Electrochemically Controlled Neurotransmitter Delivery for In Vitro Neuromodulation

The National Academies of Science and Engineering have issued a grand challenge to “reverse-engineer the brain”. Addressing this challenge requires a more complete understanding of how connections between individual neurons and groups of neurons are formed and maintained, as well as a more thorough elucidation of the signaling processes that drive neural network optimization. However, to achieve these goals, it is first necessary to develop the appropriate fundamental tools and protocols for adaptive biointerfacing, bottom-up neuroscience, and neuromodulation, which all involve a tightly coordinated synergy between sensors and specialized devices for neurotransmitter delivery. To date, extensive research efforts have been devoted to neurotransmitter sensing, but relatively little attention has been given to developing methods for electronically controlled <i>in vitro</i> neurotransmitter delivery.<br/><br/>To address this challenge, our research team has developed devices that can both sense and selectively dispense the biochemical cues that drive the formation, regulation, and modulation of <i>in vitro</i> cellular/neural networks. Here, we report electrochemically controlled dispensing of both excitatory and inhibitory neurotransmitters (<i>e.g.</i> glutamate, GABA, dopamine) from functionalized electrodes that operate at low voltage. The regulated uptake and release of neurotransmitters is mediated by electrochemically controlling the electrostatic affinity between the neurotransmitter and a polarized electrode that has been functionalized with an electroactive affinity molecule.<br/><br/>To optimize our devices, our team tested a variety of electrode functionalization approaches (<i>e.g</i>. alkane-thiol or silane chemistry, layer-by-layer or covalent modification) and a variety of electroactive affinity molecules (<i>e.g.</i> ferrocene, methylene blue, viologens). Our controlled dispensing devices function as a faradaic capacitor – when the functionalized electrode is anodically polarized, the redox affinity molecule becomes oxidized (<i>i.e.</i> positively charged), and the neurotransmitter loads on the functionalized electrode through electrostatic attraction to the oxidized redox affinity molecule. The neurotransmitter may be conveniently “stored” on the electrode by maintaining the applied electrode potential, and then released on demand by reversing the applied bias using controlled steps or pulses, which reduces the electroactive affinity molecule and releases the neurotransmitter. This controlled adjustment of applied electrode potential permits quantitative biochemical release. In addition, the electrochemical signature associated with the loading of different neurotransmitters permits sensing as well as delivery.<br/><br/>We confirm the operational principle through a variety of analytical and electroanalytical techniques, such as cyclic voltammetry, chronopotentiometry, open-circuit potentiometry, continuous enzymatic monitoring, electrochemical quartz crystal microbalance (EQCM), X-ray photoelectron spectroscopy, and triple quadrupole mass spectrometry. We then demonstrate that the functionalized electrodes may also be coupled with electrochemical transistors (ECT) for triggered neurotransmitter release in conjunction with changes in the concentration of important physiological ions (<i>i.e.</i> sodium, potassium, calcium) or biochemical messengers.<br/><br/>Finally, we will present the steps we have taken to implement these devices within organ-on-a-chip systems, and demonstrate the utility of our devices for studying the formation and longitudinal evolution of neural networks within <i>in vitro</i> neuronal colonies.