Increasing hybridization rate and sensitivity of bead-based assays using isotachophoresis

We present an electrokinetic technique to increase the reaction rate and sensitivity of bead-based assays. We use isotachophoresis (ITP) to preconcentrate and co-focus target molecules and beads into a single ITP zone. The process achieves rapid mixing, stirring, and strongly increases the binding reaction rate. We demonstrate our assay with quantitative detection of 24 nt single-stranded DNA over a dynamic range of three orders of magnitude and multiplexed detection of ten target species per sample. We show that ITP can achieve approximately the same sensitivity as a well-stirred standard reaction in 60-fold reduced reaction time (20 min versus 20 h). Alternately, compared to standard reaction times of 30 min, we show that 20 min ITP hybridization can achieve 5.3-fold higher sensitivity.