scMAR-Seq: a novel workflow for targeted single-cell genomics of microorganisms using radioactive labeling.

IMPORTANCE: A central question in microbial ecology is which member of a community performs a particular metabolism. Several sophisticated isotope labeling techniques are available for analyzing the metabolic function of populations and individual cells in a community. However, these methods are generally either insufficiently sensitive or throughput-limited and thus have limited applicability for the study of complex environmental samples. Here, we present a novel approach that combines highly sensitive radioisotope tracking, microfluidics, high-throughput sorting, and single-cell genomics to simultaneously detect and identify individual microbial cells based solely on their in situ metabolic activity, without prior information on community structure.

Quantification of Biocatalytic Transformations by Single Microbial Cells Enabled by Tailored Integration of Droplet Microfluidics and Mass Spectrometry.

Improving the performance of chemical transformations catalysed by microbial biocatalysts requires a deep understanding of cellular processes. While the cellular heterogeneity of cellular characteristics, such as the concentration of high abundant cellular content, is well studied, little is known about the reactivity of individual cells and its impact on the chemical identity, quantity, and purity of excreted products. Biocatalytic transformations were monitored chemically specific and quantifiable at the single-cell level by integrating droplet microfluidics, cell imaging, and mass spectrometry. Product formation rates for individual Saccharomyces cerevisiae cells were obtained by i) incubating nanolitre-sized droplets for product accumulation in microfluidic devices, ii) an imaging setup to determine the number of cells in the droplets, and iii) electrospray ionisation mass spectrometry for reading the chemical contents of individual droplets. These findings now enable the study of whole-cell biocatalysis at single-cell resolution.

Coupling Droplet Microfluidics with Ion Mobility Spectrometry for Monitoring Chemical Conversions at Nanoliter Scale.

We introduce the coupling of droplet microfluidics and ion mobility spectrometry (IMS) to address the challenges of label-free and chemical-specific detection of compounds in individual droplets. In analogy to the established use of mass spectrometry, droplet-IMS coupling can be also achieved via electrospray ionization but with significantly less instrumental effort. Because IMS instruments do not require high-vacuum systems, they are very compact, cost-effective, and robust, making them an ideal candidate as a chemical-specific end-of-line detector for segmented flow experiments. Herein, we demonstrate the successful coupling of droplet microfluidics with a custom-built high-resolution drift tube IMS system for monitoring chemical reactions in nL-sized droplets in an oil phase. The analytes contained in each droplet were assigned according to their characteristic ion mobility with limit of detections down to 200 nM to 1 µM and droplet frequencies ranging from 0.1 to 0.5 Hz. Using a custom sheath flow electrospray interface, we have further achieved the chemical-specific monitoring of a biochemical transformation catalyzed by a few hundred yeast cells, at single droplet level.

On-chip mass spectrometric analysis in non-polar solvents by liquid beam infrared matrix-assisted laser dispersion/ionization.

By the on-chip integration of a droplet generator in front of an emitter tip, droplets of non-polar solvents are generated in a free jet of an aqueous matrix. When an IR laser irradiates this free liquid jet consisting of water as the continuous phase and the non-polar solvent as the dispersed droplet phase, the solutes in the droplets are ionized. This ionization at atmospheric pressure enables the mass spectrometric analysis of non-polar compounds with the aid of a surrounding aqueous matrix that absorbs IR light. This works both for non-polar solvents such as n-heptane and for water non-miscible solvents like chloroform. In a proof of concept study, this approach is applied to monitor a photooxidation of N-phenyl-1,2,3,4-tetrahydroisoquinoline. By using water as an infrared absorbing matrix, analytes, dissolved in non-polar solvents from reactions carried out on a microchip, can be desorbed and ionized for investigation by mass spectrometry.

Conversion Efficiencies of a Few Living Microbial Cells Detected at a High Throughput by Droplet-Based ESI-MS.

