Faculty of Applied Sciences

Permanent URI for this communityhttp://ir-dev.dut.ac.za/handle/10321/5

Browse

Now showing 1 - 20 of 27

Analysis of nanoscale ingredients in commercial food and cosmetic products by field-flow fractionation and single particle ICP-MS
(2020-09) Naidoo, Lyndon; Bisetty, Krishna; Kanchi, Suvardhan; Sabela, Myalowenkosi Innocent
There is a growing need to disclose the possible presence of nanostructures on the labels of commercial food and cosmetic products in South Africa. Synthetic amorphous silica (SiO2) and titanium dioxide (TiO2) are the two widely used nanoparticles (NPs) selected for this study. This work was undertaken in two stages. The first part deals with an analytical method developed for the separation and characterization of TiO2 NPs capped with poly(ethylene glycol) (PEG) as a potential reference material for sunscreen analysis. The second aspect of this work focuses on SiO2, a highly attractive biomaterial widely used as a food additive (E551) to improve the flow properties of powdered food ingredients. Also, modern computational methods were implemented in both case studies, to better understand the nanoparticulate interactions with the organic substrates at an atomistic and molecular level. For the cosmetics study, asymmetric flow field-flow fractionation (AF4) coupled online to multi-angle light scattering (MALS) and dynamic light scattering (DLS) detectors were employed to assess the geometry and size of the PEGylated TiO2 NPs in terms of the evaluated radius of gyration (rg) and hydrodynamic radius (rh). Single particle inductively coupled plasma mass spectrometry (spICP-MS) and transmission electron microscopy (TEM) were implemented to provide information on the core diameter (d) of the TiO2 particle. To overcome agglomeration, Monte Carlo simulations were employed in this study to assess the effectiveness of PEG capped onto spherically constructed TiO2 anatase nanoclusters by systematically performing a series of adsorption studies. For PEG-TiO2 NPs, AF4-MALS-DLS reported rg and rh values of 28.7 nm and 40.3 nm respectively, with the shape factor (rg/rh) values generally reported in the range of 0.7 to 0.8, indicative of spherical particle geometry. SpICP-MS and TEM obtained complementary measurements of d = 32.0 nm and d = 38.4 nm, respectively. The computational modelling results demonstrated the strong binding affinity of PEG as a capping agent to TiO2, exhibiting stabilisation of TiO2 NPs in aqueous medium. Finally, the developed AF4-MALS-DLS method was applied to two commercial SPF 50 sunscreens, exhibiting promising separation and detection efficiency. These findings can contribute to regulatory measures in line with the South African National Nanotechnology Strategy for the cosmetics industry. With regards to the food application study, a multivariate method was developed for the detection and characterization of SiO2 particles also based on AF4-MALS-DLS. This analytical approach attempts to address the fate and the presence of nanoparticulate SiO2 additives (E551) in food products. The experimental design using SiO2 NP standards resulted in the following optimum conditions of the system: crossflow, 0.8 ml/min; injection time, 5 min and sonication time, 60 min. It was observed that the average geometric diameters (Dgeo) for SiO2 NPs in three selected commercial coffee creamers (A, B and C) detected by AF4-MALS were 286.7 nm, 129.8 nm and 190.7 nm respectively. Similar trends for the coffee creamers were observed for the hydrodynamic diameter (Dh) measurements using AF4-DLS i.e. 301.5 nm, 141.3 nm and 197.8 nm respectively. The rg/rh values were reported ranging from 0.7 to 0.8, indicative of spherical particle geometry. Also, the electrostatic interactions between SiO2 NPs and glucose/water mixtures, as evaluated by Monte Carlo simulations, revealed O-interactions dominating over the flat amorphous SiO2 surface. The strongest interaction observed (around - 239 kcal/mol) for the SiO2-water/glucose mixture demonstrates the hydrophilic nature of SiO2 NPs. The findings in both case studies provide fundamental information to improve the understanding of nanoparticulate interactions with additives and paves the way for the labelling of cosmetic and food products that potentially exhibit nanomaterials in complex matrices.
An assessment of the conformational profile of bombesin and its mammalian analogues using computational chemistry methods
(2011) Sharma, Parul; Bisetty, Krishna
Understanding the dynamics and mechanism of protein folding continues to be one of the central problems in molecular biology. Peptide folding experiments characterize the dynamics and molecular mechanisms of the early events of protein folding. However, generally the highly flexible nature of peptides makes their bioactive conformation assessment reasonably difficult as peptides fold at very fast rates experimentally, requiring probing on the nanosecond time resolution. On the other hand, determining the bioactive conformation of biological peptides is a requirement for the design of peptidomimetics in computer-aided drug design. Peptides offer a unique opportunity to bridge the gap between theoretical and experimental understanding of protein folding. Therefore, the present work focuses on the exploration of the conformational space of biologically active neuropeptides with the aim of characterizing their conformational profile. Specifically, bombesin, neuromedin B (NMB) and neuromedin C (NMC), have been chosen for the current investigations. These peptides are widely distributed in the gastrointestinal tract, spinal cord and brain, and are known to elicit various physiological effects, including inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. These peptides act as a growth factor in a wide range of tumours including carcinomas of the pancreas, stomach, breast, prostate, and colon. This work is intended to get some insight into the performance of different procedures used to explore the configurational space to provide an adequate atomic description of these systems. Different methodological studies involving utilization of molecular dynamics (MD), multicanonical replica exchange molecular dynamics (REMD) and simulate annealing (SA) are undertaken to explore the folding characteristics and thermodynamics of these neuropeptides. MD and REMD calculations on bombesin peptide have revealed its dual conformational behaviour never discovered before and is described in chapter 3. These results explain the known structure-activity studies and open the door to the understanding of the affinity of this peptide to two different receptors: BB1 and BB2. In the case of NMC, REMD calculations are carried out in explicit and implicit solvents, using the Generalized Born (GB) surface area, and are then complemented with two additional MD simulations performed using Langevin and Berendsen thermostats. The results obtained clearly reveal that REMD, performed under explicit solvent conditions, is more efficient and samples preferentially folded conformations with a higher content of  and γ turns. Moreover, these results show good agreement with the experimental results supporting the role of two -turns for its biological action, as reported in the literature. Finally, the results obtained from MD, REMD and SA calculations on NMB reveal that the peptide has a tendency to adopt both turns and helices suggesting its two different receptor recognizing and binding conformations during its biological action. Hence, the present work provides comprehensive information about the conformational preferences of neuropeptides which could lead to a better understanding of their native conformations for future investigations and point the way towards developing their new antagonists.
