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

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,0.6μA/μM 5-HT sensitivity,and reliable detection within a linear range of 500 nM–10μM 5-HT,can be targeted for high-concentration,high-time-resolution 5-HT monitoring.CNT films(12.5μL)with a 2-h saturation time,4.5μA/μM 5-HT sensitivity,and quantitative detection in the linear range of 100 nM–1μM 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.

3D-Printed electrochemical sensor-integrated transwell systems

This work presents a 3D-printed,modular,electrochemical sensor-integrated transwell system for monitoring cellular and molecular events in situ without sample extraction or microfluidics-assisted downstream omics.Simple additive manufacturing techniques such as 3D printing,shadow masking,and molding are used to fabricate this modular system,which is autoclavable,biocompatible,and designed to operate following standard operating protocols(SOPs)of cellular biology.Integral to the platform is a flexible porous membrane,which is used as a cell culture substrate similarly to a commercial transwell insert.Multimodal electrochemical sensors fabricated on the membrane allow direct access to cells and their products.A pair of gold electrodes on the top side of the membrane measures impedance over the course of cell attachment and growth,characterized by an exponential decrease(~160%at 10Hz)due to an increase in the double layer capacitance from secreted extracellular matrix(ECM)proteins.Cyclic voltammetry(CV)sensor electrodes,fabricated on the bottom side of the membrane,enable sensing of molecular release at the site of cell culture without the need for downstream fluidics.Real-time detection of ferrocene dimethanol injection across the membrane showed a three order-of-magnitude higher signal at the membrane than in the bulk media after reaching equilibrium.This modular sensor-integrated transwell system allows unprecedented direct,real-time,and noninvasive access to physical and biochemical information,which cannot be obtained in a conventional transwell system.