Although there are several reports demonstrating the use of aqueous two-phase systems (ATPS) for biological sample separation in miniaturized devices, they only used ATPS prepared in macro scale by following the conventional way. Formation of ATPS in a confined micro scale environment and their characteristics has not been studied yet. While ATPS formation in macro scale is supported by vigorous mixing and gravitational force to form two phases, both mixing capability and gravitational force aid are insignificant in micro scale. Hence, in this study, we investigated the formation of ATPS under these inferior conditions of micro scale. An electrowetting on a dielectric (EWOD) digital microfluidic chip was used as the confined miniaturized environment. In an EWOD device, micro/nano scale droplets are manipulated on an array of electrodes by sequential application of voltages to the electrodes. The common polyethylene glycol (PEG) and dextran (DEX) polymer/polymer ATPS was used as the model system. Nanoliter volume droplets of the two pure polymer solutions were brought into contact, mixed, and successfully formed ATPS on an EWOD chip. Furthermore, it was possible to separate the two phases into two droplets, demonstrating the promising capability of on-chip biological sample separations effectively by integrating formation of ATPS and separation process using ATPS in a single chip. This study also compares the characteristics of ATPS generated in microfluidic chip with that generated in conventional macro scale. The effect of mixing performance on the ATPS formation is studied experimentally.

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