Engineered Aptamer-Derived Fluorescent Aptasensor: the Label-Free, Single-Step, Rapid Detection of Vancomycin in...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Existing vancomycin (VCM) aptamers have compromised affinities due to stem truncation, limiting their clinical utility within the therapeutic window.
• The binding pocket of the parent VCM aptamer was not previously elucidated, hindering rational aptamer design.
• There is a need for improved VCM aptamers and sensitive detection methods for therapeutic drug monitoring.

Purpose of the Study:

• To elucidate the binding pocket of the VCM parent aptamer.
• To engineer a high-affinity and specific VCM aptamer using post-SELEX modifications.
• To develop a label-free fluorescent aptasensor for rapid VCM detection in clinical samples.

Main Methods:

• Elucidation of the VCM aptamer binding pocket.
• Post-SELEX modification strategy involving truncation and mutagenesis to refine the parent aptamer.
• Development of a portable, label-free fluorescent aptasensor utilizing competitive binding.

Main Results:

• A novel VCM aptamer (ABC20-11) was engineered with an intramolecular G-triplex, enhanced fluorescence, and significantly improved affinity (K_d = 0.591 × 10^-6 m) and specificity.
• The developed aptasensor demonstrated excellent analytical performance in serum, cerebrospinal fluid, and joint fluid.
• Linear ranges were observed from 0.5-50 × 10^-6 m (serum, joint fluid) and 0.5-40 × 10^-6 m (CSF), with low detection limits.

Conclusions:

• The engineered VCM aptamer (ABC20-11) offers superior affinity and specificity compared to previous variants.
• The label-free fluorescent aptasensor provides a sensitive and rapid method for VCM detection.
• The proposed aptasensor holds significant clinical value for real-time VCM therapeutic drug monitoring.