Development of an electrochemical immunosensor for the detection of steviol glycosides by experimental and computational methods

Abstract

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