Faculty of Applied Sciences
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Item Development of electrochemical biosensors for sweeteners using engineered nanomaterials supported by computational modelling(2024-05) Hloma, Phathisanani; Bisetty, Krishna; Sabela, Myalowenkosi Innocent; Uwaya, Gloria EbubeElectrochemical immunosensors are a powerful tool in analytical applications. The current methods for the isolation and detection of artificial and natural sweeteners suffer from challenges in sample preparation and a lack of specificity. However, electrochemical immunosensors offer a sensitive, economical, and selective analytical solution to analyse these commonly used sweeteners, such as aspartame. The author of this work developed electrochemical immunosensors for the food and beverage industries to use in the detection and measurement of aspartame, a non-nutritive sweetener, and rebaudioside A, a natural sweetener. Most artificial sweeteners are low-calorie options that are suggested for ailments linked to health. These sweeteners' ability to remain stable at even high temperatures has greatly expanded the range of meals that can use them. Aspartame and rebaudioside A have not been linked to any health risks, although regulation is still necessary because of their extensive use in the food industry. The developed immunosensors for the detection of aspartame and rebaudioside A were achieved and presented as three case studies in this study. In the first case study, the immunosensor was achieved by fabricating green synthesized PVP capped silver nanoparticles (PVP-AgNPs) with functionalized multi-walled carbon nanotubes (fMWCNTs) and immobilizing the human sweet tase receptor T1R2 in a glassy carbon electrode (GCE), resulting in GCE/PVP-AgNPs/fMWCNTs/T1R2. The electrochemical assessment of aspartame was achieved using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), respectively, under optimum pH 8 in a 0.1 M phosphate buffer with reference to the Ag/AgCl reference electrode. The electro-oxidation of ASP was noticed by a well-defined oxidation peak potential at 1.4 V. The immunosensor sensor showed a linear dynamic range of 2.89 to 27.61 μM (R 2 = 0.9170) based on differential pulse voltammetry, with limits of detection (LOD) and quantification (LOQ) (S/N = 3) of 0.40 μM and 1.34 μM, respectively. The second case study focused on the indirect electrochemical detection of rebaudioside A in the presence of ferro/ferricyanide as a redox probe. The immunosensor was developed by fabricating GCE with zeolitic imidazolate framework-67 (ZIF-67) in combination with fMWCNTs and the immobilization of the T1R2 receptor. The qualitative and quantitative analysis of rebaudioside A was done using CV, EIS, and DPV utilizing a 5 mM [Fe (CN)6] 3-/-4 redox probe. The stable electrode had an exponential dynamic range of 0.9901 µM to 8.2569 µM (R2 = 0.9996). The LOD and LOQ were computed to be 1.10 µM and 3.33 µM, respectively. This case study also used Patch Dock and PyRx to better understand the interactions between Reb A and T1R2. The final case study employed a platinum electrode (PtE) as the working electrode (WE) for the electrochemical immunosensing of aspartame. The modification of PtE involved the utilization of a nanocomposite consisting of PVP-AgNPs and reduced graphene oxide (rGO), with T1R2 immobilized. The electrochemical detection of aspartame was achieved under optimized conditions at pH 8 in a 0.1 M phosphate buffer, utilizing CV, EIS, and DPV as electrochemical tools. The PVP-AgNPs/rGO/T1R2 was used to fabricate Pt and the electrode performed well with a linear increase in oxidation peak currents as aspartame concentrations were increased from 2.38 µM to 25.78 µM (0.9529). The LOD and LOQ were calculated to be 5.85 µM and 17.73 µM, respectively. The synthesized nanoparticles and nanocomposites (PVP-AgNPs/fMWCNTs, ZIF 67/fMWCNTs, and PVP-AgNPs/rGO) were characterized using conventional techniques such as UV-Vis spectroscopy, thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), field flow fractionation (FFF), single particle inductively coupled plasma mass spectrometry (sp-ICPMS), energy-dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). In addition to the experimental results, computational chemistry methods were undertaken. These included adsorption assessments, density functional theory (DFT), and molecular docking techniques. These techniques were all aimed at achieving a deeper molecular-level understanding of the interactions among the analytes (Aspartame and Reb A), T1R2, and the nanocomposites employed in the modification of the working electrodes (GCE and Pt-E)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 Gold nanoparticle-based lateral flow kit for in vitro detection of malaria antibodies(2018) Mthembu, Christian Lungani; Mdluli, Phumlani