Development of a Polypyrrole–Chitosan Electrospun Nanofiber-Based Enzymatic Biosensor for Sensitive and Rapid Detection of Acetylcholine

The development of a highly sensitive and rapid electrospun nanofiber-based enzymatic biosensor for acetylcholine (ACh) detection is presented. This biosensor employs dual enzyme reactions involving acetylcholine esterase (AChE) and choline oxidase (ChO), immobilized on polypyrrole (PPy) and chitosan (CS)-based electrospun nanofibers. For this purpose, polypyrrole nanoparticles containing chitosan nanofibers were first electrospun on the working electrode of a screen-printed electrode (SPE). The immobilization was achieved through two methods: covalent bonding and chitosan entrapment. The electrospun nanofibers offer a large surface area and high porosity, enhancing the biosensor's sensitivity and selectivity. Structural and morphological analyses, including SEM and FTIR, confirmed the successful synthesis and integration of PPy nanoparticles within the CS nanofibers. Nanofibers with an average diameter of 280 nm were obtained. Electrochemical characterization using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) demonstrated improved conductivity and effective enzyme immobilization on the nanofiber-modified electrodes. The biosensor exhibited a linear response to ACh concentrations ranging from 10 μM to 1 mM, with a detection limit of 5 μM. Selectivity tests indicated minimal interference from other substances, and stability assessments confirmed reliable performance over 30 days. The application of the biosensor to spiked serum samples demonstrated accurate ACh detection, highlighting its potential for clinical diagnostics and neurological research. This innovative approach offers a portable, cost-effective, and efficient solution for rapid acetylcholine measurement, addressing the limitations of traditional analytical methods.