Singlet oxygen generated by PSII reaction centers of chlamydomonas reinhardtii mutants in relation to biosensoristic purposes

Dottorato di ricerca in Biotecnologie vegetaliWith the advent of the 21st century, public awareness on issues related to human and animal health, food safety, or environmental pollution has increased considerably and a great deal of research and substantial investments have focused on the development of novel tools for continuous in situ environmental monitoring. In this respect, the use of microalgae as a biological tool for monitoring and assessment of environmental toxicants is an application that has attracted much interest. Among photosynthetic organisms sensitive to pesticide classes, the unicellular green alga Chlamydomonas reinhardtii and Chlorella vulgaris are some examples revealed to be a smart bio-sensing element useful for the realization of analytical devices. Intact algae cell-based biosensors preserve photosystem functionality, and perform sensitive detection at parts per billion (ppb) levels of herbicide. However, they provide a slow response and sometimes-low sensitivity. Beside, their analytical application is sometimes hampered, among other problems, by their relative instability in non-physiological or extreme environmental conditions. The main challenge is the preservation of the algal photosynthetic functionality when integrated with non-biological electronic components or operated under fluctuating environmental conditions. In fact, photodynamic reactive oxygen species (ROS) reactions are determinative in reactions damaging the photosynthetic apparatus of Chlamydomonas reinhardtii, causing a short half-life that is a problem for biosensor applications. High light illumination of photosynthetic organisms stimulates the production of singlet oxygen (1O2) by photosystem II and causes photooxidative stress. It is known that photo-inhibitory damage of Photosystem II (PSII) under various conditions can result in the formation not only of 1O2 but also of other ROS. In Chlamydomonas reinhardtii, reducing the formation of 1O2 and ROS thus lessening the photooxidative membrane damage (including the RC protein) and increasing the stability and sensitivity for biosensor applications is of special interest. The ability to produce new algal biomediators, with a broad structural stability, can be expected to lead biosensing for food control or environmental monitoring. The main goal of this PhD thesis was increasing the stability and sensitivity of PSII from algae for the detection of different subclasses of pollutants under extreme environmental conditions (high light, salinity etc.) using various ways such as symbiosis, site directed mutagenesis of D1 protein, UV mutagenesis and synthetic biomimetic approach. Firstly, an optical biosensor based on algal biomediators for monitoring of marine pollutants was developed. The algae-protozoa symbiotic association between Chlorella vulgaris and the ciliate Tetrahymena pyriformis was deeply studied as a potential biomediator for biosensor development, showing enhanced resistance to the salinity of marine water when compared with free-living algae strains. The symbiosis was adapted to grow into microfluidic flow cells with integrated detectors for real-time detection of marine pollutants by fluorescence analysis of photosynthetic photosystem II. An integrated symbiotic association of Paramecium-Chlorella-based biosensor was successfully developed for real-time monitoring of marine water quality and evaluation of biotoxicity. Secondly, an optical/amperometric biosensor based on algal cells immobilized in calcium alginate gel was developed. Various Chlamydomonas reinhardtii strains mutated at the level of photosynthetic D1 protein were used as biomediators to quantify the capacity of the carotenoid xanthophylls to protect the photosynthetic apparatus from photoinhibition. The highly sensitive and selective biosensor was used for studies on cell physiology aimed to determine the antioxidant and light filtering effects of the xanthophyll astaxanthin. The biosensor was proved to be suitable for the determination of the exogenous supplied astaxanthin showing in a short time a reliable response with a detection limit of 3 μM. This technique revealed the photo-protective effect of astaxanthin-enriched extracts of Haematococcus pluvialis in algal cells. The results suggest that in algae astaxanthin exploits both filtering and antioxidant effects at different light intensities. This bioinspired approach can provide new insights into biological, biomedical, environmental and agricultural research applications and nutraceutical studies. We also projected and realized a biosensor platform, which combines an optical system, intimately, integrated with an array of biomediators able to provide a helpful tool for safety management of the