CRISPR-enhanced fluorescent signal amplification for ultra-sensitive detection of vancomycin

Vancomycin is a natural glycopeptide antibiotic produced by Streptomyces species. It is primarily employed to treat infections caused by drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA). However, the therapeutic concentration range of vancomycin is narrow, necessitating therapeutic drug monitoring (TDM). Currently, the commonly used vancomycin detection methods face challenges such as high equipment costs, complex operations, lengthy processing times, and poor reproducibility. To overcome these limitations, we developed a CRISPR-enhanced fluorescent (CEF) aptasensor for efficient and accurate detection of vancomycin, using signal amplification to provide critical support for TDM. In the presence of vancomycin, its specific binding to the aptamer exposes the primer strand, which is part of the self-assembled DNA tetrahedron, thereby initiating the RCA process. The product subsequently activates Cas12a, causing it to trans-cleave the signal probe ssDNA-FQ, which is modified by Cy3 and BHQ2. The fluorophore will move away from the quencher, thereby restoring fluorescence and generating a significantly amplified fluorescent signal, achieving highly sensitive detection of vancomycin. The constructed biosensor exhibits good linear response in the target concentration range of 1 fM to 10 nM, with a detection limit of 164 aM. This novel biosensor holds great promise for the accurate detection of low-abundance biomarkers like vancomycin, which is crucial for ensuring optimal dosing and preventing adverse effects such as nephrotoxicity. Moreover, the simplicity, rapidity, and cost-effectiveness of this aptasensor make it an ideal candidate for therapeutic drug monitoring and clinical diagnostics.