In situ electrochemical measurement of alkaline phosphatase activity in engineered gut models using a porous membrane electrode device.

In vitro assays utilizing human cells have attracted attention as alternatives to animal testing for drug screening. Microphysiological systems (MPS) have been proposed as cell culture platforms that more closely mimic the in vivo microenvironment. Engineered gut models have been extensively studied using human colorectal adenocarcinoma cell lines (Caco-2 cells). Alkaline phosphatase (ALP) activity indicates Caco-2 cell differentiation and is a component of the chemical barrier that maintains intestinal barrier function. However, conventional absorbance-based methods for ALP measurement can harm cells owing to prolonged exposure to alkaline conditions (approximately 60 min). In this study, we developed an engineered gut model with a porous membrane electrode for in situ measurement of ALP activity. The electrochemical sensors, positioned directly beneath the cells on porous membrane electrodes, allowed for rapid short-term measurements. ALP activity was detected after a 3 min treatment through in situ electrochemical measurements, which indicated low cytotoxicity. Therefore, we measured ALP activity multiple times using the same device over 21 days. The proposed system offers potential for in situ measurement and real-time monitoring in in vitro organ models, including MPS devices.