Final Report Summary - BEEP-C-EN (Bio-sensor for Effective Environmental Protection and Commercialization - ENhanced) Executive Summary:The objective of BEEP-C-EN is the integration of innovative biosensor research and technology and their exploitation by industry and/or socio-economic entities in the fields of environment and agro-industry. The first target application is the detection of pesticides and other organic compounds in water. The aims is building up a biosensor industrial platform, which can be easily adopted for multi-parameter/multi-sensor design and production. It consists of a series of electrochemical-optical sensors and microsystems suitable for various biomediators (proteins, enzymes, cells) and based on new technologies studied and developed by the research performers in the consortium. The transduction approach is suggested by two main biomediators properties, often exploited in biosensor operation in response to a modification of a physical-chemical condition: the variation of the fluorescence emission and the internal electrical behaviour. These changes when transduced to readable electrical signals can give complementary information: the modification of a current signal is correlated to the electrogenic property of the biomediators (e.g. inhibition of photosystem II electron transfer in presence of a pesticide), while a modification of fluorescence is often correlated to a conformational modification (e.g. interaction of photosystem II protein with ionizing radiation). The specific proposed devices are: 1) MultiLights, a modular optical transducer for autonomous measurements of fluorescence of different biomediators assembled in series; 2) MultiAmps, modular electrochemical transducer for measurements of current variations; 3) MultiTasks, a multitransduction biosensor based on simultaneous and autonomous measurement either of fluorescence either of current variations. The three instruments, widely tested on standards and real samples, demonstrated to be suitable for in field analyses, particularly important for companies operating in the field of water monitoring and are therefore addressed to a wide European and international market.Project Context and Objectives:The project deals with the integration of innovative biosensor research and technology and their exploitation by industry and/or other socio-economic entities in the fields of environment and agro-industry. The aim is building up a biosensor modular industrial platform, which can be easily adopted for the detection of pesticides, heavy metal and organic compounds in water.The main objectives of the team, consisting of 3 research institutes and 5 companies (3 SMEs and 2 end-users), are:- select the material to be used as biomediator. Different enzymes, proteins and microrganisms will be considered as sensing material for the detection of the most common pollutants found in water. Banks of mutants already under study will provide engineered organisms sensitive to pesticides (e.g. diazines, ureas, triazines), heavy metals (e.g. mercury, arsenic, chromium, lead) and CBRN (Chemical, Biological, Radiological, and Nuclear) agents. Stability and lifetime of the selected materials will be tested in order to define suitable protocols for the biomediator immobilization.-.characterize the immobilized engineered biomediators. Reproducibility, easiness and stability of immobilization procedure, response to the interested classes of compounds and biologic activity lifetime will be tested. - manufacture optical and electrochemical biosensors. Three sensors, namely MultiLights, MultiAmps and MultiTasks, will be assembled in a full system platform characterized by high specificity, high selectivity, high sensitivity, room temperature operation, simple use, low-cost, fast response time, minimum sample pre-treatment, small dimension and ease of transport for in situ measurements, real time online measurements and easy interface with integrated circuits enabling cost effective manufacturing. - perform experimental in-field validation. Selected representatives of pesticides, heavy metal and CBRN agents will be measured in a reproducible automatic mode at EADS' and Vitens' laboratories and industrial facilities.Project Results:Work performed since the beginning of the projectFrom the beginning of the project and up to March 