Mass-Activated Droplet Sorting for the Selection of Lysine-Producing Escherichia coli.

Synthetic biology relies on engineering cells to have desirable properties, such as the production of select chemicals. A bottleneck in engineering methods is often the need to screen and sort variant libraries for potential activity. Droplet microfluidics is a method for high-throughput sample preparation and analysis which has the potential to improve the engineering of cells, but a limitation has been the reliance on fluorescent analysis. Here, we show the ability to select cell variants grown in 20 nL droplets at 0.5 samples/s using mass-activated droplet sorting (MADS), a method for selecting droplets based on the signal intensity measured by electrospray ionization mass spectrometry (ESI-MS). Escherichia coli variants producing lysine were used to evaluate the applicability of MADS for synthetic biology. E. coli were shown to be effectively grown in droplets, and the lysine produced by these cells was detectable using ESI-MS. Sorting of lysine-producing cells based on the MS signal was shown, yielding 96-98% purity for high-producing variants in the selected pool. Using this technique, cells were recovered after screening, enabling downstream validation via phenotyping. The presented method is translatable to whole-cell engineering for biocatalyst production.

Evaluation of Analyte Transfer between Microfluidic Droplets by Mass Spectrometry.

Droplet microfluidics enables high-throughput experimentation and screening by encapsulating chemical and biochemical samples in aqueous droplets segmented by an immiscible fluid. In such experiments, it is critical that each droplet remains chemically distinct. A common approach is to use fluorinated oils with surfactants to stabilize droplets. However, some small molecules have been observed to transport between droplets under these conditions. Attempts to study and mitigate this effect have relied on evaluating crosstalk using fluorescent molecules, which inherently limits the analyte scope and conclusions drawn about the mechanism of the effect. In this work, transport of low molecular weight compounds between droplets was investigated using electrospray ionization mass spectrometry (ESI-MS) for measurement. The use of ESI-MS significantly expands the scope of analytes that can be tested. We tested 36 structurally diverse analytes that were found to exhibit crosstalk ranging from negligible to complete transfer using HFE 7500 as the carrier fluid and 008-fluorosurfactant as a surfactant. Using this data set, we developed a predictive tool showing that high log P and log D values correlate with high crosstalk, and high polar surface area and log S correlate with low crosstalk. We then investigated several carrier fluids, surfactants, and flow conditions. It was discovered that transport is strongly dependent on all of these factors and that experimental design and surfactant tailoring can reduce carryover. We present evidence for mixed crosstalk mechanisms including both micellar and oil partitioning transfer. By understanding the driving mechanisms, surfactant and oil compositions can be designed to better reduce chemical transport for screening workflows.

Microbowls with Controlled Concavity for Accurate Microscale Mass Spectrometry.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell-based assays.

Mapping enzyme catalysis with metabolic biosensing.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

Continuous and automated slug flow nanoextraction for rapid partition coefficient measurement.

Octanol-water partition coefficients (log Kow) are widely used in pharmaceutical and environmental chemistry to assess the lipophilicity of compounds. Traditionally log Kow is determined using a shake-flask method that uses milliliters of sample and solvent and requires hours for preparation, extraction, and analysis. Here, we report an automated system for rapid log Kow determination for an array of compounds using slug flow nanoextraction (SFNE) enabled by a microfluidic chip. In the method, an autosampler is used to introduce 1 µL of sample into a microfluidic device that segments the injected volume into a series of 4 nL slugs that are each paired to an adjacent octanol slug. Each octanol-water phase pair is compartmentalized by an immiscible fluorous carrier fluid. During flow, rapid extraction occurs at each octanol-water interface. The resulting linear array of slugs flows into an online UV absorbance detector that is used to determine concentrations in the phases, allowing the log Kow to be measured. The microfluidic device allows toggling between two-phase "aqueous plug" generation (aqueous sample separated by fluorous carrier fluid) and three-phase "phase pair" generation. In this way, online calibration for detection in the aqueous phase can be achieved. The method is applied to determining log Kow for a panel of seven pharmaceutical compounds, including complete calibration curves, at three different pHs in under 2 h using 5 µL of extraction standard and 2.9 µL of octanol per extraction standard analyzed.

A droplet microfluidic platform for high-throughput photochemical reaction discovery.

The implementation of continuous flow technology is critical towards enhancing the application of photochemical reactions for industrial process development. However, there are significant time and resource constraints associated with translating discovery scale vial-based batch reactions to continuous flow scale-up conditions. Herein we report the development of a droplet microfluidic platform, which enables high-throughput reaction discovery in flow to generate pharmaceutically relevant compound libraries. This platform allows for enhanced material efficiency, as reactions can be performed on picomole scale. Furthermore, high-throughput data collection via on-line ESI mass spectrometry facilitates the rapid analysis of individual, nanoliter-sized reaction droplets at acquisition rates of 0.3 samples/s. We envision this high-throughput screening platform to expand upon the robust capabilities and impact of photochemical reactions in drug discovery and development.

High-throughput screening by droplet microfluidics: perspective into key challenges and future prospects.

In two decades of development, impressive strides have been made for automating basic laboratory operations in droplet-based microfluidics, allowing the emergence of a new form of high-throughput screening and experimentation in nanoliter to femtoliter volumes. Despite advancements in droplet storage, manipulation, and analysis, the field has not yet been widely adapted for many high-throughput screening (HTS) applications. Broad adoption and commercial development of these techniques require robust implementation of strategies for the stable storage, chemical containment, generation of libraries, sample tracking, and chemical analysis of these small samples. We discuss these challenges for implementing droplet HTS and highlight key strategies that have begun to address these concerns. Recent advances in the field leave us optimistic about the future prospects of this rapidly developing technology.