Triple-Branch Catalytic Assembly DNAzyme Motivated DNA Tweezer for Sensitive and Reliable <i>mecA</i> Gene Detection in <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> (<i>S. aureus</i>, SA) is one of the most common bacteria in nosocomial infections. Sensitive and efficient analysis of methicillin-resistance of SA is crucial for improving the nursing performance of pneumonia. However, methicillin-resistance analysis with favorable sensitivity and specificity in an enzyme-free manner remains a huge challenge. This paper presents the development of a new fluorescent biosensor for detecting <i>mecA</i> gene using a triple-branch catalytic hairpin assembly (CHA) triggered DNAzyme switch-based DNA tweezer. The SA from the samples are immobilized on the plate's surface using the protein A antibody. The biosensor possesses several key features. Firstly, it utilizes dual signal amplification processes, specifically the triple-branch CHA and DNAzyme controlled DNA tweezer-based signal recycling, to enable <i>mecA</i> detection on the plate. This design enhances the method's sensitivity, resulting in a low limit of detection of 1.5 fM. Secondly, the biosensor does not rely on enzymes for <i>mecA</i> analysis, ensuring a high level of stability during target analysis. Lastly, the method demonstrates a remarkable selectivity by accurately distinguishing target sequences from non-target sequences. The proposed biosensor, which does not require enzymes and has a high level of sensitivity, offers a viable platform for the rapid and simple quantification of <i>mecA</i> in SA.