Highly sensitive ochratoxin A impedimetric aptasensor based on the immobilization of azido-aptamer onto electrografted binary film via click chemistry

The aptamer immbolization onto organized mixed layers of diazonium salts via click chemistry was explored. The immobilized aptamer was employed in the fabrication of a highly sensitive and reusable electrochemical impedimetric aptasensor for the detection of ochratoxin A (OTA). The screen-printed carbon electrodes (SPCEs) were first modified by electrografting of a protected 4-((trimethylsilyl)ethynyl) benzene (TMSi-Eth-Ar) layer followed by a second one of p-nitrobenzene (p-NO2-Ar) by means of electrochemical reduction of their corresponding diazonium salts, (TMSi-Eth-Ar-N2+) and (p-NO2-ArN2+). After deprotection, a layer with active ethynyl groups was obtained. In the presence of copper (I) catalyst, the ethynyl groups reacted efficiently with aptamer bearing an azide function, thus forming a covalent 1,2,3-triazole linkage. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of ferri/ferrocyanide redox probe [Fe(CN)6]4−/3− were used to characterize each step in the aptasensor development. The increase in electron-transfer resistance (Ret) values due to the specific aptamer–OTA interaction was proportional to the concentration of OTA in a range between 1.25 ng/L and 500 ng/L, with a detection limit of 0.25 ng/L.

► The SPCE was modified with a robust binary film of diazonium salts. ► Click chemistry was used to immobilize azido-aptamer onto ethynyl modified surface. ► The immobilized aptamer was used in the construction of impedimetric aptasensor for OTA detection. ► The immobilization via click chemistry improved the aptamer binding capacity and magnified the signal response.