Santa Cátia, Park Soohyun, Gejt Artur, Clark Heather A, Hengerer Bastian, Sergelen Khulan
BioMed X Institute, Heidelberg, Germany.
Faculty of Biotechnology, Mannheim University of Applied Sciences, Germany.
Analyst. 2024 Dec 16;150(1):131-141. doi: 10.1039/d4an01226g.
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.
治疗药物的实时监测对于治疗管理和药代动力学研究至关重要。我们展示了用于窄治疗窗药物万古霉素实时监测的分裂适体夹心测定法的优化和亲和力调整，采用表面等离子体共振（SPR）技术。为了通过SPR实现对小分子的可逆、无标记传感，我们通过分裂适体方法将万古霉素结合适体应用于夹心测定形式。通过评估原始适体二级结构内的多个分裂位点，我们确定位置27（P27）对于保持靶标亲和力、确保可逆性和最大化灵敏度是最佳的。该测定法在生理相关的pH和二价阳离子范围内表现出稳健的性能，并且通过圆二色光谱法证实了适体分裂片段与分析物形成特定的三元复合物。随后，我们设计了一系列在茎区互补性增加的分裂适体对，与原始P27对相比，亲和力和检测限提高了10倍。对工程化分裂适体对的动力学进行了评估，揭示了快速的结合和解离速率，证实了各变体的亲和力和检测限均有所提高。最重要的是，所有对都保持了测定法的可逆性，这对于实时监测至关重要。最后，该测定法在复杂生物基质中进一步得到验证，包括犬的脑脊液和大鼠的稀释血浆，证明了其在生物环境中的功能以及超过9小时的稳定性。我们的研究为分裂适体在小分子实时监测中的应用铺平了道路，对治疗药物监测和药代动力学研究具有潜在意义。
