Over the past few years, food safety has become a hot topic of research due to the rapid rise in foodborne diseases. Consumption of food contaminated with pathogens or toxins has caused numerous fatal health incidents worldwide. Biosensing platforms capable of early detection of food contaminants with high specificity and sensitivity are urgently required. Conventional immuno-based detection techniques, such as enzyme-linked immunosorbent assay (ELISA), utilize natural peroxidase enzymes (horseradish peroxidase: HRP), and although they have merit they also have several drawbacks such as susceptibility for inhibition of its peroxidase activity and high production cost. Artificial nanomaterials that mimic peroxidase activity have attracted interest as an alternative as they can overcome the inherent drawbacks of the natural enzymes. Advancements in nanotechnology have provided a potential solution in the search for a suitable peroxidase mimicking artificial enzyme. The unique surface morphology of gold particles (Au NPs) combined with their chemical characteristics has led to the investigation of their high catalytic efficiency as a peroxidase mimicking enzyme, so-called nanozymes. In the present study, we have biologically synthesized gold nanozymes with controlled morphology and surface chemistry. They possess ultra-active intrinsic peroxidase-like activity and can catalyse the oxidation of 3,3′,5,5′ -tetramethylbenzidine (TMB) in presence of H2O2. Synthesis of Au NPs using Prunus nepalensis fruit extract was performed after standard biochemical characterizations of fruit extract such as total phenolic content, reducing power assay. Characterizations of the biogenic Au NPs were performed using UV-visible spectrophotometer, X-Ray Diffraction (XRD) and Transmission Electron Microscopy (TEM). Analysis using Michaelis–Menten kinetics determined superior affinity of the biogenic Au NPs towards both the chromogen (TMB) and the substrate (H2O2) when compared with the natural HRP enzyme. The Michaelis–Menten constant (Km) for biogenic Au NPs and for HRP was found to be 3.23×10-2 mM and 6.083.23×10-2 mM, respectively at the same nanozyme/enzyme concentration of 100 pM and under 6% H2O2. To investigate its applicability for biosensing analytics, the biogenic Au NPs surface was modified using a direct coupling conjugation method and a monoclonal antibody specific for Mycobacterium bovis attached. The aim was to develop an immunoassay for detection of M. bovis, a zoonotic pathogen transmitted via contaminated milk and meat products. The biogenic AuNP conjugate showed ultra-active peroxidase activity (only 1.71% suppression was observed), and the immunological activity of the antibody was maintained. Our preliminary results are promising and indicate the potential for development of a cost-effective biosensing platform with high sensitivity and specificity for the detection of M. bovis using antibody conjugated Au nanozymes.
|Publication status||Published - Oct 2019|
|Event||The 9th International Symposium on Recent Advances in Food Analysis - Clarion Congress Hotel, Prague, Czech Republic|
Duration: 05 Nov 2019 → 08 Nov 2019
Conference number: 9
|Conference||The 9th International Symposium on Recent Advances in Food Analysis|
|Abbreviated title||RAFA 2019|
|Period||05/11/2019 → 08/11/2019|
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Student thesis: Doctoral Thesis › Doctor of Philosophy
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Student thesis: Doctoral Thesis › Doctor of PhilosophyFile