Neuropeptide Y Detection Using an Aptamer-Modifed Microelectrode Together with Methylene Blue As a Redox Probe

Abstract

Neuroscience research has been growing fast over the last few years. Particularly the knowledge about neurotransmitters has increased over the past 50 years, impacting the understanding of many neurological disorders, including depression, schizophrenia, Parkinson's disease, and Alzheimer's disease. Still, there is a lack of knowledge about neuropeptides' liberation and action, and most of their function is assumed by indirect measurements. Neuropeptides are classified as non-electroactive molecules, making them difficult to track; consequently, the design of an analytical technique for their measurements is imperative. Neuropeptide Y (NPY) is the most abundant neuropeptide in the brain. It is associated with a wide range of biological processes and conditions, including depression, anxiety, obesity, alcoholism, mood changes, feeding behavior, and post-traumatic stress disorder (PTSD), among others. Currently, there is no method for NPY measurement in high temporal and spatial resolution in the brain. Therefore, we proposed the design of a biological sensor to understand the mechanism in which NPY is involved. This research is focused on electrochemical methods to detect and measure NPY based on an affinity-mediated strategy. An aptamer, a short single-stranded DNA with high affinity by NPY, was attached to a Pt surface of a microelectrode, taking advantage of the affinity between Pt and a thiol group in the aptamer structure. Then, to amplify the electrochemical signal, a redox molecule such as methylene blue (MB) was conjugated to the aptamer to allow rapid measurements of NPY levels in real-time. Real-time techniques such as cyclic voltammetry and square wave voltammetry (SWV) were explored to detect NPY in artificial Cerebrospinal Fluid (aCSF). Due to MB having a characteristic signal in both techniques, we successfully measured NPY concentration in aCSF (pH = 7.4). A change in current was observed as the NPY molecules interacted with the aptamer affecting the MB electrochemical signal and producing a "signal-off" sensor. These results allow us to obtain a calibration curve that will be used as an initial platform to perform Fast Scan Cyclic Voltammetry (FSCV) measurements to increase the time resolution in our measurements.