Nanostructured NiO thin films for electrochemical and colorimetric biosensors
Creators: Tripathi, Anuja
Publisher: name: University of Alberta Library
Year: 2022

<p>Biosensors are devices that detect biological or chemical responses by generating signals that are proportional to the concentration of the analyte. An ideal biosensor should be highly sensitive, selective, and reproducible, with a low detection limit and rapid response time. Nanostructured porous materials are advancing the development of new types of electrochemical and colorimetric biosensors that can detect chemical compounds associated with food spoilage and medical conditions. In this thesis, I developed both electrochemical and colorimetric biosensors, which are united by fabrication technique and material systems.<br>
An electrochemical biosensor is a simple and low-cost sensing device. The working electrode material has a significant impact on the analytical performance of such biosensors, and methods that increase the surface area, such as nanoparticle incorporation, have been shown to improve sensor performance. Enzymes act as highly selective catalysts, which means they only speed up one type of reaction at a time and generate a product that can be used to detect desired compound selectively. Electrode incorporated with nanoparticles exhibit high surface area and porosity, which allows enhanced enzyme immobilization towards the development of a selective and sensitive sensor with high response time. Amongst several nanoparticle types, metal and metal oxides based nanoparticles have been widely investigated for serving this purpose.<br>
Colorimetric biosensing platforms, which are counterparts of electrochemical sensors, often use enzymes as catalysts, which require well-controlled operating conditions (temperature, pH, and purity) for proper functioning. However, precise control of the operating and storage conditions can be difficult to achieve. Nanozymes are high surface area nanostructures that mimic enzyme-like catalytic properties. However, most of the reported nanozymes are dispersed in solution, which makes them hard to recover after use. Moreover, nanozymes tend to have a low density of active sites which result in lesser catalytic activity than natural enzymes. <br>
This thesis investigates the glancing angle deposition (GLAD) technique to explore inexpensive transition metal oxides such as NiO for developing enzymatic electrochemical and colorimetric biosensors. GLAD is a physical vapor deposition technique that exploits atomic shadowing and dynamic motion control to engineer nanostructures with high surface area and controlled porosity. The high specific surface area leads to the high concentration of catalytic active sites, which can improve the physicochemical properties of electrochemical and colorimetric biosensors. <br>
In the first project, macroporous NiO electrodes were fabricated using the GLAD technique to develop an enzymatic electrochemical sensing of xanthine (XA), an indicator for fish freshness. GLAD NiO electrodes were used due to their high isoelectric point, biocompatibility, chemical stability, and well-defined porosity. This provided an accessible surface for enzyme (xanthine oxidase, XO) immobilization using physisorption technique which was used to selectively monitor XA. The developed XA electrochemical sensor showed the dynamic range of 0.1 µM to 650 µM, limit of detection of 37 nM, good reproducibility (relative standard deviation of ~ 4%, n=18), rapid response time (~7 s), and a high sensitivity (1.1 µA·µM-1·cm-2 in the low concentration range from 0.1-5 µM, and 0.3 µA·µM-1·cm-2 in the higher concentration range from 5-650 µM). Overall, the sensor was more sensitive in comparison to the flat NiO-based enzymatic sensor (0.03 µA·µM-1·cm-2). Moreover, the sensor showed less interference from common fish sample matrices (such as glucose, uric acid, hypoxanthine) and the common fish preservative (sodium benzoate). The sensor also did not loose its performance when stored in buffer at 4oC for over a week. <br>
Natural enzyme such as horseradish peroxidase (HRP) is used as a catalyst in the colorimetric detection of uric acid (UA), which is a biomarker for gout, arthritis, and high blood pressure. Hence, in my second work, Ni GLAD film on Si substrate was used to mimic HRP for accelerating the oxidation reaction of colorless 3,3’,5,5’-tetramethybenzidine (TMB) to blue colored (oxTMB) in the presence of hydrogen peroxide (H2O2). This reaction is dependent on UA, allowing the concentration of UA to be determined based on the solution color. The absorbance of oxidized TMB solutions was measured by UV-VIS spectroscopy to detect the concentration of UA added to the solution, while surface characterization and morphology studies of Ni GLAD films were done by XPS and SEM. In the first phase of this study, the concentration of TMB, amount of H2O2, contact time between GLAD films and reacting mixture, and pH of reactants were optimized to achieve high optical absorbance at 652 nm as measured by UV VIS (corresponding to a blue solution). As these thin films were surface-anchored structures, they could be easily recovered after use and be reused. Next, the catalytic activity of the nanostructured film was compared to a flat Ni reference sample. Results showed the excellent reusability of the GLAD film in 1.6 mM TMB and 0.29 M H2O2 solution, with LOD of 3 µM, exhibiting its practical relevancy to detect UA under physiological conditions. Furthermore, the developed colorimetric sensor was also tested against glucose and urea to examine its potential for use as a UA biomarker in clinical diagnosis. Calculated Michaelis Menten constant (Km) of the Ni GLAD film ranged ~1 mM, which confirmed high peroxidase-like activity in comparison to mono metal nanoparticles-based nanozymes. However, the fabricated Ni GLAD-based nanozyme was not up to the mark of natural peroxidase enzyme (Km = 0.43 mM). Low Michaelis-Menten constant (Km) demonstrates the high effectivity of the catalyst.  Therefore, in the third work, further enhancement of the surface energy of nanozymes were conducted via N plasma functionalization. Surface characterization and morphology studies by XRD, TEM, XPS and SEM, confirmed the presence of adsorbed N3- ions as catalytically-active centers for accelerating electron transfer. Hence, an improved catalytic activity was achieved which resulted in low UA LOD (1 µM) and improved Km (0.17 mM). The functionalized Ni GLAD nanozyme with N plasma even exhibited high peroxidase-like activity for colorimetric sensing of UA. The potential application of functionalized GLAD nanozymes was also demonstrated through a gravity-driven continuous catalytic reaction device for colorimetric sensing of UA using paper based point of care device.</p>