Environmental analytical methods
Validation of a conductometric bienzyme biosensor for the detection ofproteins as marker of organic matter in river samples
Received ,Revised , Accepted , Available online
Volume 21,2009,Pages 545-551
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This article describes a conductometric bi-layer based bienzyme biosensor for the detection of proteins as a marker of organic matter
in rivers. Proteins were chosen to be used as indicators of urban pollution. The working mechanism of the bienzyme biosensor is based
on the enzymatic hydrolysis of proteins into several fractions (peptides and amino acids), which results in a local conductivity change
depending of the concentration of proteins. In this work, we began with the optimization of biosensor response using bovine serum
albumin (BSA) as standard protein. For this objective seven enzymatic biosensors were prepared: four enzymatic sensors with only one
layer of enzyme (proteinase K, trypsin, pronase or protease X) and three other enzymatic sensors with two layers (first layer: membrane
containing proteinase K; second layer: one of the three other enzymes: trypsin, pronase or protease X). The biosensors were obtained
through the deposition of enzymatic layers and the cross-linking process between enzymes and BSA in saturated glutaraldehyde vapour.
The response of the various biosensors, described previously, were compared with the values of total organic carbon (TOC), and those of
organic nitrogen (Norg), as determined by the laboratory accredits (CEMAGREF of Lyon) using the traditional method of analysis (NF
EN 1484, infrared spectroscopy) and (NF EN 25663, mineralization/colorimetry assay) respectively for each water sample obtained
from di erent sites in Lyon (France). The linear correlations obtained with the response of the seven biosensors showed the most
important indices of correlations for the biosensor with two enzymatic layers: proteinase K + pronase (pkp). The optimum conditions
for the preparation of the pkp biosensor increased the sensitivity and gave a limit of quantification of 0.583 g/L for TOC and 0.218
g/L for Norg in water samples. This sensor shows good reproducibility (2.28%), a capacity to be used at temperatures range 10–
30°C (depending on the season) and moreover a long lifetime (5 weeks).
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