Many droplet microfluidic assays have been described in the literature over the last decade of research, however, there has been little reported industrial use of droplet microfluidics in drug discovery compound screening, and in particular that of P450 enzyme inhibition assays for profiling drug-drug interactions. This is partly for Intellectual Property reasons, since Pharmaceutical companies do not wish to give away trade secrets in a competitive market, but also because the technology is not yet 'proven' and remains in the proof-of-concept stage. In droplet microfluidics, where at least two liquid phases are encountered, it is important that leakage of material between phases is addressed. This effect has been extensively reported in the literature using fluorescent dyes, however there is very little evidence of research using large compound sets of diverse chemistry. This is probably because few researchers have access to the large pharmaceutical libraries necessary for this work. This project assessed the feasibility of translating a widely used microtitre plate-based P450 enzyme inhibition assay to droplet format; determined the extent of partitioning from droplets using a large pharmaceutical library set and attempted to model this behaviour, and thirdly, considered the pharmacological impact the droplet format may have on the assay. The P450 cytochrome 1A2 enzyme type (isoform) was chosen for translation to the micro-droplet format. Assays of this type are often conducted using fluorogenic substrates, making them favourable for relatively easy fluorescent detection in droplet format using simple optical detection assemblies. Oil selection was investigated to determine which oil systems would be better suited in respect of droplet formation. The use of surfactants in the oil phase and its impact on droplet formation was studied and the synthesis, preparation and characterisation of a custom perfluoropolyether (PFPE) surfactant ('AZF') conducted. Droplet chips were designed and fabricated to produce droplets of 200-300 µm diameter using novel channel designs and sealing techniques. The droplets were analysed by fluorescence spectroscopy using bespoke detector apparatus. Partitioning from aqueous to oil phase was studied for a small range of compounds and oils (with and without surfactant for fluorous oils). Partitioning was lowest using fluorous oils alone, and increased substantially when surfactant was included. Results from the large pharmaceutical test set suggested the percentage of compounds that may partition readily to the oil phase is low even when using surfactant. However, attempts to correlate this to known physicochemical properties and to develop a predictive model for fluorous solubility proved largely unsuccessful. Partitioning in the droplet chip using a droplet collection pooling method was difficult to quantify as a consequence of the profound impact turbulence had on partitioning. Miniaturisation of the P450 cytochrome inhibition assay to the droplet format initially gave poorly reproducible low signals. Possible causes included detector insensitivity, partitioning of reagent and/or fluorescent metabolite over longer incubation times, and binding of the 1A2 P450 cytochrome enzyme-protein at the droplet interface. Protein interaction at the droplet-oil boundary was studied by fluorescence labelling a protein contained in 200µm droplets and observing the extent of fluorescence localisation at the interface by epifluorescent and confocal fluorescence microscopy. The data from this work indicates a pronounced localisation of protein at the droplet interface, possibly leading to enzyme deactivation and the loss of signal seen for the assay in the droplet chip. A number of protein titrations were co-added to the droplets as 'blocking proteins' which were found to improve the reaction output, however were also noted to affect the pharmacology of the assay, noted by an order of magnitude shift in the reported IC50 for the test inhibitor used (fluvoxamine). The effects of compound leakage from droplets, and the possible detrimental impact on biological reagents by interaction at the droplet-oil interface, is a challenge that may limit widespread adoption of droplet MF systems in drug screening operations. Appropriate control measures and/or a means to reduce these effects are essential to enable accurate quantification with industrial drug discovery environments. The findings in this work highlight the challenges that have to be addressed for droplet microfluidic technology to be successfully incorporated into key areas of assay screening within drug discovery. In terms of further research, there is a significant requirement for the research community to delve further into these challenges and work closely with the industry sector to understand the beneficial role microfluidics can have and how to develop effective robust strategies the industry can easily adopt to progress this area of science.

<p>Microscale environments provide significant resolution and distortion challenges with respect to measurement techniques; however, with improvements to existing techniques, it is possible to gather relevant data to better understand the thermal and fluidic mechanisms at such small scales in evaporating droplets.</p> <p> </p> <p>Infrared thermography provides several unique challenges at small scales. A primary issue is that the low native resolution of traditional infrared cameras significantly hamper the collection of details of microscale features. Furthermore, surfaces exhibiting vastly different emissivities, results in inaccurate temperature measurements that can only be corrected with irradiance-based emissivity maps of the surface; however, due to the resolution limitations of infrared thermography, these emissivity maps can also display significant errors. These issues are overcome through the use of multi-frame super-resolution. The enhanced resolution allows for better capture of microscale features, therefore, enhancing the emissivity map. A quantitative error analysis of the system is conducted to quantify the feature size resolution improvement as well as the smoothing effect of super-resolution reconstruction. Furthermore, a sensitivity analysis is conducted to quantify the impact of registration uncertainty on the accuracy of the reconstruction. Finally, the improved emissivity map from super-resolution is demonstrated to show the increased accuracy over low-resolution mapping.</p> <p> </p> <p>When applied to water droplets, particularly on nonwetting surfaces, infrared thermography is confounded by the presence of nonuniform reflectivities due to the spherical curvature of the liquid-air interface. Thus, when measuring the temperature along the vertical axis of a water droplet, it is necessary to correct the reflection. Using a controlled background environment, in conjunction with the Fresnel equations, it is possible to correct the reflective effects on the interface and calculate the actual temperature profile. This allows for a better understanding of the governing mechanisms that determine the thermal transport within the droplet. While thermal conduction is the primary transport mechanism along the vertical axis of the droplet, it is determined that the temperature drop is partially dampened by the convective transport from the ambient air to the liquid interface. From this understanding revealed by the measurements, the vapor-diffusion-based model for evaporation was enhanced to better predict evaporation rates.</p> <p> </p> <p>Further exploration into the mechanisms behind droplet evaporation on nonwetting surfaces requires accurate knowledge of the internal flow behavior. In addition, the influence of the working fluid can have a significant impact on the governing mechanisms driving the flow and the magnitude of the flowrate. While water droplet evaporation has been shown to be governed by buoyancy-driven convection on nonwetting substrates, similar studies on organic liquid droplets are lacking. Particle image velocimetry is effective at generating a velocity flow field, but droplets introduce distortion due to the refraction from the spherical interface of the droplet. As such, velocity correction using a ray-tracing approach was conducted to correct the velocity magnitudes and direction. With the velocity measurements, the flow was determined to be surface-tension-driven and showed speeds that are an order of magnitude higher than those seen in buoyancy-driven flow in water droplets. This resulted in the discovery that advection plays a significant role in the transport within the droplet. As such, the vapor-diffusion-governed evaporation model was adjusted to show a dramatic improvement at predicting the temperature gradient along the vertical axis of the droplet.</p> <p> </p> <p>Armed with the knowledge of flow behavior inside droplets, it is expected that droplets with aqueous solutions should exhibit buoyancy-driven convection. The final part of this work, therefore, leverages this phenomenon to enhance mixing during reactions. Colorimetry is a technique that is widely utilized to measure the concentration of a desired sample within some liquid; the sample reacts with a reagent dye the color change is measured, usually through absorbance measurements. In particular, the Bradford assay is used to measure protein concentration by reacting the protein to a Coomassie^TM Brilliant Blue G-250. The absorbance of the dye increases, most significantly at the 590 nm wavelength, allowing for precise quantitation of the amount of protein in the solution. A droplet-based reaction chamber with buoyancy-enhanced mixing has the potential to speed up the measurement process by removing the need for a separate pre-mixing step. Furthermore, the reduced volume makes the process more efficient in terms of reactant usage. Experimental results of premixed solutions of protein sample and reagent dye show that the absorbance measurement through a droplet tracks strongly with the protein concentration. When the protein sample and dye reagent are mixed <i>in situ</i>, the complex interaction between the reactants, the mixing, and the adsorption of protein onto the substrate creates a unique temporal evolution in the measured absorbance of the droplet. The characteristic peaks and valleys of this evolution track strongly with concentration and provide the framework for measurement of concentration in a droplet-based system.</p> <p> </p> <p>This thesis extends knowledge about droplet thermal and fluidic behavior through enhanced measurement techniques. This knowledge is then leveraged in a novel application to create a simple, buoyancy-driven colorimetric reaction setup. Overall, this study contributes to the field of miniaturized, efficient reaction and measurement devices.</p>