Neuropeptides are related to many processes of human life, from hormones that carry chemical signals into the endocrine system to neurochemical messengers that work similarly to small neurotransmitters. (Duvall et al., 2019) NPY is the most abundant neuropeptide in the brain and has been related to diseases such as hypertension, obesity, schizophrenia,(Morosawa et al., 2017) fear,(Tasan et al., 2016) addiction,(Gilpin, 2012) stress, (Wagner et al., 2016) depression, pain, cancer,(Tilan and Kitlinska, 2016) memory,(Gøtzsche and Woldbye, 2016) and sleep disorders, among others.(Crespi, 2011; Kalra et al., 1990; Langley et al., 2022; Sajdyk, 2005) Currently, there is a lack of an analytical technique that directly measures NPY levels, preventing researchers from understanding the natural role of NPY in these diseases. Each microenvironment under study requires the precise measurement of the different molecules present at appropriate rates depending on the kinetics of each process.(Eugster et al., 2022) Developing analytical techniques has brought the understanding of brain chemistry in space and time, especially electrochemical techniques. Fast scan cyclic voltammetry (FSCV) and amperometry allowed the researchers to study sub-second processes related to oxidizable molecules (electroactive molecules). (Bath et al., 2000) In the last decade, enzymatic electrodes have been developed using these two techniques to measure non-electroactive molecules. For example, glucose oxidase has been used to measure glucose thanks to the formation of hydrogen peroxide, which is electroactive and can be measured by amperometry.(Lugo-Morales et al., 2013) Researchers have now measured the classic neurotransmitters, which are electroactive molecules. Neuropeptides are classified as non-electroactive molecules for which no method is available. Due to their structural differences compared to classic neurotransmitters, the enzymatic method does not work for detecting neuropeptides because it would require different oxidases for each neuropeptide. The lack of an analytical technique to measure neuropeptides with high temporal and spatial resolution has made the rapid progression in this area difficult.

Neuropeptides are constituted by short amino acid chains synthesized in the soma and dendrites. These peptides are stored in large, dense core vesicles until their release from nerve endings and dendrites. Unlike small neurotransmitters, some neuropeptides do not undergo recycling, lack a reuptake system, and are broken down by peptidases. This demands that the cell synthesize new molecules, sending them from the soma to the nerve endings and dendrites for replacement. Since other small neurotransmitters are typically released alongside neuropeptides, it becomes crucial to devise a highly selective technique for their detection and formulate a strategy to mitigate the signal interference from other neurotransmitters. Electrochemical biosensors (EB) offer a variety of platforms for performing measurements. EB are tools that help transduce biological processes into a quantitative measurement that we can analyze and constantly evolve to achieve higher sensitivity, selectivity, and stability. (Singh et al., 2021) Different strategies are used to measure molecules selectively, such as aptamers, antibodies, enzymes, and molecular imprinting polymers (MIPs). MIPs are materials that can be electropolymerized on the surface of the electrodes with recognition capabilities for specific molecules. They are created by polymerizing functional monomers around a template molecule, resulting in cavities with sizes, shapes, and functionalities complementary to the template. (Yücebaş et al., 2020) After template removal, these cavities act as highly selective binding sites for the target molecule, allowing its recognition from a complex mixture.(Ayankojo et al., 2022) MIPs offer several advantages over traditional detection techniques. Their high selectivity minimizes interference from other molecules, while their robust synthetic nature enables modifying their properties for specific applications. Additionally, MIPs can be easily scaled and immobilized on various supports (Feier et al., 2019), making them ideal for sensor development.

In a recent development, J. M. Seibold and colleagues introduced an innovative approach utilizing aptamer-modified microelectrodes for the dynamic measurement of NPY, showcasing promising potential for exceptional spatial and temporal resolution.(Seibold et al., 2023) The researchers achieved precise measurements of NPY in serum. However, it's noteworthy that the microelectrodes used are too big for brain-related analyses, ca. 4 mm in diameter. In a related breakthrough, N. K. M. Churcher and collaborators demonstrated a remarkable detection limit for NPY in sweat, reaching 10 pg/mL(Mintah Churcher et al., 2020) and 50 pg/mL(Churcher et al., 2022) using antibodies and larger electrodes. While this marks substantial progress in the field, a primary challenge persists in the inherent limitations of antibodies, which lack long-term stability. Additionally, the large size of these electrodes poses a hindrance, rendering them unsuitable for utilization in brain-related studies. Our group reported the measurement of NPY concentrations in an implantable platinum microelectrode in concentrations as low as 10 ng/mL in aCSF using aptamers. (López et al., 2021) While we tested biofouling and compared it to carbon fiber microelectrodes, metal microelectrodes still have biofouling problems that are expected to be reduced by depositing polymers on the surface.(Sun et al., 2021; Zhang et al., 2015).

In this conference paper, we use a biosensing method based on molecularly imprinted polymers (MIP) to measure the concentration of Neuropeptide Y (NPY) using carbon fiber microelectrodes. In order to test this technique, NPY1–36 has been used in all experiments. Two techniques were used: CV, which showed a decrease in the current in the presence of NPY, and EIS, we were able to observe a change in the capacitance of the microelectrode due to NPY concentration changes.