2010, CNR research group (partner no.4) worked on the screening and selection of several organisms to be used as biomediators (Task 1.1) and on the evaluation, by molecular biology studies, of the specificity of the selected microorganisms (Task 1.2). Once the most appropriate biomediators were identified, CNR worked with the purpose of optimizing, for each selected biomediator, extraction (Task 1.3) and immobilization procedures (Task 2.1) useful to define a precise protocol for the preparation of the sensing material. Tests of stability were performed in order to confirm the feasibility of each procedure, before any protocol definition and the effect of different classes of herbicides on the biomediators selected were evaluated with the aim of identifying a group of biomediators highly sensitive to these pollutants and able to reveal their presence in water. This phase of the project also involved PERPI (partner no.5) which developed the immobilization protocols of tyrosinase and acethylcholinasterase enzymes, selected as useful biomediators for the detection of phenols and organophosphorus pesticides. With the aim of realizing highly specific and efficient biomediators, PERPI developed different Molecular Imprinted Polymers (or MIPs) with high affinity for organophosphorus pesticides (Task 2.2) while CNR tested a new generation of supports for the immobilization of photosynthetic biomediators (Task 2.3) with the expected property of enhancing their light-harvesting power. These special supports, called Light Emitting Polymers (or LEPs), MIPs and all immobilization protocols were checked against stability (at various temperatures, in storage and delivery conditions), reaction time and repeatability, in order to provide definitive protocols of immobilization and analysis (Task 2.4). During the period April 2010-September 2010, the mentioned activities were successfully completed while new tasks started. Data collecting and pre-processing (Task 3.1) whose lead beneficiaries were PERPI and CNR, were done in order to improve the biosensor selectivity and allow the exact identification of the inhibitor present in a sample. With this purpose, a sensor array coupled with a chemometric tool, such as an Artificial Neural Network (ANN), was employed for data treatment. The development of software for data handling and analysis (Task 3.2) performed by BIO, was also completed in order to allow an easier analysis of the simultaneous responses coming from the different biomediators employed. In March 2011, the design of the fluorescence optical sensor (Task 4.1) and the amperometric sensor (Task 4.2) was ready. The two sensors were extensively tested from PERPI and CNR using all previously selected biomediators. The aim was optimizing the two sensors for using them in the construction of the prototypes, which were completed in June 2011, along with the fluidic system (Tasks 5.1 5.2 5.3 and 4.3). The last period of the project (July-September 2011) was mainly dedicated to the in-field tests at Vitens laboratories (task 6.1). As a completion of the work performed, a life cycle analysis was done (task 6.2) and the manuals of the instruments realized were prepared (task 6.3). A part the scientific activity itself, another import activity, corresponding to the tasks 7.1 7.2 7.3 7.4 7.5 and carried on all along the project, has been the complete management of the project, included the dissemination of the results and the creation of a website continuously updated for giving greater visibility to the project.Main results achievedDuring the two years of the project, many results have been achieved. First of all, the biomediators useful for the detection of some important water pollutants have been selected, tested and confirmed as suitable for the aims of the project. In particular, a list of mutants obtained from Chlamydomonas reinhardtii and able to recognize the presence of two classes of herbicides (triazines and ureas) has been achieved along with enzymes able to recognize phenolic compounds and organophosphorus pesticides. Protocols of preparation/extraction of the selected biomediators and protocols of immobilization for C. reinhardtii whole cells (wild type and mutant strains), spinach thylakoids, tyrosinase and acethylcholinesterase have been defined, together with stability data. Immobilization protocols