Computational and experimental studies of putative virulence factors of Mycobacterium tuberculosis H37Rv
(2017) Shahbaaz, Mohd; Bisetty, Krishna
In drug discovery and development of anti-tubercular therapeutics, it is necessary to study the physiology and genetics of the molecular mechanisms present in the Mycobacterium tuberculosis. The virulence of M. tuberculosis is attributed to its unique genome, which contains a high frequency of glycine-rich proteins and genes involved in the metabolism of the fatty acids. Consequently, the presence of a diversity of the pathogenic pathways such as acid tolerance and drug resistance mechanisms in M. tuberculosis makes the treatment of Tuberculosis (TB) challenging. However, the molecular basis of the virulence factors involved in the pathogenesis is not fully understood. Accordingly, the current study focuses on better understanding of the pathogenic proteins present in this bacterium using available computational techniques. In South Africa, there is an alarming increase in the drug-resistant TB in HIV co-infected patients, which is one of the biggest challenges to the current anti-tubercular therapies. An extensive literature search showed that the mutations in the virulent proteins of M. tuberculosis resulted in the development of drug tolerance in the pathogen. The molecular and genetic studies identified frequently occurring point mutations associated with the drug resistance in proteins of M. tuberculosis. Despite the efforts, TB infection is still increasing because different pathogenic pathways in the bacterial system are still undiscovered. Therefore, this study involves an in silico approach aimed at the identification of novel drug resistance implicated point mutations. The site- directed mutations leading to the development of resistance against four first-line drugs (Ethambutol, Isoniazid, Rifampicin, and Streptomycin) were studied extensively. In the primary investigation, pathogenic mutational landscapes were classified in the sequences of the studied proteins. The effects of these mutations on the stability of the proteins were studied using diverse computational techniques. The structural basis of the point mutations with the highest destabilizing effects was analyzed using the principles of the Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulation studies. The varied conformational behavior resulted from these predicted substitutions were compared with the experimentally derived mutations reported in the literature. The outcome of this study enabled the identification of the novel drug resistance-associated point mutations which were not previously reported. Furthermore, a detailed understanding of the conformational behavior of diverse virulent proteins present in M. tuberculosis was also generated in this study. Literature study showed that inside the host’s macrophage cells, the virulent proteins such as isocitrate lyase, lipase lipF, magnesium transporter MgtC, porin protein OmpATb, a protein of two component systems PhoP, Rv2136c and Rv3671c have an established role in the development of the acid tolerance. On the other hand, information regarding their role in the acid resistance is scarce. Accordingly, the structural basis of their role in acid resistance was analyzed using constant pH based MD simulations. In the studied proteins, the lipF and PhoP showed highest structural stability in highly acidic conditions throughout the course of MD simulations. Therefore, these proteins may play a primary role in the process of resistance. In addition to these pathogenic proteins, there is a need to identify new undiscovered virulent proteins in the genome of M. tuberculosis, which increases the efficiency of the current therapy. The knowledge generated by the analyses of the proteins involved in resistance and pathogenic mechanisms of M. tuberculosis forms the basis for the identification of new virulence factors. Therefore, an in silico protocol was used for the functional annotations and analyses of the virulence characteristics. M. tuberculosis contains 1000 Hypothetical Proteins (HPs), which are functionally uncharacterized proteins and their existence was not validated at the biochemical level. In this study, the sequences of the HPs were extensively analyzed and the functions of 662 HPs were successfully predicted. Furthermore, 483 HPs were classified in the category of the enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. The 307 HPs among this group of proteins were less precisely predicted because of the unavailability of the reliable functional homologs. An assessment of the virulence characteristics associated with the 1000 HPs enabled the classification of 28 virulent HPs. The structure of six HPs with highest predicted virulence score was analyzed using molecular modelling techniques. Amongst the predicted virulent HPs, the clone for Rv3906c purchased from the DNASU repository because of the ease of its availability. The gene of Rv3906c was isolated and cloned into a pET-21c expression vector. The analyses of the nucleotide sequence showed that Rv3906c gene (500 bp) encodes a 169 amino acid protein of molecular weight 17.80 kDa (~18.0 kDa). The sequence analyses of Rv3906c showed that the HPs showed high similarities with pullulanase, a thermophilic enzyme. The stability profile at different temperatures for Rv3906c generated using MD simulations showed that Rv3906c maintained its structural identity at higher temperatures. It is expected that this study will result in the design of better therapeutic against the infection of M. tuberculosis, as novel undiscovered virulence factors were classified and analyzed in addition to the conformational profiles of the virulent proteins involved in the resistance mechanisms.
