Measurement of Neuropeptides and Amino Acids with Carbon-Fiber Multielectrode Array Enzyme-Modified Biosensors
Authors: Zestos Alexander George , Alyamni Nadiah , Abot Jandro L. 
Published: August 2024

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. These assays typically measure small molecule neurotransmitters such as dopamine and serotonin. The carbon fiber is relatively small, biocompatible, and makes minimally invasive measurements at high spatial and temporal resolution. Carbon Fiber Multielectrode arrays have been utilized to measure multiple neurotransmitters in several brain regions simultaneously with multi-waveform application on each electrode. We have extended this work to measure larger molecule neuropeptides such as Neuropeptide Y and Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and peptide, decreased surface fouling, and codetection with other catecholamines. As both oxytocin and Neuropeptide Y contain tyrosine, the MSW was also used to detect these neuropeptides. Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied neuropeptides on rat brain slices. These results may serve as novel assays for neuropeptide detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of neuropeptides such as Neuropeptide Y and oxytocin.

Moreover, we have also developed enzyme modified microelectrodes for the measurement of glutamate (L-glutamic acid), which is an important excitatory amino acid and biomarker for epilepsy along with the inhibitory GABA. Since glutamate is not redox active at carbon electrodes, we modified CFMEs with glutamate oxidase enzyme to metabolize glutamate to hydrogen peroxide, which was then oxidized at carbon electrodes to produce readout cyclic voltammograms (CVs). The enzyme coating was optimized by varying the concentration of enzyme, chitosan binder, solvent, and deposition time. The coating was further analyzed electrochemically and imaged with scanning electron microscopy (SEM) for thickness and uniformity of surface coverages. Energy-Dispersive Spectroscopy (EDS/EDX) was utilized for chemical surface functionalization analysis. Glutamate oxidation was found to be adsorption controlled to CFMEs and characterized at various scan rates, concentrations, and stability times as well with an approximate 100 nM limit of detection. Glutamate was co-detected in complex mixtures with several monoamines such as dopamine, serotonin, norepinephrine, and others. Glutamate will furthermore be measured in several food samples and ex vivo in rat coronal brain slices and in vivo in anesthetized and freely behaving animals.