Thread-based isotachophoresis for DNA extraction and purification from biological samples

Abstract

A rapid, low-cost, and disposable microfluidic thread-based isotachophoresis method was developed for the purification and preconcentration of nucleic acids from biological samples, prior to their extraction and successful analysis using quantitative polymerase chain reaction (qPCR). This approach extracts and concentrates protein-free DNA from the terminating electrolyte buffer, via a continuous sampling approach, resulting in significant focussing of the extracted DNA upon a 6 cm length nylon thread. The platform was optimised using the preconcentration of a fluorescent dye, showing a 600-fold concentration capacity within <5 min. The system was then applied to the one-step extraction of lambda DNA – an E. coli bacteriophage – spiked into whole blood, exhibiting the exclusion of PCR inhibitors. The extraction efficiency from the thread material following concentration was consistent, between 94.4–113.9%. The determination of lambda DNA in whole blood was achieved within a linear range of 1.0–1 × 10⁵ fg μL⁻¹ (20–2 × 10⁶ copies per μL). This technique demonstrates great potential for the development of thread-based affordable analytical and diagnostic devices based upon DNA and RNA isolation.