The human sweet taste receptor (T1R2) monomer—a member of the G-protein coupled receptor family that detects a wide variety of chemically and structurally diverse sweet tasting molecules, is known to pose a significant threat to human health. Protein that lack crystal structure is a challenge in structure-based protein design. This study focused on the interaction of the T1R2 monomer with rebaudioside A (Reb-A), a steviol glycoside with potential use as a natural sweetener using in-silico and biosensing methods. Herein, homology modelling, docking studies, and molecular dynamics simulations were applied to elucidate the interaction between Reb-A and the T1R2 monomer. In addition, the electrochemical sensing of the immobilised T1R2-Reb-A complex with zinc oxide nanoparticles (ZnONPs) and graphene oxide (GO) were assessed by testing the performance of multiwalled carbon nanotube (MWCNT) as an adsorbent experimentally. Results indicate a strong interaction between Reb-A and the T1R2 receptor, revealing the stabilizing interaction of the amino acids with the Reb-A by hydrogen bonds with the hydroxyl groups of the glucose moieties, along with a significant amount of hydrophobic interactions. Moreover, the presence of the MWCNT as an anchor confirms the adsorption strength of the T1R2-Reb-A complex onto the GO nanocomposite and supported with electrochemical measurements. Overall, this study could serve as a cornerstone in the development of electrochemical immunosensor for the detection of Reb-A, with applications in the food industry.
Herein, we report on the performance of graphene oxide (GOx) and zinc oxide nanoparticles (ZnONPs) on a platinum (Pt) electrode, immobilized with the human T1R2 sweet taste receptor subunit for the detection of rebaudioside A (Reb-A). The characterization studies performed in this work confirmed the thin-layered structure of GOx and the polydispersed nature of ZnONPs. The elucidation of the mass loss observed by TGA demonstrates the stability of GOx. The cyclic voltammetry results for Pt/GOx revealed good catalytic activity over Pt/ZnONPs for adsorption of the T1R2-Reb-A complex. In addition, a series of computational modelling studies were carried out to better understand the surface adsorption phenomena of GOx and ZnONPs to mimic the layer-by-layer electrode modification strategies independently. The strongest interaction energy observed (−573 kcal mol−1) for the direct interaction of ZnONPs onto the Pt electrode surface, demonstrates a stronger adsorption in contrast to the GOx modified Pt electrode (−23 kcal mol−1). However, the overall results for the layered-nanocomposite revealed that the GOx (−256 kcal mol−1) were more strongly adsorbed in contrast to ZnONPs (−231 kcal mol−1) for the detection of the T1R2-ReB-A complex, demonstrating the reliability of our GOx electrode functionalization strategy. The results of this study can potentially be used to improve the design of rapid Reb-A sensors for the food and beverage industry.
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
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