Enhanced amperometric tyramine sensing via tyrosinase entrapment in polyethylene glycol-modified methacrylate microgels

Abstract

A highly sensitive and selective tyrosinase-based electrochemical biosensor for tyramine detection in complex food matrices was developed using polyethylene glycol-poly(methacrylic acid) microgels (PEG-pMAA) as enzyme immobilization matrix. The morphology and particle size of the obtained PPO-PEG-pMAA microgel were studied by SEM and particle analyzer system, obtained polydisperse microgels in the range of 2–18 μm with a 6.2 μm of average particle size. Incorporation of PEG during microgel polymerization provided a favorable microenvironment for enzyme activity, resulting in a 22 % higher amperometric response toward tyramine compared to microgels without PEG (PPO-pMAA), without compromising reproducibility (RSD ≤6.1 %). The PPO-PEG-pMAA biosensor’s performance was optimized in terms of cross-linking degree, PEG %, pH, potential, temperature, enzyme loading and microgel deposition. A complete enzyme entrapment at pH 6 in microgel synthesis with a cross-linking ratio of 2 %, which balanced enzyme retention and substrate accessibility is obtained. Under optimal conditions, the device exhibited a wide linear range (2.0 × 10􀀀 7 – 4.7 × 10􀀀 5 M), low detection limit (45nM) and rapid amperometric responses (50 s). The freeze-dried microgels were stable 9 months and the microgelbased biosensor retained 95–100 % of its initial response during 35 days of storage, confirming excellent stability. Interference studies showed negligible effects from the most abundant acids and amino acids present in cheese, highlighting the device’s selectivity. Application to cheese samples, including Brie and Gouda, yielded recoveries between 97.6 and 105.6 %, and allowed monitoring of tyramine accumulation during storage, demonstrating the biosensor’s reliability and suitability as a rapid and practical tool for tyramine screening in complex food matrices.