Separation of DNA molecules of different dimensions is of fundamental importance in the field of molecular biology. Pulsed field gel electrophoresis (PFGE) is an analytical technique used to separate DNA fragments inside a gel by frequently changing the direction of the electric field. While PFGE has been used for a decade, it suffers from several limitations, such as, requirement of high electric field, low throughput, and complicated recovery of the separated DNA from the gel. Moreover, due to the random structure of the gel, the fundamental basis of the separation mechanism is not clearly understood.

A novel microfluidic technique was recently developed for DNA separation based on colloidal self-assembly and pulsed electrophoresis (Fig. 1). This device employs uniformly structured matrices of nanoparticles as an alternative to the randomly structured gels.¹ In this pulsed field colloidal array electrophoresis (PFCAE) technique, the separation is achieved in a continuous-flow, giving a high throughput of sample processing due to the 3-D ordered structure of the lattice. It is evident that self assembled colloidal arrays can provide considerable flexibility in choosing various pore sizes by changing the size of nanoparticles to target different molecular size ranges.  However, in our previous study, the minimum size of the nanoparticles that provided a continuous packed array was about 300-400 nm.  In this study, nanoparticles of ca. 100 nm are used to form an ordered array to investigate the potential of   enhancing the resolution of the separation process in such structures.  To measure DNA mobility inside the nanoporous structure, particle image velocimetry (μPIV) was used. In this method, the fluid velocity is measured by taking subsequent images of the tracking particles inside the media. In our experiments, fluorescent DNA molecules themselves were considered as tracking particles. Using this method, the bulk velocity of the DNA stream along with the velocity of single DNA molecules was measured. The technique was used to determine the influence of the applied frequency, nanoparticle size, applied field strength, and DNA concentration on the fractionation efficiency.

1. Y. Zeng, and D. J. Harrison, Anal. Chem., 79, 2289 - 2295 (2007).