High-Throughput Monitoring of Bacterial Cell Density in Nanoliter Droplets: Label-Free Detection of Unmodified Gram-Positive and Gram-Negative Bacteria

Abstract

Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a highthroughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.

Emerging microfluidic technologies for microbiome research

“…However, the speed of droplet turbidity-based detection can be comparable with droplet fluorescence detection due to a similar photoelectric signal detection principle, which is significantly higher than another label-free monitoring strategy, the bright field microscopic imaging. Currently, this approach has been proven to robustly detect the growth of more than 12 bacterial species with different cell morphologies ( Liu et al, 2016 ; Hengoju et al, 2020 ; Pacocha et al, 2021 ). Obviously, it holds great promise to be applied to monitoring the growth of each microbial member in a complex community.…”

UV–Vis Spectra-Activated Droplet Sorting for Label-Free Chemical Identification and Collection of Droplets

“…Among one of the most common uses of absorbance in a life science laboratory is to monitor cell growth label-free. Recent publications have demonstrated this in droplets using light scattering 19 or capacitive sensing, 24 but they lack the full spectral data that are typically acquired in a benchtop system. To assess cell-growth measurements in our in-droplet UV−vis platform, this GFP-expressing strain of E. coli K12 MG1655 [pSEVA271-sfgfp] 32 was used.…”

“…To address this bottleneck, there has been tremendous progress in recent years in integrating droplet microfluidics with a number of label-free analytical measurements. ,,,− − ,− Notably, Holland-Moritz et al developed a mass spectrometry-activated droplet sorter that circumvents the sample-destructive nature of mass spectrometry by splitting droplets into two streams, analyzing a portion with electrospray ionization mass spectrometry in real-time, and sorting the partner droplet stream based on the mass spectra. While a very promising approach, its overall throughput of 0.7 samples per second leaves much to be desired for high-throughput screens.…”

Microfluidics for antibiotic susceptibility testing

“…We suggest that resazurin should not be a marker of choice for the detection of bacterial growth in clinical samples, and other label-free techniques should be applied. 150 3.1.2. Optical density.…”