The "channeling hypothesis" of DNA electrophoresis in sparse, ordered arrays of posts predicts that the DNA will move through the array relatively unhindered if ͑i͒ the spacing between the posts is larger than the DNA coil and ͑ii͒ the electric field lines are straight. We tested this hypothesis by studying the electrophoretic separation of a small plasmid DNA ͑pUC19, 2686 base pairs͒ and a large, linear DNA ͑-DNA, 48 500 base pairs͒ in a hexagonal array of 1 m diameter posts with a pitch of 7 m. At low electric field strengths, these DNAs are separated due to the long-lived, rope-over-pulley collisions of -DNA with the posts. The resolution is lost as the electric field increases due to the onset of channeling by the -DNA. Using a diffusive model, we show that channeling arises at low electric fields due to the finite size of the array. This channeling is not intrinsic to the system and is attenuated by increasing the size of the array. Higher electric fields lead to intrinsic channeling, which is attributed to the disparate time scales for a rope-over-pulley collision and transverse diffusion between collisions. The onset of channeling is a gradual process, in agreement with extant Brownian dynamics simulation data. Even at weak electric fields, the electrophoretic mobility of -DNA in the array is considerably higher than would be expected if the DNA frequently collided with the posts.

Separating long DNA in a microfabricated post array requires (tens of) thousands of posts in the separation channel. Moving from microposts to nanoposts is thus a fabrication challenge owing to the large area that needs to be nanopatterned. The authors implemented an oxygen plasma etching method in conjunction with conventional optical photolithography and deep trench etching that led to centimeter-long microchannels containing either 360 or 460 nm diameter posts in a hexagonal array with a 3 μm spacing. Separations of the XhoI λ-DNA digest in the device indicate that these sparse nanopost arrays are an improvement over the equivalent micropost array with only a marginal increase in fabrication cost. The fabrication method described here is broadly applicable to biological microfluidic and nanofluidic platforms that require nanoscale features with micrometer-scale spacing.

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