On-chip hydrodynamic chromatography of DNA through centimeters-long glass nanocapillaries

This study demonstrates hydrodynamic chromatography of DNA fragments in a microchip. The microchip contains a highly regular array of nanofluidic channels (nanocapillaries) that are essential for resolving DNA in this chromatography mode. The nanocapillaries are self-enclosed robust structures built inside a doped glass layer on silicon using low-resolution photolithography and standard semiconductor processing techniques. Additionally, the unique nanocapillaries feature a cylindrical inner radius of 600 nm maintained over a length scale of 5 cm. The microchip with bare open nanocapillaries is shown to rapidly separate a digest of lambda DNA in free solution (<5 min under the elution pressure of 60 to 120 psi), relying entirely on pressure-driven flows and, in doing so, avoiding the field-induced DNA aggregations encountered in gel-free electrophoresis. The nanocapillaries, despite their relatively short length, are observed to fractionate DNA fragments reasonably well with a minimum resolvable size difference below 5 kbp. In the chromatograms obtained, the number of theoretical plates exceeds 10⁵ plates per m for 3.5 and 21 kbp long DNA fragments. The relative mobility of fragments in relation to their size is found to be in excellent agreement with the simple quadratic model of hydrodynamic chromatography. The model is shown to estimate greater effective hydrodynamic radii than those of respective fragments being unconfined in bulk solution, implying increased drag forces and reduced diffusion coefficients, which is also a noticeable trend among diffusion coefficient estimates derived from the experimentally obtained plate heights. This robust mass-producible microchip can be further developed into a fully integrated bioanalytic microsystem.