Electrochemical enzymatic biosensing of neotame in sweeteners by experimental and computational methods
Authors:   , Lephalala Matshidiso 
Published: January 2022

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.