Conductometric enzyme biosensors based on natural zeolite clinoptilolite for urea determination

Highly sensitive conductometric urea biosensors were developed by exploiting the successful combination of ammonium-sieving and ion exchange properties of clinoptilolite, with a unique biorecognition capacity of urease. To optimize the performance of urea biosensors based on clinoptilolite, the dependences of their analytical signals on pH, buffer capacity and ionic strength of phosphate buffer solution (PBS) were studied. Optimum pH for urea biosensors was found within the range of pH 6.0–7.0. The dependences of biosensors responses on buffer capacity and ionic strength of PBS were of the same profile as those obtained for the urea biosensor which was not modified with clinoptilolite.Analytical characteristics of urea biosensors based on clinoptilolite were evaluated by determination of the sensitivity, linear and dynamic ranges, detection limit, the apparent Michaelis–Menten constant and the response time. The optimum features in terms of sensitivity, dynamic range, and detection limit (20.36 μS/mM, 0–64 mM, and 10−6 M, respectively) were found for the urea biosensor, based on a primary layer of clinoptilolite followed by a secondary layer of urease and clinoptilolite in a single bioselective membrane. The apparent Michaelis–Menten constants Km for the developed urea biosensors based on clinoptilolite varied from 2.73 to 5.67 mM. These values differed significantly from the value of Km for the urea biosensor which was not modified with zeolite (1.89 mM). All types of zeolite-modified biosensors showed high operational and storage stability.

Research highlights
► Combination of zeolite ammonium-sieving property together with biorecognition provided by urease increases the sensitivity of urea detection.
► Urea biosensor based on a layer of clinoptilolite shows the optimum analytical characteristics.
► Zeolite-based biosensor presents 87% of its initial activity after 5 months.