Biology subjects

Cowell, T. W.

Publications and source records attributed to Cowell, T. W..

3 recordsLinked to original sources

bioRxiv2024

Drop-by-drop Addition of Reagents to a Double Emulsion

Developments in droplet microfluidic assays have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, identification of rare variants, and more. Double emulsions (DEs) have seen new interest in recent years for their potential to enable new droplet assays and build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows, addressing inherent limitations of SEs. Despite these strengths, DEs lack important technical functions that exist in SEs such as picoinjection or any other method for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, we report a simple device achieving picoinjection of DEs. We developed strategies to enable active manipulations on DEs by converting DE inputs to SEs on chip. The released aqueous cores of the DE can be manipulated using existing SE techniques, such as reagent addition, before reforming a DE at the outlet. We identified device designs and operation conditions achieving drop-by-drop reagent addition to DEs and used it as part of a muti-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.

bioengineering↗

bioRxiv2024

Unbiased, Cell-free Profiling of Single Influenza Genomes at High-throughput

The segmented structure of the Influenza A virus (IAV) genome facilitates reassortment, segment exchange during co-infection. When divergent strains mix across human, agricultural, and wildlife reservoirs novel strains are generated, which has been the source of pandemics. Due to the limited throughput and infection-based assays, IAV reassortment studies has been limited to permissive reassortment. We have developed DE-flowSVP to achieve extremely high throughput, direct profiling of as many as 105 IAV particles in a single-day experiment and enabled quantitative profiling of reassortment propensity between divergent strains for the first time. By profiling reassortants between two naturally circulating low-pathogenicity avian IAVs, we confirmed that molecular incompatibility yields strong preference toward within-strain mixing. Surprisingly, we revealed that two-to-three particle aggregation contributed primarily to genome mixing (75-99%), suggesting that aggregation mediated by sialic acid binding by viral surface proteins provides a secondary pathway to genome mixing while avoiding the co-packaging fitness cost. We showed that genome mixing is sensitively dependent on co-infection timing, relative segment abundances, and viral surface-protein background. DE-flowSVP enables large-scale survey of reassortment potential among the broad diversity of IAV strains informing pandemic strain emergence.

molecular biology↗

bioRxiv2021

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent content mixing, which is essential for single entity analysis. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer fabrication method for DE drop makers. This design is powerful as it allows robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets effectively maintain the compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.

bioengineering↗