ABSTRACT
In developing countries, the most common diagnostic method for tuberculosis (TB) is microscopic examination sputum smears. Current assessment requires time-intensive inspection across the microscope slide area, and this contributes to its poor diagnostic sensitivity of ≈50%. Spatially concentrating TB bacteria in a smaller area is one potential approach to improve visual detection and potentially increase sensitivity. We hypothesized that a combination of magnetic concentration and induced droplet Marangoni flow would spatially concentrate Mycobacterium tuberculosis on the slide surface by preferential deposition of beads and TB-bead complexes in the center of an evaporating droplet. To this end, slide substrate and droplet solvent thermal conductivities and solvent surface tension, variables known to impact microfluidic flow patterns in evaporating droplets, were varied to select the most appropriate slide surface coating. Optimization in a model system used goniometry, optical coherence tomography, and microscope images of the final deposition pattern to observe the droplet flows and maximize central deposition of 1 µm fluorescent polystyrene particles and 200 nm nanoparticles (NPs) in 2 µL droplets. Rain-X® polysiloxane glass coating was identified as the best substrate material, with a PBS-Tween droplet solvent. The use of smaller, 200 nm magnetic NPs instead of larger 1 µm beads allowed for bright field imaging of bacteria. Using these optimized components, we compared standard smear methods to the Marangoni-based spatial concentration system, which was paired with magnetic enrichment using iron oxide NPs, isolating M. bovis BCG (BCG) from samples containing 0 and 103 to 106 bacilli/mL. Compared to standard smear preparation, paired analysis demonstrated a combined volumetric and spatial sample enrichment of 100-fold. With further refinement, this magnetic/Marangoni flow concentration approach is expected to improve whole-pathogen microscopy-based diagnosis of TB and other infectious diseases.
ABSTRACT
Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of â¼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.
