<i>In situ</i> electrochemical measurement of alkaline phosphatase activity in engineered gut models using a porous membrane electrode device

<i>In vitro</i> 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 <i>in vivo</i> 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 <i>in situ</i> 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 <i>in situ</i> 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 <i>in situ</i> measurement and real-time monitoring in <i>in vitro</i> organ models, including MPS devices.