New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS)

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Abstract

The immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the water-in-oil (W/O) emulsion system were studied. The terminal amine group was used as the important functionality for GOD immobilization on the silica substrate. When only TEOS is used as a silica source, the disordered mesoporous silica microspheres are obtained. As the molar ratio of APTMS to TEOS (RAT) increases, the surface area and pore volume of the silica particles measured by nitrogen adsorption and desorption method and SEM decrease rapidly. Particularly, the largest change of the surface morphology is observed between RAT = 0.20 and RAT = 0.25. The amount and the adsorption time of immobilized enzyme were measured by UV spectroscopy. About 20 wt% of GOD was immobilized into the silica substrates above RAT = 0.60 and was completely adsorbed into the substrate of RAT = 0.80 with lapse of 4 h after addition. In the measurement of the thermal stability, GOD dissolved in buffer solution loses nearly all of its activity after 30 min at 65 °C. In contrast, GOD immobilized on the surface-modified silica particles still retains about 90% of its activity after the same treatment. At this temperature, the immobilized glucose oxidase retained half of its initial activity after 4 h. It is shown that the suitable usage of functionalizing agent like APTMS as well as the control of surface morphology is very important on the immobilization of enzyme.

Introduction

Encapsulation and immobilization of enzymes and other biomolecules on substrates have been the focus of intense study due to the various potential applications of such materials in electrochemical sensors, biocatalysts, biosensors, and diagnostic devices [1], [2], [3], [4]. Enzymes, proteins, antibodies, antigens, and cells have been immobilized on various inorganic materials in the form of fibers, thin films, monoliths, or granules [5], [6], [7], [8]. Of the biomolecules, enzymes exhibit high selectivity and reactivity under normal conditions but are sensitive to denaturalization or inactivation by pH and temperature extremes, organic solvents and detergents, because it is difficult to maintain a three-dimensional structure and arrangement in a reaction system under such harsh conditions. To maintain the activity of enzymes under extreme conditions, immobilization of enzymes onto the polymeric organic materials such as resins, organic gels, and fibers, and the inorganic supports like porous silica gels have been studied by many research groups [9], [10], [11], [12], [13].

Recently, chemically inert inorganic oxide sol–gel materials with negligible swelling effects, controllable surface parameters, and high purity have attracted great interest as a new class of host materials that are well suitable for immobilization and encapsulation of enzymes at room temperature. In particular, mesoporous silicas such as the MCM-type materials and SBA-type materials have been studied as the appropriate host matrices for immobilizing enzymes because they have large pore volumes and controllable pore sizes comparable to the diameter of enzymes. Takahashi et al. [14] reported that enzymes were selectively adsorbed to FSM-16 and MCM-41 prepared with a cationic surfactant and was not adsorbed significantly to SBA-15 prepared with a non-ionic surfactant. Butler and co-workers [15] describe the results that mesocellular form (MCF) is a promising material for immobilizing enzymes, due to its large pore structure and high loading capacity compared to other mesoporous materials, such as MCM-48, SBA-16, and SBA-15.

Surface modification using organic functional groups has been found to be useful for the immobilization and encapsulation of enzymes to the surface of the silica materials [16], [17], [18]. Organic functional groups have been incorporated into mesoporous silica by post-modification, known as post-synthetic grafting, or co-condensation of trialkoxyorganosilanes with tetraethylorthosilicate (TEOS). Post-synthetic grafting is a method more commonly used in performing surface modification by covalently linking organosilane species with surface silanol groups [19]. On the other hand, the co-condensation method was first reported by two research groups in 1996 [20], [21]. This method allows modification of the surface of the mesoporous materials in a single step by copolymerization of organosilane with silica or organosilica precursors in the presence of a surfactant [20], [21], [22], [23], [24], [25], [26], [27], [28]. This approach enables a higher and more homogeneous surface coverage of organosilane functionalities [28].

The commonly used immobilization methods may be subdivided into three general classes. First of all, the immobilization of enzymes can be made by physisorption [29], [30], [31]. In this case, the weak interaction such as hydrogen bonding, van der Waals, or electrostatic forces occurs between the enzymes and the matrix. Another method for immobilizing enzymes in supporting materials consists of chemisorption [32], [33]. This second pathway involves a covalent link between enzyme and matrix. Most of the surface of the matrix has to be functionalized before the immobilization. Finally, the enzymes can be incorporated in surfactant or non-surfactant templated mesoporous materials through a direct one-step immobilization [34], [35], although only a few papers about this kind of encapsulation have been reported.

Glucose oxidase (GOD) is one of the most conspicuously studied enzymes [36], [37]. This is because of its utility in the selective measurement of β-d-glucose, an analyte of broad analytical and pharmaceutical interest. The use of insoluble glucose oxidase provides some advantages that overcome limitations emerged when using soluble enzyme in solution: an increase in the retention of enzyme activity with time and easy separation and recovery with minimum contamination. Many glucose biosensors based on the entrapment of glucose oxidase and/or (per)oxidase in sol–gel silica materials have been reported [38], [39], [40], [41].

In this study, we report the immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the W/O emulsion system. As the molar ratio of APTMS to TEOS increases, the surface area and pore parameters of the silica particles measured by nitrogen adsorption and desorption method decrease rapidly. In general, when the pore size of silica particles is small, compared with the size of the enzyme, the low enzyme loadings and slow enzyme immobilization rates are observed [31], [32], [42]. However, in our study, as the surface area and pore size decrease by the increase of the molar ratio of APTMS to TEOS, the enzyme loadings inversely increase. GOD immobilized on the non-porous silica microspheres show the much higher thermal and pH stability than pure GODs in solution. In addition, the desorption of enzymes encountered with the direct immobilization of enzymes on mesoporous silicas is not observed.

Section snippets

Materials

Tetraethylorthosilicate (TEOS, 98%), (3-aminopropyl)trimethoxysilane (APTMS), hydroxyproyl cellulose (HPC, average Mw ca. 370,000), and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (P123) were purchased from Aldrich. Glucose oxidase (GOD), horseradish peroxidase (POD), glucose, o-dianisidine and sorbitan monooleate (Span 80) were obtained from Sigma. The 1-octanol and ammonium hydroxide (NH4OH) were purchased from Junsei Chemical Company. Ethanol (95%) as a

Synthesis of silica particles in W/O emulsion

Since Stöber et al. [43] introduced a method for the preparation of monodisperse silica particles from aqueous solutions of silicon alkoxides containing ammonia, the sol–gel method has been successfully used for the preparation of many inorganic particles.

In contrast to the homogeneous system, emulsion medium of heterogeneous system has been utilized to obtain micro-sized silica particles. An emulsion is the stable suspension of particles of liquid of a certain size within a second, immiscible

Conclusions

Glucose oxidase (GOD), one of the most conspicuous enzyme, was immobilized onto the mesoporous and the non-porous silica microspheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the W/O emulsion system. Water droplets in the emulsion serve as a micro-reactor for particle growth, thus reflecting the shape, size, and size distribution of particles. When only TEOS is used as a silica source, disordered mesoporous silica microspheres

Acknowledgement

This work was supported by the research fund of Hanyang University (HY-2006-I).

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