A dual-mode biosensor for rapid Mycobacterium tuberculosis complex diagnosis: Combining rolling circle amplification with visual and fluorescent readout

Abstract

Effective and early diagnosis of tuberculosis (TB) is crucial, yet it remains a significant challenge in developing countries with limited healthcare facilities. To address this, we developed an innovative, affordable, and practical diagnostic method. This study focuses on a biosensor that combines Rolling Circle Amplification (RCA) with phenol red and carbon nanodots (CNDs). The novelty of this approach lies in the integration of a synergistic dual-mode detection strategy, seamlessly coupling fluorometric precision via pH-sensitive CNDs with direct naked-eye visual readout using phenol red. RCA was selected for its simplicity, high specificity, and strong amplification capability, while phenol red and CNDs act as dual-mode sensors, detecting the pH changes that occur during the RCA reaction. The system demonstrated high sensitivity with a limit of detection (LOD) of 1.83 × 10 3 copies/mL (fluorescence) and 10 4 copies/mL (visual). It also showed high specificity with no cross-reactivity against common bacteria. Validation with real patient sputum samples confirmed its ability to detect MTBC DNA, with results that correlated well with the GeneXpert Ultra test, proving its clinical relevance. This innovative biosensor offers a promising and accessible solution for rapid, sensitive, and specific TB diagnosis, directly addressing the critical needs of resource-limited settings and ultimately contributing to better patient outcomes globally.

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