To develop novel, highly selective and sensitive biosensing systems for the on-line detection of nitrogen oxy-anions (nitrate and nitrite) in environmental waters (including natural, potable and wastewaters).
1. The purification and large scale preparation of nitrate and nitrite reductases from bacterial sources to act as the biological recognition centre for the development of the biosensor systems.
2. Development of novel immobilisation techniques based on self-assembled monolayers, membrane entrapment and sol-gel encapsulation of the engineered reductases.
3. Design and development of biosensor systems using optical absorption and surface plasmon resonance techniques.
4. Assessment of the biosensor systems in real water samples (including rivers, lakes, reservoirs and wastewaters) in consultation with national water authorities of the participants.
The need for a rapid, on-line method for the detection of nitrogen oxy-anions (nitrate and nitrite) in European Union waters has become essential with increasing use of nitrate fertilisers which cause eutrophication and increase the risk of intestinal cancers through the formation of nitrosamines from nikites and nitrates released into the environment.
We propose to develop highly selective and sensitive biosensor systems which can be used on-line to monitor nitrogen oxy-anions in natural, potable and waste waters. Such a device is not available at present and current analytical methods for the detection of nitrates and nitrites have severe limitations. The problems are particularly severe when the sample is an industrial effluent or sewerage.
The biological recognition molecules we intend to use are nitrate and nitrite reductases derived from bacterial sources, which have been shown to be more specific than their plant derived counterparts when binding with their respective nitrogen oxy-anion. Importantly, in preliminary studies, conducted over the past nine months, these metalloprotein reductases have proven to be extremely stable biological molecules and are therefore ideal for use in a biosensing system. The reductases will be engineered to enable immobilisation either as a self-assembled monolayer, entrapped on a membrane or encapsulated in a sol-gel matrix. Full assessment and characterisation of the reductase enzymes in solution and in their immobilised state will be performed to determine the optimum conditions for use as a biosensing recognition centre. The biosensor devices will be based on electronic absorption spectroscopic techniques, as the nitrate and nitrite reductases display a spectroscopic change when the nitrogen oxy-anion reversibly binds to and oxidises the enzyme. Additionally, surface plasmon resonance of a self-assembled monolayer of the reductases will also be investigated as a biosensor. The performance of the biosensor devices will be assessed both in the laboratory and in on-line field measurements in consultation with National Water Authorities. The partners include an optical physicist, chemists, a biologist and a biotechnologist.
Therefore, the collaborators are well placed to realise the aims and objectives of this research proposal.
Funding SchemeCSC - Cost-sharing contracts