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Protein Biophysics in Hollow-Core Photonic Crystal Fibres: Label-Free Monitoring of Biomolecules in Optofluidic Waveguides https://doi.org/10.17863/CAM.101735 Heck, Jan Euser, Tijmen Knowles, tuomas Proteins are the central building blocks of life, and experimental methods for their study are fundamental to deepening our under- standing of biology and the life sciences. When studying protein interactions, we seek new experimental methods that can detect and monitor proteins in solution, at small volumes and low con- centrations. Of particular interest are optical methods that leave the system undisturbed and allow for in-situ measurements over a span of time. Indirect optical detection, using fluorescent stains or genetically-incorporated fluorophores as proxies, has enabled these aims in many applications. However, these labels have been shown to alter protein behaviour, such as the protein–protein interactions that determine their aggregation mechanisms. In this PhD dissertation, we study the use of hollow-core photonic crystal fibre (HC-PCF), a class of optofluidic waveguides, to optically detect and monitor proteins in microfluidic environments without labels. Within this waveguiding microfluidic fibre geometry, we monitor the label-free optical extinction and fluorescence of different proteins in the ultraviolet spectrum, in native and aggregated forms, and over extended periods of time as they undergo aggregation. Specifically, we developed and demonstrate here three applica- tions that form the experimental chapters of this dissertation. First, we quantify serum proteins by exciting and collecting their in- trinsic protein fluorescence in the deep ultraviolet (𝜆exc = 280 nm, 𝜆ems = 350 nm) within a HC-PCF (49 nL cm−1) under continuous flow at sub-micromolar concentrations. Second, we adapt and re- design this system to enable long-pathlength (17 cm), ultraviolet (365 nm) extinction measurements on protein aggregates. Finally, combining these two techniques into a multiplexed measurement, we demonstrate the ability to monitor the aggregation kinetics of silk protein over several days in both extinction and fluorescence. University of Cambridge 2023-07-20 Thesis Doctoral Doctor of Philosophy (PhD) eng EPSRC Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1) https://www.repository.cam.ac.uk/handle/1810/357612 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/126f5d0c-0588-400d-b5a0-17cc7c52575f/download fc883e1c220742f5cfaf1cf57c7818b4 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ad115a19-a4df-4848-aecb-88e9238c2446/download 87eda9de84448d1f82354d60eee3eb5f https://creativecommons.org/licenses/by-sa/4.0/ microfluidics photonics hollow-core photonic crystal fibre hollow-core photonic crystal fiber optics biophysics proteins waveguide optofluidics label-free protein biophysics photonic crystal optical fiber optical fibre