Genetically Encoded Förster Resonance Energy Transfer-Based Biosensors Studied on the Single-Molecule Level

ACS Sens. 2018 Aug 24;3(8):1462-1470. doi: 10.1021/acssensors.8b00143. Epub 2018 Jul 18.


Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for the quantification of ligand molecules change the magnitude of FRET between two fluorescent proteins upon binding a target metabolite. When highly sensitive sensors are being designed, extensive sensor optimization is essential. However, it is often difficult to verify the ideas of modifications made to a sensor during the sensor optimization process because of the limited information content of ensemble FRET measurements. In contrast, single-molecule detection provides detailed information and higher accuracy. Here, we investigated a set of glucose and crowding sensors on the single-molecule level. We report the first comprehensive single-molecule study of FRET-based biosensors with reasonable counting statistics and identify characteristics in the single-molecule FRET histograms that constitute fingerprints of sensor performance. Hence, our single-molecule approach extends the toolbox of methods aiming to understand and optimize the design of FRET-based biosensors.

Keywords: chomophore maturation; conformational change; crowding sensor; glucose sensor; single-molecule FRET.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biosensing Techniques / methods*
  • Fluorescence Resonance Energy Transfer*
  • Glucose / analysis*
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / metabolism
  • Polyethylene Glycols / chemistry


  • Luminescent Proteins
  • Polyethylene Glycol 6000
  • Polyethylene Glycols
  • Glucose