Optimization of a microfluidic microarray device for the fast discrimination of fungal pathogenic DNA.

A microfluidic microarray device, which has been developed for parallel DNA detection, is now further optimized for more rapid and sensitive DNA detection and for the single-base-pair discrimination of two fungal pathogenic PCR products. Two poly(dimethylsiloxane) (PDMS)-based microfluidic chips consist of radial and spiral microchannels in which flexible probe creation and convenient sample delivery have been achieved by centrifugal pumping. The microarray hybridizations occurred at the cross sections within the spiral channels intersecting the preprinted radial probe lines. The centrifugal pumping method showed advantages over the vacuum suction method in terms of parallel solution delivery and less signal variations between replicate samples. The effect of microchannel depth was studied, and hybridization time is predictable at a certain rotation speed. Cy5 dye labels were proved to show much higher hybridization efficiency as well as less photobleaching effect as compared with the fluorescein dye labels used in our previous work. With these optimized conditions, the method was applied to the detection of three fungal pathogenic polymerase chain reaction (PCR) products with a sample load of 0.2 ng (in 1 microl). Furthermore, the single-base-pair discrimination between the PCR products of two relevant Botrytis species (B. cinerea and B. squamosa) was achieved in a duration as short as 3 min.