Isoelectric focusing and isotachophoresis on integrated microchips

Bingwen Liu

Progress in proteomic research has increased the demand for high-throughput analysis of biological samples. The workhorse for protein separation and identification is still mainly the traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). However, the peak capacity and dynamic range provided by current analytical techniques, including 2-D PAGE, fall short of the needed resolution for very complex proteomic mixtures. This dissertation will address integration of various electrophoretic modes, development of new focusing methods, as well as the relevant issues such as microchip fabrication and sample zone dispersion. Poly(dimethylsiloxane) (PDMS) and Poly(methyl methacrylate) (PMMA) microchips were fabricated for different application purposes. Isotachophoresis (ITP) and isoelectric focusing (IEF) were integrated on a valved PDMS chip. ITP was used to preconcentrate and desalt samples, which were then resolved by IEF. Effects of various experimental parameters on separation performance, such as salts, joule heating, and channel geometry, were identified. Valve incorporation in the chip helped reduce the intermixing between ITP and IEF buffers. High-strength PMMA microchips were fabricated and bonded with a solvent-assisted thermal bonding method. The chips could tolerate high pressures up to1000 psi. ITP-capillary zone electrophoresis (CZE) inline integration separation was demonstrated in a dog-leg-channel PMMA chip. More than one million theoretical plates per meter were achieved. Dispersion in counter-flow ITP was studied using numerical simulations, and validated by experimental results. The dispersion in counter-flow ITP in an open capillary was found to be strongly related to analyte molecular diffusivity. Good agreement between simulation results and experimental output was observed. In order to overcome the severe dispersion in an open capillary, counter-flow ITP was conducted in a porous poly(acrylamide-co-N,N'-methylenebisacrylamide) monolithic column. In the monolithic column, the parabolic flow profile could be turned to a plug-shaped flow profile. Counter-flow ITP in the monolith showed less dispersion, when compared to that in the open capillary. Counter-flow ITP was demonstrated to concentrate dilute and abundant samples as well as stack zone separation using 2-(N-morpholino)ethanesulfic acid (MES) as a spacer.

Isoelectric focusing and isotachophoresis on integrated microchips

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