Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

• Published in: Analytica chimica acta 2008-03, Vol.610 (1), p.97-104
• Main Authors: Wang, Lin, Li, Paul C.H., Yu, Hua-Zhong, Parameswaran, Ash M.
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Base Sequence; Biological and medical sciences; Biotechnology; Centrifugal pumping; Chemistry; Dimethylpolysiloxanes - chemistry; DNA Primers; DNA, Fungal - analysis; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Fungal pathogen; Fungi - genetics; General, instrumentation; Genetic engineering; Genetic technics; In vitro gene amplification. Pcr technique; Methods. Procedures. Technologies; Microfluidic microarray; Microfluidics - instrumentation; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2007.12.048

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 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.