have been studied taking into account the Biosensor srl instruments design. Molecular imprinted polymers (MIPs) with high affinity for different organophosphorus pesticides have been found, but following the decision made during the Progress Meeting on the April 2010 in France, this part of the project has been stopped, since the high specificity and affinity of imprinting polymers for single molecules, instead of classes of compounds, has not been found useful and necessary for the project. Moreover, studies for the coupling of LEPs with photosynthetic materials, have been also performed with the aim of enhancing the light-harvesting power of the photosynthetic material and its stability, but the obtained results showed that no improvement occurred by using those polymers and for this reason their use was abandoned.After preliminary analyses on water samples provided by Vitens, the ability of the selected material to detect the presence of pollutants was tested, demonstrating the suitability of the selected biomediators for the construction of the needed biosensors. Another interesting point of the project was the creation of a database useful for performing traditional and multivariate analysis, and the development of Neural Networks and genetic algorithms modeling performed by PERPI, with the aim of inserting all collected data from real samples, into a database available to all partners through Internet. The core of the project, however, was the design of the amperometric and optical sensors realized by the cooperation of BIO, DROP, UNI and EADS. The sensors have been extensively tested by PERPI and CNR revealing to be highly sensitive for the detection of different pollutants. This point was the starting point for the construction of the respective prototypes: MultiAmps, MultiLights and MultiTasks. The three prototypes have been previously tested at BIO and CNR laboratories, and then, sent to Vitens laboratories, have been used for the in-field tests on real samples, resulting highly robust, affordable, sensitive, accurate and precise. Once confirmed the suitability of the developed prototypes, detailed manuals of use have been prepared making the instruments completely accessible to any potential user and allowing them to be put on the market.For details, please see the files in attachments: "Work performed during the 1st year of the project" and ""Work performed during the 2nd year of the project". Potential Impact:The most important expected results were: 1) obtaining an array of various organisms characterized by high specificity for different groups of pollutants and 2) using such organisms as biomediators for the detection of water pollutants performed by optical and amperometric biosensors. At the end of the project, it can be said that both of them have been successfully achieved offering a platform of new biosensors able to detect the presence of herbicides, organophosphorus pesticides and toxic phenolic compounds in the same water sample, at the same time. The developed biosensors are based in fact on a selection of highly specific biomediators able to detect, each, very low concentrations of pollutants. The obtained prototypes provided portable and easy to use biosensors with unique features of automation and microfluidics, making them particularly suitable for the in-situ control of water pollution. This point represents the main advantage of the instruments developed and, at the same time, clarifies which can be the socio-economic impact of these instruments and their societal implications. The possibility of using the developed biosensors for in situ analyses, in fact, means the possibility of avoiding the daily freight of samples collected from the sources, to the laboratories specialized in quality control, and this is a basic point for saving money and time. Moreover, the miniaturization of the instruments reduces not only the amount of sample necessary for the analyses, but also the amount of solvents needed, making them devices with low environmental impact, a very important factor today.1. Giardi MT, Scognamiglio V, Rea G, Rodio G, Antonacci A, Lambreva M, Pezzotti G, Johanningmeier U (2009). Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii. Biosensors and Bioelectronics 25: 294-300.2. Rea G, Polticelli F, Antonacci A, Scognamiglio V, Katiyar P, Kulkarni SA, Johanningmeier U, Giardi MT (2009) Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring. Protein Sci. 18(10):2139-2151.3. Scognamiglio V, Raffi D, Lambreva M, Rea G, Pezzotti G, Johanningmeier U, Giardi MT (2009) Chlamydomonas reinhardtii genetic variants as probes for fluorescence sensing system in detection of pollutants. Anal Bioanal Chem. 394(4):1081-1087.4. Approaches in Biosensing Applications. Zourob (ed.), Recognition Receptors in Biosensors, DOI 10.1007/978-1-4419-0919-0_15 # Springer ScienceþBusiness Media, LLC 2010, Springer-Verlag New York5. V Scognamiglio, G Pezzotti, I Pezzotti, J Cano, K Buonasera, D Giannini, MT Giardi (2010) Biosensors for effective environmental and agrifood protection and commercialization: from research to market. Microchimica Acta 170: 215-2256. K Buonasera, G Pezzotti, V Scognamiglio, A Tibuzzi, MT Giardi (2010) A new platform of biosensors for pre-screening of pesticide residues to support laboratory analyses. Journal of Agricultural and Food Chemistry 58: 5982-5990PERPI:Oral communications :1) G. Istamboulié, T. Noguer. Biocapteur associant l'acétylcholinestérase et la phosphotriestérase pour un contrôle environnemental des insecticides organophosphorés. 40ème Congrès Scientifique du Groupe Français des Pesticides, Banyuls/mer (France), 26-28 Mai 2010 2) G. A. Alonso, G. Istamboulie, R. Muñoz, T. Noguer, J.-L. Marty. Determination of pesticides mixtures using an acetylcholinesterase-based biosnesor array and hardware artificial neural network implementation. XV Crossborder Meeting of Sensors and Biosensors, Sant Carles de la Ràpita (Spain), 16-17 Septembre 2010 (Oral communication)3) R. ROUILLON, M-T GIARDI et R. CARPENTIER « Les biocapteurs photosynthétiques : conception, utilisation, avantages et inconvénients ».2ème Colloque International sur Chimie, Environnement et développement DurableRabat, 20-21 Octobre 2011Posters :1) G. Istamboulie, G.A. Alonso, J.L. Marty, R. Munoz, T. Noguer. Genetically-engineered enzymes and hardware artificial network implementation for the highly sensitive and selective détection of insecticides. 2nd International Conference on Bio-Sensing Technology, Amsterdam, 10-12 Octobre 2011.2) E. Moczko, G. Istamboulie, C. Blanchard, T. Noguer. Electrochemical biosensor for the detection and quantification of phenolic environmental pollutants. 2nd International Conference on Bio-Sensing Technology, Amsterdam, 10-12 Octobre 2011 The website of the project is cited on PERPI website (http://images.univ-perp.fr/en_cours.html).CNR:Oral communications:1) K. Buonasera, M. Lambreva, I. Pezzotti, G. Pappalettera, P. Boffi, G. Pezzotti, M.T. Giardi. "Laser-based fluorescence sensor for the assessment of herbicide levels in cultivable areas". XIV International Symposium on Luminescence Spectrometry, July 13-16, 2010, Prague, Czech Republic 2) K. Buonasera, M. Lambreva, A. Antonacci, G. Rodio, S. Pastorelli, I. Husu, J.B. Cano, I. Pezzotti, G. Pezzotti, V. Scognamiglio, M.T. Giardi, G. Rea - "Biosensori innovativi per il rilevamento di erbicidi e distruttori endocrini e la promozione dell'agricoltura biologica" - Primo Congresso Nazionale della Rete Italiana per la Ricerca in Agricoltura Biologica, RIRAB, Novembre 7-8, 2011, Catania, Italy 3) Antonacci A. Convegno "Dal Comitato Pari Opportunità al Comitato Unico di Garanzia", CPO-CNR, 5 Luglio 2011, Roma, Italia. Abstracts: Viviana Scognamiglio, Giuseppina Rea, Irene Rambaldi, Amina Antonacci, Maya Lambreva and Maria Teresa Giardi. New biomimetics molecules for biosensing applications. 2nd International Conference on Bio-Sensing Technology 2011, 10-12 October 2011, Amsterdam, The Netherlands. Poster:1) E. Touloupakis, I. Husu, V. Scognamiglio, K. Buonasera, G. Rodio, G. Pezzotti, J. Cano, M.T. Giardi. "New Platform of Biosensors for Pesticide Prescreening". Pesticides 2010, 6th European Conference on Pesticides and Related Organic Micropollutants in the Environment and 12th Symposium on Chemistry and Fate of Modern Pesticides, September 5-10, 2010, Matera, Italy 2) K. Buonasera, J. Griffiths, D. Hernández, T. Noguer, T. Ziemann, G. Pezzotti, et al. - "A new biosensor for the screening of toxic compounds in water samples" - 2nd International Conference on Bio-sensing Technology, October 10-12, 2011, Amsterdam, The NetherlandsISI PUBLICATIONS1. Buonasera K, Lambreva M, Rea G, Touloupakis E, Giardi MT. (2011) Technological applications of chlorophyll a fluorescence for the assessment of environmental pollutants. Anal Bioanal Chem. 401(4):1139-51. 