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Sensors
Volume 25
Issue 18
10.3390/s25185903
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP

by
Autumn Cholger
1,†,
Jason M. Conley
1,†,
Elaine Colomb
1,
Olivia de Cuba
2,
Jacob Kress
2 and
Mathew Tantama
2,*
1
Department of Chemistry & Interdisciplinary Life Science Program, Purdue University, West Lafayette, IN 47907, USA
2
Department of Chemistry & Biochemistry Program, Wellesley College, Wellesley, MA 02481, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sensors 2025, 25(18), 5903; https://doi.org/10.3390/s25185903 (registering DOI)
Submission received: 15 August 2025 / Revised: 18 September 2025 / Accepted: 18 September 2025 / Published: 21 September 2025
(This article belongs to the Section Biosensors)
Versions Notes

Abstract

Adenosine triphosphate (ATP) is readily released into the extracellular space as an autocrine and paracrine purinergic signaling molecule. We originally reported a genetically encoded, fluorescent protein-based Förster Resonance Energy Transfer (FRET) biosensor that can detect micromolar levels of extracellular ATP. Through mutagenesis of the ATP binding site and optimization of cell-surface display, here we report the development of a second-generation biosensor called ECATS2 with greater than three-fold higher affinity for extracellular ATP. We found that the tether length between the FRET biosensor and the cell surface anchor is critical to optimization of its performance. Furthermore, we demonstrate that the improved sensor can detect extracellular ATP release upon hypoosmotic stress in cultured astrocytes. This new sensor contributes an improved tool for the ratiometric detection of extracellular ATP dynamics and purinergic signaling.
Keywords: extracellular ATP; purinergic; FRET biosensor; fluorescent protein extracellular ATP; purinergic; FRET biosensor; fluorescent protein

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MDPI and ACS Style

Cholger, A.; Conley, J.M.; Colomb, E.; de Cuba, O.; Kress, J.; Tantama, M. An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP. Sensors 2025, 25, 5903. https://doi.org/10.3390/s25185903

AMA Style

Cholger A, Conley JM, Colomb E, de Cuba O, Kress J, Tantama M. An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP. Sensors. 2025; 25(18):5903. https://doi.org/10.3390/s25185903

Chicago/Turabian Style

Cholger, Autumn, Jason M. Conley, Elaine Colomb, Olivia de Cuba, Jacob Kress, and Mathew Tantama. 2025. "An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP" Sensors 25, no. 18: 5903. https://doi.org/10.3390/s25185903

APA Style

Cholger, A., Conley, J. M., Colomb, E., de Cuba, O., Kress, J., & Tantama, M. (2025). An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP. Sensors, 25(18), 5903. https://doi.org/10.3390/s25185903

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