A rapidly-prototyped microfluidic device for size-based nucleic acid fractionation using isotachophoresis

We present a novel microfluidic device for size-based nucleic acid (NA) fractionation using isotachophoresis (ITP) and an ionic spacer. Our rapid-prototyped laser-cut plastic device has easily modifiable channel dimensions, can process up to 10 μL of sample, and contains an in-line extraction reservoir for minimally-disruptive manual collection of size-fractionated NAs. We designed custom buffering reservoirs using 1 mL pipette tips to provide high buffering capacity and prevent bubbles from entering the microfluidic channels. We demonstrated the utility of the device by implementing a proof-of-concept assay in which NAs were preconcentrated (via ITP) and then segregated by size (using the ionic spacer and sieving matrix) to generate two separate fractions, the first comprised of small (<50 nt) NA, and the second comprised of NAs of all sizes. Through this approach, we demonstrated size-based fractionation of both DNA and RNA samples (a mixture of synthetic ssDNA molecules, and a commercially-available RNA molecular weight standard, respectively). Our results indicate that this simple, rapid (≤10 min), and label-free approach is a promising and cost-effective alternative to the commercially-available size-selection kits currently on the market. We discuss the design and features of the device, as well as challenges which must be met in the future in order to further improve its performance and utility.