The Phototrophic Prokaryotes

Abstract

A computational approach for the design of a molecularly imprinted polymer (MIP) specific for Cyanobacterial toxin microcystin-LR is presented. By using molecular model-ing software, a virtual library of functional monomers was designed and screened against the target toxin, employed as a template. The monomers giving the highest binding energy were selected and used in a simulated annealing (molecular dynamics) process to investigate their interaction with the template. The stoichiometric ratio observed from the simulated annealing study was used in MIP preparation for microcystin-LR. The monomers were copolymerized with a cross-linker in the presence of the template. A control (blank) polymer was prepared under the same conditions but in the absence of template. A competitive assay with microcystin-horseradish peroxi-dase conjugate was optimized and used to evaluate the affinity and cross-reactivity of the polymer. The performance of the artificial receptor was compared to the performance of monoclonal and polyclonal antibodies raised against the toxin. The results indicate that imprinted polymer has affinity and sensitivity comparable to those of polyclonal antibodies (the detection limit for microcystin-LR using the MIP-based assay was found to be 0.1 µg L-1), while superior chemical and thermal stabilities were obtained. Moreover, cross-reactivity to other toxin analogues was very low for the imprinted polymer, in contrast to the results achieved for antibodies. It is anticipated that the polymer designed could be used in assays, sensors, and solid-phase extraction. Cyanobacteria such as Microcystis, Anabaena, Nodularia, Nos-toc, and Oscillatoria are aquatic microorganisms, often known as "blue-green algae", that produce toxic cyclic heptapeptides (mi-crocystins) and pentapeptide (nodularin) during the period of bloom formation. 1 Toxins from Cyanobacteria are responsible for intermittent but repeated cases of sickness and death in aquaculture species, livestock, wildlife, and humans. 2,3 At present, analysis of these toxins is largely achieved by bioassay, liquid chromatography (HPLC), or immunoassay. 4 The difficulty of raising antibodies against toxins and the continuing trend to reduce the use of animals for antibody production has stimulated research and development of synthetic receptors for toxins. In the past few years, molecular imprinting has been considered as one of the simplest, most straightforward, and cost-effective methods to develop artificial receptors for toxic organic compounds. The technology of molecular imprinting was developed in 1972 5 and has been advancing with a wide range of applications appearing in the literature. 6-11 The basic concept of imprinting involves three stages: (i) selection of components (functional monomers, analyte as a template, solvent, cross-linker, and initiator); (ii) formation of a functional monomer-template complex in solution; and (iii) polymerization process. The polymer obtained is then washed to remove the template and cavities with shape, and functionalities complementary to the target analyte are left behind. A crucial element for the success of the imprinting procedure is the creation of a strong monomer-template complex, whose complex should be preserved during the entire polymer-ization step. However, due to the exothermic nature of the polymerization reaction and to conformational changes of the monomers and template, it is inevitable that some percentage of the preformed complexes will be destroyed or changed in the resulting polymer. Since these changes can be sufficiently minimized when a strong interaction between template and monomers is achieved, the choice of monomers is of utmost importance. Thermodynamic calculations and combinatorial screening approaches have been successfully used to identify the best monomer candidates for imprinting. 12-15 The work became difficult * Matuschewski, H.; Schedler, U.; Wilpert, A.; Piletska, E. V.; Thiele, T. A.; Ulbricht, M.