The recent rapid growth of microfluidic applications has witnessed the emergence of several particle flow focusing techniques for analysis and/or further processing. The majority of flow focusing techniques employ an external sheath fluid to achieve sample flow focusing independent of the flow rate, in contrast to sheath-free techniques. However, the introduction of a sheath fluid to surround the sample fluid has complicated the device design and fabrication, generally involving multi-layer fabrication and bonding of multiple polydimethylsiloxane (PDMS) layers. Several promising efforts have been made to reduce the complexity of fabrication. However, most of these methods involved the use of inertial/Dean effects, which in turn demanded the use of higher sample flow rates. In this paper, we report a method of flow focusing that uses a sheath fluid to enclose the sample in a single layer of PDMS, and that possesses applicability for a wide range of sample flow rates. This method of flow focusing uses abrupt channel depth variation and a shift of one of the sample-sheath junctions (termed as 'junction-shift') against the direction of the sample flow. This configuration serves to manipulate the sample fluid with respect to the sheath fluid and achieve the desired flow focusing. This design facilitates the attainment of 3D flow focusing in two sequential steps (depth-wise and then along the lateral direction) and in distinct regions, hence enabling the regions to be used in imaging and non-imaging flow cytometric applications, respectively. Simulations were performed to characterize and determine the optimum set of design parameters. Experimental demonstrations of this technique were carried out by focusing fluorescein dye and blood cells in flow.
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