Acoustofluidics-based enzymatic constant determination by rapid and stable in situ mixing

Microfluidic mixings create considerable opportunities for microscale exploration of biomacromolecule function and local reaction kinetics. However, a robust and efficient way for localized mixing performance is not yet well established. In this study, we describe a simple and reliable method for rapid, stable, and controllable in situ mixing using a single-layer acoustofluidic system with a microneedle. We developed the single microstructure for region-confined mixing, which can be controlled dynamically by intentional acoustic activation. Various acoustofluidic impacts including driving frequency, microstructure design, driving voltage, and flow control on homogeneous mixing were systematically investigated. The microneedle-based device allows a fast (∼ 85 ms) mixing operation. Robust and high reusability of acoustofluidic system was experimentally demonstrated with up to 200 times of repetitive mixing and 100 min of continuous mixing. Furthermore, the proof-of-concept applications including real-time characterization of fluorescein quenching and β-glucuronidase-catalyzed hydrolysis of 4-methyl-umbelliferyl-β-d-glucuronide were successfully accomplished in the in situ mixing platform. We believe this microfluidic system could be suitable for investigation of different instantaneous chemical/biochemical events associated with various molecular interactions and reactions. Also, the acoustofluidic approach is potentially valuable to the development of mixing-based integrated microfluidic systems for applications in molecular science and life science.