High Resolution Separation of Proteins in a Polymeric Micro-Fluidic Chip

Abstract

An integrated micro-fluidic chip has been developed using Poly-di-methyl siloxane (PDMS) to separate proteins by isoelectric focusing (IEF). Soft lithography techniques, which offer rapid prototyping, easy multilayer fabrication, mass production capability and biocompatibility, were utilized to fabricate various parts of the micro-fluidic chip. Separately molded PDMS layers were bonded together to form three-dimensional microfluidic chips. The microfluidic chips were prepared for IEF by conditioning the channel with 1 M NaOH and then loading it with a solution of fluorescent proteins made using 0.4% MC, 4% broad-range ampholyte and 0.018 mg/ml protein in 18 MOhm water. Relatively large reservoirs on the acidic and basic ends of the channel were filled with anolyte (50 mM phosphoric acid) and catholyte (50 mM sodium hydroxide), respectively, and then current was applied along the axis of the channel until one or more bands of protein focused, usually in just a few minutes even at relatively low voltages. The focused bands were generally well-formed with sharp edges and were less than 100 microns across yielding a putative peak capacity in excess of 100 peaks in a 2-cm long channel.