(579c) On-Chip Aqueous Two-Phase Extraction for Protein Isolation from Cell Lysate

High-throughput protein expression and purification is typically limited by lack of protein purification techniques that can handle small amounts of starting material in a continuous fashion. Microfluidic technologies can enable microliter- or nanoliter-scale cell cultures at one end, as well as sensitive detection and analysis of proteins at the other end, but integrated approaches for purifying proteins between the cell culture and analysis steps are still required.

Aqueous two-phase systems formed by mixtures of PEG and salt or PEG and Dextran have long been used for large-scale bioseparations. Mixtures of PEG and potassium phosphate have been used industrially as a first step to isolate desired recombinant proteins from other proteins and cell debris. Certain proteins such as β-galactosidase exhibit a strong preference for the PEG-rich phase, and can even be used as fusion tags to change the partitioning behavior of other proteins of interest.

Scaling down of aqueous two-phase extraction represents a promising approach for performing extraction of desired proteins from cell lysate. Aqueous two-phase systems are characterized by low interfacial tension between the two phases, allowing formation of a stable interface between the two phases in laminar flow on a microfluidic device. A stream containing cell lysate is hydrodynamically focused into to a narrow zone between a PEG-rich stream and a phosphate-rich stream at the chip inlet, and individual components must only diffuse a short distance (on the order of 10 microns) to partition into one phase or the other. Residence time is a critical parameter, and partitioning behavior has been studied as a function of channel length as well as flow rate. Simple modeling of mass-transfer was used to optimize residence time and microfluidic chip architecture. On-chip extraction has been used to observe partitioning of β-galactosidase, as well as β-galactosidase fusion proteins, away from other proteins, nucleic acids, cell debris. Multiple stages of separation, e.g. with PEG-salt followed by PEG-Dextran, are also possible. Aqueous two-phase extraction can be integrated with on-chip cell lysis, allowing for a microfluidic approach for processing microscale cell cultures prior to protein characterization or proteomic studies.