Deep eutectic solvent-based molecularly imprinted polymers for selective extraction and HPLC-SEC determination of pepsin in human saliva

Early and non-invasive detection of gastroesophageal reflux disease (GERD) remains challenging, highlighting the need for selective and environmentally sustainable analytical methods for salivary biomarkers. In this study, eco-friendly molecularly imprinted polymers (MIPs) based on deep eutectic solvents (DESs) were developed for the selective extraction of pepsin enzyme from human saliva. Two DES systems were designed: a DES functional monomer composed of methacrylic acid and choline chloride, and a DES comonomer consisting of itaconic acid and [2-(methacryloyloxy)ethyl] trimethylammonium chloride. Surface imprinting polymerization was performed on silica nanoparticles using pepsin as the template, ethylene glycol dimethacrylate (EGDMA) as the crosslinker, and ammonium persulfate as the initiator in phosphate buffer. The resulting DES-MIPs exhibited preferential recognition toward pepsin, achieving a binding capacity (Q) of 385 mg g−1, significantly exceeding that of non-imprinted polymers (NIPs) (205 mg g−1), with negligible interference from other salivary enzymes such as amylase and lipase. Structural and thermal characterization using FT-IR, TGA, DSC, and SEM confirmed the successful formation and stability of the imprinted polymers. Adsorption kinetics followed a pseudo-second-order model (R2 = 0.999), while the adsorption isotherm fitted the Langmuir model (R2 = 0.906), indicating monolayer chemisorption on a homogeneous surface. After extraction, pepsin was eluted with phosphate buffer and quantified by HPLC-SEC. The integrated DES-MIP-HPLC-SEC method showed linearity between 0.5 and 150 μg mL−1 with a limit of detection of 0.15 μg mL−1. The method demonstrated strong green analytical performance, achieving a 0.76 AGREEmip score and 80% GAPI score, confirming its sustainability.