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)