Scanning electron microscopy study of protein immobilized on SIO₂ sol-gel surfaces

 

 

O.B.G.Assis

EMBRAPA Instrumentation Research Center, Cx. P. 741, Fax (16) 2725958, 13560-970, São Carlos – SP, São Paulo, Brazil, E-mail: odilio@cnpdia.embrapa.br

 

 

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ABSTRACT

Uniform attachment of enzymes to solid surfaces is essential in the development of bio and optical sensor devices. Immobilization by adsorption according to hydrophilic or hydrophobic nature is dependent on the charges and defects of the support surfaces. Sol-gel SiO₂ densified glass surfaces, frequently used as supports for protein immobilization, are evaluated via scanning electron microscopy. The model protein is globular enzyme lysozyme, deposited by adsorption on functionalized surfaces. Formation of a protein layer is confirmed by FTIR spectroscopy, and the SEM images suggest discontinuous adsorption in areas where cracks predominate on the glass surface.

Keywords: protein immobilization, enzyme, scanning electron.

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INTRODUCTION

Biosensors based on immobilized enzymes have many applications in areas such as industry, biochemistry and immunology (Marconi, 1978; Cheetham, 1985), where a wide range of fixation techniques have been developed and are continuously being improved. Of these techniques, covalent coupling on inorganic supports is one of the most commonly used due to its stability in achieving a good enzyme attachment (Weetall, 1993).

The simplest and cheapest way to introduce molecules into inert supports or insoluble matrices and preserve their natural state is by simple adsorption through hydrophobic or hydrophilic interactions. The important feature of this technique is that it usually requires no reagents in the process. Additionally, most of the enzymes and other active biopolymers of medical interest have amphifilic properties with predominantly positive or negative spatial charge distributions and therefore a tendency to be attracted and adsorbed on charged interfaces through electrostatic interaction.

Nevertheless, the bonds which link the enzymes to the support, in any kind of immobilization should be sufficiently stable under the conditions of catalytic application.

Ceramic and glassy materials are chemically inert and have been extensively used as solid supports suitable for immobilizing materials from aqueous solutions (Assis & Claro, 1999; Trevisan et al., 2000). When submitted to specific chemical treatment, the ceramic or glass hydrophilic or hydrophobic properties may increase or even change due to chemical modification of the surface (Weetall, 1993). The increase in the hydrophilicity or hydrophobicity of surface enhances the stability of enzyme coupling, maximizing sensor applications.

Glass produced by chemical derivatization also has the advantage of easy surface functionalization by alteration of charges. This modification may enhance its organophilicity and consequently effectiveness in adsorbing a variety of organic molecules, especially when dissolved in water (Avnir et al., 1994; Assis & Alves, 1999).

Glass prepared by the sol-gel method has been successfully used as platforms or matrices for enzyme and microorganism entrapment (Avnir et al., 1994; Chai & Ben-Nissan, 1999). Owing to their purity and porosity control, sol-gel glass films can be used as supports for coupling enzymes that are especially suitable as chemical and biological sensors for diagnostic applications.

Specifically, sol-gel glass has advantages over conventional ceramic or metallic surfaces that include its high biocompatibility, controlled density and ability to achieve faster response time on typical sensor substrates such as optical fibers or wave guides (Seddon, 1998).

The efficiency of a sol-gel based sensor, however is, strongly dependent on glass surface uniformity which plays an important role in the quality of enzyme attachment and sensitivity.

The purpose of this work is to conduct a simple study using scanning electron microscopy (SEM) of the effects of glass surface continuity on the formation of protein layer by simple adsorption. The globular enzyme lysozyme, used as model protein, was studied on functionalized SiO₂ sol-gel sintered glass with the aim to develop future biosensors.

 

MATERIALS AND METHODS

Silica gel was prepared from tetramethoxysilane (TMOS) precursor diluted in methanol (H₃COH) and formic acetic acid (H₃CCOOH) in proportions of 1:1:0.1 by volume. The densification cycle followed the conventional sequence used for the sol-gel process, viz., drying at 60 ^oC for 15 minutes followed by elimination of solvent at 450 ^oC (1 h.) and finally densification at 550 ^oC (1 h.). Densification was carried out in air and the specimens were allowed to cool slowly in the furnace to avoid thermal shock. The glass underwent functionalization treatment, which consisted in a series of surface cleanings by ultra-sonic baths in warm acid solution, in a previously established chemical sequence (Kern, 1984; Bernardes-Filho et al., 1997). This treatment essentially consists in a cleaning procedure that enhances the negative surface charges of the glass and the corresponding hydrophilicity support index. The enzyme lysozyme, LYZ (from Sigma), was immobilized by direct immersion in a surfactant solution at high concentration (10^-2 Ml^-1). The pH was 6.4, ensuring predominantly positive charges of the protein molecules. Immersion time was 12 minutes. FTIR reflectance spectroscopy of covered and bared surfaces was carried out in a BOMEN DA8 system. Glass with and without the enzyme-adsorbed layer was observed under SEM.

 

RESULTS AND DISCUSSION

The immobilization of LYZ is confirmed by FTIR spectroscopy as shown in Figure 1. In the scanning wavelength interval from 400 to 2000 cm^-1, it is possible to assess typical silicon vibration binding types (Sakka & Yoko, 1992), as identified in the small box in Figure 1. The pattern in wavelengths of 400 to 2000 cm^-1 corresponds to the glass support after immobilization of the enzyme. The patterns are in the carbonylic area where amide I, II and III bands are identified (Forato et al., 1998). In this plot, the amide II band is assumed to be composed of an overlapping of glass-LYZ spectra.

 

 

For microscopic analysis, two types of resultant SiO₂ support surface were selected for study:

i) an apparently uniform flat surface, characterized by areas of full density and

ii) areas with a discontinuous structure, where cracks predominate along the surface.

Cracks and irregular features were expected to be found in SiO₂ glass processed with sol-gel, since this characteristic is currently observed in glass coatings under different conditions and for different substrate systems (Lima-Neto et al., 1994; Simões et al., 2000).

These cracks have been attributed to a differential shrinkage and also to internal stress yielded from the differences in thermal contraction in different areas in the gel.

Figure 2 shows a series of SEM micrographs of the SiO₂ film surfaces under both conditions, before and after enzyme deposition. As can be seen from the selected photographs, a high density of organic material is adsorbed on the glass surface, where a continuous film structure predominates on the flat surface, as shown in Figure 2A and 2B.

 

 

In the areas with cracks (forming valleys as large as 1 mm), the deposition was irregular or even absent. Since deposition is based on electrostatic interaction, the physical characteristics of the support surface were shown to play an important role in the immobilization process.

Interactions involving organic molecules and inorganic surfaces are related to their chemical and structural affinities and how these features are distributed throughout the material. Imperfection in the glass support will substantially affect the continuous fixation of enzyme which will be reflected by the efficiency of the reactor or biosensor signal. Therefore for good results efforts have to be done in processing glass absent of cracks or by careful selecting of areas on supports surface.

 

ACKNOWLEDGEMENTS

The author is grateful to CNPq and Embrapa for their financial support.

 

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Received: May 26, 2002
Accepted: April 29, 2003