Selby; Mlambo, Mbuso; Gumede, HalalisaniThis study involves the development of three-dimensional dual lateral flow diagnostic assays. These assays were fabricated with quick response (QR) barcodes to ease the accessibility and transfer test data. The assays were designed to also improve the collection and transfer of survey from point-of-care facilities to centralized laboratories, thus, these would help to speed-up response to disease out-break. The study introduces the fabrication of two barcode based malaria diagnostic in the field of diagnostics. Two lateral flow kits were modified with two QR barcodes and three QR barcodes encoded with Google analytics codes for the detection and real-time tracking of malaria lateral flow which was designed to detect Plasmodium lactate dehydrogenase (pLDH). The fabrication of test kit was achieved by attaching two and three QR barcodes into two different test kits which were encoded with websites that were linked to Google analytics website as a tracking and performance monitor. Gold nanoparticles (AuNPs) were used as a substrate, where optical and structural properties were studied using UV/Visible spectroscopy, fluorescence spectroscopy, and transmission electron microscopy (TEM). The anti-mouse IgG antibody was used as a secondary antibody to act as control and the anti-(pLDH) was stripped on the test line. Phosphate buffer was used as a mobile phase solution. The antibody binding with pLDH antigen showed red test line indicating a positive test. Two diagnostic kits for rapid detection of pLDH were developed and validated for the detection of malaria antigen with lowest detectable recombinant concentration of 10 ng.mL-1. The diagnostic kits were incorporated with two and three optimally angled QR barcodes for identifying positive and negative. The second three QR barcode embedded test kit identified positive, negative and invalid using tracked website. These QR barcodes enabled massive results and tracking with precise location of the test through Google Analytics.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 graphene based aptasensors for early detection of prostate cancer by experimental and computational techniques(2017) Putri, Athika Darumas; Bisetty, Krishna; Tiwari, AshutoshHigh prevalence and mortality cases of prostate cancer (PCa) have increased around the world, particularly in developing countries. Several forthcoming factors have been revealed nowadays, one of them is due to the incapability of the diagnostic methods to produce reliable results, which impacts negatively on cancer-treatment. However, a sensitive diagnosis of PCa cells remains a challenge in the field of biosensors. Emerging whole-cell detection as biosensing targets has opened up avenues for successful cancer diagnostics, due to high selectivity among other cells. A switchable and flexible surface-based graphene material is one of the techniques that revolutionized smart biodevice platforms in biosensor technology. In this present study, a covalently linked poly-(N-isopropylacrylamide) (PNIPAM) to graphene oxide surface has been employed as “on/off”-switchable aptamer-based sensor for the detection of PC3 whole-cancer cell. The constructed surface has benefitted from PNIPAM, as the thermal-stimulus agent, which allows the coil-to-globule transitions by triggering temperature changes. When the system is above its lower critical solution temperature (LCST) of 32oC, PNIPAM will exist as hydrophobic -globular state providing an “on” binding region for the whole-cell, reaching the interactions on the biosurface. The “off” binding systems is only possibly when the PNIPAM turns into extended-state by lowering its temperature below LCST. The first principle studies have successfully characterized the electronic behavior with particular emphasis of PNIPAM monomer functions along with the description of the structural energetics of complex through density functional theory (DFT). Docking studies have further been performed to predict a plausible binding aptamer toward the protein-representative PCa cell. To better understand the prospect of an aptamer-based tunable biosensor, molecular dynamics (MD) highlighted the behavior of PNIPAM-grafted GO in exhibiting a globular and extended conformations at above and below LCST, permitting the biomolecules to interact with each other as well as to avoid interactions, respectively. Experimental studies have been included to validate the theoretical predictions by fabricating real-biosensor systems using electrochemical impedance technique, resulting a low-detection limit down to 14 cells/mL. Engagement between theoretical and experimental studies delivered an enhanced tunable-biosensor performance for the detection of whole cell prostate cancer.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.