3D neuronal monitoring platforms for electrochemical sensing of neurotransmitters in vitro

Neurological diseases and mental disorders pose a major challenge due to their high prevalence and progressive neuronal degeneration, which often disrupts neurotransmitter release. Among the most critical neurotransmitters are glutamate and dopamine, both deeply involved in key neurophysiological processes and strongly associated with disorders like Parkinson’s, Alzheimer’s, and epilepsy. To better understand these mechanisms under physiologically relevant conditions, it is essential to develop platforms capable of detecting neurotransmitters in environments that mimic the human brain. In this context, this thesis presents innovative 3D platforms that not only support the growth of human neuronal networks but also enable real-time, in situ monitoring of neurotransmitter release.

A robust chemical and genetical differentiation protocol was established to generate glutamatergic and dopaminergic neurons from human induced pluripotent stem cells (iPSCs). Remarkably, incorporating multi-walled carbon nanotubes (MWCNTs) significantly enhanced neuronal maturation, both phenotypically and functionally. These nanomaterials boosted the expression of key neuronal markers such as vesicular glutamate transporter 1 (vGLUT1) and tyrosine hydroxylase (TH), indicating enhanced subtype specification. Additionally, calcium imaging revealed a marked increase in spike frequency, confirming greater neuronal excitability and network activity in the presence of MWCNTs.

A planar electrochemical sensor was designed to detect dopamine and glutamate in vitro. Glutamate was detected using chronoamperometry with cobalt phthalocyanine and glutamate oxidase-functionalized MWCNTs, reaching a detection limit of 136.00 µM. MWCNTs modified with gold nanoparticles enabled dopamine detection through differential pulse voltammetry, achieving a detection limit of 62.20 nM. Notably, the sensor successfully detected dopamine released by iPSC-derived dopaminergic neurons, identifying both spontaneous and stimulated release using potassium chloride and benztropine. These findings establish a novel platform for monitoring dopamine release in mature neuronal cultures.

To further bridge the gap between sensing and biological relevance, a 3D scaffold composed of brain extracellular matrix and MWCNTs crosslinked with genipin was engineered. This biohybrid structure supported iPSC differentiation, with MWCNTs significantly boosting TH expression in dopaminergic neurons and more than doubling vGLUT1 expression in glutamatergic neurons. As a 3D sensor, the dopamine 3D platform achieved a detection limit of 218.52 μM, while the hydrogen peroxide sensor reached 42.00 µM. While both sensors demonstrated high reproducibility and repeatability, their performance in terms of limit of detection and sensitivity did not surpass the planar platforms. Nonetheless, these 3D systems represent a promising foundation for integrated neurotransmitter sensing at cellular level. Future studies should focus on enhancing the scaffold’s conductivity, immobilizing the enzyme within the 3D structure, and further improving their reliability and applicability in complex biological environments.