Study of zeolite influence on analytical characteristics of urea biosensor based on ion-selective field-effect transistors
© Shelyakina et al.; licensee Springer. 2014
Received: 3 December 2013
Accepted: 27 February 2014
Published: 17 March 2014
A possibility of the creation of potentiometric biosensor by adsorption of enzyme urease on zeolite was investigated. Several variants of zeolites (nano beta, calcinated nano beta, silicalite, and nano L) were chosen for experiments. The surface of pH-sensitive field-effect transistors was modified with particles of zeolites, and then the enzyme was adsorbed. As a control, we used the method of enzyme immobilization in glutaraldehyde vapour (without zeolites). It was shown that all used zeolites can serve as adsorbents (with different effectiveness). The biosensors obtained by urease adsorption on zeolites were characterized by good analytical parameters (signal reproducibility, linear range, detection limit and the minimal drift factor of a baseline). In this work, it was shown that modification of the surface of pH-sensitive field-effect transistors with zeolites can improve some characteristics of biosensors.
Zeolites are microporous crystalline solids with well-defined structures. Generally, they contain silicon, aluminium and oxygen in their framework and cations, water and/or other molecules within their pores. Many of them occur naturally as minerals and are extensively mined in different parts of the world. Others are synthetic, and are made commercially for specific applications, or produced by research scientists trying to understand more about their chemistry .
A defining feature of zeolites is that their frameworks are made up of four connected networks of atoms. One can think about this in terms of tetrahedral, with a silicon atom in the middle and oxygen atoms at the corners. These tetrahedra can then link together by their corners to form a rich variety of beautiful structures. The framework structure may contain linked cages, cavities or channels, which are of the proper size to allow small molecules to enter .
The shape-selective properties of zeolites are also the basis for their use in molecular adsorption. The ability to adsorb preferentially certain molecules, while excluding others, has opened up a wide range of molecular sieving applications. Sometimes, it is simply a matter of the size and shape of pores controlling access into the zeolite. In other cases, different types of molecule enter the zeolite, but some diffuse through the channels more quickly, leaving others stuck behind, as in the purification of para-xylene by silicalite .
Cation-containing zeolites are extensively used as desiccants due to their high affinity for water, and also find application in gas separation, where molecules are differentiated on the basis of their electrostatic interactions with the metal ions. Conversely, hydrophobic silica zeolites preferentially absorb organic solvents. Zeolites can thus separate molecules based on differences of size, shape and polarity.
Nowadays, zeolites are of great interest due to their high surface areas, rigid and well-defined pore structures, thermal stabilities and tailorable surface charges with respect to other types of nanomaterials. Such properties of zeolites make them inevitable candidates for preparing ion-selective membranes for potentiometric cation sensing .
The hydrophilic character of zeolites also makes them very suitable materials for the co-immobilization of enzymes and mediators in the preparation of biosensors. The number and type of surface hydroxyl groups, which are important for immobilization applications, can be simply controlled by applying different heat treatment procedures. Finally, zeolites are known to be stable in both wet and dry conditions and well-tolerated by microorganisms, leading to an enhanced compatibility with biochemical analyses. All of these properties make zeolites unique nanomaterials and promising candidates for the immobilization of biological molecules and for advanced analytical tasks .
At present, some variants of biosensors containing zeolite crystals are known. As shown in , zeolites of various kinds can be effectively used for the glucose oxidase immobilization while developing glucose amperometric biosensors to optimize their sensitivity, selectivity and stability. As reported in [6, 7], zeolites are used as alternative carriers for the enzyme immobilization while creating conductometric biosensors. Diverse variants of co-immobilization of urease and BEA-zeolites onto the surface of conductometric transducers were analyzed in respect to the improvement of analytical characteristics of biosensors for urea determination . Promising results were obtained when zeolites were used for the development of urease biosensors based on pH-sensitive field-effect transistors [6, 9–14].
There is also information on the benefits of application of zeolites as adsorbents for enzyme immobilization . This option of immobilization allowed obtaining biomembranes on the surface of conductometric transducers without use of toxic substances.
In our work, we proposed a method of enzyme adsorption on zeolite monolayers for the creation of potentiometric biosensor. We have chosen the urease since it is one of the most studied and stable enzymes in biosensorics. In addition, the results obtained on urea biosensors can be applied to any other enzyme systems.
