Development of a fluorescent biosensor for the personalized healthcare of diseases.
Creators: name: Sundaramoorthy, Uthayasuriya
Publisher: name: The Australian National University
Year: 2023

description: This is a series of comprehensive investigations into developing a new electrochemical sensing platform and linker library for fusion proteins using a periplasmic-binding protein-based biosensor. This work aims to enhance biosensors' performance by understanding their molecular mechanisms and developing new approaches to improve their selectivity and sensitivity. It involves multiple strategies, including analysing the sensing mechanism, exploring different models and theories related to electrochemical techniques, and conducting linker studies on the fluorescent biosensor. Specifically, it focused on three constructs: Cys-GlyFS (GRIP), Cys-LivK (Leucine), and D-serine (DalS). Based on these studies, I was able to identify fundamental phenomena that are crucial in fusion proteins. First, cysteine tag binding attachments to the screen-printed gold electrode for the recognition of gold nanoparticles; second, the benefits of electrochemical sensing techniques are highly sensitive and selective to the solute binding proteins because the redox probe reactions cause significant changes in the current by modulating the conformational changes in the binding domain when target analyte binds to it; Finally, linker studies involves varying the length and composition of the linker region between the sensing and reporter domains to optimise the performance of the biosensor. By systematically varying the linker library, we can identify optimal linker sequences that produce the desired sensitivity, selectivity, and dynamic range for a desired biosensor. In Chapters 2-3, I focus on Cys-GlyFS and Cys-LivK, performing detailed sensing analysis of the electrochemical detection technique that allows for developing highly sensitive and specific biosensors by integrating solute binding protein-based biosensors. This platform has been used to detect small molecules, specifically leucine and glycine, which are biomarkers for the early stage of type-I diabetes mellitus. In the future, optimisation of our biosensors could combine solute-binding protein-based biosensors in electrochemical detection techniques to detect point-of-care medical diagnostic and environmental monitoring. This is followed by re-engineering a solute periplasmic binding protein-based receptor to detect glycine amino acids specifically. We made a minor modification by removing fluorescent probes and binding them to in-house produced gold carbon screen-printed electrodes to monitor whether we require a fluorescent probe for sensing the target molecule. Finally, using these different models resulted in a total loss of sensing capacity. This work highlights the importance and necessity of the spacer and mobile domains to create a new sensor design. Chapter 4 describes the effects of linker libraries on the construction of fusion proteins, where two or more proteins are connected to engineer new functions. To improve sensor performance and probe the effect of different linkers on the performance of fluorescent sensors, we focused on characterising the dynamic ranges of the various sensor architectures. My contribution to this work included designing the linker library and the analysis of the fluorescence titration. Finally, the last chapter focused on the conclusions and future directions of the biosensors to detect specific small molecule biomarkers.