Protein-folding-based in-vivo biosensors

摘要我们的总体研究目标是开发一种微创的，基于受体的 技术能够有效地监测任何特定分子的浓度（无论其化学性质如何）， 反应性）在活体中。为此，我们已经证明了电化学， 基于适体（E-AB）的传感器，用于监测血管中原位的一系列药物和代谢物， 清醒、自由活动大鼠的大脑，秒分辨率和测量持续时间为小时。一个潜在 然而，该平台的显著限制是其依赖于核酸适体，即有限的化学物质， 其复杂性无疑将最终限制易于使用 approach.作为回应，我们建议在这里开发这类传感器中的第一个，而不是采用蛋白质 作为他们的识别元素。具体来说，我们提出了两个目标，将显着降低风险的发展 基于蛋白质折叠的电化学传感器，为它们的发展奠定了基础， 分子监测方法。首先，我们将扩展到单个原理验证示例（传感器 使用FynSH 3结构域作为我们的受体制造），我们迄今为止已经实现了针对三种 另外，临床上重要的靶点（脓毒症诊断细胞因子IL-6，化疗和免疫调节因子IL-6，免疫调节因子IL-10，免疫调节因子IL-11，免疫调节因子IL-12，免疫调节因子IL-13，免疫调节因子IL-14，免疫调节因子IL-15，免疫调节因子IL-16，免疫调节因子IL-18，免疫调节因子IL-19。 调节剂甲氨蝶呤和免疫抑制剂环孢菌素）作为证据，证明我们的设计策略是 将军第二，我们将调整这些传感器，我们已经证明，这些传感器能够在多小时内 将体外未稀释全血中的性能与体内更具挑战性的测量条件相比较。如果 如果成功，这里描述的R21规模的项目将为R 01级研究计划奠定基础， 将该技术与体外和体内蛋白质选择相结合， 真实的实时测量许多临床上重要的分子，包括窄治疗指数药物和 蛋白质生物标志物指示许多严重的、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.