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Volume 3, Pages 585-592
Peer-Reviewed Protocol

Optical Enzyme Sensor for Urea Determination via Immobilized pH Indicator and Urease onto Transparent Membranes

Department of Biotechnology, University of Chemical Technology and Metallurgy, Blvd. 8 Kl.Ohridski, 1756, Sofia, Bulgaria

Academic Editor: David Rickwood

Copyright © 2003 Milka Krysteva and Mohamed Al Hallak.


Transparent triacetylcellulose membranes with immobilized pH indicator (neutral red) as well as with simultaneously immobilized urease and neutral red were used as optical sensors for determination of urea concentrations in model solutions. Decomposition of urea with the enzyme urease is accompanied by evolution of ammonia. This leads to the changes of the neutral red absorption, which is proportional to the substrate (urea) within certain concentration limits in model solution. As a result of the investigation, standard curves were plotted for determination of urea over the range of 1 to 500 mM using immobilized indicator and free urease. Simultaneous immobilization of indicator and urease permitted determination of urea in the interval 50 to 500 mM. The membrane used contained 0.169 U urease activity on an area of 1.7 cm2. The standard curves were plotted using the linear region of the kinetic curves for the corresponding substrate concentrations. A possible scheme of the interaction between the activated triacetylcellulose membrane and the indicator and enzyme is proposed. The membranes obtained are suitable for repeated ecological applications where urea is to be determined.