Study of recent Prussian Blue-based and uric acid biosensors with development of an enzymatic biosensor for human saliva detection of uric acid

A key point of clinical research is the improvement of the quality of life, whose fundamental requirements are prevention and monitoring. The development of tools for rapid and accurate detection of biomarkers is essential in this regard. The possibility for such devices to be used directly in the physician study or the comfort of people homes would be an additional benefit, designing it as point-of-care or PoC tools. Electrochemical biosensors, especially screen-printed ones, gather several features which make them particularly feasible for application as PoC devices, especially considering their cost effectiveness, high performance and good sensitivity comparable to that of traditional methods, avoiding lengthy and laborious procedures, large sample volumes and pre-treatments. Furthermore, electrochemical biosensors can be designed to be disposable, easy-to-use and low-cost manufacturing, which facilitates large-scale production. For such reasons, biosensor development is spreading and has a strong impact on applications not only in clinical and biomedical but also in defence, food analysis and environmental fields. Contemporary, the use of nanomaterials has hugely gained attention in the electrochemical field thanks to their high surface-to-volume ratio, which strongly improves the electrochemical performances and the loading capacity of biorecognition elements on the sensing surface of biosensors. This thesis is focused on two strategies to realize an enzymatic biosensor aimed at detecting uric acid in human saliva. The relevance of detecting this biomarker relies on its main role as a scavenger in our body and the manifestation of severe diseases when its functionality and levels are impaired, often evolving into chronicity. In part I, or the Background section, a definition and classification of biosensors are provided, especially focusing on electrochemical ones realized with enzymes. In particular, screen-printed electrodes are briefly discussed, reporting materials employed for fabrication and nanomaterials used for their functionalization and consequent improvement of analytical performances, especially Carbon Black and Prussian Blue nanomaterials. Finally, a short description of the electrochemical techniques used: cyclic voltammetry and chronoamperometry. In part II or Prussian Blue: a walkthrough since its discovery to the spreading of PB-based electrochemical biosensors in three main areas of interest, an in-depth study is reported regarding Prussian Blue nanomaterial in all its features, mainly focusing on the ameliorations provided in biosensors realized using this nanomaterial, successfully utilized for the detection of analytes of interest in the biomedical, environmental, and food analysis fields. In part III, or the outstanding role of carbon black in enhancing the performance of electrochemical biosensors, we'll explore carbon black amorphous material, focusing on its morphology, physicochemical properties, and fields of application. We'll examine the structure, characterized by aggregated nanoparticles and high surface area, which enhances interactions with other materials. The material electrical conductivity and stability make it a crucial component in many fields. Particularly, we'll delve into its role in electrochemical biosensors, where carbon black boosts sensitivity and facilitates rapid electron transfer, making it ideal for applications like glucose monitoring, pathogen detection, environmental sensing, and medical diagnostics. We aim to highlight how these properties contribute to its effectiveness in (bio) sensor applicationsn. In part IV, Nanomaterials and paper-based electrochemical devices: merging strategies for fostering sustainable detection of biomarkers, a review article of which I am co-first author, is quoted. This work examined how in recent decades, nanomaterials have significantly advanced biosensors by enhancing sensitivity, selectivity, robustness, and reproducibility. The sustainability trend has further driven the development of eco-friendly electrochemical paper-based devices, capable of detecting target analytes with high sensitivity in complex matrices. These devices are cost effective, require no external equipment due to paper capillary action, allow reagent-free operation by loading reagents onto the paper, and enable easy multistep analyses via the origami approach. Benefits offered by nanomaterials, are leading to a new generation of paper-based electrochemical biosensors for biomedical applications and the reported review covers the past decade progress, highlighting the functionalities from integrating nanomaterials with paper-based biosensors for biomarker detection. The part V, or trends and advancements in electrochemical devices for uric acid detection in the most common human diagnostic fluids: new perspectives toward the development of point of-care tools concerns about trends in the development of electrochemical sensors and biosensors for uric acid detection, which are critically compared, especially focusing on the biological matrices which have been validated. At last, part VI, or designing enzymatic biosensors for point-of-care application and monitoring of uric acid in human saliva, a comparative electrochemical study is performed for two biosensor configurations aimed at detecting uric acid in human saliva. The study was performed using screen printed electrodes modified with a nanoparticle dispersion of Carbon Black and Prussian Blue and applying the biorecognition element, which is the uricase enzyme, in two ways that characterize the different configurations. On one side, the paper pad with adsorbed uricase is applied on the modified screen-printed electrode, resulting in a hybrid origami configuration; on the other, the enzyme is directly immobilized on the previously modified sensing surface. This thesis is then concluded with closing remarks and future perspectives.