Sequential DNA hybridisation assays by fast micromixing

Abstract

The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article. It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm. It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means. The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only. DNA oligomers (20-mers) of different sequences were injected on the chip for mixing. The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes. Two modes of operation were investigated. First, the samples were injected into the micromixing device at a high flow rate of 40 µl min⁻¹. When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2–15 s, as defined by the channel geometry. Using this continuous flow approach, photobleaching of fluorophores could be avoided. In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5–20 s. Single base-pair mismatches were successfully identified under low salt conditions. In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed. Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5–7 s, in some cases even below 1 s.