Improved electrochemical competitive immunosensor for ochratoxin A with a biotinylated monoclonal antibody capture probe and colloidal gold nanostructuring

Abstract

We report a sensitive electrochemical immunosensor for Ochratoxin A (OTA), which is a frequent mycotoxin contaminant in cereals and other kinds of agricultural commodities, based on a biotinylated monoclonal antibody against OTA (mAbOTA–bi) and the avidin–biotin coupling of the tracer extravidin–horseradish peroxidase (ea–HRP). The analytical performance has been improved with respect to a previously developed immunosensor based on a polyclonal antibody against OTA and a secondary aIgG antibody labeled with alkaline phosphatase as tracer. The immunosensor relies on indirect competitive assay format after the passive physical adsorption of the antigen conjugated to bovine serum albumin (OTA–BSA) or bound to gold nanoparticles (OTA–BSA–AuNPs), and the screen-printed technology for voltammetric (DPV) measurements. The new design is simpler (only one capture probe), requires less time (only one incubation time with antibody), and shows an increased slope at the linear range of the calibration plot due to higher affinity of the monoclonal antibody compared to the polyclonal one. The newly designed immunosensor has a linear dynamic range of 0.15 to 9.94 ng mL⁻¹ of OTA (R ≥ 0.9900), lower detection limit (0.10 ng mL⁻¹ OTA), and a variability between assays of about 10%. The immunosensor was validated with certified wheat samples after extraction of OTA in acetonitrile : water (6/4) (v/v), and allows the determination of OTA in concentration levels well below those permitted in cereals under European Union recommendations (3 ng g⁻¹).