Entrapment of enzyme-linked magnetic nanoparticles within poly(ethylene glycol) hydrogel microparticles prepared by photopatterning
Introduction
The immobilization of enzymes onto or into solid supports has been an active research topic in enzyme technology. Various organic and inorganic materials have been used as supports for immobilization of enzymes via covalent attachment or/and physical entrapment. Often, these enzyme-functionalized supports are used in analytical devices or as catalysts in industrial processes [1], [2].
Of the various immobilization methods, entrapment of enzymes within a hydrogel matrix has attracted the most attention [3], [4], [5], [6], [7]. Hydrogels are crosslinked polymers with a three-dimensional network arrangement and are able to retain the large amounts of water needed for enzyme activity. The soft, hydrated environment of a swollen hydrogel can provide entrapped enzymes with near-physiological conditions that minimize denaturation and allow them to retain their full biological function. Hydrogels can also provide a protective environment for the entrapped enzyme and inhibit degradation and non-specific adsorption [8], [9], [10]. Hydrogels are usually formed by free radical polymerization and can be made in either a bulk form or as nano/microparticles [11]. The bulk gels are easy to handle and to study, but they have very slow response time. Hydrogel nanoparticles react quickly to external stimuli, but are too small for some applications. As a result, micrometer-sized hydrogel particles are most commonly used for enzyme immobilization. Micron-sized spherical hydrogel particles are conventionally prepared by emulsion techniques, suspension polymerization, or by using shear flow in microfluidic channels [12], [13], [14], [15], [16], [17].
Despite the many advantages associated with enzyme-entrapped hydrogel microparticles, loss in the activity of the entrapped enzymes is inevitable because the crosslinked hydrogel often hinders diffusion of substrates. Diffusion of substrate through hydrogels can be enhanced by increasing the water content or porosity of the hydrogel [18], [19], [20], but these approaches can result in increased enzyme leaching from hydrogel. Such leaching limits the lifespan of the device.
The objective of this study is to immobilize enzyme into hydrogel microparticles with higher water content and larger mesh size, while preventing enzyme leaching. The goal of this effort is to minimize diffusion limitations for substrates and subsequently improve the rate of enzymatic reactions. To achieve this end, we immobilized enzyme on the surface of magnetic nanoparticles (MNPs) and subsequently entrapped enzyme-linked MNPs within hydrogel microparticles. The size of the MNPs was larger than the mesh size of the hydrogel, such that enzymes immobilized on MNPs could not leach from the hydrogel microparticles. MNPs were chosen because they have a high specific binding area per unit mass, and provide the additional advantage of allowing rapid and simple separation of immobilized enzyme from mixtures by applying a magnetic field [21], [22], [23], [24]. Poly(ethylene glycol)- (PEG) based hydrogels were chosen for enzyme entrapment because the physical properties of PEG hydrogels (water content, mesh size, etc.) can be easily controlled by varying the molecular weight (MW) of PEG [25]. Instead of using conventional methods, PEG hydrogel microparticles were prepared by photopatterning to demonstrate the ability to produce non-spherical hydrogel particles.
Section snippets
Materials
Poly(ethylene glycol) diacrylate (PEG–DA) (MW 575), poly(ethylene glycol) (PEG) (MW 3400, later converted to PEG–DA by a published protocol [26]), acryloyl chloride, triethylamine, hexane, tetrahydrofuran (THF), iron oxide (Fe3O4) magnetic nanoparticles (average size 30 nm), o-dianisidine, 3-aminopropyltriethoxysilane (APTES), 2-hydroxy-2-methylpropiophenone, peroxidase (POD, Type I, from horseradish, 80 unit/mg solid), and 1-vinyl-2-pyrrolidinone were purchased from Aldrich (Milwaukee, WI,
Preparation of enzyme-linked MNP
Silane coupling agents are often used to link reactive functional groups to the surface of MNPs [27]. For this study, silanization was used to introduce amine groups that could serve as sites for enzyme immobilization. After APTES modification, peroxidase (POD) was covalently attached to the surface of the MNPs by an imine linkage formed between the aldehyde group and the primary amine on POD. Surface modification of MNPs with APTES and subsequent POD immobilization were confirmed by XPS and
Conclusion
This paper describes the preparation of hydrogel microparticles entrapping enzyme-linked MNPs. The surface of the MNPs was modified with APTES, and it was demonstrated that surface modification significantly improved the covalent attachment of POD when compared to unmodified MNP. Various shapes and sizes of hydrogel microparticles were fabricated using a photopatterning technique and different molecular weights of PEG. Hydrogel-entrapped enzymes maintained their activity over the course of one
Acknowledgements
This work was supported by Korea Science and Engineering Foundation (KOSEF) funded by MEST (R11-2007-050-03002-0 and R01-2007-000-11495).
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