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In the regime of inertial microfluidics, the secondary flow is widely adopted to reduce the number of the equilibrium position and improve the focusing performance of particles. At the same time, secondary flow can also enhance the mixing effect and may deteriorate particle focusing due to the induced rotating streams, especially for the particle with a small size. In a double-layered microchannel with slanted groove structures, it has been demonstrated that the generated secondary flow at a high flow rate could focus the particle with a large size (> 8 µm). However, the manipulation of small-size particles (< 8 µm) was unsuccessful as the effects of secondary flow on the small-size particles were not strong enough. In this work, to manipulate the small-size particle, we proposed a scheme to utilize a top sheath flow to enhance the focusing efficiency of the structure-induced secondary flow. The effects of the total flow rate and the flow rate ratio between the sheath and sample flow were investigated comprehensively in a large range. The 4.8 µm particle could be manipulated effectively at different flow rates with the assistance of appropriate sheath flows. Besides, the effects of other factors, such as the quantity of the expansion groove structure, and particle concentration and size, on particle focusing performance were also investigated. We found that the particles with the diameter of 2.9 µm can also be effectively focused within the double-layered microchannel. Furthermore, we demonstrated the continuous plasma extraction from the undiluted whole blood using this proposed technique to validate its potential biological applications. The results show that the purity of plasma extracted could reach up to ~ 99% after a single process. As such, this demonstrated that sheath flow-assisted particle manipulating method can overcome the limitations of conventional design and offer much better performance for controlling smaller particles. Such a platform may enable great potential in the applications of biological and diagnostic assays, for bioparticles smaller than the normal cell size.READ MORE