Engineered Aptamer‐Derived Fluorescent Aptasensor: the Label‐Free, Single‐Step, Rapid Detection of Vancomycin in Clinical Samples

Currently, the reported vancomycin (VCM) aptamers, including the 3- (K_d = 9.13 × 10^-6 m) and 4-truncated variants (K_d = 45.5 × 10^-6 m), are engineered via stem truncation of the VCM parent aptamer, which inevitably compromises their affinities, thus affecting their clinical application within the VCM therapeutic window of 6.9-13.8 × 10^-6 m. Herein, the binding pocket of the VCM parent aptamer is elucidated for the first time and we implemented the Post-SELEX modification strategy involving truncation and mutagenesis to refined the VCM parent aptamer. This yielded a VCM aptamer (ABC20-11) with an intramolecular G-triplex, an enhanced thioflavin T (ThT) fluorescence intensity, and an improved affinity (K_d = 0.591 × 10^-6 m) and specificity (one-methyl level) for VCM. Utilizing a portable fluorescence detector specifically designed for rapidly detecting VCM concentration and leveraging the competitive binding between VCM and ThT to ABC20-11, a label-free fluorescent aptasensor is developed. This aptasensor exhibits exceptional analytical performances across various clinical samples (serum, cerebrospinal fluid, and joint fluid), with corresponding linear ranges of 0.5-50, 0.5-40, and 0.5-50 × 10^-6 m and detection limits at 0.11, 0.12, and 0.16 × 10^-6 m, respectively. Consequently, the proposed VCM aptasensor displays considerable clinical value and potential for use in rapid VCM detection.