Electrochemical Detection of Peanut Allergen Ara h 1 Using a Sensitive DNA Biosensor Based on Stem–Loop Probe

A novel electrochemical DNA sensor was developed by using a stem–loop probe for peanut allergen Ara h 1 detection. The probe was modified with a thiol at its 5′ end and a biotin at its 3′ end. The biotin-tagged “molecular beacon”-like probe was attached to the surface of a gold electrode to form a stem–loop structure by self-assembly through facile gold–thiol affinity. 6-Mercaptohexanol (MCH) was used to cover the remnant bare region. The stem–-loop probe was “closed” when the target was absent, and then the hybridization of the target induced the conformational change to “open”, along with the biotin at its 3′ end moved away from the electrode surface. The probe conformational change process was verified by circular dichroism (CD); meanwhile, electron-transfer efficiency changes between probe and electrode were proved by electrochemical impedance spectroscopy (EIS). The detection limit of this method was 0.35 fM with the linear response ranging from 10^–15 to 10^–10 M. Moreover, a complementary target could be discriminated from one-base mismatch and noncomplementarity. The proposed strategy has been successfully applied to detect Ara h 1 in the peanut DNA extracts of peanut milk beverage, and the concentration of it was 3.2 × 10^–13 mol/L