Genetically Encoded Biosensors

Figure 1. Microscope image of HeLa cells expressing FRET sensors in growth conditions.
Figure 2. Sensor response to metal chelating agents and added zinc ions.
Figure 3. Experimental workflow.
Figure 4. Single cell measurements immediately and several seconds after added zinc ions reveal sensor heterogeneity within the microfluidic device. Pair-matched response (inset).

Genetically encoded biosensors are important tools for real-time measurement of the distribution of metal-ions (e.g. Ca2+, Zn2+) and other analytes in living cells (Figure 1).  Among a handful of design platforms, the fluorescent protein (FP)-based ratiometric sensors have the distinctive advantage of being able to compensate for potential signal variations caused by cellular fluorophore concentration and excitation laser intensity. As shown in Figure 2, these sensors report the concentration change of intra- or extracellular metal ions by recruiting one (or a few) metal ion(s) to their metal sensing domains, inducing a conformational change which leads to a change in fluorescence resonance energy transfer (FRET) efficiency between the donor (CFP) and acceptor (YFP).

Over the years, most efforts in rational sensor design and optimization have encountered unpredictable and surprising results, due to poorly understood interactions between molecular components of the FRET constructs and the cellular environment. One goal of this project is to develop genetically encoded metal sensors with dramatically improved optical, physical, and chemical properties and to enable high speed event detection at low signal levels in living cells. Using a directed evolution approach to systematically explore the landscapes of the sensor constructs, this study will also provide insight into the rational design of biosensors.

The experimental method mimics the typical calibration experiment utilized by sensor designers to determine full response by measuring the minimum FRET signal and the FRET at later times reaching saturation. We design targeted libraries of the sensor constructs and express the library members in living cells (Figure 3). Using microfluidic technology developed in our lab, the library-encoded cells are loaded into a microfluidic system in which cellular responses are chemically induced and optically measured, and are screened and sorted at high-throughput. Micron-scale laminar flow and droplet generation techniques offered by microfluidics enable us to achieve rapid reaction initiation, accurate timing control, high-throughput detection of the sensor response, and sorting at the single cell level (Figure 4). Our interest extends from the fundamental molecular dynamics, such as the mechanism of metal-ion sensing, to the important cellular processes of metal homeostasis, such as the dynamics of metal-ion permeation through the plasma membrane, as well as the improvement of these sensors for measurements in a variety of cell organelles with differing chemical environments.