The materials used in this study were as follows: urease activity 66 U/mg, urea, glutaraldehyde (GA), glycerol and bovine serum albumin (BSA) (fraction V) from ‘Sigma-Aldrich Chemie’ (Munich, Germany). Silicalite, zeolite L, nano zeolite beta and calcine nano zeolite beta were synthesized in the Middle East Technical University (Ankara, Turkey). KH2P04, NaOH and other substances used in the work were of domestic production and of chemical purity.
Design of potentiometric transducer and measuring device
Device for measurements
Immobilization of biological elements in glutaraldehyde vapour
To prepare the enzyme membrane, 5% urease solution was mixed with 5% BSA solution in 20 mM phosphate buffer (pH 7.4), which contained 10% glycerol (to stabilize the enzyme during immobilization and prevent early drying of the solution deposited on the transducer surface). To obtain the reference membrane, 10% BSA solution was prepared in 20 mM phosphate buffer containing 10% glycerol. A drop of the enzyme-BSA mixture was deposited on one fragment of the transducer-sensitive surface (selective membrane) and the BSA solution without enzyme on another (reference membrane). For membrane polymerization, the transducers were placed in atmosphere of saturated GA vapour for 20 to 25 min. The latter reacts with the available amino groups of proteins, contributing to the formation of cross links of Schiff base type (−N = CH−) in the membrane.
After immobilization, the transducers were dried in air and washed from GA excess in buffer solution for 10 to 15 min. Before measurements, the sensors with deposited biomaterial were kept for some minutes in the working buffer until a stable signal baseline is obtained.
Procedure of zeolite synthesis
To synthesize the silicalite solution, we used 1TPAOH:4TEOS:350 × H2O. When hydrolysing tetraethoxysilane (TEOS) with tetrapropylammonium hydroxide (TPA-OH), we obtained a homogeneous solution by constant stirring for 6 h at room temperature. The crystallization took place at 125°C during 1 day. The material, which did not react, was removed from the solution by centrifugation. The size of silicalite particles was approximately 400 nm.
Nano beta zeolite
The molar composition of the nano beta zeolite is 0.25 Al2O3:25 SiO2:490 H2O:9 TEAOH. Silica source was TEOS (98%, Aldrich). Aluminium isopropoxide (98%, Aldrich), tetraethylammonium hydroxide (TEAOH) (20 wt.% in water, Aldrich) and double-distilled water were used as the other reactants. Ageing was continued under static conditions for 4 h with clear solution. The crystallization was completed within 17 days under static conditions at 100°C in Teflon-lined autoclaves. The product was purified by centrifugation . Approximately, the particle size of nano beta zeolite is 60 nm.
The molar composition of the nano zeolite L is 0.08Al2O3:1SiO2:0.5K2O:10H2O. Aluminium powder is dissolved in KOH solution. Colloidal silica (Ludox HS-40, Dupont, Wilmington, DE, USA) was then added under vigorous stirring, and the gel was stirred at room temperature for 5 min. The crystallization continued for 6 days in Teflon-lined autoclaves under static conditions at 170°C. Approximately, the particle size of nano zeolite L is 60 nm.
Modification procedure for zeolite monolayers
Firstly, we tried to attach the zeolite on the electrodes surface but failed in our attempts. Then, we used a layer of poly(ethyleneimine) (PEI) between zeolite and electrode surface. In this case, zeolite attachment was attained but there was a homogeneity problem. To solve it, we used mucasol (1/6 v/v) in distilled water, which gave good results due to changing surface hydrophility.
The electrode surfaces were dip-coated with mucasol for 15 min, rinsed with copious amount of distilled water and dried under air. For the formation of homogeneous layers of PEI, both dip coating and spin coating techniques had been tried. Since spin coating gave more homogeneous layers, it was continued to be used. The effect of PEI solvent type (hot water and ethanol), PEI concentration (0.5%, 1%, 3%), spin coating time (3,000 rpm 15 s, 7 s) and calcination temperature (100°C, 90°C, 50°C) were investigated. The obtained monolayers were checked by microscope. The suitable conditions for zeolite monolayer production were chosen as follows: spin coating with 0.5% PEI in ethanol at 3,000 rpm during 15 s and calcination at temperature 90°C for 30 min.
The synthesized zeolites were directly attached to the obtained electrode surfaces simply by rubbing zeolites with a finger, a technique called direct attachment. These electrodes were used in further studies.
