Advances in small-scale cell culture have made it possible to perform highly parallel experiments in which hundreds of proteins or protein variants can be expressed simultaneously. These techniques can be useful for fields as diverse as drug discovery, fundamental proteomics, or engineering of enzymes with enhanced or novel activities. Traditional bench-scale methods such as chromatography for purifying proteins are laborious and do not scale well, and would be impractical for purifying small amounts of protein from many cultures for high-throughput applications.

To this end, we have developed a microfluidic technique for isolating specific proteins from cell lysate. The process is based on continuous-flow aqueous two-phase extraction using a PEG-potassium phosphate system. The low interfacial tension of the two-phase system enables stable side-by-side laminar flow of the two phases over a wide range of flow conditions. The aqueous two-phase system is mild, preserving protein structure, allowing us to recover intact, functional proteins and enzymes from the process. Aqueous two-phase systems are highly tunable, allowing specific recovery of nearly any protein of interest while rejecting undesired proteins and cell debris. We have also demonstrated simple engineering of proteins to modify their partitioning behavior and enhance recovery. The objective of the separation is typically to remove unwanted or interfering proteins or activities; the extraction can also serve as an initial, crude purification prior to a second orthogonal purification technique. The fluid mechanics and mass transport of the two-phase flow are interesting and varied, and we present simple modeling and experiments to predict the performance of the process.

The microfluidic extraction approach has several advantages. The process operates in a continuous-flow, rather than batch mode, enabling automation. No packed particles, chromatography supports, or other heterogeneous materials are required, which greatly simplifies chip setup and operation. The small scale of the microfluidic device allows us to processes microliter and sub-microliter samples in minutes, eliminating wasteful, oversized bench scale processing. Parallel processing can be enabled by simply adding additional microchannels. Finally, the process can be integrated into a multi-step lab-on-a-chip platform as an intermediate purification step for true high-throughput analysis.