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Gut–brain axis (GBA) communication relies on serotonin (5-HT) signaling between the gut epithelium and the peripheral nervous system, where 5-HT release patterns from the basolateral (i.e., bottom) side of the epithelium activate nerve afferents. There have been few quantitative studies of this gut-neuron signaling due to a lack of real-time measurement tools that can access the basolateral gut epithelium. In vitro platforms allow quantitative studies of cultured gut tissue, but they mainly employ offline and endpoint assays that cannot resolve dynamic molecular-release patterns. Here, we present the modification of a microporous cell culture membrane with carbon nanotube-coated gold (Au-CNT) electrodes capable of continuous, label-free, and direct detection of 5-HT at physiological concentrations. Electrochemical characterization of single-walled carbon nanotube (SWCNT)-coated Au electrodes shows increased electroactive surface area, 5-HT specificity, sensitivity, and saturation time, which are correlated with the CNT film drop-cast volume. Two microliters of CNT films, with a 10-min saturation time, 0.6 μA/μM 5-HT sensitivity, and reliable detection within a linear range of 500 nM–10 μM 5-HT, can be targeted for high-concentration, high-time-resolution 5-HT monitoring. CNT films (12.5 μL) with a 2-h saturation time, 4.5 μA/μM 5-HT sensitivity, and quantitative detection in the linear range of 100 nM–1 μM can target low concentrations with low time resolution. These electrodes achieved continuous detection of dynamic diffusion across the porous membrane, mimicking basolateral 5-HT release from cells, and detection of cell-released 5-HT from separately cultured RIN14B cell supernatant. Electrode-integrated cell culture systems such as this can improve in vitro molecular detection mechanisms and aid in quantitative GBA signaling studies.

Electrochemical measurement of serotonin by Au-CNT electrodes fabricated on microporous cell culture membranes