The acoustofluidic-focusing microfluidic chip was fabricated with three layers: a silicon layer and two borosilicate glass layers, as shown in fig. S5. The microchannel was patterned in the silicon layer and packaged by the glass layers. Using this design, we achieved both transparency for the optical interrogation and high acoustic impedance. The thicknesses of the borosilicate glass layers and silicon layer were 300 and 200 μm, respectively. Since the width of the microchannel was designed as 200 μm, we obtained a square-shaped section of the microchannel at the optical interrogation point. The details of the fabrication process are described as follows (28). (i) The borosilicate glass was sandblasted using an etching mask of an SCM250 (Nikko-Materials Co. Ltd., Japan), which is a negative photoresist. In this process, we obtained ports for the inlet and outlet of the microchannel. (ii) We removed the patterned SCM250 layer. (iii) The other borosilicate glass and silicon were bonded by using the anodic bonding technique. Then, an SU-8 (Nihon Kayaku Co. Ltd., Japan) layer was patterned on the surface of the silicon layer. (iv) The silicon layer was etched by using the deep reactive etching technique, which allowed us to fabricate the vertical sidewalls for the microchannel. (v) The borosilicate glass with the ports and patterned silicon layer were bonded by using the anodic bonding technique. (vi) A piezoelectric actuator was bonded by using a cyanoacrylate adhesive.

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