As a prerequisite for a successful combination of genetic engineering and biosensor development the current expression systems for peroxidase have to be optimised. This will be achieved by periplasmic expression. With these techniques a peroxidase chip (P-chip) will be developed which serves as a general detection unit, both for enzyme biosensor and immunosensors. The expected high sensitivity of the P-chip for hydrogen peroxide will be exploited in a biosensor by coimmobilisation of an oxidase which confers the substrate specificity. As analyses with relevance as food and-water contaminants in the Eastern countries the herbicides Simazine and Metsulfuron-methyl are chosen to demonstrate the practicability of the P-chip in the development of immunosensors. In a P-chip irnmunosensor the space-oriented peroxidase is coimmobilised with monoclonal antibodies for the respective herbicide which constitute the recognising element. Monoclonal antibodies with high affinity and specificity will be selected for this purpose. In this case a glucose oxidase-hapten conjugate serves as tracer.
Biosensor have the potential to reduce time and costs of the analysis of food components and contaminations and are particularly suited for routine analysis and field testing. This project focuses on the development of amperometric biosensor that are based on direct electron transfer from the electrode to the enzyme and are thus independent on addition of mediators. The usefulness of peroxidase for direct electron transfer has been demonstrated earlier, here it is aimed to drastically improve the efficiency of the electron transfer by controlling the orientation of the peroxidase on the electrode surface. Two routes will be explored to achieve this goal. The availability of a set of epitope specific antibodies allows the immobilization of the peroxidase in different orientations. On the other hand, based on the results of the epitope mapping and modelling studies loop regions will be defined that can be mutated without disturbing the general structure of the enzyme. Replacement of these loops with short histidine stretches should enable the immobilization of the mutated peroxidase on an electrode surface that is modified with chelating molecules. Similarly, by site directed mutagenesis single cysteines will be introduced at different locations of the enzyme surface in such way, that they are exposed to the solvent. By coupling with thiol specific crosslinking reagents a panel of site specific hapten-peroxidase conjugates will be prepared and tested.
Funding SchemeCSC - Cost-sharing contracts
221 00 Lund
621 32 Brno