The label-free and sensitive detection of synthesis products from single microbial cells remains the bottleneck for determining the specific turnover numbers of individual whole-cell biocatalysts. We demonstrate the detection of lysine synthesized by only a few living cells in microfluidic droplets via mass spectrometry. Biocatalyst turnover numbers were analyzed using rationally designed reaction environments compatible with mass spectrometry, which were decoupled from cell growth and showed high specific turnover rates (∼1 fmol/(cell h)), high conversion yields (25%), and long-term catalyst stability (>14h). The heterogeneity of the cellular reactivity of only 15 ± 5 single biocatalysts per droplet could be demonstrated for the first time by parallelizing the droplet incubation. These results enable the resolution of biocatalysis beyond averages of populations. This is a key step toward quantifying specific reactivities of single cells as minimal functional catalytic units.

Fluorescence lifetime-activated droplet sorting in microfluidic chip systems.

We present a highly efficient microfluidic fluorescence lifetime-activated droplet sorting (FLADS) approach as a novel technology for droplet manipulation in lab-on-a-chip devices. In a proof-of-concept study, we successfully applied the approach to sort droplets containing two different fluorescent compounds on the basis of their corresponding fluorescence lifetime. Towards this end, a technical set-up was developed enabling on-the-fly fluorescence lifetime determination of passing droplets. The herein developed LabVIEW program enabled fast triggering of a downstream dielectrophoretic force sorting functionality depending on average fluorescence lifetimes of individual droplets. The approach worked reliably at individual substrate concentrations from 1 nM to 1 mM. This not only allowed reliable sorting of droplets containing species with different fluorescence lifetimes but also enabled differentiation of mixtures in individual droplets.

Publisher Correction: Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

An integrated chip-mass spectrometry and epifluorescence approach for online monitoring of bioactive metabolites from incubated Actinobacteria in picoliter droplets.

We present a lab-on-a-chip approach for the analysis of secondary metabolites produced in microfluidic droplets by simultaneous epifluorescence microscopy and electrospray ionization mass spectrometry (ESI-MS). The approach includes encapsulation and long-term off-chip incubation of microbes in surfactant-stabilized droplets followed by a transfer of droplets into a microfluidic chip for subsequent analysis. Before the reinjected droplets are spaced and electrosprayed from an integrated emitter into a mass spectrometer, the presence of fluorescent marker molecules is monitored nearly simultaneously with a custom-made portable epifluorescence microscope. This combined fluorescence and MS-detection setup allows the analysis of metabolites and fluorescent labels in a complex biological matrix at a single droplet level. Using hyphae of Streptomyces griseus, encapsulated in microfluidic droplets of ~ 200 picoliter as a model system, we show the detection of in situ produced streptomycin by ESI-MS and the feasibility of detecting fluorophores inside droplets shortly before they are electrosprayed. The presented method expands the analytical toolbox for the discovery of bioactive metabolites such as novel antibiotics, produced by microorganisms.

Detection of antibiotics synthetized in microfluidic picolitre-droplets by various actinobacteria.

The natural bacterial diversity is regarded as a treasure trove for natural products. However, accessing complex cell mixtures derived from environmental samples in standardized high-throughput screenings is challenging. Here, we present a droplet-based microfluidic platform for ultrahigh-throughput screenings able to directly harness the diversity of entire microbial communities. This platform combines extensive cultivation protocols in aqueous droplets starting from single cells or spores with modular detection methods for produced antimicrobial compounds. After long-term incubation for bacterial cell propagation and metabolite production, we implemented a setup for mass spectrometric analysis relying on direct electrospray ionization and injection of single droplets. Even in the presence of dense biomass we show robust detection of streptomycin on the single droplet level. Furthermore, we developed an ultrahigh-throughput screening based on a functional whole-cell assay by picoinjecting reporter cells into droplets. Depending on the survival of reporter cells, droplets were selected for the isolation of producing bacteria, which we demonstrated for a microbial soil community. The established ultrahigh-throughput screening for producers of antibiotics in miniaturized bioreactors in which diverse cell mixtures can be screened on the single cell level is a promising approach to find novel antimicrobial scaffolds.