In microfluidic systems, liquid flow structure near the substrate is crucial for regulating the mechanical microenvironment of cells cultured on the surface and mass transport toward the substrate. Therefore, externally induced flow is expected not only to enhance surface reactions but also to modulate cellular responses through controlled shear stress. However, the direct evaluation of the velocity component normal to the substrate remains challenging in monolithic microfluidic devices. In this study, a three-dimensional (3D) flow velocity measurement method based on orthogonal-plane micro-particle image velocimetry (micro-PIV) was developed to evaluate alternating-current electrothermal flow modulation. The 3D velocity field was reconstructed from two orthogonal velocity fields obtained by scanning with different observation planes: one parallel to the bottom and the other perpendicular to the bottom through a sidewall of a polydimethylsiloxane microchannel. The validity of the orthogonal-plane micro-PIV was confirmed through measurements of three-component velocities in a tilted rectangular channel. Using this method, alternating-current electrothermal (ACET) flow generated by temperature-dependent gradients in fluid properties under an electric field was investigated. The results revealed a 3D circular stirring flow that transports fluid from the bulk region toward the surface. This flow structure enhances the transport of suspended species toward the interface and is expected to modulate the local shear environment, demonstrating that ACET-driven 3D flow can simultaneously enhance surface reactions and regulate the cellular mechanical environment in microfluidic systems.

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