Computational and micro-analytical techniques to study the in vitro and in silico models of novel therapeutic drugs
(2016) Gumede, Njabulo Joyfull; Bisetty, Krishna; Sagrado, Sagrado
In drug discovery and development projects, metabolism of new chemical entities (NCEs) is a major contributing factor for the withdrawal of drug candidates, a major concern for other chemical industries where chemical-biological interactions are involved. NCEs interact with a target macro-molecule to stimulate a pharmacological or toxic response, known as pharmacodynamics (PD) effect or through the Adsorption, Distribution, Metabolism, and Excretion (ADME) process, triggered when a bio-macromolecule interacts with a therapeutic drug. Therefore, the drug discovery process is important because 75% of diseases known to human kind are not all cured by therapeutics currently available in the market. This is attributed to the lack of knowledge of the function of targets and their therapeutic use in order to design therapeutics that would trigger their pharmacological responses. Accordingly, the focus of this work is to develop cost saving strategies for medicinal chemists involved with drug discovery projects. Therefore, studying the synergy between in silico and in vitro approaches maybe useful in the discovery of novel therapeutic compounds and their biological activities. In this work, in silico methods such as structure-based and ligand-based approaches were used in the design of the pharmacophore model, database screening and flexible docking methods. Specifically, this work is presented by the following case studies: The first involved molecular docking studies to predict the binding modes of catechin enantiomer to human serum albumin (HSA) interaction; the second involved the use of docking methods to predict the binding affinities and enantioselectivity of the interaction of warfarin enantiomers to HSA. the third case study involved a combined computational strategy in order to generate information on a diverse set of steroidal and non-steroidal CYP17A1 inhibitors obtained from literature with known experimental IC50 values. Finally, the fourth case study involved the prediction of the site of metabolisms (SOMs) of probe substrates to Cytochrome P450 metabolic enzymes CYP 3A4, 2D6, and 2C9 making use of P450 module from Schrödinger suite for ADME/Tox prediction. The results of case study I were promising as they were able to provide clues to the factors that drive the synergy between experimental kinetic parameters and computational thermodynamics parameters to explain the interaction between drug enantiomers and thetarget protein. These parameters were correlated/converted and used to estimate the pseudo enantioselectivity of catechin enantiomer to HSA. This approach of combining docking methodology with docking post-processing methods such as MM-GBSA proved to be vital in estimating the correct pseudo binding affinities of a protein-ligand complexes. The enantioselectivity for enantiomers of catechin to HSA were 1,60 and 1,25 for site I and site II respectively. The results of case study II validates and verifies the preparation of ligands and accounting for tautomers at physiological pH, as well as conformational changes prior to and during docking with a flexible protein. The log KS = 5.43 and log KR = 5.34 for warfarin enantiomer-HSA interaction and the enantioselectivity (ES = KS/KR) of 1.23 were close to the experimental results and hence referred to as experimental-like affinity constants which validated and verified their applicability to predict protein-ligand binding affinities. In case study III, a 3D-QSAR pharmacophore model was developed by using 98 known CYP17A1 inhibitors from the literature with known experimental IC50 values. The starting compounds were diverse which included steroidal and non-steroidal inhibitors. The resulting pharmacophore models were trained with 69 molecules and 19 test set ligands. The best pharmacophore models were selected based on the regression coefficient for a best fit model with R2 (ranging from 0.85-0.99) & Q2 (ranging from 0.80-0.99) for both the training and test sets respectively, using Partial Least Squares (PLS) regression. On the other hand, the best pharmacophore model selected was further used for a database screening of novel inhibitors and the prediction of their CYP17A1 inhibition. The hits obtained from the database searches were further subjected to a virtual screening workflow docked to CYP17A1 enzyme in order to predict the binding mode and their binding affinities. The resulting poses from the virtual screening workflow were subjected to Induced Fit Docking workflow to account for protein flexibility during docking. The resulting docking poses were examined and ranked ordered according to the docking scores (a measure of affinity). Finally, the resulting hits designed from an updated model from case study III were further synthesized in an external organic chemistry laboratory and the synthetic protocols as well as spectroscopic data for structure elucidation forms part of the provisional patent specification. A provisional patent specification has been filed (RSA Pat. Appln. 2015/ 07849). The case studies performed in this thesis have enabled the discovery of non-steroidal CYP17A1 inhibitors.
