AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 AWACSS Dr. Dale M. Willard, Universität Tübingen (Gauglitz Group) Contract #: EVK1-CT Duration: 3 Year, RTD Project (Starting March, 2001) Visit: Automated Water Analyser Computer Supported System

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Motivation

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Goal: Develop a cost-effective, on-line, water monitoring biosensor that will measure a variety of small organic pollutants in short-time with remote control and surveillance. Expanded multi-analyte analysis capability allowing for up to 32 simultaneous analyte measurements. Immunochemical reagents are being developed for a host of small organic water pollutants. Novel design approaches to the optical detection and fluidics including miniturized integrated optics and microfluidics. Reduced reagent volumes. Limits of detection matching EU prescribed requirements (~1 ppb). Intelligent remote surveillance and control that will allow for unattended continuous monitoring. Instruments will be rugged and easy to handle by untrained personnel. Monitoring of Estuaries  Origin of Pollution Monitoring of Coasts  Direction of Pollution Monitoring of Side Streams  Origin of Pollution Monitoring of Industrial Plants  Contamination

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Methodology: Immunoassay coupled with Total Internal Reflection Fluorescence Laser IO-Chip Waveguide Detection PC Samples Waste Signal Collection Fluidics Analytederivative Analyte Antibody Fluorophor Analytederivative Analyte Antibody Fluorophor Analyte Antibody Fluorophore * * Flow Transducer * * Flow Remaining free antibodies are captured and detected. * * Flow * * Samples are pre-incubated with labelled antibodies.

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 AWACSS Technology Miniaturisation (μL volumes) Quick Analysis (ca. 15 min.) Multi-analyte Detection 2D Array Detection 32 Analysis Spots Microfluidics Integrated Optics

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Active Areas of Work Instrument Design Immuno- chemistry Market Survey & Tutorial Water Monitoring Software & Network Integrated Optical Chip

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Instrument Design 1st Year Goal: Design and order the equipment necessary to assemble the AWACSS instruments. Progress: Microfluidics designed with preliminary testing: small volumes & Poiseuille flow. Designed or selected pumps, valves, lasers, detectors, power supplies, filters, circuit boards, computers. Autosampler chosen. Preliminary testing of electronics.

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Integrated-Optical (IO)-Chip Development Goal: Develop IO-Chips capable of simultaneously illuminating 32 analysis windows in a miniaturised format. Advantages: Single mode waveguides allow for efficient and consistant analysis window illumination. Compact dimensions allow for microfluidic design and reduce sample & reagent volumes. Spatially defined but resolved analysis windows allow efficient detection and suppress signal crosstalk. Progress: Protype IO-Chips have been designed and fabricated. Preliminary tests are now being carried out.

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Software and Network Local station Local Stations Multi-analyte detection Data acquisition Data evaluation Local station control Control Station Global survey and early warning Global trend analyses Local station control Detection limit control prescribed by EU Directives Local station Progress: Measurement station software modules are selected and requirements are defined. The first generation software simulator is complete. The network design is defined.

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Immunochemistry Transducer * * Flow * * * * 1) Synthesis and Isolation of Antibodies and Analyte Derivatives. Polyclonal from Livestock Antibodies Isolated with ELISA 2) Assay Development. Binding efficiency Cross-reactivity Fluorescently labelled 3) Transducer Spotting & Final Testing. Spotting protocol established Final Testing will conclude project

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Targeted Analytes under Development Herbicides 2,4-D Atrazine Simazine Isoproturon Diuron Fenuron Methoxuron Alachlor Propanil Trifluralin Glyphosate Fungicides Pentachlorophenol Pentachlorobenzene Insecticides Chlorpyrifos Endosulfan Endocrine Disrupting Compounds Steriod Estrogens: Progesterone, Estradiol Bisphenol A Irgarol Nonylphenol Antibiotics Sulphonamide Tetracyclines Suspected Carcinogins Pyrene Trichloroethylene BTEX DEHP Industrial Wastes/Chemical Markers MTBE Sulphonate Detergents Caffeine Assays in Phase 2

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Water Monitoring IIQAB, Spain (Llobregat River) TZW, Germany (Rhine River) EI, Slovak Republic (Nitra River) Goals: Cataloguing and monitoring local water quality using standard methods (HPLC). Data helps community and project establish trends and identify problems. Establishing monitoring sites for final instruments. Defining requirements of end-users, validating design issues, and testing final instruments.

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Water Monitoring Progress Cross-referencing exercise was performed and standardised protocols for sampling, storing, shipment, and extraction developed. Target sites for field deployment: Saint Joan Despi (Llobregat River, Spain), Worms (Rhine River, Germany), Novaky Chemical Plant (Nitra River, Slovak Republic). Data Collection: 12 measurement sites, 36 compounds 9 compounds recorded significant levels Atrazine Conc. in Rhine over 3 Years Matrix Effect Studies: Turbity, Temperature, pH, Ionic Strength Temperature of Rhine through Calender Year 5 °C 25 °C

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Other Results Market Analysis Survey conducted of European and American water monitoring programs. Survey found total micropollutant water sample examination market to be >30 million €. Information broken down according to regions and compounds. Water Framework Directive 2000/60/EC is found as bench mark legal act. (13 AWACSS compounds are listed as "dangerous substances".) Multimedia Tutorial Efficient Training Cost-savings

AWACSS (EVK1-CT )ENVSENS, Lecce, Italy, 2002 Partners Eberhard-Karls-University Tübingen: Project Management and Surface Immunoassay Development Central Research Laboratory: Instrument Hardward Design Siemens: Electronics, Internet Communication, and Market Analysis. Optoelectronics Research Centre: Integrated Optical Chips Environmental Institute, Kos: Instrument Design and Field Tests- Slovakia King‘s College London: Immunochemistry Institute of Chem. & Environ. Chem. IIQAB: Instrument Design and Field Tests-Spain DVGW-Technologiezentrum Wasser: Instrument Design and Field Tests- Germany Funding: This Project is funded under the 5th Framework Programme: Energy, Environment, and Sustainable Development. Visit: