Development of electrophoretic and biosensor methods applied to high intensity sweeteners
Authors:   , Bathinapatla Ayyappa 
Published: August 2021

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.