BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells

Abstract

ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.

RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

“…This approach may be useful for end-users performing experiments under conditions involving harsh pH changes. Furthermore, when considering dual-imaging of ATP in both the cytoplasm and mitochondria of the same cell, the combination of mitoMaLionR and MaLionG is likely to be the most appropriate, given that ATP levels in mitochondria are relatively lower than in the cytoplasm [26] . In fact, MaLionG ( K d = 1.1 mM) was able to sense the elevation of cytoplasmic ATP levels during accelerated glycolysis in HeLa cells as shown in Figure 3b, whereas MaLionR ( K d = 0.34 mM) was unable to sense this (Figure S15), most likely due to saturation of fluorescence under high cytoplasmic ATP levels.…”

Recent Progress in Optical Sensors for Biomedical Diagnostics

“…LUMABS with furimazine substrates compares well against ELISA [204] and is compatible with several different antibodies [205]. Furimazine systems have also been employed for intracellular ATP sensing [206], voltage indication in live cells [207] as well as metal ion detection and bioimaging [207][208][209].…”

“…LUMABS with furimazine substrates compares well against ELISA [204] and is compatible with several different antibodies [205]. Furimazine systems have also been employed for intracellular ATP sensing [206], voltage indication in live cells [207] as well as metal ion detection and bioimaging [207][208][209]. [186]; (b) Vancomycin conjugated magnetic particles for gram-positive bacteria detection [187]; (c) Magnetic nanoliposomes for bioluminescent protein sensing [196]; (d) Magnetic sandwich assay for procalcitonin detection [197]; (e) Furimazine-based antibody detection using LUMABS [202]; (f) Obelin as a photoprotein in the sensing of anti-myelin basic protein autoantibody [210].…”

RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

“…This approach may be useful for end‐users performing experiments under conditions involving harsh pH changes. Furthermore, when considering dual‐imaging of ATP in both the cytoplasm and mitochondria of the same cell, the combination of mitoMaLionR and MaLionG is likely to be the most appropriate, given that ATP levels in mitochondria are relatively lower than in the cytoplasm . In fact, MaLionG ( K d =1.1 m m ) was able to sense the elevation of cytoplasmic ATP levels during accelerated glycolysis in HeLa cells as shown in Figure b, whereas MaLionR ( K d =0.34 m m ) was unable to sense this (Supporting Information, Figure S15), which is most likely due to saturation of fluorescence under high cytoplasmic ATP levels.…”