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Project title:  BIOSENSORS BASED ON COVALENTLY IMMOBILIZED ENZYMES ON POLYMERS FOR MONITORING THE NITRITES AND NITRATES FROM HUMAN WATER CONSUMPTION
Acronym:  BIOENZINIT
Project number:  52-159
Financing:  Governmental budget - Ministerul Educatiei, Cercetarii si Tineretului, Programul 4 - PARTENERIATE IN DOMENIILE PRIORITARE
Project budget (including other sources):  2.000.000 lei
Contract budget (only governmental source):  2.000.000 lei
Contract period:  2008 - 2011
Contracting Authority:  National Center of Programme Management (CNMP)
Coordinator:  National Institute of Research and Development for Chemistry and Petrochemistry - ICECHIM


PROJECT ABSTRACT:

Nitrates and nitrites are largely used as preservatives and fertilizing agents. However, a continuous exposition of these ions to humans can have serious health implications. Particularly, nitrites can react irreversibly with hemoglobin to produce methemoglobin, therefore reducing blood capacity to transport oxygen. The disease is known "as blue baby syndrome". Also, the nitrite can form nitrosoamines in the digestive tract, with cancer risk.

The European Community by the European Drinking Water Directive from 1998 has established the maximum admissible levels of nitrate and nitrite in drinking water at 50 and 0.1 mg/L, respectively. This level is now imposed also in Romania, by the law 458 from 8 July 2002.

Nitrite determination is mainly carried out in specialized laboratories, but these centralized and sophisticated analytical systems are representing delays in solving problems in emergency situations. The project target is to elaborate the production technologies for 2 biosensors based on covalently immobilized enzymes on polymers (one for nitrate and another for the nitrite content). This will allow continuous monitoring, in real time, at distance, of both anion concentrations in waters for human consumption (for dinking and foods) and hereby to prevent some diseases of the population. It is intended to perform easier and continuously environment monitoring using biochemical sensors.

Biosensor technologies are widely used for the detection and quantitative determination of biologically active compounds from complex biological and environmental samples. One of the most popular configurations of biosensor consists of enzyme immobilized within a polymer, which is generated over an electrode. The enzymes immobilized in films on electrode surfaces should exhibit well-defined and direct electrochemical responses, retain their native structure and bioactivity, and demonstrate good stability. The majority of biosensors reported for the measurement of nitrate or nitrite ions are molecular-based systems using a nitrate reductase respectively nitrite reductase enzymes purified from plant, fungal, or bacterial sources. The nitrate reductase catalyses the reduction of nitrate to nitrite. The nitrite reductase catalyses the reduction of nitrite to ammonium in a six-electron transfer reaction.

In most of the biochemical sensors, the enzyme is physically immobilized, by adsorption or by entrapment, both methods being deficient: in the first case because of the enzyme elution and in the second case because only a small amount of the enzyme is exploited (much being entrapped in the middle of the composite and having no access to the reaction place).

The novelty and originality of the project proposal is that it is intended to covalently immobilize the enzyme on the surface of hydrophilic polymers, creating in this manner a much more favorable microenvironment enabling the enzyme to adopt an optimal configuration of the active centre. The enzyme immobilization on a hydrophilic polymer is very important in the case of the nitrate and nitrite anions, because they are found in aqueous phase and especially in case of nitrites from the water for human consumption, whose concentration is very low (0,1 mg/l). It is a very difficult problem, because, beside with the covalent immobilization on hydrophilic polymers there must be used only those polymers having good adhesiveness to the electrode surface and which can be deposed in very thin films, or those which are electro conducting in order to allow the detection.

The project comprises 4 steps. It is proposed a successive steps research of the phenomena and processes related to: the covalent immobilization of enzymes, enzyme activity characterization of the nanocomposites, biosensor preparation, the structural and physical -chemical characterization of the new nanocomposites and the use of the new devices in the nitrate and nitrite monitoring in the waters for human consumption. The lab technologies will be experimented in order to obtain an optimal level, dissemination on large scale of the results will be done and the original results will be patented.

The project targets as main results, on one hand the increase of food safety and of the population health and on another hand the growing of the technical and qualitative level of autochthonous output of the advanced materials, promoting the rising of the competitiveness and the improvement of the market position of the interested beneficiary and of other potential beneficiaries, especially SME.