Core—Shell Droplets for Parallel DNA Ligation of an Ultra-Micro Volume Using an EWOD Microfluidic System

An electrowetting-on-dielectric (EWOD) microfluidic system of coplanar electrode type was designed for parallel DNA ligation of an ultra-micro volume, which was applied to droplet manipulation containing DNA, a ligation enzyme and a multi-salt reaction buffer. For environmental reasons, we applied a concept of a water–oil core–shell droplet; such droplets of volume ∼0.3 μL were generated from reservoirs between one plate with coplanar electrodes and another plate with a hydrophobic surface free of electrodes. The experimental results show that the best mixing sequence for the DNA ligation involves mixing first the insert DNA and vector DNA, and then adding the ligase solution. The waiting time was less than 5 min. In one cloning test, the total usage of reagents in an ultramicro-volume EWOD chip was 2.1 μL with no wasted volume, for comparison with 85% waste according to the standard protocol was 15 μL. The results showed also that four parallel DNA ligations were accomplished without damage to a chip and without biomaterial annulment.