Integrated isotachophoretic stacking and gel electrophoresis on a plastic substrate and variations in detection...

In this study, we demonstrated an integrated ITP-gel electrophoresis (GE) device on a plastic substrate, in which 50 nL of samples could be hydrodynamically or electrokinetically injected and enriched by ITP into narrow bands and then subsequently introduced into a homogeneous GE channel for separation and detection. This microchip design rendered a simple introduction scheme for creating sandwiched stacking buffer system and flexibilities in choosing separation and stacking buffers independently. We used gel sieving buffers which compositions were different from those for stacking buffers to separate DNA and protein molecules based on sizing mechanism. Compared to conventional microchip GE, the sensitivity of microchip ITP-GE was estimated to increase by one to two orders of magnitude based on the dilution factor of the injected sample and the S/N ratio detected from the electropherogram. Moreover, it is interesting to note that ITP stacking leads to a preferential enhancement for analytes with lower concentrations compared to those with higher concentrations. Therefore, a reduction in the detection dynamic range for ITP-GE was gained. We demonstrated that ITP-GE could lead to 2-4-folds of reduction in the signal dynamic range for two PCR products in a mixture. Such advantage is demonstrated to be useful for the detection of two products amplified from a multiplex PCR in which one product is poorly amplified compared to the other.