Computational studies of anti-cancer Aurein peptides
(2015-01-14) Manhas, Neha; Bisetty, Krishna
Peptide folding is a very complicated and dynamic process taking place in all living systems. The understanding of a bioactive conformation of the peptides is very important to understand their biological functions and underlying mechanism of action. However, the high flexible nature of peptides makes this process difficult as they can adopt thousands of conformations within the fraction of a second. The usage of experimental techniques in the characterization process is also limited due to several associated complications including synthesis, isolation and crystallization of peptides. The present computational methodologies, on the other hand, are solid enough to provide detailed complementary information about the intrinsic conformational features of peptides by mimicking their physiological conditions. In the present work, molecular dynamics (MD) computational method was used to explore the configurational space of three Aurein peptides, namely Aurein 2.3, Aurein 2.4 and Aurein 2.5. These peptides are secreted by the amphibian skin when they are exposed to external stimuli. These peptides have been reported to possess anti-cancer and anti-bacterial activity with minimum resistance compared to the available drugs. However, despite their medicinal significance, the precise three dimensional structures of Aurein 2.4 and Aurein 2.5 are not as yet known. First, a validation study was performed on Aurein 2.3 to check the efficiency of the computational protocol. The results obtained revealed the presence of -helicity in all residues of the Aurein 2.3, in accordance with its experimental structure. A similar protocol was further used to explore the conformational profiles of the remaining two peptides (Aurein 2.4 and Aurein 2.5) under implicit and explicit solvent conditions. The results obtained revealed that both these peptides exhibit -helical character in all residues although in varying percentages. The -helical region in the case of Aurein 2.4 was localized predominantly in the central residues extending towards its N-terminal residues, whereas it was flanked by N-terminal and the central residues in Aurein 2.5. However, -helicity was completely absent in the explicit solvents, and the peptides preferred to stay either in -turns or extended forms. Hence, the present work provides comprehensive information about the conformational preferences of Aurein peptides which could lead to a better understanding of their native conformations for future investigations and point the way towards developing their new agonists.
Computational studies of the folding patterns of small and medium-size polypeptides
(2010) Mokoena, Paul; Bisetty, Krishna; Perez, J. J.; Corcho, F. J.
This study involved a series of molecular dynamics (MD) simulations applied to case studies of small and medium-size polypeptides to assess the thermodynamics of their folding characteristics. Peptide folding is a complex and vital phenomenon taking place in all living systems. Bioactive conformational structures of folded peptides need to be well characterized before using them in computer-aided drug design. The computational procedure was validated on the 10-residue long chignolin-like synthetic mini-protein (CLN025). For this peptide, replica exchange molecular dynamics (REMD) calculations were carried out in explicit and implicit solvents using the generalized Born (GB)/surface area (SA) approximation with different sets of force field parameters. Following this validation procedure, case studies of the folding conformations of peptides of different lengths including the 5-residue met-enkephalin, the 27-residue pituitary adenylate-activating polypeptide 27(PACAP27) and the 28-residue vasoactive intestinal peptide (VIP) were undertaken. The latter two peptides are multifunctional hormones that mediate diverse biological functions, such as the cell cycle, cardiac muscle relaxation, immune response, septic shock, bone metabolism, and endocrine function. Results obtained indicate that when explicit water, methanol and DMSO solvents were used, it appeared that methanol (MeOH) and dimethylsulphoxide (DMSO) afforded met-enkephalin the ability to form more intra-hydrogen bonds than water, producing type I and type III β-turn structures; thus enhancing the helical conformation of the peptide. MD trajectories of longer polypeptides (VIP and PACAP27) were also populated with type I and type III β-turns, which occurred consecutively; with α- and 310-helices occurring from the middle of each peptide towards the C-terminal. Characterization of implicit solvent results, reveal that these simulations have been able to reproduce the same type of conformers obtained by experimental NMR studies published in literature, which structurally resemble the native conformation of the bioactive peptides. These conformational structures will be applied as lead agents in computer-aided drug design. One of the major achievements of this study is the ability to optimize and validate the force field parameter sets to describe the thermodynamic properties of peptide systems in an unbiased manner, a non-trivial task for even the smallest of peptides. These findings re-affirm the notion that computational methods have matured enough to model dynamic biological phenomena such as peptide folding, a feat previously thought to be impossible.
Computational studies on the identification and analyses of p53 cancer associated mutations
(2017) Cele, Nosipho Magnificat; Bisetty, Krishna; Hassan, Md. Imtaiyaz
P53 is a tumour suppressor protein that is dysfunctional in most human cancer cells. Mutations in the p53 genes result in the expression of mutant proteins which accumulate to high levels in tumour cells. Several studies have shown that majority of the mutations are concentrated in the DNA-binding domain where they destabilize its conformation and eliminate the sequence- specific DNA-binding to abolish p53 transcription activities. Accordingly, this study involved an investigation of the effects of mutations associated with cancer, based on the framework of sequences and structures of p53 DNA-binding domains, analysed by SIFT, Pmut, I-mutant, MuStab, CUPSAT, EASY-MM and SDM servers. These analyses suggest that 156 mutations may be associated with cancer, and may result in protein malfunction, including the experimentally validated mutations. Thereafter, 54 mutations were further classified as disease- causing mutations and probably have a significant impact on the stability of the structure. The detailed stability analyses revealed that Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro, Cys238Pro, Gly262Pro and Cys275Pro mutations caused the highest destabilization of the structure thus leading to malfunctioning of the protein. Additionally, the structural and functional consequences of the resulting highly destabilizing mutations were explored further using molecular docking and molecular dynamics simulations. Molecular docking results revealed that the p53 DNA-binding domain loses its stability and abrogates the specific DNA-binding as shown by a decrease in binding affinity characterized by the ZRANK scores. This result was confirmed by the residues Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro and Cys238Pro p53-DNA mutant complexes inducing the loss of important hydrogen bonds, and introduced non-native hydrogen bonds between the two biomolecules. Furthermore, Molecular dynamics (MD) simulations of the experimental mutant forms showed that the structures of the p53 DNA-binding domains were more rigid comparing to the wild-type structure. The MD trajectories of Val134Ala, Arg213Gly and Gly245Ser DNA-binding domain mutants clearly revealed a loss of the flexibility and stability by the structures. This might affect the structural conformation and interfere with the interaction to DNA. Understanding the effects of mutations associated with cancer at a molecular level will be helpful in designing new therapeutics for cancer diseases.
