PCR-Free Self-Calibrated Ratiometric Electrochemical Genosensor Utilizing a Dual-Signal Amplification Approach for Genomic Detection of Mycobacterium tuberculosis

Abstract Tuberculosis (TB) remains a critical global health challenge. While many electrochemical (EC) biosensors have been developed for Mycobacterium detection, most relied on PCR amplification strategies. This study introduced a PCR-free, self-calibrated ratiometric electrochemical (REC) genosensor leveraging dual-signal amplification for the detection of Mycobacterium tuberculosis (MTB). Electroactive silver nanoparticles (AgNPs) were modified onto screen-printed gold electrodes, followed by chemisorption of a methylene blue (MB)-tagged peptide nucleic acid (PNA) probe targeting MTB genomic DNA. MTB presence triggered structural changes in the probe, reducing the MB redox current (IMB) as the detection signal, while the Ag current (IAg) diminished as an internal calibration marker. This ratiometric mechanism minimized system errors, enhancing detection reliability. The genosensor achieved a broad detection range (3.5 fM–35 nM) with a detection limit of 1.59 fM for non-amplified MTB DNA, showing high specificity, reproducibility, and accuracy comparable to standard diagnostic methods. It effectively detected MTB in simulated sputum samples, demonstrating its potential for reliable, rapid, and amplification-free TB diagnostics.