Development, optimization and validation of an electrochemical immunosensor for determination of total aflatoxins in pistachio

Aflatoxins (AFs) can be produced by fungi in nuts such as pistachios, groundnuts, and cashews. As ingestion of even low amounts of AFs can lead to adverse effects for humans, their levels in food are strictly regulated. Electrochemical immunosensing, providing sensitive, simple, and cost-effective instrumentation, has emerged as an advantageous alternative to standard AFs analysis, which is typically based on liquid chromatography with fluorescence or mass spectrometry detection.Approaches for AFB1 detection, the most common in food and the most potent genotoxic and carcinogenic aflatoxin, have been widely reported. However, total AFs (sum of AFB1, AFB2, AFG1 and AFG2) determination, also required by the legislation, is scarcely reported with electrochemical immunosensors.We present here a novel sensor to detect traces of total AFs in pistachio and its cross-validation using a confirmatory method. The technology consists in an immunosensor on a screen-printed carbon electrode, featuring a competitive assay, followed by electrochemical detection. The optimized sensor covered a linear range of 0.01-2 mu g L-1 with good reproducibility (RSD: 2%). The limit of detection was estimated at 0.017 mu g L-1 and 0.066 mu g kg- 1 in PBS buffer and in pistachio matrix respectively, the latter being well below the maximum level for total AFs in pistachio set by the European legislation. The system exhibited excellent selectivity against Zearalenone and Ochratoxin and other possible interferences (glucose, Na+, K+), as well as a long-term stability of at least 30 days at room temperature. The methodology comprised extraction of AFs from pistachio using immunoaffinity columns and showed good recovery (87-106%), in addition to being validated with a LC-MS/MS method. Overall, the developed immunosensor is simpler, cheaper and more rapid, and exhibits sensitivity comparable to ELISA and LC-MS/MS, making our approach a promising tool for total AFs screening at the point -of-use.