RIANA ( RIver ANAlyser )

EUROPEAN UNION ENVIRONMENT AND CLIMATE PROGRAMME 1994 - 1998 / (AREA 2)
ENVIRONMENTAL TECHNOLOGIES (INSTRUMENTS, TECHNIQUES AND METHODS FOR MONITORING THE ENVIRONMENT)
DURATION: 3 YEARS (MAY/1996 - APRIL/1999)

Project Coordinator:
Prof. Dr. G. Gauglitz, Institute of Physical and Theoretical Chemistry, University of Tübingen

Objectives Analytes Methodology Project Consortium

Objectives
The objective of this project is to develop a system for monitoring organic compounds present in polluted river water and in surface water. The core device shall be a multiresidue bioanalytical device, allowing testing in one sample a multitude of simultaneously present agents. In accordance with the objectives of the Environmental Workprogramme the system envisaged is expected to be more cost effective than reference techniques, to allow the detection of a broader range of analytes with a single device than established reference techniques, and to avoid most of the sample pretreatment common in current environmental multiresidue analysis with concomitant advantages in speed.
The main characteristics are:

• The use of a single sample to detect a plurality of analytes. Two separate panels of five analytes from the list given below will be chosen for the purpose of this project.
• The use of antibodies to detect selectively analytes from a complex sample matrix.
• A generic approach allowing the user to choose a panel of analytes from a large number of analytes available.
• A handling protocol involving the addition of only one set of reagents for a multitude of analytes, and sacrificing the biological layer/material after each test.
• Fast operation, that is 15 minutes or less per cycle time and in consequence 15 minutes or less time to result/reading.
• A capability of system automation for use in on-line monitoring applications.
• An optical transducer element based on fluorescence detection.
• A limit of detection of 1 ppb. It will be tried to reduce this figure to 0.1 ppb or below with the final version of the demonstrator set up within the project.

10 analytes will be selected for the project.

Compounds in this list arise from production processes as well as from transformation of substances intentionally introduced to the environment (agrochemicals) and are of environmental concern. These substances (hydroxylated, sulfonic and acidic compounds, often bound to the humic acid fraction so that extraction and separation procedures give poorly defined results) are not easily assessed by existing analytical technology:


List of analytes

1. Trichlorobenzene (isomers),
2. Pentachlorophenol,
3. Trichlorophenol,
4. Aldicarb sulphoxide,
5. Hydroxycarbofuran,
6. Desethylatrazine,
7. Deisopropylatrazine,
8. Glyphosate
9. 2-(chloro-methyl)-phenoxypropionic acid
10. Nonylphenol

Project Methodology
To achieve the objectives given above, the system envisaged requires a number of well integrated building blocks and methodologies:

• Analyte specific elements will be antibodies raised against the analytes of choice. Polyclonal antibodies will be prepared according to established protocols. Specificity will be matched to the environmental problem addressed by appropriate immunisation protocols and/or blending of antibodies. The performance of immunochemistry will be assessed in a conventional ELISA testbed.
  
• For the detection of analytes a competitive test scheme will be implemented. ELISA results will serve as a reference. Competition will occur between the free analyte and compounds of the biochemical immobilisation system conjugated with analyte derivatives. The detection will occur in a heterogeneous format, i. e. at the transducer surface.
• Spatially resolved surface modification strategies (covalent and non-covalent) will be developed for the stable and reproducible patterned modification of the transducer surface to establish spatially resolved multi-reaction areas corresponding to the number of analytes. (Optical) surface analytical techniques and functional testing will be used to characterise surface chemistry.
• An auxiliary immobilising system consisting of highly specific auxiliary bioaffinity compounds (antibodies or bioaffinity ligands) will serve to target the detection compounds to selected areas of the transducer surface. This novel idea is owned by GEC Marconi Ltd and is currently subject to patent applications (Ref. No. 9216450.8/ 9216683.4/ 9219743.4/ 9315995.2 in GB, 93917967.7 in Europe and 08-318,825 in the USA).
• The transducer system will be based on fluorescence detection. A micro-optical/integrated optical transducer element will be used to allow effective excitation and spatially resolved detection of fluorophores bound to the surface. Mathematical modelling and systematic analysis of experimental results will be used to optimise the device.
• The system setup will start with well defined interfaces (soft- and hardware) between compounds. Commercially available building blocks or units from commercially sold systems will be used where available. A system analysis will precede the implementation of an advanced demonstrator.
• Validation will be carried out by two partners with a high degree of experience in analysing complex environmental samples. The RIANA system will be tested in comparison with established reference techniques and procedures (instrumental analysis). Reference materials that were developed under the Measurement and Testing programme of the EU will be used. Where standardised methods do not exist neither at the national level nor at the European level US-EPA protocols will be adapted.

Project Consortium

Major subcontractor

The partners collaborating in this project have been selected to contribute specific expertise required for a successful realisation of project aims. This includes an organisation with expertise in immunochemistry, immunoanalytics and organic chemistry (GEC); a group experienced in the field of surface chemistry for immunoprobe development (TU); a group with expertise in the modelling and development of micro-optical/integrated optical transducers (ORC); two groups are contributing expertise and experience in the field of optical immunoprobes (ORC, TU). The expertise and knowledge required in engineering complex systems is contributed by an instrument manufacturer (PE) and a user with own instrument development capabilities (AR). Finally close and intimate knowledge of environmental requirements, and established analytical techniques is crucial for sound validation of the results achieved at different levels. This experience is contributed by two partners from academia and "users" (CID) and (AR).

The management of the project will be handled by Tübingen University.