Multidimensional Protein Separations in a Plastic Microfluidic Network

Abstract

The field of microfabrication of bioanalytical devices has grown significantly over the last decade from academic research to several commercially available systems. The performances of the microfluidic systems in terms of reproducibility, resolution, sensitivity, and speed can be achieved by applying these technologies to complex biological samples.

A commonly used capillary fitting is employed for housing miniaturized membrane chromatography for performing reversed-phase peptide separations. Separation performance of cytochrome C digest in miniaturized membrane chromatography is compared with the results obtained from μ-LC and capillary LC. The use of miniaturized membrane chromatography allows significant reduction in sample consumption together with enhanced detection sensitivity.

In order to further reduce sample consumption and dead volume, an isoelectric focusing separation and dynamic sample introduction are demonstrated in a microfluidic microchannel. The dynamic sample introduction in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. It enhances sample loading and therefore the concentrations of focused analytes by approximately 10-100 fold in comparison with conventional isoelectric focusing.

An integrated 2-D protein separation system provides significant resolving power for complex protein mixtures. Non-native IEF is chosen for the first separation dimension and gel electrophoresis for the second dimension. Once the focusing is complete, the focusing proteins at IEF microchannel are simultaneously transferred using an electrokinetic method from the first dimensional microchannel into an array of the second dimensional microchannels for achieving parallel size-depended separation on each sampled fraction. Although this simple study involves a limited number of second dimensional microchannels, the ability for a microfluidic platform to perform parallel 2-D separations of complex protein samples has been successfully demonstrated.

This study investigates that microfabricated systems have the potential to automate and combine high throughput multidimensional protein separations in a microfluidic network. It is crucial to combine various microfluidic components, which enable all required proteome technologies in an integrated platform for a true lab-on-a-chip.