Enzyme immobilization on the surface of zeolite monolayers
To produce the enzyme membrane, 5% urease solution in 20 mM phosphate buffer (pH 7.4) was prepared. To obtain the reference membrane, 5% BSA solution in 20 mM phosphate buffer was prepared. A drop of enzyme solution was deposited on one section of the transducer-sensitive surface, covered with zeolite, on another - BSA solution without enzyme (reference membrane).
After immobilization, the transducers were dried in air and washed from unbound components of membranes in buffer for 10 to 15 min. Before measuring, the sensors with deposited biomaterial were kept for some minutes in the working buffer until a stable signal baseline is obtained.
Procedure of measurement by biosensor device
Measurements were conducted in 5 mM phosphate buffer, pH 7.4, at room temperature using a cell measuring system. The substrate concentration in the cell was specified by the addition of different aliquots of the substrate stock solutions to the working buffer. The experiments were performed in at least three repetitions. Nonspecific changes in the output signal because of fluctuations in temperature, pH and electrical breakthrough were significantly reduced due to the differential mode of measurements.
Results and discussion
The aim of our work was to improve analytic characteristics of the enzyme-based biosensors. We have chosen the urease, since it is one of the most studied and stable enzymes in biosensorics. We studied the method of urease adsorption on the surface of pH-sensitive field-effect transistors (ISFET) using monolayer of different types of zeolites: silicalite, nano beta zeolite, zeolite nano L and calcinated nano beta zeolite. Obtained results were compared with results of biosensors based on urease immobilized in GA vapour.
This reaction results in the change of pH inside selective membrane which is recorded by pH-sensitive field-effect transistors.
As seen, the best signal reproducibility manifested the sensor obtained by immobilization on a monolayer of nano zeolite beta; the worst was obtained for immobilization in GA vapour. Thus, the conclusion can be made that immobilization by deposition of selective membrane on the zeolite-modified surface results in noticeable improvement of reproducibility and thus to the enzyme stabilization.
As seen, the slightest data dispersion (i.e. the best inter-reproducibility) at both urea concentrations was revealed for the biosensor obtained by urease immobilization on the surface of monolayer of silicalite and zeolite L. The inter-reproducibility for the biosensors immobilized by adsorption on the surface of monolayers of nano zeolite beta and nano calcined zeolite beta was almost the same; the worst was observed for biosensors based on urease immobilized in GA vapour.
Comparison of operational characteristics of biosensors created using different types of urease immobilization
Type of immobilization
Drift of base line (μA/min)
Detection limit (mM)
RSd of reproducibility of signal (%)
RSd of inter-reproducibility of immobilization (%)
Linear range (mM)
In GA vapour
0 to 0.5
On the surface of silicalite monolayer
0 to 1.5
On the surface of nano beta zeolite monolayer
0 to 1.0
On the surface of zeolite L monolayer
0 to 1.5
On the surface of calcinated nano beta zeolite monolayer
0 to 1.0
The obtained data show that adsorption on zeolites can promote a linear range, a decrease in the error of response reproducibility and in baseline drift. Nevertheless, the biosensors obtained by traditional immobilization in GA vapour can be characterized by lower baseline noise.
Thus, the technique of enzyme adsorption on zeolite monolayers allows us, on the one hand, to avoid using toxic substances (glutaraldehyde, etc.), on the other - to obtain potentiometric biosensors with improved analytical characteristics.
We investigated a possibility of application of a new method of immobilization of enzyme urease on the surface of pH-sensitive field-effect transistors by adsorption on zeolite monolayers. In the experiment, we used different types of zeolites: nano beta, calcinated nano beta, silicalite and nano zeolite L. As a control, we used the method of immobilization in glutaraldehyde vapour (without zeolite). It is shown that the enzyme immobilization on monolayers of silicalite or nano zeolite L results in increasing biosensor linear range up to 1.5 mM. We found that the biosensors based on adsorbed urease were characterized by better signal reproducibility during work and better reproducible stable immobilization of the biological material. The biosensors obtained with new methods were compared by various parameters: noise, baseline drift, minimum determination limit, error of signal reproducibility, error of immobilization reproducibility and width of the linear range of operation.
It was found that the use of monolayers of different zeolites as a carrier for adsorption of the enzyme for the creation of potentiometric biosensors can result in increasing linear range of their operation, reducing the minimum limit of urea determination, improved response reproducibility and inter-reproducibility and decreasing time of analysis.
The authors gratefully acknowledge the financial support of this study by the European IRSES-NANODEVISE Project. 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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