Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii.

Homology-based protein modelling and computational screening followed by virtual mutagenesis analyses were used to identify functional amino acids in the D1 protein of the photosynthetic electron transfer chain interacting with herbicides. A library of functional mutations in the unicellular green alga Chlamydomonas reinhardtii for preparing biomediators was built and their interactions with herbicides were calculated. D1 proteins giving the lowest and highest binding energy with herbicides were considered as suitable for preparing the environmental biosensors for detecting specific herbicide classes. Arising from the results of theoretical calculations, three mutants were prepared by site-directed mutagenesis and characterized by fluorescence analysis. Their adsorption and selective recognition ability were studied by an equilibrium-adsorption method. The S268C and S264K biomediators showed high sensitivity and resistance, respectively, to both triazine and urea classes of herbicides. When immobilized on a silicon septum, the biomediators were found to be highly stable, remaining so for at least 1-month at room temperature. The fluorescence properties were exploited and a reusable and portable multiarray optical biosensor for environmental monitoring was developed with limits of detection between 0.8 x 10(-11) and 3.0 x 10(-9), depending on the target analyte. In addition, biomediator regeneration without obvious deterioration in performance was demonstrated.