Eine kritische Evaluierung FRET-basierter Biosensoren als Werkzeuge für die quantitative Metabolitanalytik

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In recent years, a significant number of genetically-encoded fluorescence biosensors have been developed, enabling real-time detection of signaling intermediates and metabolites with subcellular spatial resolution. Many of these biosensors utilize Foerster Resonance Energy Transfer (FRET) and serve as the foundation for non-invasive cell-based assays to monitor signal transduction in living cells. This work focuses on two FRET-based biosensors composed of periplasmatic binding proteins from E. coli (PBP) fused to derivatives of green fluorescent protein (GFP). Structural changes in the PBPs upon metabolite binding alter the orientation and distance between the fluorescent proteins, thereby affecting the FRET signal. The study investigates two established sugar sensors for maltose and glucose detection, previously used in various cell types. To evaluate these biosensors for quantitative metabolite analyses, the impact of environmental conditions—such as pH, buffer salts, ionic strength, temperature, and intracellular metabolites—on signal intensity and Kd-values was thoroughly examined. Additionally, the effects of molecular crowding agents like polyethyleneglycol and Ficoll were explored, revealing that sensor-specific parameters are influenced by medium viscosity. The results indicate that both biosensors are significantly affected by these factors, leading to variations in FRET signals and Kd-values. This highlights the risk