Nondestructive In-Situ Measurement of Alkaline Phosphatase Activity of Human Intestinal Organoids in Hydrogel Domes...

Human intestinal organoids serve as valuable models in regenerative medicine and drug screening. Nondestructive evaluation of both the hydrogel domes and the organoids cultured within them, including assessment of alkaline phosphatase (ALP), a key differentiation marker, remains a critical analytical challenge. To address this, we have developed a scanning electrochemical microscopy (SECM)-based approach that enables in-situ, real-time monitoring of ALP activity of organoids in hydrogel domes. Based on the recorded oxidation currents of enzymatic product of ALP, we have quantitatively determined ALP activity at the single-organoid level, reporting for the first time the enzyme activity in units per organoid (where 1 U corresponds to 1 μmol of product formed per minute). This innovative SECM strategy provides a noninvasive and spatially resolved analytical platform that is expected to advance organoid-based drug screening and transplantation studies.