Microfluidic 2-D PAGE using multifunctional in situ polyacrylamide gels and discontinuous buffers

Abstract

A two-dimensional microfluidic system is presented for intact protein separations combining isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) employing in situ photopolymerized polyacrylamide (PAAm) gels. The PAAm gels are used for multiple functions. In addition to serving as a highly-resolving separation medium for gel electrophoresis, discrete polyacrylamide gel plugs are used to enable the efficient isolation of different on-chip media including anolyte, catholyte, and sample/ampholyte solutions for IEF. The gel plugs are demonstrated as on-chip reagent containers, holding defined quantities of SDS for on-chip SDS-protein complexation, and enabling the use of a discontinuous buffer system for sample band sharpening during SDS-PAGE. The 2-D chip also employs several unique design features including an angled isoelectric focusing channel to minimize sample tailing, and backbiasing channels designed to achieve uniform interdimensional sample transfer. Separation results using E. coli cell lysate are presented using a 10-channel chip with and without the discontinuous buffer system, with resolving power more than doubled in the former case. Further improvements in separation resolution are demonstrated using a 20-channel chip design.