Real-Time Neurotransmitter Sensing in Midbrain Organoids Using 3D Carbon Cup Electrodes
Real time, chemically selective detection of neurotransmitter release in brain organoids remains a challenge due to their complex three dimensional architecture and limited optical accessibility. We introduce 3D printed carbon cup electrodes (CCEs) -pyrolyzed, multi level pillar structures - designed to confine a single organoid, increase electroactive surface area, and enable amperometric recordings of oxidizable neurotransmitters. The CCEs exhibit excellent electrochemical performance and reproducibility, supporting diffusion controlled dopamine redox behavior without interference from culture medium. Using ventral midbrain (vMB) organoids at two maturation stages (Day 30 and Day 90), we demonstrate real time detection of potassium evoked dopamine release directly from organoids. Mature organoids exhibit higher peak currents, correlating with increased tyrosine hydroxylase (TH) expression confirmed by immunocytochemistry. Embedding organoids in Matrigel significantly attenuates dopamine signals, indicating a diffusion barrier that limits stimulant access and/or dopamine transport to the electrode. Repetitive stimulations reveal progressive depletion of dopamine stores, consistent with diminished vesicular release. Chemical perturbations using 6 hydroxydopamine and verapamil further validate the platform by producing substantial reductions in dopamine release accompanied by loss of TH neurons. These results establish CCEs as a powerful tool for label free, real-time monitoring of neurotransmission in human organoids, with potential for multiplexed sensing and integration of enzymatic detection strategies for non electroactive neurotransmitters.