Protein-folding-based in-vivo biosensors

总结。我们的首要研究目标是开发一种基于受体的微创 能够有效地监测任何特定分子(无论其化学成分)的浓度的技术 反应性)在活体中。为此，我们已经展示了电化学的能力， 基于适体的(E-AB)传感器，用于在血管和组织中监测一系列药物和代谢物 清醒的、自由活动的大鼠的大脑，分辨率为秒，测量持续时间为小时。一个潜在的 然而，该平台的重大限制是它对核酸适体的依赖，核酸适体是一种有限的化学物质 其复杂性无疑将最终限制可使用 接近。作为回应，我们建议开发这一类中的第一个传感器，而不是使用蛋白质 作为它们的识别元素。具体地说，我们提出了两个目标，这将显著降低发展的风险 基于蛋白质折叠的电化学传感器，为其发展成为一种强大的新技术奠定了基础 分子监测的方法。首先，我们将超越单一的原则证明示例(传感器 使用FynSH3结构域作为我们的受体)，到目前为止，我们已经意识到传感器对三个 其他临床上重要的靶点(败血症诊断细胞因子IL-6、化疗和免疫 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 将军。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持续数小时 在体外未稀释的全血中的性能，到体内发现的更具挑战性的测量条件。如果 成功，这里描述的R21规模的项目将为R01级研究计划奠定基础 将这项技术与体外和体内蛋白质选择相结合，创造出支持连续、 许多临床重要分子的实时测量，包括窄治疗指数药物和 蛋白质生物标志物，指示许多严重、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.