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

Carnegie Institution Of Washington, Washington DC

Investigators

Abstract

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.