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

Homology-based protein modelling and computational screening followed by virtual mutagenesis analyseswereused to identify functional amino acids in the D1 protein of the photosynthetic electron transferchain interacting with herbicides. A library of functional mutations in the unicellular green alga Chlamydomonasreinhardtii for preparing biomediators was built and their interactions with herbicides werecalculated. D1 proteins giving the lowest and highest binding energy with herbicides were considered assuitable for preparing the environmental biosensors for detecting specific herbicide classes. Arising fromthe results of theoretical calculations, three mutants were prepared by site-directed mutagenesis andcharacterized by fluorescence analysis. Their adsorption and selective recognition ability were studiedby an equilibrium-adsorption method. The S268C and S264K biomediators showed high sensitivity andresistance, respectively, to both triazine and urea classes of herbicides. When immobilized on a siliconseptum, the biomediators were found to be highly stable, remaining so for at least 1-month at roomtemperature. The fluorescence properties were exploited and a reusable and portable multiarray opticalbiosensor for environmental monitoring was developed with limits of detection between 0.8×10-11and 3.0×10-9, depending on the target analyte. In addition, biomediator regeneration without obviousdeterioration in performance was demonstrated