Theses and dissertations (Applied Sciences)
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Item Development of electrochemical sensors for the detection of mycotoxins in food matrices using functionalised nanocomposites(2024-05) Naidoo, Lyndon; Bisetty, Krishna; Meier, Florian; Uwaya, Gloria EbubeThe analysis of pathogens in foods is of critical importance to ensure consumer safety and quality assurance, as contaminants pose serious risks to public health. Mycotoxins are naturally occurring carcinogenic toxins that arise from specific strains of fungi as they contaminate food. They are found in a wide variety of grains, cereals, and dairy products, causing cancer in both humans and animals. Thus, there is a growing demand for simple, sensitive and inexpensive sensors for mycotoxin detection in lieu of conventionally employed large-scale instrumentation. In this study, the development of electrochemical sensors for the detection of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in foods was investigated and presented as three case studies, respectively. In the first case study, an ultrasensitive aptasensor was developed for the indirect detection of AFB1 in the presence of a ferri/ferrocyanide ([Fe(CN)6]3-/4-) redox probe solution. The sensor was constructed by immobilizing an anti-AFB1 aptamer (Apt) to a carboxylated multiwalled carbon nanotube (cMWCNT) and iron oxide (Fe3O4) nanoparticle (NP) composite using a glassy carbon electrode (GCE). This resulted in the development of the GCE/cMWCNTsFe3O4 NP/Apt sensor. An electrochemical response was exhibited from AFB1 applying cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), respectively, utilizing a [Fe(CN)6]3-/4- redox probe prepared in phosphatebuffered saline (PBS) solution with reference to the Ag/AgCl reference electrode under optimized conditions. DPV findings reported very low limits of detection (LOD) and quantification (LOQ) of 0.43 fg mL-1 and 1.44 fg mL-1 respectively in comparison to current literature, over a calibration range of 0.50 fg mL-1 to 5.00 fg mL-1. For real sample analysis, excellent spike recoveries from 95% to 105% were obtained for corn and rice flour. Density functional theory (DFT) was used to propose a reaction scheme by ascertaining the electronic properties of the redox-active functional groups of AFB1. This supported the experimental anodic response findings of DPV. The second case study focused on how PEGylated Fe3O4 NPs and cMWCNTs fabricated on a GCE could be used for the design of an electrochemical sensor for ZEN analysis. The qualitative and quantitative analyses of ZEN were completed using CV, EIS and DPV, respectively, under optimized conditions in a sodium phosphate buffer solution. The developed sensor reported significantly low LODs and LOQs of 0.34 fg mL-1 and 1.12 fg mL-1 respectively, over a calibration range of 1.00 fg mL-1 to 10.00 fg mL-1 by DPV. Excellent spike recoveries ranging from 92% to 106% were obtained for rice and corn flour. The Monte Carlo (MC) adsorption simulation studies predicted the strong interaction of ZEN with the constructed sensor. In the final case study, an OTA electrochemical sensor was designed using a nickel metalorganic framework (Ni-MOF) and carboxylated reduced graphene oxide (cRGO) on a GCE. The detection of OTA was achieved under optimized conditions in PBS solution with the developed GCE/Ni-MOF/cRGO electrode, employing CV, EIS and DPV as electrochemical tools. Applying DPV, the sensor reported very low LODs and LOQs of 3.29 fg mL-1 and 10.97 fg mL-1 respectively, over a calibration range of 10.00 fg mL-1 to 90.00 fg mL-1. Regarding real sample analysis, excellent spike recoveries from 95% to 105% were obtained for corn and rice flour. Molecular dynamics (MD) studies predicted that the Ni-MOF exhibited a strong electrostatic interaction with the OTA analyte, in agreement with the experimental findings. The synthesized nanomaterials (cMWCNTs-Fe3O4 NP, PEG-Fe3O4 NPs/cMWCNTs, and NiMOF/cRGO) utilized in this study were characterized by an array of techniques, including single particle inductively coupled plasma-mass spectrometry (spICP-MS), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), multidetector asymmetrical flow field-flow fractionation (AF4), and Fourier transform infrared spectroscopy (FTIR). Finally, computational modelling studies