EAGER: A Microfluidic Platform for Accelerated Construction of Nanosensors for High-Resolution Analysis of Hormone Levels in Vivo

在植物中，细胞和器官之间的通信主要通过小分子信号发生，并且这种通信是将发育和环境信号整合到连贯的生长和生殖策略中所必需的。更详细地了解植物中的小分子信号传导将允许合理地改进作物和农业实践。理解小分子信号传导的一个主要限制是缺乏测量细胞或亚细胞区室中小分子浓度波动的工具。小的信号分子（例如激素）与特定的受体蛋白结合，然后改变形状（构象）。改变的受体构象导致进一步的信号传导，最终导致对原始激素信号的适当响应。原则上，如果可以跟踪构象变化，则由激素结合诱导的受体蛋白质构象变化可以用作激素浓度的代表。 这种跟踪可以使用Förster共振能量转移（FRET）纳米传感器以高分辨率完成。FRET纳米传感器是在小分子结合时改变构象的荧光蛋白。这种构象变化导致FRET效率的变化，可以测量光学和非侵入性。至少目前，这种传感器的设计是经验性的，对于单个传感器需要多年的开发和优化，而不能保证成功。该项目的中心目标是使用专为高通量蛋白质合成和荧光分析而设计的微流控芯片快速开发一套新的激素纳米传感器。数千种潜在的激素传感蛋白将在微流控芯片上合成，然后在暴露于植物激素后测试FRET变化。可以在体外跟踪激素浓度的蛋白质将被部署在植物中，以验证它们可以用于体内动态激素测量。激素传感器是一个关键和必要的工具，在这个项目中开发的纳米传感器将提供给科学界。快速传感器开发的新平台将有助于构建用于测量更多小分子的纳米传感器，从而大大扩展了用于研究信号感知和转导的潜在工具集。该项目还提供了一个独特的机会，交叉培训激素生物学和生物工程博士后研究员。最后，来自当地一所少数民族比例较高的高中的学生将参加该项目，并将测试一种简单的低成本基于数码相机的FRET成像系统，用于非侵入性激素分析。

In plants, communication between cells and organs occurs predominantly via small molecule signals, and this communication is required to integrate developmental and environmental signals into a coherent growth and reproductive strategy. More detailed understanding of small molecule signaling in plants will allow for rational improvements to crops and agricultural practices. A major limitation for understanding small molecule signaling is the lack of tools for measuring the fluctuations of small molecule concentrations in cells or subcellular compartments. Small signaling molecules (e.g. hormones) bind to specific receptor proteins, which then change shape (conformation). The altered receptor conformation leads to further signaling that eventually results in appropriate responses to the original hormone signal. In principle, the receptor protein conformation change induced by hormone binding can be used as a proxy for hormone concentration if the conformation change can be tracked. This tracking can be accomplished with high-resolution using Förster resonance energy transfer (FRET) nanosensors. FRET nanosensors are fluorescent proteins that change conformation upon small molecule binding. This conformational change results in FRET efficiency changes that can be measured optically and non-invasively. At least presently, the design of such sensors is empirical, requiring years of development and optimization for a single sensor without a guarantee of success. The central aim of this project is to rapidly develop a suite of new nanosensors for hormones using a microfluidic chip designed for high-throughput protein synthesis and fluorescence analysis. Thousands of potential hormone sensing proteins will be synthesized on microfluidic chips and then tested for FRET changes after exposure to plant hormones. Proteins that can track hormone concentration in vitro will be deployed in plants to verify that they can be used for dynamic hormone measurement in vivo. Hormone sensors are a critical and needed tool and the nanosensors developed in this project will be made available to the scientific community. The novel platform for rapid sensor development will be useful for constructing nanosensors for measuring many more small molecules, thereby dramatically expanding the potential tool sets available for studying signal perception and transduction. The project also provides a unique opportunity to cross-train a postdoctoral fellow in hormone biology and bioengineering. Finally, students from a local high school with a high minority representation will participate in the project and will test a simple low-cost digital camera-based FRET imaging system for non-invasive hormone analysis.