Re-modeling ELISA kits embedded in an automated system suitable for on-line detection of algal toxins in seawater

The increasing demand by citizens and environmental organization for the protection, preservation, and possible restoration of the marine environment has made seawater protection one of the urgent priorities of the European Union. In this contest, extensive monitoring and surveillance are required to correctly assess the current status of marine environment, paying attention not only to traditional chemical parameters, but also to the so-called “emerging pollutants”. Among these, marine biotoxins represent a serious hazard, because they can accumulate in fish and mussels and enter the food chain. The detection of marine toxins released by algae has mostly been directed on the analysis of fish/shellfish homogenate rather than seawater samples. To fill this gap, in this work, we propose the re-modeling of ELISA kits to set-up non-automated direct competitive ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assays for the detection of Domoic Acid (DA), Okadaic Acid (OA) and Saxitoxin (STX) in seawater. For each toxin, linear working range, sensitivity and detection limit (0.03, 0.05, 0.01 ng/mL for DA, OA and STX, respectively) of the ELIMC assays were calculated. After evaluation of seawater matrix effect and recovery study, ELIMC assays were integrated within a novel automated networked system (ASMAT - Analytical System for Marine Algal Toxins) based on the micro Loop Flow Reactor (μLFR) technology, suitable for on-line monitoring of marine toxins. Once established the best operational conditions, ASMAT was calibrated towards DA, OA and STX verifying its capability to detect sub-ppb levels of the target toxins. Analysis of DA, OA and STX in real samples of marine water, sampled in Alonnisos (Greece) were carried out in laboratory using ELIMC assays, ASMAT and ELISA Kits, for a comparative evaluation. Finally, on-line suitability of ASMAT as an early warning alarm system was demonstrated by a field installation within a floating platform in the port of La Spezia (Italy).