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 …