were undertaken to establish a synergy with the experimental approaches employed in each case study. These methodologies included DFT, docking studies, MC adsorption and MD simulations, which were aimed at predicting and assessing the atomic and molecular interactions between the mycotoxins and their respective electrode systems.Item Electrochemical enzymatic biosensing of neotame in sweeteners by experimental and computational methods(2020) Lephalala, Matshidiso; Bisetty, Krishna; Kanchi, Suvardhan; Sabela, Myalowenkosi I.An enzymatic biosensor comprises of an enzyme, which recognizes and then reacts with the target analyte producing a chemical signal. In this type of sensor, an electrode is a key component that is employed as a solid support for the immobilization of biomolecules and electron movement. This work focuses on two case studies to assess the signal enhancing strategy that can potentially be used to quantify Neotame (NTM) in food and non-alcoholic beverages. The first case study involves a highly sensitive electrochemical enzymatic biosensor for the detection of NTM in the soft drinks developed, based on multiwalled carbon nanotubes (MWCNTs) decorated with aloe vera-derived gold nanoparticles (AuNPs) and carboxylesterase (CaE) enzyme. This electrochemical biosensor showed high sensitivity with a limit of detection (LOD) and limit of quantification (LOQ) of 27 μg L-1 and 83 μg L-1, respectively. The calibration plot revealed a linear dependence of the cathodic peak current on the NTM concentration profile with anR2 of 0.9829, indicating an improved electrocatalytic property of the glassy carbon electrode. The viability of the proposed strategy was confirmed by assessing the interactions between the enzyme and the analyte using computational methods. The density functional theory (DFT) calculations of NTM showed a HOMO–LUMO energy gap of -0.46618 eV, indicating that NTM can act as a good electron donor. Moreover, adsorption and enzyme-analyte docking studies were carried out to better understand the redox mechanism. These outcomes showed that NTM formed hydrogen bonds with LEU 249, GLU251, and other amino acids of the hydrophobic channel of the binding sites, making it easier for the redox reaction to take place for the detection of NTM. The results confirmed that the aloe vera-derived AuNPs are good platforms for immobilizing CaE because of their high surface area, encouraging an electron transfer from NTM to form a substrate-enzyme complex, contributing to improved biosensing signals. The second case study deals with an enzymatic biosensor developed, based on graphene oxide (GO) anchored with honey-derived nickel nanoparticles (NiNPs) and alcohol oxidase (AOx) enzyme. The biosensor showed high sensitivity with a limit of quantification (LOQ) of 47 μg L-1 and a limit of detection (LOD) of 15 μg L-1, respectively. The calibration curve of the cathodic peak current on the analyte concentration profile showed an improved electrocatalytic property with an R2 of 0.9926. The interactions between the enzyme and analyte were assessed using computational tools to confirm the viability of the proposed biosensor. A HOMO– LUMO energy gap of -0.46618 eV was confirmed using density functional theory (DFT) calculations, this suggested that NTM has great potential to act as an electron donor. Analyte-enzyme and adsorption docking studies were carried out for a better comprehension of the redox reaction mechanisms. These outcomes indicated that NTM forms hydrogen bonds with TRP 47, ARG 56, VAL 328, PRO 55, and other amino acids, thus assisting the redox reaction for the determination of NTM. The results confirmed that the honey-derived NiNPs have a high surface area, which acted as a good platform to immobilize AOx so that the electrons can be transferred from NTM to form a substrate-enzyme composite to give out an improved biosensing signal. Moreover, the magnified catalytic activity of these two biosensors for the determination of NTM in soft drinks showed great potential in the beverage industry.Item Development of a third-generation electrochemical enzyme-based biosensor for a scalable detection of oxygen in power generation cells(2019-12) Jiyane, Sphumelele Nomnontho; Bisetty, Krishna; Sabela, M. I.; Kanchi, S.Pencil graphite electrodes (PGEs) are another form of carbon electrodes with