Label-free electrochemical biosensing of MDR-TB genes using graphite-ZnO nanofibers on glassy carbon electrodes

The rising global burden of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for simple, affordable, and reliable diagnostic technologies. In this work, a low-cost, label-free DNA biosensor is developed using graphite-zinc oxide (Graphite-ZnO) nanofibers fabricated by electrospinning and integrated onto glassy carbon electrodes. The biosensor is specifically designed for the detection of drug-resistance-associated genes of Mycobacterium tuberculosis (MTB), with particular emphasis on the embC gene, which is linked to both active and latent TB infections. Single-stranded oligonucleotide probes targeting the embC gene were carefully designed and synthesized, followed by the optimization of surface immobilization protocols to ensure stable and efficient probe attachment onto the nanofiber-modified electrodes. The synthesized nanofibers were structurally and morphologically characterized at a scale of up to 20 μm, confirming the formation of a uniform and interconnected fibrous network suitable for biosensing applications. Electrochemical evaluation of DNA hybridization events was carried out using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The biosensor demonstrated sensitive detection performance, achieving limits of detection of 0.665 pM/mL, 0.326 pM/mL, and 0.51 pM/mL using CV, DPV, and EIS, respectively. The practical applicability of the proposed biosensor was further validated through hybridization assays using DNA extracted from urine samples, confirming its potential as a non-invasive and point-of-care diagnostic platform for MDR-TB detection.