Ratiometric self-calibration strategy based on conductive hydrogen bond organic framework for reliable biosensing of peanut allergen Ara h1

: Ara h1, the primary peanut allergen, is responsible for most allergic reactions, prompting the need for precise detection methods to safeguard those with peanut allergies. Herein, we developed a novel self-calibrating ratio electrochemical immunosensor for ultrasensitive Ara h1 detection, leveraging a conductive porous hydrogen-bonded organic framework (HOF) for signal amplification. Concretely, a capture substrate was fabricated on the electrode surface by compositing ferrocene-covalently modified silica with gold nanoparticles, serving the dual functions of coupling Ara h1 aptamers and generating a ferrocene electrochemical signal. A negatively charged HOF effectively adsorbed positively charged thionine through electrostatic attraction and others, producing a second electrical signal and loading Ara h1 antibodies. In the presence of Ara h1, the aptamers and antibodies specifically bind to target, forming a sandwich structure on the electrode surface that activates ratiometric sensing. The biosensor rendered a wide linear detection range (5∼1250 ng/mL) and a low limit of detection (1.29 ng/mL). The biosensor's potential and reliability for real sample detection were confirmed through tests on 20 real samples and spiking experiments. The proposed ratiometric detection strategy provides a practical tool for allergen labeling and allergy prevention.