good mechanical strength and comparable electrical properties. Moreover. their low cost makes them an excellent alternative to more conventional electrodes. especially in disposable applications. In this study. the PGEs were constructed with a 2 mm diameter pencil graphite with hardness 4H. HB. and 4B. The electrodes were cleaned and modified with 1 mg/ml of graphene oxide (GO) to enhance the surface area of the electrodes. The PGE-GO was further reduced electrochemically using Na2S2O4 from -1.2 V to 0.8 V at 50 mV/s for 50 cyclic voltammetry scans in the presence of oxygen. using K3Fe(CN)6 / K4Fe(CN)6 as a redox couple. The performance of the PGE was evaluated with multi-walled carbon nanotubes and nanomaterials with various linking agents. A further evaluation was conducted with multi-copper oxidase (MCO) enzymes (Bilirubin oxidase (BOx) and Laccase oxidase) applied for the bio-catalytic reduction of oxygen. The outcome of this study showed that the modification with GO revealed redox peaks 3.6 times higher than the bare PGE. The immobilization of MCO was confirmed by cyclic voltammetry in the presence of a phosphate buffer. Furthermore. the amperometric measurements of O2 at a reducing potential of +0.34 V. showed linearity up to 0.36 mM and sensitivity of 520 μA/(mM.cm2) to O2. Furthermore. computational adsorption studies were performed for the layer-by-layer electrode modification steps. The adsorption simulations revealed a lowering of the energy favored between the designed electrode layers. suggesting a most favorable interaction for the GO/MWCNTs/PBSE/BOx layer. Overall the computational data correlated well with experimental work. Notably. the layer-by-layer adsorption of the GO/MWCNTs/PBSE/BOx showed excellent affinity 11.4 M−1 between PBSE and the enzyme interaction. The direct electron transfer (DET) of the enzymatic reaction integrated with nanotechnology. has led to a small. portable and renewable power generating device. Thus this study addresses the demand for implantable medical devices. in the absence of an external power source.Item Development of an electrochemical immunosensor for the detection of steviol glycosides by experimental and computational methods(2020-04) Hloma, Phathisanani; Bisetty, Krishna; Sabela, M. I.; Kanchi, S.An electrochemical immunosensor employs antibodies as a capture and detection mechanism to produce an electrical charge for the quantitative analysis of target molecules. The current analytical methods for the separation and detection of stevia glycosides can be tedious in terms of sample preparation and the lack of selectivity. However, electrochemical immunosensors provide selective, sensitive and costeffective detection routes for these widely consumed sweeteners. In this study, the author developed an electrochemical immunosensor for the detection and quantification of steviol glycosides, a non-nutritive sweetener widely employed in the food and beverage industries. Most of the artificial sweeteners are low-calorie sweeteners recommended for health-related illnesses. The stability of these sweeteners at even high temperatures has increased their applications in foodstuffs widely. Constant exposure to these sweeteners is somehow associated with health complications, as some are cancer-causing agents. Although there are no reports on stevia glycosides as a health risk sweetener, its widespread use in the food industry needs to be regulated. Herein, the developed immunosensor was achieved by fabricating the platinum electrodes with graphene oxide (GO) assimilated in Zinc Oxide nanoparticles (ZnONPs) with multiwalled carbon nanotubes (MWCNTs) and immobilized with the human sweet receptor subunit T1R2. The electrochemical detection of the natural sweetening compound, Rebaudioside A (Reb A) was evaluated qualitatively and quantitatively using cyclic and differential pulse voltammetry, respectively under optimised conditions in pH 11 borate buffer from -0.4 V to 0.8 V vs Ag/AgCl. The GO/MWCNT/ZnONPs nanocomposite was characterized using High-resolution Transmission Electron microscope (HR-TEM), Thermogravimetric Analysis (TGA), Attenuated Total Reflection Mode Fourier transform infrared (ATR-FTIR) and UV-VIS spectroscopy characterization techniques. Also, asymmetric flow-field-flow fractionation and centrifugal flow-field-flow fractionation