TWO-DIMENSIONAL CHIP-BASED PROTEIN ANALYSISUSING COUPLED ISOELECTRIC FOCUSING AND CAPILLARY ELECTROPHORESIS

摘要

A chip-based acrylic microfluidic device that sequentiallycouples isoelectric focusing (IEF) and capillary electrophoresis(CE) has been designed and demonstrated. To our knowledge, thisis the first time such an implementation of on-chip twodimensional(2-D) electrophoresis has been presented. Bothdimensions in this liquid-phase system were characterized using afull-field CCD imaging approach. Commercial ampholytes wereused in microchannel-based IEF to sustain a stable pH gradient.Analysis of the concentrating IEF step revealed substantiallyreduced electroosmotic mobilities, as compared to typicalmobilities for acrylic microchannels filled with standard buffersolutions. Due to this residual electroosmotic flow, IEF specieswere simultaneously rapidly focused and slowly mobilized intointersections. After IEF separation and 100x pre-concentration,voltage switching was used to electrokinetically inject portions of amulti-protein mixture into the second separation, which wasampholyte-based CE. Mobility information was obtained from thesecond dimension. Results are presented in a 2-D area plot. Withoptimization, this architecture has the potential to be a basis forhigh-throughput, high-resolution protein and peptide analysis.