Integration of microfluidic sample preparation with PCR detection to investigate the effects of simultaneous DNA-Inhibitor separation and DNA solution exchange.

In this paper, we applied a curved-channel microfluidic device to separate DNA from PCR-inhibitor-containing water and simultaneously wash them into clean water for detection using a portable PCR thermocycler. Environmental DNA (eDNA) sampling has become an effective surveying approach for detecting rare organisms. However, low concentration eDNA molecules may be masked by PCR inhibitors during amplification and detection, increasing the risk of false negatives. Therefore, technologies for on-site DNA separation and washing are urgently needed. Our device consisted of a half-circle microchannel with a DNA-inhibitor sample inlet, a clean buffer inlet, and multiple outlets. By using the flow-induced inertial forces, 10 μm DNA-conjugated microparticles were focused at the inner-wall of the curved microchannel while separation from 1 μm inhibitor-conjugated microparticles and DNA washing were achieved simultaneously with the Dean flow. We achieved singleplex focusing, isolation and washing of 10 μm particles at an efficiency of 94.5 ± 2.0%. In duplex experiments with 1 μm and 10 μm particles, larger particles were washed with an efficiency of 92.1 ± 1.6% and a purity of 79 ± 2%. By surface-functionalizing the microparticles with affinity groups against Atlantic salmon DNA and humic acid (HA), and processing samples of various concentrations in our device, we achieved an effective purification and detection of DNA molecules using the portable PCR thermocycler. Our method significantly decreased PCR quantitation cycles from Cq > 38 to Cq = 30.35 ± 0.5, which confirmed enhancement of PCR amplification. The proposed device takes a promising step forward in sample preparation towards an integrated device that can be used for simultaneous purification and solution exchange of DNA in point-of-need environmental monitoring applications.