progression of biofilm formation and how it impacts antibiotic resistance 42. This concept could be extended to test various antimicrobial coatings and their properties.

A method to monitor the level of oxygen in microdroplets is presented. Optical sensor nanoparticles are dispersed in the aqueous phase of the microfluidic droplets for culturing bacteria. The oxygen sensor nanoparticles consist of phosphorescent indicator dye embedded in poly(styrene-block-vinylpyrrolidone) nanobeads. The nanoparticles are excitable by red light and emit in the near-infrared spectra region which minimizes background fluorescence from biological matter. The biocompatibility of the nanoparticles was proven. Nanoparticles sensors were read out by adapted miniaturized oxygen meters. The instruments can be easily integrated into the microfluidic system by placing it next to the tubing and measuring through the tubing wall. The phosphorescence lifetime-based measurement circumvents the drawbacks of intensity-based measurements and enables the determination of the absolute oxygen concentration in individual moving droplets. The technique can also be used for monitoring the growth of bacteria in microdroplets. We demonstrate simultaneous measurement of concentration of oxygen and optical density (OD) from micro cultures of E. coli and M. smegmatis.

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.

Heteroresistance is a phenomenon where isogenic bacteria population exhibits a diverse antibiotic resistance pattern at sub-population or single cell level. The sub-populations with higher resistance can remain undetected with conventional diagnostics which makes them subsequently harder to treat. Such surviving phenotypically heterogeneous sub-populations are also a potential hotbed for novel mutations, thus increasing the resistance permanently in bacteria. Droplet microfluidics gives tools for high-throughput analysis of bacteria and their response to antibiotics at single cell level, which is difficult to obtain with traditional agar plate technologies. In here we show for the first time the precise digital quantification of drug resistance profile in isogenic population at single cell level. We also see that the inhibiting amount of drug per bacteria remains quite stable regardless of bacteria density. Interestingly, the bacteria clump together preferably near these sub-inhibitory conditions. The technology and findings we describe here provide novel quantitative insight into the heteroresistance which is a key step in understanding the pathways leading to drug resistance. This knowledge is crucial in the context of global drug resistance threat as it can help us to find tools to prevent further escalation of drug resistance.

Networks of droplets, in which aqueous compartments are separated by lipid bilayers, have shown great potential as a model for biological transmembrane communication. We present a microfluidic system which allows for on-demand generation of droplets that are hydrodynamically locked in a trapping structure. As a result, the system enables the formation of a network of four droplets connected via lipid bilayers and the positions of each droplet in the network can be controlled thanks to automation of microfluidic operations. We perform electrophysiological measurements of ionic currents indicating interactions between nanopores and small molecules to prove the potential of the device in screening of the inhibitors acting on membrane proteins. We also demonstrate, for the first time, a microfluidic droplet interface bilayer (DIB) system in which the testing of inhibitors can be performed without direct contact between the tested sample and the electrodes recording picoampere currents.

We demonstrate the utility of non-contact printing to fabricate the mAST—an easy-to-operate, microwell-based microfluidic device for combinatorial antibiotic susceptibility testing (AST) in a point-of-care format. The wells are prefilled with antibiotics in any desired concentration and combination by non-contact printing (spotting). For the execution of the AST, the only requirements are the mAST device, the sample, and the incubation chamber. Bacteria proliferation can be continuously monitored by using an absorbance reader. We investigate the profile of resistance of two reference Escherichia coli strains, report the minimum inhibitory concentration (MIC) for single antibiotics, and assess drug–drug interactions in cocktails by using the Bliss independence model.

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