A computational study of Trishomocubane amino acid dipeptide
(2004) Govender, Poomani Penny; Bisetty, Krishna; Kruger, H. G.
4-amino-(D3)-trishomocubane-4-carboxylic acid (tris-amino acid) is a constrained a-amino acid residue that exhibits peculiar conformational characteristics. The aim of the present study is to provide a deeper understanding of these features, which can be used as a guide when chOOSing@shomocubane as suitable building blocks for peptide design. The Ca carbon of@ishomocubane forms part of the cyclic structure, and consequently a peptidic environment was simulated with an acetyl group on its N-terminus and a methyl amide group on its C-terminus. This study involved a complete exploration of the conformational profile of (Yishomocubane using computational techniques.The parm94 parametization of the AMBER oio forc@eld was used to explore the conformational space of the peptide,Q)\xEFshomocubane. The Ramachandran maps computed at the molecular mechanics level' with the parm94 forc@\xEFeld parameters compared reasonably with the corresponding maps computed at the Hartree Fock (HF) level, using the 6-31G* basis set. The results of this study revealed that the conformational profile of the @ishomocubane peptide can be characterized by four low energy regions, viz., C7ax, C7eq, 310 and al helical structures.
Determination of capsaicin using carbon nanotube based electrochemical biosensors
(2016) Mpanza, Thabani Eugene; Bisetty, Krishna; Singh, Parvesh
This 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.
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.
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 industry
Development of electrochemical biosensors for sweeteners using engineered nanomaterials supported by computational modelling
(2024-05) Hloma, Phathisanani; Bisetty, Krishna; Sabela, Myalowenkosi Innocent; Uwaya, Gloria Ebube
Electrochemical 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)
Development of electrochemical immunosensors for detection of Tau protein : computational and experimental studies
(2019-11) Harilal, Calvin Carl; Bisetty, Krishna; Kanchi, Suvardhan
Tau protein is a microtubule-associated protein (MAP) found in neuronal cells of the central nervous system. In recent years it has become an important biomarker for neurodegeneration and pathologies of the nervous system, thereby necessitating novel approaches for its detection. This study involves the development of two immunosensors for the detection of tau protein. The study makes use of nanomaterials and antibody transducers as signal enhancing strategies. Both sensors rely on indirect detection of tau protein as a copper(II) complex using a Cu(II)/Cu (I) redox probe. The electrochemical immunoassay is based on the immobilisation of anti-tau antibodies onto a gold working electrode that has been modified with nanomaterials using N- Hydroxysuccinimide (NHS) binder. The first sensor makes use of gold nanoparticles (AuNPs) and the second utilises a nanocomposite of graphene oxide (GO) decorated with silver nanoparticles (AgNPs). Cyclic voltammetry (CV) was used to optimise the electrochemical signal of the tau protein, while quantitative analyses were achieved by differential pulse voltammetry (DPV) and square wave voltammetry (SWV) under the established optimised conditions. Results for the quantitative experimental studies revealed relatively low detection limits for both sensors. The lowest of these detection limits were obtained for DPV analysis of using sensor 1 which produced an LOD of 3.31 nM and an LOQ of 11.04 nM. For sensor 2 the SWV analysis produced the lowest LOD and LOQ of 1.73 nM and 5.76 nM respectively. Computational chemistry methods implemented at the DFT level were used to support the developed electrochemical sensor. The molecular docking results showed relatively good binding affinity of -4.72 kcal/mol between the NHS and the antibody. A 100 ns MD simulation showed a good free binding energy value of -20.51 kcal/mol at pH 7, in accordance with the optimum pH implemented in the experimental work. Furthermore, adsorption studies were performed between the citrate coated nanoparticles on the Au electrode and NHS/anti-tau antibody/tau complex. The energy adsorption simulations revealed the energy favoured interaction between the designed layers with the stabilizing energy changes from -- 23.74 to -142.96 kcal/mol for sensor 1 and for sensor 2 it changed from -7.6 to -127.82 kcal/mol. Overall the computational data correlated well with experimental work. The two novel immunosensors developed in this work, give new insights into electrochemical and computational methods for the detection of proteins, and could lead to the fabrication of a device for point-of-applications in early diagnosis of neurodegenerative disorders.
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 Ebube
The 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.
