Real-time monitoring of vancomycin using a split-aptamer surface plasmon resonance biosensor.

Real-time monitoring of therapeutic drugs is crucial for treatment management and pharmacokinetic studies. We present the optimization and affinity tuning of split-aptamer sandwich assay for real-time monitoring of the narrow therapeutic window drug vancomycin, using surface plasmon resonance (SPR). To achieve reversible, label-free sensing of small molecules by SPR, we adapted a vancomycin binding aptamer in a sandwich assay format through the split-aptamer approach. By evaluating multiple split sites within the secondary structure of the original aptamer, we identified position 27 (P27) as optimal for preserving target affinity, ensuring reversibility, and maximizing sensitivity. The assay demonstrated robust performance under physiologically relevant ranges of pH and divalent cations, and the specific ternary complex formation of the aptamer split segments with the analyte was confirmed by circular dichroism spectroscopy. Subsequently, we engineered a series of split-aptamer pairs with increasing complementarity in the stem regions, improving both the affinity and limit of detection up to 10-fold, as compared to the primary P27 pair. The kinetics of the engineered split-aptamer pairs were evaluated, revealing fast association and dissociation rates, confirming improved affinity and detection limits across variants. Most importantly, the reversibility of the assay, essential for real-time monitoring, was maintained in all pairs. Finally, the assay was further validated in complex biological matrices, including the cerebrospinal fluid from dogs and diluted plasma from rats, demonstrating functionality in biological environments and stability exceeding 9 hours. Our study paves the way for applications of split-aptamers in real-time monitoring of small molecules, with potential implications for therapeutic drug monitoring and pharmacokinetic studies.