equipped with a UV-vis and multi-angle angle light scattering detectors were used to separate and characterize the size distribution of the synthesised ZnO nanostructures. The field flow fractionation (FFF) is one of the efficient separation techniques known, and centrifugal flow fieldflow fractionation separates different particle sized nanoparticles by density, thus determining size variation within the synthesised batch. The results obtained using FFF were compared and validated with the conventional characterisation techniques described above. Computational studies were used to supplement experimental results using docking and adsorption methods. Adsorption studies were carried out to better understand the mechanistic aspects between T1R2, the nanocomposite used to modify the platinum working electrode, and the analyte Reb A. Docking studies between the T1R2 receptor and the steviol glycosides were used to explore the interaction and mechanism of the immunosensor detection. The results of this study may contribute to the development of an immunosensor that can potentially be used to quantify steviol glycosides in the food and beverage industryItem Laccase-mediated biotransformation of phenolic compounds for the synthesis of new antioxidants(2020) Mazibuko, Bodine; Kudanga, TukayiThe increased incidences, mortality rate and economic impact of noncommunicable diseases (e.g. high blood pressure and diabetes) associated with oxidative stress, have led to the higher demand for antioxidant supplements for their prevention. The use of naturally occurring antioxidants is becoming a more attractive option due to the health risks associated with synthetic antioxidants. Phenolic compounds from plants have been shown to have antioxidant properties with the potential to be used as substitutes to synthetic antioxidants. However, monomeric phenolic compounds have several short comings such as low bioavailability, poor solubility, and low antioxidant capacity while some have pro-oxidant properties at high concentrations. Hence there has been increasing research focused on the biotransformation of these phenolic antioxidants through enzymatic oligomerisation to higher molecular weight compounds with improved antioxidant capacity and stability. Of the investigated enzymes, laccases have shown the most promise owing to their green catalytic properties. Their reaction mechanism involves the use of molecular oxygen as a co- substrate in oxidising phenolic compounds to corresponding radicals, with water as the only by- product. This study focused on the synthesis of antioxidants with enhanced antioxidant capacity using a laccase from Trametes pubescens as biocatalyst. To establish the potential of the phenolic compounds for use as substrates for the coupling reactions, a preliminary screening process was done. Guaiacol, caffeic acid, vanillic acid, eugenol, catechol, gallic acid, ferulic acid and quercetin hydrate were identified as suitable substrates for the laccase enzyme. However, only products from eugenol, coumaric acid and quercetin could be isolated, hence coupling reactions were carried out using these substrates in monophasic systems. Reaction products were monitored using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purification was carried out using preparative TLC and characterisation using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The antioxidant capacities of reaction products were determined using ABTS (2,2’-Azinobis 3- ethylbenzthiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric- reducing antioxidant power) assays. Quercetin hydrate oxidation produced one product which was purified and characterised. The product had an Rf of 0.68, tR 13.567 and m/z 601 in negative mode, indicating that it was a dimeric form of quercetin. Oxidation of ρ-coumaric acid resulted in the production of two products designated P1 (Rf 0.47) and P2 (Rf 0.42). Further characterisation was done on product P2 since product P1 could not be successfully purified. P2 had a retention time of 11.295 and m/z 325, indicating that it was a dimer of ρ-coumaric. The ρ-coumaric dimer had an enhanced antioxidant capacity, approximately 2-fold, 3-fold and 6-fold higher compared to the substrate, as demonstrated by the ABTS, DPPH and FRAP assays, respectively. A symmetrical 5-5 eugenol dimer (m/z 