A novel urea conductometric biosensor based on zeolite immobilized urease

Abstract

A new approach was developed for urea determination where a thin film of silicalite and zeolite Beta deposited onto gold electrodes of a conductometric biosensor was used to immobilize the enzyme. Biosensor responses, operational and storage stabilities were compared with results obtained from the standard membrane methods for the same measurements. For this purpose, different surface modification techniques, which are simply named as Zeolite Membrane Transducers (ZMTs) and Zeolite Coated Transducers (ZCTs) were compared with Standard Membrane Transducers (SMTs). Silicalite and zeolite Beta with Si/Al ratios 40, 50 and 60 were used to modify the conductometric electrodes and to study the biosensor responses as a function of changing zeolitic parameters. During the measurements using ZCT electrodes, there was no need for any cross-linker to immobilize urease, which allowed the direct evaluation of the effect of changing Si/Al ratio for the same type of zeolite on the biosensor responses for the first time. It was seen that silicalite and zeolite Beta added electrodes in all cases lead to increased responses with respect to SMTs. The responses obtained from ZCTs were always higher than ZMTs as well. The responses obtained from zeolite Beta modified ZMTs and ZCTs increased as a function of increasing Si/Al ratio, which might be due to the increased hydrophobicity and/or the acid strength of the medium.

Introduction

Enzyme-based biosensors have been of intense investigation [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. The research has focused on enhancement of the sensitivity, detection limit, selectivity and the storage stability of these electrochemical biosensors. To be able to tailor these properties, variety of modification methods on electrochemical biosensor surfaces are proposed [2], since an important method in the development of enzyme-based biosensor is known to be the immobilization of enzymes on the transducer surfaces [3]. Electrode surfaces of the biosensors can be modified with different nanotechnology products such as sol–gels [4], nanotubes [5], polymers [6], and zeolites [2], [7], [8], [9], [10].

The use of zeolites in combination with enzymes have been of interest for a while due to some of their particular properties, such as tailorable surface groups, controlled hydrophilic/hydrophobic properties, shape, charge, and size selectivities, and their ability to regulate acidity for bi-functional enzymatic-acid catalysis. Furthermore, they are stable at high temperatures, insoluble in organic solvents, and resistant to harsh experimental conditions. Thus, they have been used to control the micro-environment of enzymes [11]. With such interesting properties, zeolites can offer themselves as alternative materials to be used for functionalizing solid substrates in a controlled manner. This can be of interest in the field of biosensors, and especially for conductometric ones, since they can be designed as integrated microbiosensors, which results into significantly reduced background conductivity due to the influence of temperature variations and other factors [12]. However, in order to use zeolites as alternative materials for enzyme immobilization and integrate them into such biosensor devices, the possibility to develop a simple and general technique to engineer the electrode surfaces for immobilization of biomolecules should be investigated. In this way, the potential advantageous roles for integrating zeolites can be explored by adsorbing enzymes on the surface of appropriate zeolites to obtain microdevices with high-sensitive biocatalytic function and long life biosensor property. The major challenge to use zeolites in such integrated devices and applications is its powder form upon synthesis. For using zeolites in these fields, zeolite films have to be constructed on the electrodes with controllable thickness and such electrodes are called as Zeolite-Modificated Electrodes (ZMEs). There have been different studies related with the construction of ZME's [13], [14], [15]; however studies reporting biosensor responses obtained from ZME's are scarce. Alain Walcarius made an attempt to classify the wide range of different fabrication procedures into 7 classes in his review [7]. These methodologies basically consist of adding/mixing zeolite particles with different composite materials, such as polymers and carbon paste and covalently tethering clay particles to the electrode surfaces. Furthermore, there has been no study on such systems in conductometric biosensors, which usually show certain limitations due to their signal to noise ratio. It is known that the need to use buffers for traditional conductometric applications in solution results in a drop in sensitivity, especially in the presence of non-reacting ions in the solution [12].

In our previous report, such a traditionally formed ZME was tested using conductometric electrochemical biosensors for the first time [16]. The advantage of such membranes is that there is no enzyme leakage from the membrane that results in a more stable electrode. However, it is time consuming and usage of another chemical makes the system more expensive. In literature, there are various reports about physical adsorption of urease on the transducer solid substrates with some supporting materials such as sol–gels [3], [4], polymeric membranes [12], [17], and microcapsules [18], [19]. This approach can increase the adsorption capability of the solid substrate, but also reduce the catalytic activity [20] with an increased response time [4]. To our knowledge, the immobilization of urease on nanozeolite assembled electrodes for conductometric biosensors has not been explored.

In the present work, we introduce a new approach for urease determination where thin films of silicalite and zeolite Beta were obtained on the gold electrodes of conductometric biosensors. The responses, as well as the operational and storage stabilities were compared with the results obtained using traditional ZME's that were constructed for comparison purposes. The novelty of the current system with respect to the traditional ones is that there is no need for an extra membrane and the biosensor works as a result of the simple sorption of urease on the zeolite thin film. For that purpose, the modified electrodes were firstly developed by using silicalite samples and afterwards, zeolite Beta samples with different silicon to aluminum (Si/Al) ratios were used to modify the electrodes to compare the varying responses as a function of tailored electrode surfaces. The newly obtained ZME's that were constructed from the zeolite thin films on gold electrodes were compared with the “zeolite mixed” membrane transducers (ZMTs) and “un-modified” ones (SMTs). In this way, it was possible to investigate the effect of modification type on the biosensor responses using two different types of zeolites. Furthermore, Si/Al ratio was changed using the same type of zeolite, zeolite Beta, which allowed us to evaluate the effect of changing hydrophobicity of the transducer surfaces on the biosensor responses for the first time using conductometric biosensors.

The conductometric responses of such electrodes modified with zeolites were tested using the enzymatic reaction of urease:

$${({\text{NH}}_2)}_2\text{CO}+2{\text{H}}_2\text{O}+{\text{H}}^{+}\stackrel{\text{urease}}{⟶}2\text{N}{\text{H}}_4^{+}+\text{HC}{\text{O}}_3^{-}$$

This reaction results in a change of charged ions which results in local alteration of conductivity in the solution. This allows usage of conductometric electrodes as transducers. Accordingly, in the first part of this work, a conductometric biosensor was constructed using silicalite for the first time and different biosensor characteristics were compared with the membrane type electrode configurations, such as “Zeolite Membrane Transducers” (ZMTs) and “Standard Membrane Transducers” (SMTs), of conductometric electrodes that does and does not include silicalite particles, respectively.