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Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

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An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP.

Autumn Cholger¹, Jason M Conley¹, Elaine Colomb¹

¹Department of Chemistry & Interdisciplinary Life Science Program, Purdue University, West Lafayette, IN 47907, USA.

Sensors (Basel, Switzerland)

|September 27, 2025

Summary

Researchers developed ECATS2, a new biosensor for extracellular adenosine triphosphate (ATP). This improved tool offers higher affinity for detecting purinergic signaling and ATP release in cells.

Keywords:

FRET biosensor extracellular ATP fluorescent protein purinergic

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Area of Science:

Biochemistry
Cell Biology
Neuroscience

Background:

Extracellular adenosine triphosphate (ATP) acts as a key purinergic signaling molecule.
Previous genetically encoded Förster Resonance Energy Transfer (FRET) biosensors could detect micromolar levels of extracellular ATP.

Purpose of the Study:

To develop a second-generation biosensor with enhanced affinity for extracellular ATP.
To optimize the biosensor for improved detection of purinergic signaling dynamics.

Main Methods:

Site-directed mutagenesis of the ATP binding site.
Optimization of cell-surface display and tether length.
Development of the ECATS2 FRET biosensor.

Main Results:

The ECATS2 biosensor exhibits over three-fold higher affinity for extracellular ATP compared to previous sensors.
Tether length was identified as a critical factor for biosensor performance optimization.
ECATS2 successfully detected extracellular ATP release in cultured astrocytes under hypoosmotic stress.

Conclusions:

ECATS2 represents a significant advancement in tools for detecting extracellular ATP.
The enhanced sensor enables more sensitive ratiometric detection of purinergic signaling.
This tool facilitates the study of extracellular ATP dynamics in various biological contexts.