- Nano Express
- Open Access
A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite
© Velychko et al. 2016
- Received: 30 November 2015
- Accepted: 15 February 2016
- Published: 25 February 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.
- Recombinant urease
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 , 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 . 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 , spectrophotometry [4, 5], and fluorometry . 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 . 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 , physical adsorption , 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 ; 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 aimed at the development of the biosensor for highly accurate and stable determination of urea in a wide range of concentrations. For the purpose, it was proposed to use recombinant urease adsorbed on the surface of zeolite-modified conductometric transducers.
The enzyme urease (EC 126.96.36.199) from E. coli was used in the work, activity 150 U/mg, produced from “USBiological” (USA). Bovine serum albumin (BSA, fraction V) and urea were obtained from “Sigma-Aldrich Chemie” (Germany). Working buffer was phosphate buffer (KH2P04-Na0H), pH 7.4, from “Helicon” (Moscow, Russia). Other inorganic compounds used were of analytical reagent grade.
Silicalite was synthesized in the Middle-East Technical University (Ankara, Turkey). To synthesize the silicalite crystals, the gel 1TPAOH:4TEOS:350 H2O was prepared. To obtain the formula, tetraethoxysilane (TEOS) and tetrapropylammonium hydroxide (TPAOH) were mixed with distilled water under constant stirring for 6 h at room temperature. The crystallization took place at 125 °C for 18 h. The resulting solid material was washed four times with distilled water under centrifugation. The products were dried at 100 °C overnight. The size of silicalite particles was approximately 250 nm.
The conductometric transducers used in the work were produced at V.Ye. Lashkarev Institute of Semiconductor Physics, NASU (Kyiv, Ukraine) in accordance with our recommendations. They were 5 × 30 mm in size and consisted of two pairs of identical gold interdigitated electrodes on the sital substrate. The transducer design, preparation, and application are presented in detail in .
Scheme of Experimental Setup for Conductometric Measurements
The portable device MXP-3 (6) is connected to the electrical supply network via adapter (7), to the sensor block—with wires via contact (8), and to the personal computer (9) with a suite of related software—via contact (10). The current of frequency 37 kHz and amplitude 14 mV was used. First, conductometric transducer (1) was connected to the holder (2) and an initial baseline was obtained. Then, the tested substance was added to the working cell. The responses were recorded on a personal computer screen.
Preparation of Bioselective Elements
The procedure of urease adsorption on silicalite was developed earlier . The transducers previously coated with silicalite were used; 0.15 μl of 5 % recombinant urease solution in 20 mM phosphate buffer, рН 6.5, was deposited onto one pair of electrodes and 0.15 μl of 5 % BSA in the analogous buffer—onto the other (reference) pair. Afterwards, the transducers were exposed to complete air-drying (for 17 min). Neither glutaraldehyde nor any other auxiliary compounds were used. Next, the transducers were submerged into the working buffer for 20 to 30 min to wash off the unbound enzyme. After experiments, the transducer surface was cleaned from silicalite and adsorbed urease with ethanol-wetted cotton.
Procedure of Measurement
The measurements were carried out in 5 mM phosphate buffer, pH 6.5, at room temperature in an open cell with constant stirring. The necessary substrate concentration in the working cell was obtained by addition of aliquots of the substrate stock solutions. All experiments were conducted in four series. The non-specific changes in output signal associated with the fluctuations in temperature, environmental pH, and electrical noise were avoided due to the differential mode of measurements.
Characterization of Silicalite
Characteristics of KK46 silicalite
Analytical Characteristics of Biosensor
In the course of enzymatic reaction, the local concentration of ions in the enzyme membrane increases. This changes the solution conductivity, which is registered by the conductometric transducer . These changes and, consequently, biosensor responses are proportional to the concentration of urea.
Effect of Solution Parameters on Value of Biosensor Response
As known, the conductometric method is based on the measuring of changes in the sample solution conductivity. This change in conductivity may depend on both the enzymatic reaction itself and the characteristics of solution in which this reaction occurs. So, first, an influence of the solution parameters (ionic strength, buffer capacity, protein concentration in the solution) on the value of sensor response was studied.
Operational Stability and Response Reproducibility of Biosensor
The biosensor based on recombinant urease adsorbed on silicalite was developed to determine the concentration of urea in biological samples. To adapt the biosensor to the work with real samples, its sensitivity to urea was tested depending on the concentration of the working buffer and the salt and protein concentration in the samples. The biosensor developed using the proposed method of immobilization was characterized by high operational stability over 19 days. A significant extension of the linear range of urea determination was demonstrated. This enables an analysis of the samples with high urea concentrations without significant dilution.
The developed biosensor with improved analytical characteristics may be used in biomedical and clinical diagnostics.
The authors gratefully acknowledge the financial support of this study by the STCU Project 6052 “Enzyme multibiosensor system for renal dysfunction diagnosis and hemodialysis control.” Furthermore, this study was partly supported by the National Academy of Sciences of Ukraine in the frame of Scientific and Technical Government Program “Sensor systems for medico-ecological and industrial-technological requirement: metrological support and experimental operation.”
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