milk. Optical feature selection of various enzymes was performed for monitoring compounds of various chemical classes and metabolic markers of cow’s wellness. The detection of selected analytes was evaluated through biomediators of Chlamydomonas reinhardtii cells, and acetylcholinesterase, tyrosinase, urease, β-galactosidase and D-lactate dehydrogenase enzymes. The analyses were performed by fluorescence that for the algal cells was based on chlorophyll a fluorescence emission while for enzymes was guaranteed by the use of fluorescein 5(6)-isothiocyanate or 5(6)-carboxynaphthofluorescein. Furthermore, a novel fluidic setup based on simple fluid mechanics laws has been realized with no moving components when pump is on. It yields a fluctuation-free outcome signal that we compare with those produced by commercial peristaltic pumps. Evidence of the readout amperometric enhanced signal is shown thus confirming the successful implementation of more reliable biosensors and electrochemical signal transduction devices. Such stable and oscillation free amperometric signals are very desirable in view of biosensors integration in environmental monitoring platforms equipped with advanced communication technologies. In addition, two set of C. reinhardtii mutants, able to quench 1O2 and other ROS produced under extreme conditions, were produced in collaboration with U. Johanningmeier (Uni of Halle). First set was mutated by site-directed mutagenesis (antiox-mutants): antioxidant peptides able to quench 1O2 and variety of free radicals introduced in the D1 protein of Photosystem II. The second set was produced by UV-mutagenesis. The mutated strains were characterized to select more resistant strains to be used as biological element for environmental monitoring under extreme conditions. In this study, total lipid and phenol content of two set of Chlamydomonas reinhardtii mutants (Antiox and UV mutants) were determined. The in-vivo and in-vitro antioxidant capacity of the antiox-mutants were evaluated using different assay and in the presence of 1O2 precursors. The mutants have reduced lipid and phenol content compared to the wild type Chlamydomonas reinhardtii. However, they showed better resistant to stress conditions such as high light, 1O2 and H2O2. UV mutants have showed longer half-life. The produced mutants revealed to be a promising candidate for improved environmental monitoring on earth. We also described the use of biomimetic peptides of the photosynthetic plastoquinone binding niche of the green alga Chlamydomonas reinhardtii for pesticide measurement in environmental samples. Three biomimetic peptides containing the plastoquinone-binding site in a loop shaped by two alpha helices were designed and characterized. Natural sequence of 70 aminoacid-peptide was modified to increase the solubility in aqueous solvents by adding two histidine in the N- and C-terminus. A cysteine was included in two of the modified peptides (S264C or S268C) and labeled with commercial carboxylated quantum dots (peak emissions 710 nm) by carbodiimide reaction coupling and fluorometric detection was performed. The results confirmed that mutation S264C conferred resistance to atrazine and diuron, while the change S268C increased the sensitivity. This work demonstrates the interest to replace whole microalgae cells or their photosynthetic apparatus (thylakoids, isolated reaction centers) by much smaller fragments such as D1 peptide mimics. This approach might improve the system in terms of sensitivity, but also in terms of stability (oligopeptides being much more stable than the whole three-dimensional protein molecule) and facilitate the commercialization for environmental monitoring. English is an international language that allows us to communicate with scientists from all over the world and writing a manuscript is important since it is a form of written communication in science. In this occasion, as a part of my training to improve my writing skills and enhance my knowledge on biosensors, we presented a review focuses on bioassay development based on the simultaneous use of quantum dots and magnetic beads. Due to the outstanding characteristics of both particles for biosensing applications and the large number of publications using a combined approach, we aim to provide a comprehensive overview of the literature on different bioassays. The improvement of current approaches together with novel multiplex detection systems and nanomaterials-based research, including the use of multimodal nanoparticles, will contribute to simpler and more sensitive bioanalyses. The optical and electrochemical biosensors, that were constructed and optimized within this doctoral thesis, have the advantage of being user friendly, low cost and portable analytical tools, which facilitates a future transfer to the market of environmental and agrifood monitoring