An ultrasensitive lateral flow strip assay based on MXene@PtCu nanozymes for visual biosensing of Burkholderia pseudomallei EV-sRNA

Burkholderia pseudomallei (B. pseudomallei) is the pathogen of fatal tropical infectious disease melioidosis. This study screened sRNA BprsO from extracellular vesicles (EVs) of B. pseudomallei as a new biomarker and constructed a lateral flow strip assay (LFSA) with dual signal amplification for its visual biosensing by coupling catalytic hairpin assembly (CHA) with MXene@PtCu nanozymes. The platinum nanoparticles (Pt NPs) in the nanozymes provided a large number of active sites for the assembly of nucleic acid and antibodies, and enhanced the catalytic activity of Cu NPs through their synergistic effect. The large specific surface area of MXene avoided the aggregation of Pt and Cu NPs for improving the signal output. Moreover, CHA technology not only recycled the target, but also successfully introduced biotin-labeled probes to the nanozymes surface, ultimately converting the nucleic acid concentration into easily accessible visual detection signals. Benefiting from the twofold amplification of MXene@PtCu and CHA, the proposed colorimetric assay exhibited superior performance for BprsO detection with a dynamic range from 50 aM to 5 nM, and a detection limit of 5 aM. Notably, the method was successfully implemented in the context of LFSA, thereby illustrating its capacity for the visual detection of BprsO at a concentration as low as 15 fM and achieving a detection sensitivity of 1.26 fM. This work validates that the screened BprsO can serve as a specific biomarker for B. pseudomallei, and proposes a strategy with high sensitivity and specificity that offers a robust visual platform for early diagnosis and monitoring of B. pseudomallei.