2. Cano J, Giannini D, Pezzotti G, Rea G, Giardi MT. (2011) Space impact and technological transfer of a biosensor facility to Earth application. Recent Patents on Space Technology. Vol.1 Issue no. 1.3. Christos Boutopoulos, Eleftherios Touloupakis, Ittalo Pezzotti, Maria Teresa Giardi and Ioanna Zergioti (2011). Direct laser immobilization of photosynthetic material on screen printed electrodes for amperometric biosensor. APPLIED PHYSICS LETTERS 98, 0937034. Eleftherios Touloupakis, Katioa Buonasera, Christos Boutopoulos, Ioanna Zergioti, Maria Teresa Giardi and (2011). A photosynthetic biosensor with enhanced electron transfer generation at the electrodes realized by laser printing technology, in revision in Analytical Chemistry and BiochemistryBOOK CHAPTERSG. Rea, F. Polticelli, A. Antonacci, M. Lambreva, S. Pastorelli, V. Scognamiglio, V. Zobnina and M.T. Giardi. Computational biology, protein engineering, and biosensor technology: a close cooperation for herbicides monitoring. Herbicides, Theory and Applications. Invited book chapter 2011: INTECH ISBN 978-953-307-975-2.UNI:1) 8th Spring Meeting of the International Society of Electrochemistry, Columbus (Ohio, USA) Advances in Corrosion Science for Lifetime Prediction and Sustainability 3-5 May 2010 2) Biosensors 2010, 26-28 May Glasgow (UK) (http://www.biosensors-congress.elsevier.com) 3) RSE-SEE 2, Second Regional Symposium on Electrochemistry Belgrade Serbia (http://www.rse-see.net) 06-10 June4) ELECTROCHEM 2010, Wolverhampton (UK) (http://www.soci.org/General-Pages/Display 14-15 September 2010)5) 27-30 September 61st Annual Meeting of the ISE Electrochemistry from Biology to Physics, Nice (France) (http://event10.ise-online.org/index.php)6) 3-7 October 6th Workshop of SECM in Frejus, France7) 11-15 October Electorochemical Society Fall Meeting in Las Vegas USA8) 10 March 2011 Biosensor Workshop "From Concept to Manufacture" in Cranfield, Bedfordshire, UK.9) 3-6 April Deutsches Biosensor Symphosium Heilbad Heiligenstad, Germany.10) 8-11 May ISE Spring Meeting in Turku, Finland.11) 16-18 May, Eirelec '11 in Adsare , Country Limerick, Ireland.12) 5-6 September ELECTROCHEM 2011 Bath, UK13) 9-14 October ECS Fall Meeting in Boston MA, USA.14) 10-12 October International Conference Biosensing Technology 2011, Amsterdam, The Netherlands.BIO: 1. May 2010, "Biosensors 2010" Congress, Glasgow (United Kingdom)2. 10-12 October International Conference Biosensing Technology 2011, Amsterdam, The Netherlands.3. Giardi Maria Teresa: Scognamiglio V. , Boutopoulos C., Lavecchia T., Rodio G., Pezzotti G., Pezzotti I., Zergioti I., Giardi M.T. Micro Electrodes Array (MEA) technology: a multi-biomediator and multi-detection biosensor for agrifood and environmental management. GS2011 - IV Workshop del Gruppo Sensori della Divisione di Chimica Analitica della Società Chimica Italiana 15-17 giugno 2011 - Aula Magna - Facoltà di AgrariaPoster:K. Buonasera, J. Griffiths, D. Hernández, T. Noguer, T. Ziemann, G. Pezzotti, et al. - "A new biosensor for the screening of toxic compounds in water samples" - 2nd International Conference on Bio-sensing Technology, October 10-12, 2011, Amsterdam, The Netherlands Award for best poster3) E. Touloupakis, I. Husu, V. Scognamiglio, K. Buonasera, G. Rodio, G. Pezzotti, J. Cano, M.T. Giardi. "New Platform of Biosensors for Pesticide Prescreening". Pesticides 2010, 6th European Conference on Pesticides and Related Organic Micropollutants in the Environment and 12th Symposium on Chemistry and Fate of Modern Pesticides, September 5-10, 2010, Matera, Italy Award of the best poster for the common biosensor made by the SMEs:October 2011, 2nd International Conference on Bio-sensing Technology, October 10-12, 2011, Amsterdam, The Netherlands: winner of the Award for Outstanding Poster Presentation. Poster title: A new biosensor for the screening of toxic compounds in water samples (by K. Buonasera, J. Griffiths, D. Hernández, T. Noguer, T. Ziemann, G. Pezzotti, et al.). List of Websites:A website with has been created, the second month of the project, with the following address: http://www.beep-c-en.com. The site has been continuously updated during the last two years.
