DNA-Based EC-SERS Biosensing of Single Nucleotide Mutation in Rifampicin-Resistant Mycobacterium Tuberculosis using Cerium Oxide Adsorbed ITO Slides

Abstract

Tuberculosis (TB), caused by the bacteria Mycobacterium tuberculosis, is one of the major contributors to mortality worldwide. Rifampicin is one of the first-line antituberculosis drugs, however, due to inappropriate use of the drug, Rifampicin resistance has emerged due to the mutation in the rpoB gene of Mycobacterium Tuberculosis. In this study, an ultrasensitive and label-free electrochemical-surface enhanced Raman spectroscopy (EC-SERS) dual approach biosensor has been developed for the detection of single nucleotide mutation in the rpoB gene of Mycobacterium tuberculosis, which is based on the uniform coating of Cerium Oxide nanoparticles on the surface of 3-aminopropyl trimethoxysilane (APTMS) functionalized ITO slides. Particularly, ITO electrodes were modified by Cerium Oxide nanoparticles to enhance the Raman intensity and to facilitate the immobilization of mutation 532 specific ssDNA probes via Ce-S bonds. The synthesised CeO2 nanoparticles were analysed using scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX) and Fourier transform infrared spectroscopy (FTIR). The hybridization between the single-stranded DNA (ssDNA) probe and target DNA (tDNA) was investigated using surface-enhanced Raman spectroscopy (SERS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV) techniques. The EC-SERS biosensor demonstrated a high correlation coefficient of R2= 0.989 for DPV and R2= 0.985 for SERS when tested with varying amounts of target DNA, under optimal conditions. The biosensor is capable of differentiating non-complementary DNA from target DNA that is fully matched, given optimum conditions.