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

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

Author information

1
I. Physikalisches Institut (IA) , RWTH Aachen , 52074 Aachen , Germany.
2
ICS-5: Molecular Biophysics , Forschungszentrum Jülich , 52425 Jülich , Germany.
3
IBG-1: Biotechnology , Forschungszentrum Jülich , 52425 Jülich , Germany.
4
Groningen Biomolecular Sciences and Biotechnology Institute , University of Groningen , 9700 AB Groningen , Netherlands.

Abstract

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

PMID:
29979038
DOI:
10.1021/acssensors.8b00143
[Indexed for MEDLINE]

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