Label-Free Optical Readout of Bacteria Density in Nanoliter Droplets

Summary form only given. Droplet microfluidics is an important technique, used for antibiotic susceptibility testing and analysing bacteria at the single cell level. Optical screening methods allow for fast and reliable readout of bacteria density in droplets, however most of those optofluidic platforms are based on fluorescent light detection. This limits the applicability of the method only to genetically modified and labelled bacteria strains [1]. Here, we demonstrate a label-free readout of bacteria density in nanoliter droplets. This is achieved by detection of light scattered by bacteria inside the droplet. We demonstrate the we can differentiate empty droplets and the ones where bacteria proliferated. Additionally, we included a fluorescence detection path to verify our readout. The experiments are performed on the example of Escherichia coli proliferation. Droplets are generated using microfluidic chip in flow-focusing junction, where a continuous oil phase containing surfactant divides a sample into nanoliter compartments [2] and screened in similar chip with an inlet for an optical fiber. A 475 nm laser beam is focused by a 20x objective in the middle of the detection channel. The scattered light is collected by a multimode fiber (which simplifies the setup and makes it more robust) and detected by an APD. A band-pass filter is used to filter out the fluorescence and pass the scattered light only. Whereas fluorescence signal collected by the objective is reflected by a dichroic mirror, falls into a photomultiplier in confocal configuration. Both collected signals are recorded simultaneously and processed by a custom -written LabVIEW software. First the setup is tested on the sample containing droplets with growth medium only. This result in the detection of uniformly distributed peaks, each corresponding to a single droplet. The average peak intensity is 37 mV and 35 mV in scattering and fluorescence channels, respectively. Next, screening of the library containing large number of bacteria in growth medium (overnight culture, 10 8 -10 9 CFU/ml) results in detection of higher peak intensities, reaching 48 mV in scattering and 404 mV in fluorescence channels. Next, we tested the performance of the system using a model cell library constructed by mixing droplets with medium (70%) and containing bacteria (30%). The fluorescence light signal showed the detection of 30.5% positive hits, while scattered light single showed 32.5%, which both are very close to the theoretical value. A histogram of scattered light further confirms that negative population of droplets without any cells is easily distinguishable from positive population containing high concentration of bacteria.