. Since hydrogel materials are challenging to accurately align by acoustofluidic focusing, we optimized the flow rate as well as the amplitude and frequency of acoustic standing waves to achieve precise and dense particle alignment. The precise alignment of SHMPs in microchannels allowed reliable code recognition and fluorescence signal detection. Also, densely aligned SHMPs offered an 8-fold higher analysis throughput than previously studied flow-through methods, even at low flow rates. The synergistic effects of sufficient dwell time provided by acoustofluidic focusing at low flow rates and the high sensitivity of SHMPs achieved a 61.5-fold enhancement in the detection limit compared to commercial devices using flow-through analysis. The feasibility of multiplex detection was further validated through spike-in experiments, yielding recovery rates within 100 ± 20% for all four protein targets. Finally, we assayed cytokines in clinical samples from drug allergy and healthy groups and successfully identified significant differences in the cytokine levels.