Acoustic Streaming Tunnel Enables Particle Velocity Stretching in Multiplex Flow Cytometry

Multiplexed flow cytometry, known for its powerful high-throughput identification capability, is widely applied across various biomedical and clinical fields. However, classical flow cytometry relies on multichannel lasers and detectors, which are significant in cost and size, limiting their application in miniaturized assays. Herein, we developed an acoustic streaming-based flow cytometry technique that focuses on multisized microbeads flowing sheathlessly. This method enables the discrimination of particle types and the quantification of target protein concentrations using only a single detector. Microbeads of different sizes exhibit distinct behaviors in the continuous acoustic streaming tunnel, leading to an increased velocity difference during their transition under the laser spot. Consequently, a size detection method based on "velocity stretching" has been established. A multiplex assay of three proteins: cardiac troponin I, creatine kinase-MB and myoglobin, in acute myocardial infarction is performed to validate the feasibility and evaluate the performance of the system. This new multiplexed flow cytometry strategy is expected to enable low-cost and onsite detection of multiple biomarkers.