Biological and artificial receptors in affinity sensor for water toxins detection

Abstract

Molecular recognition is the basis for many of the chemical and biochemical phenomena occurring in living organisms. For example, antibodies, which are one of the different classes of natural receptor molecules, are capable of selectively recognising a specific target molecule or structure. They are therefore routinely utilised as analytical reagents in clinical and research laboratories. The design and synthesis of biomimetic recognition systems, capable of binding target molecules with affinities and specificities comparable to natural receptors, is regarded as one of the greatest challenges in bioorganic chemistry. Molecularly imprinted polymers (MIPs) have been shown to mimic the binding sites of antibodies and are, therefore, constantly gaining in interest for applications based on specific molecular recognition. This project aimed to develop affinity sensors for the detection of algal and cyanobacterial toxins such as microcystin-LR and domoic acid in water samples. Following the investigation, a heterogeneous direct competitive enzyme-linked immunosorbent assay (ELISA) format for microcystin detection was developed. The system was then transferred to an affinity membrane sorbent based ELISA. This was an amenable format for immunoassay incorporation into a disposable amperometric immunosensor device. A three-electrode system immunosensor was fabricated using thick film screenprinting technology. Amperometric HRP transduction of hydrogen peroxide catalysis, at low reducing potentials, versus Ag/AgCl reference and carbon counter electrode, was facilitated by hydroquinone mediated electron transfer. A detection limit of 0.5 pg V1 for microcystin-LR was achieved. The work undertaken also describes the design and synthesis of biomimetic recognition systems based on MIP, capable of binding target molecules with affinities and specificities on a par with natural receptors. A MIP synthetic receptor selective for microcystin-LR was studied using an enzyme-linked competitive assay and found to be comparable to polyclonal antibodies, whilst the MIP had superior stability over natural receptors. Methacrylic acid based MIP had a detection limit of 1 pg I'1, approxilately twenty times higher than that of anti microcystin-LR polyclonal antibody. A molecularly imprinted polymer was also directly synthesised by grafting on the gold chip of a surface plasmon resonance (SPR) based bioanalytical instrument system: the BIAcore 3000™. Such a chip based platform allowed a simple test of the specific MIP receptor for the marine toxin domoic acid. A fiill characterisation of the grafting procedure was initially carried out on a bare gold surface, and each step of the polymerisation was investigated by contact angle measurements and AFM imaging. The surface photo-initiated MIP film was obtained and its thickness and homogeneity evaluated. Domoic acid is a molecule that is too small for direct analysis, hence a competition reaction was performed in presence of the conjugate DA-HRP and a detection limit of 2 fig f 1 could be achieved with the BIAcore 3000™ system.