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Toxic secondary metabolites produced by fungi (aka mycotoxins) are often identified as contaminants in cereals or other food and pose a severe health risk, since they can be carcinogenic, neurotoxic, mutagenic or harmful in other respect. The highly nephrotoxic ochratoxin A (OTA) is one of the most important and widespread mycotoxins found in grains such as rye, wheat or oats. Thus, the fast and cost-effective detection of OTA is of great importance, to ensure that the toxin concentration is below the legal limits. Currently, the mycotoxin detection in mill operations is typically performed by means of the antibody-based ELISA method, which requires extensive laboratory equipment and specialized personnel. It was the aim of this study to develop an ELISA-inspired sensor method enabling the on-site quantification of OTA. Based on a classical competitive ELISA, an electrochemical detection system was developed. Anti-OTA-antibodies were captured on magnetic beads on which the competitive affinity reaction between OTA and an OTA- horseradish peroxidase (HRP) tracer was performed. Here the OTA concentration is inversely proportional to the amount of the bound OTA-HRP tracer, which was electrochemically quantified. For this purpose, oxidized tetramethylbenzidine (TMB), which is enzymatically produced by the reduction of H2O2 by the OTA-HRP tracer, is quantified by amperometry with screen-printed electrodes in a flow system. With this method a detection limit of 250 ng L-1 could be achieved which meets the current requirements for the detection of OTA in food and feed.
Immunomagnetic ochratoxin a assay with electrochemical 3,3′,5,5′ tetramethylbenzidine detection
(2021)
The demand for miniaturized analytical devices monitoring important parameters in the food and medical industry has increased strongly in the past decades. With fast progress, smart technologies are finding their way into our everyday life. For the future, it is, therefore, a major goal to also link analytical methods with smart technologies to create user-friendly on-site devices. In food industry the monitoring of harmful substances such as dioxins, heavy metals or mycotoxins plays a key role, since the European Commission prescribes legal limits for various food products and beverages[1]. Therefore, companies often have their own laboratories and trained personnel. For one of the most abundant and toxic mycotoxins, Ochratoxin A (OTA) we want to present an electrochemical detection system in which the read-out can be performed with a smartphone connected via Bluetooth to a miniaturized potentiostat. The recognition of OTA is performed with specific antibodies in a competitive assay format. Anti-OTA-antibodies were captured on magnetic beads on which the competitive binding between OTA and an OTA horseradish peroxidase (HRP) tracer was performed. To quantify OTA, the enzymatic reaction of the tracer with 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 is employed. Oxidized TMB, which is enzymatically produced by the reduction of H2O2, is quantified by amperometry with screen-printed electrodes in a custom-made flow system. The results of the electrochemical detection method are in good correlation with the photometric detection of TMB. To demonstrate the applicability, we tested our system with OTA-spiked beer and performed the measurement via smartphone.