Triple recognition mediated split-DNAzyme catalyzed DNA walker coupling personal glucose meter for accurate and direct Methicillin-resistant Staphylococcus aureus (MRSA) analysis

Abstract Urinary tract infections involving methicillin-resistant Staphylococcus aureus (MRSA) pose significant clinical challenges, necessitating timely diagnosis and appropriate therapeutic intervention. The development of accurate and sensitive detection methods for MRSA is therefore of considerable clinical importance. This study introduces a split-DNAzyme-based DNA walking system that employs triple recognition for the precise and direct identification of intact MRSA cells. The assay utilizes three functional probes—tracker/protein A antibody-conjugated magnetic nanoparticles (MNPs), S1/aptamer targeting PBP2a, and S2/cholesterol—to specifically recognize protein A, penicillin-binding protein 2a (PBP2a), and the bacterial lipid bilayer, respectively. By integrating proximity ligation with a DNA walker mechanism, the system performs an “AND” logic-gated analysis of three key MRSA biomarkers, enabling highly sensitive detection of the pathogen. Furthermore, the incorporation of a personal glucose meter (PGM) for quantifying glucose signals generated by the biosensor significantly enhances the portability of the assay. The proposed method achieved a detection limit as low as 2.7 cfu/mL, which is attributed to the efficient signal amplification of the DNA walker and the enhanced targeting capability of the multivalent recognition system. The strategy demonstrated markedly improved accuracy in MRSA identification compared to conventional approaches. This integrated biosensing platform may broaden the repertoire of current bacterial detection methodologies and inspire the development of novel diagnostic and therapeutic tools.