Development of electrophoretic and biosensor methods applied to high intensity sweeteners
(2015) Bathinapatla, Ayyappa; Dovey, M.; Bisetty, Krishna
Materials which show sweetness are classified as nutritive sweeteners and non-nutritive sweeteners or artificial sweeteners. In the present work, capillary electrophoresis and electrochemical biosensors have been used to analyse and quantify the natural and chemical artificial sweeteners in different food samples. The experimental work was further supported by computational studies. Capillary electrophoresis (CE) is a technique in which charged molecules can efficiently be separated in a buffer solution within a capillary tube under the influence of a strong electric field. While in the case of a biosensor, the analyte interacts with the bioreceptor and the resulting output is measured by a specially designed transducer. Steviol glycosides (rebaudioside A and stevioside) are natural sweeteners, extracted from Stevia rebaudiana Bertoni belonging to the Asteraceae family. On the other hand, neotame and sucralose are chemical sweeteners manufactured from their structural analogues aspartame and sucrose, respectively. Accordingly in this work, two CE modes, namely electro kinetic chromatography–capillary electrophoresis (EKC–CE) and an indirect UV-Capillary zone electrophoresis were used for the evaluation of analytes studied. Steviol glycosides (rebaudioside A and stevioside) and neotame diastereomers (L,L and D,D) were analysed using EKC-CE in the presence of a chiral separating agent β-cyclodextrin (TM-β-CD). However, since sucralose demonstrates chromophore-like properties, an indirect UV-CZE method was therefore developed using simple amines (morpholine, piperidine, ethylamine and triethylamine) as the background electrolytes (BGE). The optimum separation conditions in EKC-CE were; UV detection at 210 nm, 50 mM phosphate buffer, 30 mM TM-β-CD, 20 kV applied voltage, 5 s hydrodynamic injection and pH of 8.0 and 5.5 (for steviol glycosides and neotame), respectively. On the other hand, optimum separation conditions for the indirect UV-CZE method were; UV detection at 230 nm, 0.2 M morpholine buffer at pH 12.0, +20 kV applied voltage, 30 0C cassette temperature and 6 s sample injection. Furthermore, a highly sensitive and novel electrochemical biosensor was developed using platinum and glassy carbon electrodes fabricated with different nanomaterials. Accordingly, cytochrome c/graphene oxide – gold NPs/multiwalled carbon nanotubes (MWCNTs) modified platinum electrodes were used for the analysis of rebaudioside A. Similarly, copper NPs capped with ammonium piperidine dithiocarbamate-MWCNTs-β-cyclodextrin and laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) immobilized graphene oxide-p-aminothiophenol capped ZnO NPs nanocomposites modified with glassy carbon electrodes were developed for the determination of neotame and sucralose, respectively. The electrochemical behaviour of these sweeteners towards the developed sensors was tested by using cyclic voltammetry and differential pulse voltammetry under optimum experimental conditions (pH, scan rate, accumulation time, accumulation potential, pulse amplitude, voltage step and voltage step time). The prepared nanocomposites were characterized using thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and transmission electron microscopy (TEM) techniques. It was found that the developed electrochemical biosensors showed excellent catalytic activity towards the determination of natural and chemical sweeteners in commercially available food samples. Additionally, a comparative study between capillary electrophoresis and biosensor methods revealed that at optimum experimental conditions, typical detection limits ranging from 0.02017 to 0.07386 mM for steviol glycosides, 0.01857 to 0.08214 mM for neotame diastereomers and for sucralose 0.2804 mM were achieved. In contrast to CE methods, biosensor methods attained very low detection limits of 0.264 µM, 0.013 mM and 0.325 µM for rebaudioside A, neotame and sucralose, respectively. The unique properties of the nanomaterials in combination with electro chemical techniques provided best results with shorter analysis time in contrast to the conventional separation methods. Finally, the computational molecular modelling tools were used to better understand the results obtained from the separation mechanisms using capillary electrophoresis. The interaction of β-cyclodextrin with steviol glycosides/neotame diastereomers and sucralose with the amine buffers were studied and the computational results were in good agreement with the elution orders observed in capillary electrophoresis. Furthermore, docking studies were performed to predict the binding affinity interactions between the artificial sweeteners and biomolecules (cytochrome c and laccase) to understand a molecular level.
Electrochemical and molecular modelling studies to assess the photoreactive properties of Efavirenz
(2022-09) Mthiyane, Thethiwe Promise; Bisetty, Krishna; Jordaan, M. A.; Uwaya, Gloria Ebube
Efavirenz (EFV) is commonly used as an antiretroviral drug to treat HIV/AIDS and is known to undergo photoreactions that could be exploited for photodegradation applications. In addition, there is limited information on the photoreactivity of EFV. This work focuses on two case studies to assess the photocatalytic properties of EFV supported by experimental and molecular modelling (commonly referred to as computational chemistry). The first case study deals with the design of an innovative electrochemical sensor for the detection of EFV, using titanium dioxide nanoparticles (TiO2-NPs) doped on glassy carbon electrode (GCE) with nafion as an anchor agent (GCE/TiO2-NPs-nafion). TiO2-NPs were synthesized using Eucalyptus globulus leaf extract and characterized using Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and energy-dispersive spectroscopy (EDS). The electrochemical and sensing properties of the developed sensor for EFV were assessed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronoamperometry. The oxidation peak current response for EFV on the GCE/TiO2-NPs-nafion electrode was greater compared to the bare and modified GCE/TiO2-NPs electrodes. A linear dynamic range of 4.5 to 18.7 µM with a 0.01 µM limit of detection was recorded on the electrode using DPV. The electrochemical sensor demonstrated good selectivity as well as practicability for the detection of EFV drugs with excellent recoveries ranging from 92.0-103.9%. The density functional theory (DFT)-based quantum chemical modelling was used to establish the chemical reactivity for EFV, suggesting the benzoxazine ring as the active site. Monte Carlo (MC) simulations revealed a strong electrostatic interaction on the GCE/TiO2-NPs-nafion-EFV (substrate-adsorbate) system. The results showed good agreement between the MC computed adsorption energies and the experimental CV results for EFV. The stronger adsorption energy of nafion onto the GCE/TiO2-NPs substrate contributed to the catalytic role in the signal amplification sensing of EFV. The second case study deals with the assessment of the photocatalytic degradation of EFV in combination with green synthesized TiO2-NPs. The photocatalytic activity of TiO2-NPs was examined by the degradation of EFV in an aqueous medium and a maximum degradation efficiency of 91.77% was observed at a reaction time of 5 h. In addition, the electronic spectra of the EFV complex bound to single TiO2-NPs in a gas- and solution-phase were investigated using time-dependent density functional theory (TD-DFT) calculations. The calculated spectra obtained in this work were benchmarked against the gas-phase photodecomposition of the EFV- TiO2-NPs complex using UV-vis spectrophotometry. Overall, the results show that the biosynthesized TiO2-NPs have the potential for sensing pharmaceutical applications and their degradation. The results provide an effective way to explore the design of new 2D materials for the sensing of EFV, which is highly significant in the field of medicinal and materials chemistry.
