title: Static self-directed sample dispensing into a series of reaction wells on a microfluidic card for parallel genetic detection of microbial pathogens.
abstract: A microfluidic card is described for simultaneous and rapid genetic detection of multiple microbial pathogens. The hydrophobic surface of native acrylic and a novel microfluidic mechanism termed "airlock" were used to dispense sample into a series of 64 reaction wells without the use of valves, external pumping peripherals, multiple layers, or vacuum assistance. This airlock mechanism was tested with dilutions of whole human blood, saliva, and urine, along with mock samples of varying viscosities and surface tensions. Samples spiked with genomic DNA (gDNA) or crude lysates from clinical bacterial isolates were tested with loop mediated isothermal amplification assays (LAMP) designed to target virulence and antibiotic resistance genes. Reactions were monitored in real time using the Gene-Z, which is a portable smartphone-driven system. Samples loaded correctly into the microfluidic card in 99.3% of instances. Amplification results confirmed no carryover of pre-dispensed primer between wells during sample loading, and no observable diffusion between adjacent wells during the 60 to 90 min isothermal reaction. Sensitivity was comparable between LAMP reactions tested within the microfluidic card and in conventional vials. Tests demonstrate that the airlock card works with various sample types, manufacturing techniques, and can potentially be used in many point-of-care diagnostics applications.
authors.affiliation: Civil and Environmental Engineering, Michigan State University, East Lansing, MI, 48824, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Stedtfeld
authors.forename: Yen-Cheng
authors.initials: YC
authors.lastname: Liu
authors.forename: Tiffany M
authors.initials: TM
authors.lastname: Stedtfeld
authors.forename: Tanja
authors.initials: T
authors.lastname: Kostic
authors.forename: Maggie
authors.initials: M
authors.lastname: Kronlein
authors.forename: Onnop
authors.initials: O
authors.lastname: Srivannavit
authors.forename: Walid T
authors.initials: WT
authors.lastname: Khalife
authors.forename: James M
authors.initials: JM
authors.lastname: Tiedje
authors.forename: Erdogan
authors.initials: E
authors.lastname: Gulari
authors.forename: Mary
authors.initials: M
authors.lastname: Hughes
authors.forename: Brett
authors.initials: B
authors.lastname: Etchebarne
authors.forename: Syed A
authors.initials: SA
authors.lastname: Hashsham
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005820:Genetic Testing / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D046228:Microarray Analysis / Q000295:instrumentation*; D008904:Miniaturization; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial