| Summary: | Droplet-based microfluidic systems have recently been developed to overcome the problems of slow mixing and dispersion associated with traditional microfluidic systems. By utilising flow instabilities between two immiscible phases, droplets can be generated using normal microfluidic formats. Further, aqueous solutions can be confined and mixed within droplets, resulting in rapid homogenisation and no dispersion. Accordingly, droplet-based microfluidic systems have been utilised in various applications in a high-throughput manner. However, the techniques and methods for droplet formation, manipulation and detection have been continuously studied and improved upon to develop, prepare, manipulate and implement droplet systems for real-world applications. Since droplets can be controllably produced with variable reagent compositions at high generation frequencies (1 kHz or above), on-line detection and characterisation of every high-speed droplet is one of the most important challenges associated with droplet analysis. The ability to extract information from each droplet microreactor is crucial for applications in high-throughput analysis and screening. An appropriate detection technique able to extract the vast amount of information produced in such systems is key in unlocking the full capabilities of droplet-based. In this work, a custom built confocal spectroscopic system was coupled with a droplet-based microfluidic system to conduct high-sensitivity and high-throughput biological experiments. The integration of a confocal system allows for online characterisation of individual droplets in terms of their size, formation frequency, fluorescence intensity and population. The combination of a droplet-based microfluidic system and the confocal detection setup has been successfully used to demonstrate a few high-throughput chemical and biological applications. For example, the droplet system was utilised to demonstrate high-throughput single cell encapsulation, characterisation and quantification for the first time. In addition, highthroughput binding assays and kinetic measurements using a well-known streptavidin-biotin binding model and a protein-protein interaction were performed. Furthermore, a novel approach for fluorescence lifetime imaging (FLIM) was developed and used to analyse mixing patterns within droplets. Specifically, data from FLIM measurements were extracted to determine spatially localised fluorescence lifetimes within droplets and thus a twodimensional map of droplet mixing. Finally, the droplet-based microfluidic approach was exploited to perform biological analysis at the single molecule level.
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