dc.description.abstract | In Vitro Compartmentalization (IVC) is a technique that utilizes water-in-oil emulsion droplets of fL – nL volume to compartmentalize and perform biological reactions and assays in parallel. At its highest capacity it has the capability to create >10^10 droplets in just 1 mL of sample volume, meaning that 10^10 reactions can be performed in a single microcentrifuge tube. This saves time and material cost in comparison to conventional microtiter plate techniques. This
has made IVC ideal for use in a variety of areas requiring analysis of large libraries of samples, including directed evolution of proteins and RNAs, amplification of complex gene libraries using
PCR, and screening of large libraries for rare mutations. Our research focuses on the use of IVC
to perform assays within droplets generated by microfluidics. Biocompatible surfactants must be
developed alongside droplet generation techniques in order to ensure droplet stability and
optimal assay performance. When combined, the chapters presented in this work provide a platform for the formation and passivation of sub-nanoliter droplets for the performance of several high-throughput biological assays. Chapter 1 provides information on the principles of
IVC, droplet microfluidics, surfactants, and PCR amplification. In Chapter 2, we introduce two
passively-controlled emulsion generator devices for rapid formation of monodisperse emulsion droplet populations. Single-channel and multi-channel emulsion generators are operated using
only a handheld, glass syringe to pull a vacuum at the outlet of each microfluidic device. In addition, wide-field and single-droplet imaging techniques are introduced for obtaining data from
emulsion droplets. Chapter 3 details a technique for the formation of a biocompatible surfactant without synthesis, exploiting the direct interaction between commercially-available primary amines and carboxylated perfluorocarbon surfactants. This interaction was confirmed using the
analytical techniques of FT-IR, Mass Spectrometry, and NMR, as well as with qualitative
observations of emulsion formation under various physical and chemical stressors. Droplets formed using this surfactant were tested for assay biocompatibility with DNA amplification
using both PCR and RPA, and with a novel proximity FRET assay for the detection of insulin. These results showed that the interaction is sufficient for performance of these assays in emulsion droplets, and compares well in efficiency to other synthesized surfactants. Chapter 4 discusses the use of bead-based assays as complements to droplet compartmentalization. Microbeads
modified with DNA have been used by other researchers to capture and detect various targets, including cancerous cells in the blood, DNA for sequencing, and for aptamer selection. PCR is often used to amplify DNA onto the surface, and the beads can be rapidly analyzed and sorted into discrete populations using FACS technology. Compartmentalizing beads into emulsion droplets ensures parallel and efficient amplification of a single DNA sequence onto a single bead, forming clonal bead populations. We have developed several methods for
attachment of DNA to micro-beads and successfully amplified them using PCR to cover the
beads with many copies of a single DNA sequence. Finally, Chapter 5 provides conclusions and future applications for this work, including a high-throughput aptamer selection method using beads and droplet microfluidics, surface-based proximity assays that exploit the surfactant
interactions discussed in chapter 3, and multi-islet secretion measurements using the pFRET assay in droplets and previously-developed microfluidic techniques for measuring murine
pancreatic islet secretions. | en_US |