Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Abstract

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were "printed" on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 microL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.