A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

Nanoscale Research Letters, Feb 2016

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05–15 mM, and a lower limit of urea detection was 20 μM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis.

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A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

Velychko et al. Nanoscale Research Letters A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite T. P. Velychko 0 1 О. О. Soldatkin 1 V. G. Melnyk 2 S. V. Marchenko 1 S. K. Kirdeciler 3 4 B. Akata 3 4 A. P. Soldatkin 0 1 A. V. El'skaya 1 S. V. Dzyadevych 0 1 0 Taras Shevchenko National University of Kyiv , Volodymyrska Street 64, 01003 Kyiv , Ukraine 1 Institute of Molecular Biology and Genetics of NAS of Ukraine , Zabolotnogo Street 150, 03143 Kyiv , Ukraine 2 Department of Electrical and Magnetic Measurements, Institute of Electrodynamics of National Academy of Sciences of Ukraine , 56, Peremohy Ave., Kyiv-57 03680 , Ukraine 3 Central Laboratory, Middle East Technical University , Ankara 06531 , Turkey 4 Micro and Nanotechnology Department, Middle East Technical University , Ankara 06531 , Turkey Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05-15 mM, and a lower limit of urea detection was 20 μM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis. Silicalite; Enzyme; Biosensor; Recombinant urease; Conductometry Background Urea [(NH2)2CO] is synthesized in the liver and is the final product of detoxification of endogenous ammonia, which is formed due to the decay of proteins and other nitrogen-containing compounds. The synthesized urea is released from the liver into the blood and transported to the kidneys where it is filtered and excreted with the urine. Normally, the urea concentration in humans ranges from 2.5 to 7.5 mM [ 1 ], but the rate of its synthesis, and thus the concentration, increase partially if either the protein-rich food is used, or endogenous catabolism is enhanced under the conditions of starvation, or the tissues are damaged, etc. However, a drastically elevated level of urea (50–150 mM) in the blood plasma indicates a kidney dysfunction. Such abnormal level of urea may be reduced to 10 mM by hemodialysis or peritoneal dialysis [ 2 ]. Therefore, determination of the urea concentration is of vital importance in biomedical and clinical assays. To this end, numerous methods are developed including gas chromatography [ 3 ], spectrophotometry [ 4, 5 ], and fluorometry [6]. The disadvantages of the above methods are dependence of the results on the sample pretreatment, long-time procedure, the need for highly qualified personnel, and impossibility of online measurements. An alternative to the above methods is the use of biosensors—miniature analytical devices without the drawbacks listed. Numerous biosensors have been developed to date for urea analysis in biological samples including potentiometric [ 7–9 ], conductometric [ 10–12 ], and amperometric [ 13–15 ]. However, all of them have two significant disadvantages. First, they have rather a narrow linear range of determination and it is a characteristic trait of urease-based biosensors, which are used in urea assays. To solve this challenge, earlier, we have proposed recombinant urease from E. coli with high Km to shift the linear range to higher urea concentrations [ 16 ]. Another drawback of the known urea biosensors is associated with the immobilization of biological material on the surface of transducers. Urease can be immobilized by covalent binding [ 17 ], physical adsorption [ 18 ], binding with polymers [ 14, 19, 20 ], or coupling to the transducer surface [ 21, 22 ]. Some problems are intrinsic for these methods. They are as follows: the loss of enzyme activity, unstable reproducibility of biosensor signals, and toxicity of the compounds, which induce the binding. The latter is a particular problem in the determination of the enzyme activity in biological samples. To overcome these difficulties, zeolites were proposed as carriers for enzyme adsorption. The zeolites are slightly toxic and highly resistant to mechanical, chemical, and thermal injuries [23]; therefore, the zeolite-based biosensors can be used for multicomponent biological samples. This method of immobilization demonstrated promising results in a number of enzyme biosensors [ 24–26 ]. To create the biosensor for urea determination in biological samples, it was necessary to address the described problems simultaneously. This study was aime (...truncated)


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T. P. Velychko, О. О. Soldatkin, V. G. Melnyk, S. V. Marchenko, S. K. Kirdeciler, B. Akata, A. P. Soldatkin, A. V. El’skaya, S. V. Dzyadevych. A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite, Nanoscale Research Letters, 2016, pp. 106, Volume 11, Issue 1, DOI: 10.1186/s11671-016-1310-3