Protein-folding-based in-vivo biosensors

概括。我们的总体研究目标是开发一种基于受体的微创方法 能够有效监测任何特定分子（无论其化学性质如何）浓度的技术 反应性）在活体内。为此，我们已经展示了电化学的能力， 基于适配体 (E-AB) 的传感器可在血管和原位监测一系列药物和代谢物 清醒、自由活动的老鼠的大脑，分辨率为秒，测量持续时间为数小时。一个潜在的 然而，该平台的显着局限性是它对核酸适体的依赖，这是一种有限的化学物质 其复杂性无疑将最终限制使用该技术进行检测的目标数量 方法。作为回应，我们在此建议开发此类中的第一个传感器，而不是使用蛋白质 作为他们的识别要素。具体来说，我们提出了两个目标，将显着降低发展风险 基于蛋白质折叠的电化学传感器的研究，为其发展成为强大的新型电化学传感器奠定了基础 分子监测方法。首先，我们将超越单一的原理验证示例（传感器 使用 FynSH3 结构域作为我们的受体制造）迄今为止，我们已经发现传感器针对三种 另外，临床上重要的目标（脓毒症诊断细胞因子 IL-6、化疗和免疫细胞因子） 调节剂甲氨蝶呤和免疫抑制剂环孢菌素）作为我们设计策略的证据 一般。其次，我们将调整这些传感器，我们已经证明这些传感器能够连续工作数小时 体外未稀释全血的性能，以适应体内发现的更具挑战性的测量条件。如果 成功后，此处描述的 R21 规模项目将为 R01 级研究计划奠定基础 将该技术与体外和体内蛋白质选择结合起来，创建支持连续、 实时测量许多临床重要分子，包括窄治疗指数药物和 蛋白质生物标志物表明许多严重的、快速进展的疾病的状态。

Summary. Our overarching research goal is the development of a minimally invasive, receptor-based technology able to monitor the concentration of effectively any specific molecule (irrespective of its chemical reactivity) in the living body. To this end, we have already demonstrated the ability of electrochemical, aptamer-based (E-AB) sensors to monitor a range of drugs and metabolites in situ in the blood vessels and brain of awake, freely moving rats with seconds resolution and measurement durations of hours. A potentially significant limitation of the platform, however, is its reliance on nucleic acid aptamers, the limited chemical complexity of which will no doubt ultimately restrict the number of targets amenable to detection using the approach. In response, we propose here to develop the first sensors in this class that instead employ proteins as their recognition elements. Specifically, we propose two aims that will significantly de-risk the development of protein-folding-based electrochemical sensors, setting the stage for their development as a powerful new approach to molecular monitoring. First, we will expand beyond the single, proof-of-principle example (a sensor fabricated using the FynSH3 domain as our receptor) we have realized to date to sensors against three additional, clinically important targets (the sepsis-diagnostic cytokine IL-6, the chemotherapeutic and immune modulator methotrexate, and the immunosuppressant cyclosporine) as evidence that our design strategy is general. Second, we will adapt these sensors, which we have already shown are capable of multi-hour performance in undiluted whole blood in vitro, to the more challenging measurement conditions found in vivo. If successful, the R21-scale project described here will lay the foundation for an R01-level research program that couples this technology with in vitro and in vivo protein selection to create sensors supporting the continuous, real time measurement of many clinically important molecules, including narrow-therapeutic-index drugs and protein biomarkers indicative of the status of many grievous, rapidly progressive diseases.