Dynamic energy metabolism physiological biosensing via integrated sensitive bivariate sensors and electronics for in situ monitoring of scalable cell models.

Energy metabolism is a fundamental physiological process essential to sustain basic cellular life activities through nutrient uptake and metabolite production. Classic metabolic analysis techniques, including molecular imaging, metabolomics, and cellular assays, provide valuable insights but are often limited by phototoxicity, invasive labeling, and poor temporal resolution derived from the endpoint methods. Hence, this work develops a noninvasive bivariate sensing platform to compatibly measure dynamic energy metabolism of multiscale cell models, characterized by the metabolic fluxes of mitochondrial respiration and glycolysis. The system integrated electrochemical sensors for dynamic amperometric detection of dissolved oxygen and potentiometric detection of pH, enabling multiplexed oxygen consumption rate and extracellular acidification rate quantitative recordings. Optimization of sensitive membranes and biocompatible materials provides superior sensitivity and stability. Moreover, we integrated the sensing platform with a micro-volume chamber (<200 μL) under low-buffering-capacity medium to amplify metabolite variations. The formation of a confined microchamber enables metabolite accumulation, while reopening it restores the bulk culture environment, permitting repeated measurements and enhancing data reliability. The platform achieves sensitive metabolic flux analysis of HEK-293T cells under pharmacological administration, providing characteristic bioenergetic profiles in mitochondrial stress and glycolytic stress, with good operational stability (>95% signal retention after seven days) and reproducibility (RSD < 2.5%). Furthermore, we constructed three-dimensional HEK-293T spheroids, where metabolic fluxes were effectively measured and showed high comparability to planar monolayers, demonstrating the compatibility in scalable microtissue models. This work established a robust and high-performance analytical platform that satisfies the critical demand for reliable, real-time metabolic flux analysis of complex biological systems.