Advancing next-generation proteomics: A polymer-patterned microchamber enables integration of the distinct protein separations comprising two-dimensional electrophoresis

Abstract

We introduce a new microfluidic approach for rapid, integrated two-dimensional electrophoresis (2DE), which uses a fundamentally different design strategy for integrating two the distinct assay stages of isoelectric focusing (IEF) and size-based electrophoretic separations. A novel device microchamber architecture coupled with spatially heterogeneous polymers enables the separated species from the 1st dimension to be transferred for sizing separations without discretization into individual side channels. The carrier ampholyte based pH gradient is confined to the microchamber by incorporating immobilines into the polyacrylamide (PA) gel regions flanking the microchamber. The resulting pH is both linear and stable, with cathodic drifts <3μm/min. Sample loading and focusing is rapidly achieved in <30min. Species with isoelectric points (pI) of <0.1pH units can be resolved and we estimate peak capacities of ∼100 in the 1st dimension. Protein transfer (enabled by ionic mobilization) and separation are rapid (<10min) and preserve 1st dimension separation information with band broadening factors <1.3x and position drifts of <50μm.