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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-015-9994-1) contains supplementary material, which is available to authorized users.

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device

“…When it is not present, annealing conditions are not favored – especially at the high reaction temperature (65 °C), thus resulting in a significant delay in amplification time. Primers for the isothermal amplification were designed according to requirements for a one h loop-mediated isothermal amplification (LAMP) (Mori et al 2001; Notomi et al 2000), which utilizes Bst polymerase, an enzyme minimally inhibited by complex sample components (Koloren et al 2011; Stedtfeld et al 2015; Stedtfeld et al 2014). The forward primer (F) and forward-inner-primers (FIP) have the same sequences as universal strand 1, so are unable to bind and begin amplifying until the overhang connection has allowed the formation of its complementary strand.…”

“…The described method appears to be well-suited for detection of microRNA via direct amplification from unprocessed biological samples, as Bst polymerase is less influenced by inhibition compared to qPCR (Koloren et al 2011; Stedtfeld et al 2015; Stedtfeld et al 2014). To our knowledge, this is the most rapid method (< 60 min) described for direct quantitative detection of microRNAs in body fluids (Table 1).…”

“…Cycling included 58 s incubation at 65 °C followed by a plate read, and repeated for 60 cycles. For reactions in the microfluidic chip, primers were dispensed into wells, dried at 70 °C for 5 min, and sealed with optical adhesive film (Stedtfeld et al 2015) (Applied Biosystems; Foster City, CA). Pluronic and BSA were not used in experiments conducted in the microfluidic chip.…”

“…The work described here is its first demonstration for microRNA detection. Features of this real time device include: i) simple microfluidic chips consisting of up to 64 reaction wells each with 1 to 20 µl (Stedtfeld et al 2015) ii) real time monitoring of amplification in less than 1 h, iii) potential for wireless communication, automated data processing and reporting using a smartphone user interface, and iv) a hand-held format with internal rechargeable battery.…”

Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

“…Disposable chips were made by etching channels and wells into black acrylic (1.58 mm thick) via a 40-W CO 2 laser (Full Spectrum). The etched chips were cleaned and prepared, as previously described (29). Briefly, the chips were cleaned with distilled water, soaked in 70% ethanol for 10 min, and dried for 10 min at 70°C.…”