Production and characterization of chlamydomonas mutants for optical and electrochemical bio-sensing.

The increased concern about ecological damages and human health threats deriving by persistent water and soil contaminations boosted the emerging of the bio-sensing technologies as reliable, fast and efficient tool for large-scale and in field monitoring of various chemical species. Photosynthetic microalgae are among the most preferred microorganisms for environmental monitoring and screening of food and agricultural products for hazards compounds. The unique features and structural constituents of the photosynthetic systems make them a suitable sensing element, largely due to their ability to conduct charge separation and electron transfer sensitive to the presence of different classes of pesticides, heavy metals, some drugs and explosive compounds. However, the photosynthetic bio-recognition elements have some limitations related to inadequate stability and sensitivity, which negatively affects the biosensor performance. This work aimed at overcoming the principal bottlenecks of photosynthesis based biosensors by improving the resistance of bio-sensing element to oxidative damage and its affinity to different classes of pollutants. Novel bio-sensing elements for the detection of herbicides were generated by an in vitro directed evolution strategy targeted at the photosystem II D1 protein of Chlamydomonas reinhardtii, using exposures to radical-generating ionizing radiation as selection pressure [1]. This approach proved to be successful in identifying D1 mutations conferring enhanced stability, tolerance to free-radicals-associated stress and competence for herbicide perception [2]. In parallel, computational methods were exploited to identify single aminoacidic substitutions in the D1 protein conferring an increased affinity to the herbicide, atrazine, and prove of concept was achieved by studies on de novo generated D1-site-directed mutants [3]. This research is focused on the various strategies for production and characterization of D1 Chlamydomonas mutants suitable for optical and electrochemical bio-sensing of herbicides contaminants in nanomolar concentration range.