325, [M] =326), bis-eugenol, was produced from eugenol oxidation. Maximum product formation (50% yield) was obtained in a monophasic system with 40% v/v dioxane as co-solvent after incubation for 18 h. The bis- eugenol dimer had an improved antioxidant capacity of up to three and four times that of eugenol as demonstrated by the ABTS and DPPH assays, respectively. In conclusion, two dimers with high antioxidant capacity were successfully produced, purified and characterised. The study has demonstrated the potential of the T. pubescens laccase as a catalyst for the synthesis of phenolic compounds with enhanced antioxidant capacity.Item Fabrication of sensors for the sensitive electrochemical detection of anti-tuberculosis drugs(2018) Chokkareddy, Rajasekhar; Redhi, Gan G.; Kumar, Bhajanthri NateshIn this work, electrochemical biosensors have been developed and quantified the pyrazinamide, isoniazid, rifampicin, ethambutol and streptomycin drugs in various pharmaceutical samples. Electrochemical methods are versatile and powerfull analytical technique of immense value in the area of pharmaceutical analyses. In addition, due to the similarity in the biological and electrochemical reactions, it can be expected that the reduction-oxidation mechanisms occur at the electrode surface. The biologically stimulated molecules can be examined by electroanalysis and they are also outstanding tools for the detection of pharmaceutical complexes in various matrices. Although in the case of a biosensors, the analyte interacts with bioreceptor and the resultant output is measured by a specifically designed transducer. Additionally, a reliable highly sensitive and novel biosensor was developed by using a glassy carbon electrode modified with various nanomaterials. Hence horseradish peroxidase (HRP) - Multiwalled carbon nanotubes (MWCNTs)-Titanium oxide nanoparticles (TiO2NPs) fabricated glassy carbon electrode (GCE) were used for the determination of isoniazid. Similarly, copper oxide nanoparticles (CuONPs)-MWCNTs immobilized with Cytochrome c (Cyt c) on glassy carbon electrode were established for the detection of pyrazinamide. Furthermore, iron oxide nanoparticles (Fe3O4NPs) and MWCNTs composite were immobilized with Coenzyme q (Coen- q) on glassy carbon electrode for the detection of rifampicin. In addition, Cyt c immobilized with ZnONPs and MWCNTs on glassy carbon electrode for the determination of streptomycin. Finally, the glassy carbon electrode fabricated with zinc oxide nanoparticles (ZnONPs) and reduced graphene oxide (RGO) nano composite, was further immobilized with HRP to enhance the electrochemical performance of the modified electrode for the determination of ethambutol. Electrochemical behaviour of these first line anti TB drugs to the developed biosensors were examined by using cyclic voltammetry and differential pulse voltammetry under the optimum experimental conditions such as scan rates, pH, accumulation potential, pulse amplitude, accumulation time, voltage step time, voltage step and deposition time respectively. The prepared biosensors and nanocomposites were characterized by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), thermo gravimetry (TGA) and x-ray diffraction (XRD). It was observed that electrochemical methods provided good and effective techniques for the determination of isoniazid, pyrazinamide, rifampicin, ethambutol and streptomycin. Compared to the other analytical methods, the limit of detection and limit of quantifications were found to be 0.0335 μM and 0.1118 μM for isoniazid, 0.0038 μM and 0.0129 μM for pyrazinamide, 0.032 µM, and 0.413 µM for rifampicin, 0.0214 μM and 0.6713 μM for ethambutol, and 0.0028 μM and 0.5628 μM for streptomycin respectively.Item Fabrication of electrochemical biosensors for the determination of phenolic compounds by experimental and computational methods(2018) Kunene, Kwanele Winterose; Bisetty, Krishna; Kanchi, SuvardhanThe polyphenolic compounds of interest, bisphenol A (BPA) and its analogue bisphenol S (BPS) used in the plastic industry to manufacture baby bottles and beverage containers, were used in this study. They are generally used in the manufacture of polycarbonates, epoxy resins and unsaturated polystyrene resins. There is a growing concern in the public and scientific community about these organic compounds due to their endocrine