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

Abstract

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.

Ionic strength effects on electrophoretic focusing and separations

“…a diffuse boundary between the LE and the TE zones). This type of focusing is common in biological applications such as immunoassays and DNA or RNA separations, where sample concentrations are inherently low [34,35].…”

Transport and separation of micron sized particles at isotachophoretic transition zones

“…3 Note that ITP is not only used for small ions but has also proven useful for the analysis of nanometer sized objects. For example, it was used as a preconcentration technique for RNA and proteins in context with gel electrophoresis, 4 for purification of nucleic acids from whole blood samples, 5 or for the study of plasma lipoproteins. 6,7 Furthermore, ITP can also be combined with micellar electrophoresis.…”

Recent Progress of On-line Sample Preconcentration Techniques in Microchip Electrophoresis

“…[41][42][43][44][45] On the other hand, a multi T-channel chip is used to inject L, sample and T solutions successively in the ITP and tITP analysis. In the recent years, α-fetoproteins, 46 DNA ladder, 47,48 and PCR products 49 are well concentrated and resolved by employing the multi T-channel chips. As a very unique approach, Bottenus et al have reported the application of a microchip with two reductions in the cross-sectional area along the axial direction of the channel to the ITP preconcentration of the biomarker cardiac troponin I and a fluorescent protein R-phycoerythrin.…”