A POC label-free biosensor to detect point mutations in the rpoB gene of Mycobacterium tuberculosis from RR-TB raw sputum samples.

Rifampicin-resistant and multidrug-resistant tuberculosis (RR/MDR-TB) remain pressing global health concerns. Conventional diagnostics such as culture and GeneXpert are limited by high cost, infrastructure requirements, and lengthy procedures, highlighting the need for rapid and reliable molecular detection platforms. We developed an electrochemical biosensor for the selective detection of wild-type and clinically relevant point mutations (codons 516 and 531) in the rpoB gene of Mycobacterium tuberculosis. Thiolated gene-specific ssDNA probes were immobilized on gold nanoparticle-modified carbon screen-printed electrodes. The working principle of the designed biosensor relies on hybridization between the immobilized ssDNA probes and their complementary target sequences, which induces a marked decrease in electrochemical signals measured using Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV), thereby confirming high mutation specificity. The biosensor achieved detection limits of 6.01 pM and 6.48 pM for codons 516 and 531, respectively, with strong linearity (R = 0.963 and 0.957). It successfully identified MDR-TB directly from raw sputum samples, eliminating the need for sample pretreatment. This highly sensitive (∼6 pM), mutation-specific electrochemical platform offers an accurate and rapid molecular diagnostic for RR/MDR-TB. Its ability to distinguish specific mutations and operate directly on clinical samples underscores its promise for point-of-care application.