Continuous-flow bioseparation using microfabricated anisotropic nanofluidic sieving structures.

The anisotropic nanofluidic-filter (nanofilter) array (ANA) is a unique molecular-sieving structure for separating biomolecules. In this protocol we describe the fabrication of planar and vertical ANA chips and how to perform continuous-flow bioseparation using them. This protocol is most useful for bioengineers who are interested in developing automated multistep chip-based bioanalysis systems and assumes previous cleanroom microfabrication knowledge. The ANA consists of a two-dimensional periodic nanofilter array, and the designed structural anisotropy of ANA causes different-sized or charged biomolecules to follow distinct trajectories under applied electric fields, leading to efficient continuous-flow separation. Using microfluidic channels surrounding the ANA, the fractionated biomolecule streams are collected and routed to different fluid channels or reservoirs for convenient sample recovery and downstream bioanalysis. The ANA is physically robust and can be reused repeatedly. Compared with the conventional gel-based separation techniques, ANA offers the potential for faster separation, higher throughput and more convenient sample recovery.