A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000 TM . The gold surface was first functionalised with a selfassembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000 TM . Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip 2 did not affect its recognition properties and continuous measurement was possible over a period of at least two months.

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The current work describes the raising of polyclonal antibodies (pAbs) against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. They were screened for sensitivity and selectivity using a competitive enzyme-linked immunosorbent assay (ELISA). The antiserum produced against a keyhole limpet haemocyanin (DA-KLH) conjugate displayed a high affinity for free DA. The optimized horseradish peroxidase (HRP) ELISA had a detection limit of 0.6 ng mL À1 (ppb) and a working range of MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 100160.8-300 ppb DA applying a streptavidin-biotin amplification system (ABC system). Furthermore this antiserum did not cross-react with similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid. When the ELISA was compared using an alkaline phosphatase (AP) label we found the system to behave in a similar manner to the optimized HRP system but the linear range was smaller in the high DA concentration range. These pAbs were then used in the optimization of a screen-printed electrode (SPE) system for measurement of DA. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at þ300 mV vs. Ag/AgCl, produced by the enzyme AP, was used for signal measurement. The sensor incorporates a relevant range for toxin detection, by which humans become ill (Iverson, F.; Truelove, J. Toxicology and seafood toxins: domoic acid. Natural Toxins 1994, 2, 334-339.) with detection limits achieved at SPE to the order of ppb. The SPE system is simple and cost-effective due to its disposable nature, and analysis time is complete in 30 min. In addition, recovery experiments on DA for both ELISA and SPE highlighted the functionality of these systems yielding a AE12% deviation for the true value for the ELISA using AP and AE25% for the sensor.

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