Nanopillar array chip integrated with on-line stacking for fast DNA separation with high sensitivity and high resolution

Recent developments of nanofabrication techniques have created a trend switching from randomly ordered polymeric matrices, such as gel, to highly ordered sieving nanostructures in the separation of biomolecules. These nanostructures have enormous potential for fast separation of biomolecules, while nanostructure-based separation techniques suffer from critical scaling problems; they are efficient in handling less than nanoliter amounts of sample fluids, but most biomolecule samples are available in a liquid volume that is over several microliter, leading to a reduction in sensitivity and resolution. In this study, we developed a nanopillar array chip integrated with an easy and rapid on-line stacking method and achieved fast DNA separation with high sensitivity and high resolution. The developed on-line stacking method is based on the balance of two forces driven by electric fields: electroosmotic flow (EOF) and electrophoresis. The EOF mobility from the microchannel to the nanopillar-channel is drastically decreased, while, on the other hand, electrophoresis has constant mobilities in the whole length of the channels. The on-line stacking was realized at the well-balanced position of the two forces, and the on-line stacking using the nanopillar array chip can also be achieved within 10 s by just applying electric voltages without any other special reagents and materials. After applying on-line stacking using the nanopillar array chip, the relative fluorescence intensity increased 1,000-fold, and the resolution was twice as good as that without on-line stacking.