Formation and Passivation of Sub-Nanoliter Droplets for High Throughput Biological Assay Platforms

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.