Competitive electrochemical immunosensors, based on disposable screen-printed electrodes (SPCEs), have been developed for the determination of the mycotoxin ochratoxin A (OTA). Two indirect immunoassays schemes were assessed using polyclonal antibodies and through the physical adsorption of OTA conjugated to albumin from bovine serum (OTA-BSA) and newly synthesized OTA-BSA bound to gold nanoparticles (OTA-BSA-AuNPs) onto the working electrode surface. After the competition step, detection was facilitated by a secondary aIgG antibody labelled with alkaline phosphatase and differential-pulse voltammetry using a-naphtyl-phosphate as substrate. The performance of the optimized immunosensors in terms of sensitivity, reproducibility and selectivity was studied. The linear working range of the described biosensors ranged between 0.9 and 9.0 ng mL À1 for the OTA-BSA based immunosensor and between 0.3 and 8.5 ng mL À1 for the gold nanostructured immunosensor, with a limit of detection (LOD) equal to 0.86 ng mL À1 (RSD ¼ 10.6%) and 0.20 ng mL À1 (RSD ¼ 8.0%) of OTA, respectively. The nanostructured immunosensor was applied to a certified wheat standard and a non-contaminated wheat material spiked with OTA, obtaining recoveries from about 104 AE 0.07% to 107 AE 0.08%. The influence of the wheat matrix on the analytical performance of the immunoassay was also studied.
A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA-bovine serum albumin (OTA-BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz-crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA-BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA-BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen-antibody complexes, regenerating the biorecognition surface.
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