Electrochemical aptasensor for the detection of mycotoxins in food samples by experimental and computational methods
(2021) Kunene, Kwanele; Bisetty, Krishna; Kanchi, Suvardhan; Sabela, Myalowenkosi Innocent
Mycotoxins are secondary metabolites of fungi that are present in various foodstuff and feed commodities. A large number of mycotoxins exist, however only a limited number represent a major damages and toxic properties. Amongst them, the aflatoxins and ochratoxins are deemed to be the most poisonous and extensively circulated in the world and then, represent a real hazard to both human and animal. Depending on several factors like the consumption levels, exposure time, mechanisms of action, digestion and defense mechanisms, mycotoxins stimulate a wide spectrum of toxicological effects leading to both acute and chronic diseases, liver and kidney failure, skin rash, cancer, immune suppression, birth defects or even death. To address the harmful impact of mycotoxin contaminants in food and feed, health authorities in various countries world-wide have established guidelines in order to protect human and animal from the possible damages instigated by these toxins. Authorities such as the European Commission, US Food and Drug Administration (FDA), World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) set up maximum level regulations for main mycotoxins in foods and feeds. To accomplish the expectations of these regulation levels, there is a great need for the development and validation of modern, uncomplicated, rapid, and detailed methodologies for the detection of toxins. In this study, various approaches for the rapid, inexpensive and ultrasensitive biosensors for the detection of two major mycotoxins were developed. The electrochemical-based aptasensor and immunosensor were developed for the determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in different food products. The fabricated biosensors demonstrated good practical analytical feasibility for mycotoxins detection in real samples such as WeetBix, yoghurt, coffee and in wine samples with excellent recoveries and RSD values. To avoid fouling on the sensor surface by the constituents present in real samples, the carbon screen printed electrode (C-SPE) and carbon felt electrode (CFE) surfaces were modified with different nanomaterials such as silver nanoparticle (AgNPs), palladium nanoparticles (PdNPs), palladium doped boron nitride (PdNPs-BN) and titanium nanoparticles doped with boron nitride BN-TiO2. In addition, the aptamers and antibodies were immobilized on the modified electrode in order to enhance the selectivity of the sensor towards the detection of OTA and AFB1. The electrochemical aptasensor for OTA permitted for highly sensitive detection in Weet-Bix with a wide linear range (0.002 - 0.016 mg L-1) and limit of detection of 7×10-4 mg L-1. It is worth prominence that it is the first time that carbon screen printed electrode (C-SPE) modified with AgNPs was used, opening new pathways for highly precise analysis. Experimental results were further supported computationally for a better understanding of the interaction between the aptamer and the analytes. Computational results were in good agreement with experimental results. The same procedure was also established in voltammetric detection of AFB1 using CFE modified with BN-TiO2 (CF/BN-TiO2). A wide concentration range of 2.5 - 20 ng mL-1 with an excellent LOD of 0.002 ng mL-1 for AFB1 was obtained. For the case study of wine samples tested for AFB1 detection, a simple but very effective pretreatment method was effectively applied. The addition of acetonitrile to the wine reduces the non-specific interactions that might be accountable for inactivation of antibody and blocking of the sensor surface. Furthermore, the PdNPs-BN enhanced the electrical signal and the sensor sensitivity. Attained results allowed for AFB1 detection at concentrations range from 1.0 - 10 ng mL-1 with limit of detection of 0.832 ng mL-1 . In the case study of the electrochemical immunosensor for the detection of OTA in coffee, a linear detection range of 0.5 - 20 ng mL-1 was achieved with LOD of 0.096 ng mL-1 . The fabricated aptasensors and immunosensors in this study combines the most desirable characteristics of a good biosensor such as high sensitivity, inexpensive, rapid, and simple but portable method make proposed approaches an important and very promising tools for extensive biosensing applications.
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.