disrupting activity and negative toxic impact on the wildlife. This has encouraged scientists to embark on research to find a sensitive and selective technique that will adequately determine these compounds even in trace amounts. The experimental research strategy adopted in this work was supported by computational methods. This work was conducted in two stages; Firstly, a sensitive EC biosensor was developed using a carbon screen printed electrode fabricated with the combination of silver doped zinc oxide nanoparticles with multiwalled carbon nanotubes (MWCNTs) and laccase enzyme. The EC behaviour of BPA towards the fabricated biosensor was investigated using cyclic voltammetry and differential pulse voltammetry under optimum experimental conditions. Secondly, a novel and selective PEC sensor was developed for the first time to detect BPS based on the vertically aligned ZnO nanorods (ZnO NRs) with a molecularly imprinted polypyrrole (PPy). Amperometric, cyclic voltammetry and impedance spectroscopy were used for the investigation of the photo induced electrochemical behaviour of BPS. Different characterisation techniques such as ultra-violet visible spectroscopy, fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, photoluminescence, Raman spectroscopy, grazing incidence X-ray diffraction and diffuse reflectance were used to characterize the synthesized nanostructures. Results revealed that the fabricated EC and PEC sensors exhibited good catalytic activity towards the determination of BPA and BPS respectively, in samples extracted from plastic water bottles. For the EC method, a low detection limit of 0.08 μM for BPA in a linear range 0.5 to 2.99 µM was detected. However, in the case of BPS, a highly selective PEC method was attained linearly ranging from 2.5 to 12.5 µM with a much higher limit of detection of 0.7 μM. Experimental results were further supported computationally for a better understanding of the optical properties of ZnO NRs-polypyrrole complex. Computational results were in good agreement with experimental results.Item Determination of capsaicin using carbon nanotube based electrochemical biosensors(2016) Mpanza, Thabani Eugene; Bisetty, Krishna; Singh, ParveshThis study involves the development of a sensitive electrochemical biosensor for the determination of capsaicin extracted from chilli pepper fruit, based on a novel signal amplification strategy. The study therefore, seeks to provide a sensitive electro-analytical technique to be used for the determination of capsaicin in food and spicy products. Electrochemical measurements using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) modes were utilized in order to understand the redox mechanism of capsaicin and to test the performance of the developed biosensor supported with computational techniques. In this work two different enzymes, Phenylalanine ammonia lyase (PAL) and Glucose oxidase (GOx) were used for electrode modifications respectively. For this purpose three different types of working electrodes namely: glassy carbon electrode (GCE), platinum electrode (Pt-E) and gold electrode (Au-E) were used and their performances were compared. For the first time, the three electrodes were modified with PAL and GOx enzymes on multiwalled carbon nanotubes used in this study and characterized by attenuated total reflectance infrared spectroscopy, transmittance electron microscopy and thermo-gravimetric analysis supported by computational methods. The comparison of the results obtained from the bare and modified platinum electrodes revealed the sensitivity of the developed biosensor with modified electrode having high sensitivity of 0.1863 µg.L-1 and electron transfer rate constant (ks) of 3.02 s-1. To understand the redox mechanism completely, adsorption and ligand-enzyme docking simulations were carried out. Docking studies revealed that capsaicin formed hydrogen bonds with Glutamates (GLU355, GLU541, GLU586), Arginine (ARG) and other amino acids of the hydrophobic channel of the binding sites which facilitated the redox reaction for detection of capsaicin. These results confirm that the PAL enzyme facilitated the electron transfer from the capsaicin ligand, hence improving the biosensing response. Our results suggest potential applications of this methodology for the determination of capsaicin in the food industry.