title: The Development of a MWCNT-DAO Biosensor for the Detection of Cadaverine in the Assessment of Periodontal Disease
Creators: name: Amin, M
Publisher: name: Liverpool John Moores University
Year: 2023

description: Periodontal disease is one the most common afflictions of human populations and is a major challenge in both the developed and developing world. Cadaverine is a biomolecule which has implications in a myriad of human diseases, in particular periodontal disease. Porphyromonas gingivalis has been suggested as one of the key microorganisms in periodontal disease, and its pathogenesis has been extensively researched. It has a number of metabolites which contribute towards its pathogenicity, and among them, cadaverine has seen particularly detrimental effects in both oral and systemic health. Traditional testing methods for periodontal disease do not provide clinicians with active disease state measurements, but instead provide information on the history of the disease. Thus, the development of a real-time biosensor, providing point-of-care information regarding the metabolite status of the oral cavity would be of significant impact for oral disease diagnostics. This thesis reports the development and testing of a newly designed working electrode fitted to a flexible screen-printed sensor platform for the detection of the biogenic amine, cadaverine. The results in chapter 3 showed that DAO enhanced MWCNT dispersion and increased overall electrode surface topographies, resulting in a more wettable electrode for faster, more efficient electron transfer kinetics. Scanning electron microscopy, UV-Vis, Fourier transform intra-red spectroscopy, raman spectroscopy, and energy diffraction x-ray spectroscopy confirmed the functional group changes the MWCNT’s underwent during modification. Investigations into the modified biosensors heterogenous electron transfer rates were carried out and determined an increase to the peak to peak separation, possible due to the additional modification layers on the electrode surface, and the presence of a binder. Chapter 4 investigated electrochemical efficacy of the MWCNT-DAO biosensor against cadaverine in stock solutions, and artificially simulated saliva. The biosensor demonstrated positive concentration dependant correlations towards cadaverine from a range of 3 µg/ml to 150 µg/ml. Similarly in artificial saliva, the biosensors efficacy remained consistent, and presented a potential for the biosensor to function outside of stock sample solutions. The cytotoxic effects of P. gingivalis and cadaverine were investigated in Chapter 5. Initial monocyte differentiation into macrophages was confirmed using flow cytometry. Scanning electron microscopy was used to demonstrate the phagocytic effects of macrophages towards P. gingivalis and resulted in phagocytosis being visualised by the pseudopodia-like appendages engulfing the bacterium. Cell viability, and cell migration assays, and showed significant reductions in the viability and migratory effects of human epithelial keratinocyte and M0 macrophage cells in response to a P. gingivalis infection, and as a response to increasing cadaverine levels, above the physiological normal thresholds. The findings from this study present a MWCNT-DAO biosensor which was able to detect cadaverine at concentrations which are respective to those in periodontal disease, in stock solutions and in simulated human saliva. Furthermore, the cytotoxic effects of cadaverine and its precursor bacteria, P. gingivalis were elucidated, and showed significant detrimental effects towards human macrophage and human epithelial keratinocyte cells. thus, the biosensor developed in this study may be used as a tool for determining the extent of a patient’s periodontal disease, using a rapid, cost effective and point-of-care biosensor.