Robust polymeric microchannel coatings for microchip-based analysis of neat PCR products.
Several silica coatings have been evaluated for replicate PCR product analysis in capillaries and electrophoretic microchips. Silica coatings are an essential component to many electrophoretic separations, and this importance is magnified in microchips, where separation distances are minimized. Increasing the resistance of coatings to separation conditions improves the reproducibility and longevity of the coated microchip, which allows for the full potential of these devices (rapid separations, high through-put, and longevity) to be realized. In this study, several coating parameters have been evaluated experimentally and through the literature to produce a coating with high resistance to the separation conditions of interest, neat PCR product separations. Coating degradation induced under these conditions was tested for several coatings, and the influence of surface hydroxylation, surface hydration, silanization solvent, silanizing reagent, catalysis, endcapping, and polymerization procedure are discussed. Under the testing conditions, a coating (coating E) prepared by silanization with chlorodimethyloctylsilane in toluene with a polymer layer of poly(vinylpyrrolidone) attached by a hydrogen abstraction method [Srinivasan, K.; Pohl, C.; Avdalovic, N. Anal. Chem. 1997, 69, 2798-2805] was most resistant. This coating was tested for longevity on electrophoretic microchips and was compared to the traditional coating of polyacrylamide. The coatings produced similar resolution and efficiencies; however, coating E provided more reproducible migration times and had performed for 635 analyses when testing was terminated. This procedure provides a reproducible, resistant surface coating, thus allowing for replicate analysis of neat PCR product on microchips.
Munro
N J
NJ
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Hühmer
A F
AF
Landers
J P
JP
D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation; D008832:Microchemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011108:Polymers
D011108:Polymers