DNAzyme-activated rolling circle amplification cascade nanomachine for sub-femtomolar detection of tuberculosis single-nucleotide mutation

Abstract

Background

Tuberculosis (TB) remains a global health crisis, with drug-resistant strains posing significant diagnostic challenges due to the high cost and performance limitations of current methods. Rifampicin-resistant TB (RR-TB), approximately 97% of which are associated with rpoB 531T single-nucleotide mutation, presents a critical target for TB control. Padlock probe ligation-based rolling circle amplification (RCA) enables discrimination of single-nucleotide variants, rendering it a promising approach for detecting TB drug-resistance mutations. However, padlock probe ligation-based RCA achieves only sub-picomolar limits of detection, limiting its application.

Results

Herein, we present a multifunctional DNA nanomachine designed for the rapid detection of the Mycobacterium tuberculosis (MTB) rpoB 531 (TCG to TTG) mutation. This nanomachine integrates a DNAzyme-mediated cleavage reaction and RCA within a spatially confined architecture. Specifically, by co-assembling a DNAzyme substrate strand, a DNAzyme catalytic strand, a preformed circular template, and an RCA primer into a single nanostructure, we established a confined reaction microenvironment that minimizes diffusion distances, enabling accelerated reaction kinetics while effectively reducing cross-talk between cascade steps. Leveraging the programmability of DNA nanomachines with detection probe-functionalized magnetic nanoparticles for real-time optomagnetic sensing, our biosensor achieved a detection limit of 0.3 fM with a total assay time of 100 min and exhibited a dynamic detection range spanning 6 orders of magnitude. The biosensor performance was validated using synthetic wild-type sequences, spiked serum samples, and clinical sputum DNA extracts.

Significance and novelty

By spatially confining cascade DNA reactions within a single nanostructure, we overcame intrinsic limitations of homogeneous cascade reactions in complex matrices, enabling autocatalytic signal recycling, RCA, nanoparticle clustering, and real-time quantification. This approach offers a robust and cost-effective solution for point-of-care RR-TB diagnosis and demonstrates the potential for detecting other single-nucleotide mutations.