Use of hydrogel microstructures as templates for protein immobilization
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1095-EE08-08
Use of hydrogel microstructures as templates for protein immobilization Won-Gun Koh, and Dae Nyun Kim Chemical Engineering, Yonsei University, Seoul, 120-749, Korea, Republic of ABSTRACT In this study, protein micropatterns were created on the surface of three-dimensional hydrogel microstructures. Poly(ethylene glycol)(PEG)-based hydrogel microstructures were fabricated on a glass substrate using a poly(dimethylsiloxane) (PDMS) replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica, respectively. Bovine serum albumin (BSA), a model protein, was covalently immobilized to the surface of the hydrogel microstructure via a 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker, which has a phenyl azide group and a protein-binding N-hydroxysuccinimide group on either end. The immobilization of BSA on the PEG hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase (GOX) was immobilized on the surface and peroxidase (POD) was encapsulated. INTRODUCTION Protein micropatterning refers to the organization of proteins on surfaces with microscale resolution and has become very popular over the past decade due to the importance in biochipbased analytical systems [1-4]. Many research groups have put tremendous effort on developing techniques that are compatible for micropatterning proteins on solid surfaces and various patterning technologies such as direct dispensing or spotting, photolithography, and soft lithography have demonstrated successful protein localization with micro/nanometer scale [1,59]. To date, most of the research has focused on generating two-dimensional micropatterns of proteins on hard, planar materials such as glass, silicon and gold. However, the amount of protein that can be attached to a fixed area on the planar substrate is limited. Furthermore, dehydration and denaturation of proteins could be minimized by the use of a hydrogel, which is a 3-dimensional polymeric structure that swells in water or other biological fluids [10,11]. In contrast to physical adsorption or protein tethering on a hard surface, the soft and hydrated environment of a swollen hydrogel could provide proteins with near-physiological conditions that minimize denaturation and aid in performing full biological functions. Among the various hydrogels, poly(ethylene glycol) (PEG)-based hydrogels have been widely used in biology and medicine due to their high water content, hydrophilicity, and biocompatibility [12-16]. The transparent nature of PEG hydrogels ma
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