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

Abstract 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.