Magnetic Beads-Based Electrochemical Label-Free DNA-Bioassay for the Detection of Peanut Allergen Ara h2 in Food Matrices

The reliable detection of the peanut allergen Ara h2 in processed foods remains a major challenge, since thermal and high-pressure treatments can alter protein structure and limit the performance of immunoassays. DNA-based methods provide a robust alternative to this approach. In this work, a highly sensitive label-free electrochemical genoassay for Ara h2 DNA detection was developed using streptavidin-coated magnetic beads (MBs). A biotinylated capture probe (CP) immobilized on the MBs’ surface enabled specific target recognition through a sandwich hybridization strategy with a secondary probe, allowing for direct electrochemical detection without enzymatic labels. Two transduction strategies were evaluated: (i) electrochemical impedance spectroscopy (EIS) with ferri/ferrocyanide as a redox probe, and (ii) differential pulse voltammetry (DPV) using methylene blue. The ferri/ferrocyanide-based EIS approach showed the best sensitivity and discrimination between hybridized and non-hybridized states. A linear dependence was observed with the concentration of the synthetic Ara h2 target over the 0.05 to 20 nM range, with a detection limit of 0.025 nM. CP-MBs showed good stability for at least 20 days. Applicability was demonstrated in soy beverages, rice beverages, and low-fat cow’s milk, with recoveries close to 100% and negligible matrix effects.