Developing a combined microfluidic and hydrogen platform for single-cell assays

Thesis

Microfluidic technologies can be utilised for the compartmentalisation of a bulk reaction into millions of water-in-oil droplets. Adapting bulk reaction methods to microfluidic environments enables sequences of multiple reactions to occur simultaneously on separate target molecules. Individual cells or strands of genetic material can be encapsulated within these droplets to compartmentalise biological assays for single cell resolution. Microfluidic bioassays can be used for the detection or sequencing of rare targets such as HIV provirus. This thesis develops robust experimental methods for several key steps in a proposed combined microfluidic and hydrogel platform for single-cell assays. While this work is shown with the context and motivation of characterising the HIV latent reservoir, the experimental systems developed could be used for a range of bioassays.

We develop a methodology to create hydrogel microspheres from water-in-oil droplets which are capable of trapping DNA at a single-cell resolution while allowing washing with reaction buffers. In essence these would act as micro-scale analogues to bulk size-based filtration techniques. We propose the encapsulation of cells and sol-phase hydrogel within water-in-oil droplets. Cell lysis and cross-linking of the hydrogel would trap the genomic DNA within hydrogel microspheres. Washing and re-encapsulation of the microspheres alongside the reagents required for polymerase chain reaction (PCR) would produce a new suspension. Successful PCR would produce a fluorescent droplet when the target DNA is present. Sorting these droplets allows for the sequencing of only genomic material containing target DNA.

We start by developing a reliable methodology to produce alginate and agarose microspheres with the physical and chemical properties required for the proposed assay. We investigate the viscoelastic properties of bulk alginate and agarose hydrogels and then predict how the composition of microspheres affects their pore size and trapping ability. We develop robust methodologies for cell lysis, the trapping of genomic DNA within hydrogel microspheres, and the re-encapsulation of these microspheres. We compare the efficiency of PCR within different hydrogel networks, showing that alginate networks inhibit PCR, while agarose networks allow successful PCR. We discuss the work required to achieve successful PCR within alginate networks. Finally, we quantify the ability of the microspheres to trap particles, cells and DNA. We show how the composition of alginate hydrogel affects their trapping ability and investigate the diffusion of DNA through microspheres of various hydrogel compositions. We conclude by investigating the pore size of microspheres using fluorescent and confocal imaging techniques.

Authors: Beck, C

• DOI: 10.5287/ora-go05y7eao
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: HIV assays; droplet microfluidics; single-cell
• Subjects: Chemistry, Physical and theoretical; Microfluidics