Kozhemyakin Roman¹, Yamamoto Saki², Imran Sehrish³, Barbosa Tiago⁴, Meier Jonas⁵, Chevalier Lucille⁶
ABSTRACT:
Background: Understanding the dynamic regulation of metabolic pathways in living cells is essential for deciphering cellular physiology, disease mechanisms, and therapeutic responses. Traditional methods often rely on endpoint measurements or destructive sampling, limiting temporal resolution and physiological relevance. The development of genetically encoded and synthetic biosensors has enabled real-time, non-invasive monitoring of specific metabolites, enzymatic activities, and signaling events within live cells with unprecedented spatial and temporal resolution. Methods and Results: This review highlights key strategies and recent advances in the engineering and application of biosensors for metabolic pathway analysis in living systems. Biosensors based on Förster resonance energy transfer (FRET), fluorescence intensity, circularly permuted fluorescent proteins (cpFPs), and luminescence have been successfully applied to monitor ATP, NAD⁺/NADH, lactate, pyruvate, glucose, and other metabolic intermediates. Biosensor design involves careful consideration of target specificity, dynamic range, signal-to-noise ratio, and cellular localization. Real-time imaging of metabolic flux has revealed compartmentalized and oscillatory patterns of metabolite distribution, uncovering new regulatory mechanisms in cancer metabolism, stem cell differentiation, and immunometabolism. Advances in microscopy, such as fluorescence lifetime imaging (FLIM) and light-sheet microscopy, further enhance resolution and minimize phototoxicity during long-term observation. Conclusion: Biosensors represent a transformative toolkit for metabolic research, offering live-cell, real-time insights into cellular biochemistry. Their continued refinement and integration with systems biology platforms will enable comprehensive understanding of metabolic dynamics in health and disease.