Double emulsion droplet microfluidics and high-throughput measurements of single viruses

Droplet microfluidics relies on the rapid partitioning of fluids into monodisperse emulsions. By dividing a fluid into millions of nearly identical compartments, heterogeneous biological suspensions can be isolated for sensitive high-resolution analysis. Through compartmentalization emulsion droplets efficiently process large numbers of single cells or viruses in parallel. Despite this inherent utility of droplets, emulsion-based workflows either require clever labeling schemes to preserve single droplet resolution during a pooled analysis or some method of drop-by-drop screening. A large portion of this work focuses on double emulsification as a mechanism to allow microfluidic drops to be rapidly screened using flow cytometry. By converting standard single emulsion (SE) drops into to water-in-oil-in-water double emulsion (DE) drops, the properties of the individual compartments are retained or enhanced while becoming suspended within an aqueous medium. DE formation is generally more complex but allows for each drop to be screened or sorted using flow cytometry. A simple and easy to use DE drop generation device was designed, fabricated, and tested to enable routine DE production. The coalescence properties of SE and DE droplets were compared. DEs were advantageous due to their resistance to content mixing and ability to maintain a monodisperse size distribution. Protocols for DE drop profiling by flow cytometry are also explored. To expand the potential workflows that are possible in DEs, a device capable of drop-by-drop addition of reagents to a double emulsion was designed and tested. An emulsion-switching junction was used to convert close-packed DE drops into a spaced stream of SE drops corresponding to the aqueous cores of the initial DE. This allows for all existing SE droplet techniques to be applied to the aqueous cores of DE droplets. After manipulation, the double emulsion is regenerated. This device enables multi-step reactions in DE drops. Leveraging flow cytometry and DE microfluidics, a high-throughput single influenza genotyping platform was also developed. This work enabled the direct, culture-free measurement of tens to hundreds of thousands of single IAV particles. We were able to profile genome mixing during a co-infection event between two naturally circulating strains of avian influenza. This type of measurement was not previously possible. Approaches towards whole genome sequencing of single IAVs were also investigated. A droplet platform was also developed to cultivate the thermophilic archaea Sulfolobus islandicus and the viruses that infect it, with applications in enhanced metagenomics. Additionally, drop microfluidic synthesis of monodisperse microparticles enabled increased multiplexing of biomarker detection in a Coulter-counting-based electrical point-of-care measurement device. By leveraging droplet compartmentalization, throughput, and monodispersity, the types of possible measurements of biological systems are expanded.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Cowell, Thomas Whiting

Contributors dc:contributor

• Han, Hee-Sun
• Kenis, Paul J. A.
• Kong, Hyunjoon
• Sweedler, Jonathan V.

• Droplet Microfluidics
• Double Emulsions
• Flow Cytometry
• Single Virus
• Influenza
• Sulfolobus
• Hydrogel Microparticles

Statement dc:rights

• Copyright 2023 Thomas Cowell

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/122239

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F122239