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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 10 3  to 10 6  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.

Controlling Droplet Marangoni Flows to Improve Microscopy-Based TB Diagnosis