Received: 13 January 2009, Revised: 11 March 2009, Accepted: 5 April 2009, Published online in Wiley InterScience: 26 May 2009 Fabrication of poly(ethylene glycol)-based hydrogels entrapping enzyme-immobilized silica nanoparticles Eunji Jang a , Saemi Park a , Sangphil Park a , Yeol Lee a , Dae-Nyun Kim a , Bumsang Kim b and Won-Gun Koh a * In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3-aminopropyltriethoxysilane (APTES), and the resultant SNP-immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K m of SNP-immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long-term stability test demonstrated that SNP-immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non-entrapped SNP-immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six-fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP-immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright ß 2009 John Wiley & Sons, Ltd. Keywords: enzyme immobilization; silica nanoparticles; poly(ethylene glycol) hydrogel; hydrogel microarray; enzyme-catalyzed reaction INTRODUCTION The immobilization of enzymes on solid supports is an area of intense research due to the widespread application of immobilized enzymes in many analytical devices and as catalysts in industrial processes. [1,2] Various immobilization strategies have been developed using different solid support materials (inorganic materials, synthetic polymers, and natural macromolecules) and immobilization methods (physical adsorption, microencapsula- tion, covalent bonding, or matrix entrapment) to optimize the catalytic features of enzymes. [3–10] In recent years, there has been rapid development and extensive application of inorganic nanomaterials in the field of enzyme immobilization because of their mechanical and chemical stability, high surface- to-volume area, and reduced mass transport limitation of inorganic nanomaterials. [11] Among various inorganic materials, silica nanoparticles (SNPs) have been extensively explored for the immobilization of enzymes because they are easily prepared with monodispersed size, are suitable for many surface immobil- ization methods, are environmentally more acceptable, are structurally more stable, and are chemically resistant to organic solvents and microbial attacks. [12–16] Because of these properties, SNPs have been used effectively for the immobilization of not only enzymes but also various biomolecules for assorted applications ranging from biosensors to interfacial interaction studies. [17] Although covalent immobilization to SNPs surfaces via organic or polymeric linker has been frequently used for enzyme immobilization to utilize benefits of SNPs, this method has several limitations. First, the enzymes are directly exposed to external conditions, and may be easily inactivated by non-specific adsorption of various substances. Second, the immobilized proteins may dehydrate and denature due to rapid evaporation (www.interscience.wiley.com) DOI: 10.1002/pat.1455 Research Article * Correspondence to: W.-G. Koh, Department of Chemical and Biomolecular Engineering, Yonsei University, 134 Sinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea. E-mail: wongun@yonsei.ac.kr a E. Jang, S. Park, S. Park, Y. Lee, D.-N. Kim, W.-G. Koh Department of Chemical and Biomolecular Engineering, Yonsei University, 134 Sinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea b B. Kim Department of Chemical Engineering, Hongik University, 72-1 Sangsu-dong Mapo-gu, Seoul 121-791, Republic of Korea Contract/grant sponsor: Korea Science and Engineering Foundation (KOSEF) funded by MEST; contract/grant number: R11-2007-050-03002-0. Contract/grant sponsor: Active Polymer Center for Pattern Integration; contract/grant number: R01-2007-000-11495-0. Contract/grant sponsor: Seoul Research and Business Development Program; contract/grant number: NT080584. Polym. Adv. Technol. 2010, 21 476–482 Copyright ß 2009 John Wiley & Sons, Ltd. 476