Over the last twenty years, biosensors exploiting Förster Resonance Energy Transfer (FRET) have become a useful tool to measure a variety of biological processes. Due to their ability to detect nanoscale changes in distance, they allow for the quantitative measurement of molecular binding events and changes in protein structure. This is possible because changes in distance between the two fluorophores alter the energy transfer between them, producing a ratiometric readout of protein activity or an analyte concentration. Two popular biosensor designs have emerged that exploit this phenomenon: a single peptide (single chain) design that places the protein/analyte binding domain between the fluorophores on the same peptide and a dual chain design that places each fluorophore on a separate peptide which dimerize due to protein activity or analyte presence. Each of these designs has its own supposed benefits and drawbacks. The single chain sensor is easier to design and provides more control over the ratio and distribution of fluorophores in the cell, but has a limited dynamic range as the fluorophores cannot move very far apart to reduce the energy transfer between them. The dual chain sensor allows for larger fluorophore separation, producing a larger dynamic range, but at a cost of reduced sensitivity and increased spatial noise compared to the single chain sensor. While these principles are often held to be true, there has been no systematic trial to quantify these design constraints. In this study, we quantify the differences in dynamic range and sensitivity between dual and single chain sensors using Rac1 as a target analyte. In addition, we quantitatively explore several bicistronic expression systems to provide for a more equal expression of dual chain sensor peptides. Our work provides crucial information to better inform the design and optimization of FRET-based biosensors.

Quantifying tradeoffs in FRET-based biosensor design

256th National Meeting (2018)

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