Experimental and computational studies of a fungal chitinase
(2015) Khan, Faez Iqbal; Bisetty, Krishna; Singh, Suren; Permaul, Kugen
Chitin, the second most abundant natural biopolymer, is composed of repeating units of N-acetyl-β-D-glucosamine and primarily forms the structural component of protective biological matrices such as fungal cell walls and exoskeletons of insects. Chitinases are a ubiquitous class of extracellular enzymes that have gained attention in the past few years due to their wide range of biotechnological applications, especially in the field of agriculture for bio-control of fungal phytopathogens. They play an important role in the defense of organisms against chitin-containing parasites by hydrolyzing the β-1,4-linkages in chitin and hence act as anti-fungal as well as anti-biofouling agents. Moreover, the effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance and thus, chitinases offer a potential alternative to the use of chemical fungicides. In recent years, thermostable enzymes isolated from thermophilic microorganisms have gained widespread attention in industrial, medical, environmental and biotechnological applications due to their inherent stability at high temperatures and a wide range of pH optima. Determination of the three- dimensional structure of a protein can provide important details about its biological functions and its mode of action. However, despite their significance, the precise three-dimensional structures of most of the chitinases, including those isolated from Thermomyces lanuginosus is not fully characterized so far. Hence, the main focus of the present study was to gain a better understanding of the structural features of chitinases obtained from this thermostable fungus using both experimental and computational techniques, and their relationship with their activity profiles. The genes encoding thermostable chitinase II from T. lanuginosus were isolated and cloned in both E. coli as well as the Pichia pastoris expression system. Analysis of the nucleotide sequences revealed that the chitinase II gene (1196 bp) encodes a 343 amino acid protein of molecular weight 36.65 kDa whereas the chitinase I gene (1538 bp) encodes a 400 amino acid protein of molecular weight 44.14 kDa. In silico protein modeling was helpful in predicting the 3D models of the novel chitinase II enzyme, followed by the prediction of its active sites. The presence of Glu176 was found to be essential for the activity of chitinase II. Similarly, analysis of chitinase I revealed several active sites in its structural framework. A 10 ns Molecular dynamics (MD) simulations was implemented to assess the conformational preferences of chitinases. The MD trajectories at different temperatures clearly revealed that the stability of the enzymes were maintained at higher temperatures. Additionally, a constant pH molecular dynamics simulations at a pH range 2-6 was performed to establish the optimum activity and stability profiles of chitinase I and chitinase II. For this purpose, the Molecular Dynamics simulations were carried out at fixed protonation states in an explicit water environment to evaluate the effect of the physiological pH on chitinase I and II enzymes obtained from T. lanuginosus. The results suggest a strong conformational pH dependence of chitinases. These enzymes retained their characteristic TIM Barrel fold at the respective protonated conditions, thus validated the experimental outcomes. Further, the different stability and flexibility predictions were used to assess the relation of point mutations and enzyme stabilities. Our results pave the way to engineer new and better thermostable enzymes.
Experimental and computational studies on sensing of DNA damage in Alzheimer's disease
(2017) Murti, Bayu Tri; Bisetty, Krishna; Tiwari, Ashutosh
DNA damage plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) therefore, an innovative ss-DNA/dopamine/TiO2/FTO electrode strategy was developed to detect the genotoxicity upon photocatalytic reactions. This study involves a computational and electrochemical investigation towards the direct measurement of DNA damage. Computational chemistry was useful to resolve the intricate chemistry problems behind electrode constructions. The computational protocols were simultaneously carried out comprising of density functional theory (DFT) calculations, Metropolis Monte Carlo (MC) adsorption studies, and molecular dynamics (MD) simulations. The DFT calculations elucidated the structural, electronics, and vibrational properties of the electrode components resulting in a good agreement with the experimental parameters. The MC simulations carried out using simulated annealing predicted the adsorption process within layer-by-layer electrode as well generating reliable inputs prior to MD simulations. A 100 ns MD simulations were performed using a canonical ensemble provided information on the thermodynamics parameters such as total energy, temperature, and potential energy profiles, including radius of gyrations and atomic density profiles. Binding energies calculated from the MD trajectories revealed increasing interaction energies for the layer-by-layer electrode, in agreement with the electrochemical characterization studies (i.e. gradual decrease of cyclic voltammogram (CV) as well as increasing diameter of electrochemical impedance spectroscopy (EIS) semicircle upon electrode modification). The higher binding energies may lead to smaller changes in the electrochemical polarizability which directly affect to the decreasing of redox peak current and charge transfer resistance enhancement. Instead, HOMO-LUMO DFT levels are also taken into account to explain electron transfer phenomena within layer construction leading to the alteration of CV behaviours. Experimentally, the ss-DNA was electronically linked to TiO2/FTO surface through dopamine as a molecular anchor. Electrochemical measurements using cyclic voltammetry and EIS were employed to characterize the electrode modifications. The square wave voltammetry was subsequently used to measure the DNA damage and the potency of antioxidant treatment using ascorbic acid (AA) due to its ability in protecting the DNA from the damages. The presence of AA significantly protected the DNA from the damage, therefore was able to be used as a potential treatment in AD. Theoretically, guanine residues predicted by DFT as the most reactive sites of the ss-DNA involved in the genotoxic reactions. Overall, the theoretical studies successfully validated the experimental study as well as providing the molecular basis of interaction phenomena towards electrode constructions. Our results highlight the potential application of this methodology to screen the genotoxicity in Alzheimer’s, suggesting the important role of theoretical studies to predict the molecular interaction and validation of the DNA-based sensors and bioelectronics.