Real-Time Assessment of Spatial and Temporal Coupled Catalysis within Polyelectrolyte Microcapsules Containing Coimmobilized Glucose Oxidase and Peroxidase Erich W. Stein, ²,‡ Dmitry V. Volodkin, ² Michael J. McShane,* ,‡ and Gleb B. Sukhorukov ²,§ Max-Planck Institute of Colloids and Interfaces, Golm/Potsdam, 14476, Germany, and Institute of Micromanufacturing, Louisiana Tech University, Ruston, Louisiana 71272 Received May 1, 2005; Revised Manuscript Received January 6, 2006 The encapsulation of biological enzymes within polyelectrolyte microcapsules is an important step toward microscale devices for processing and analytical applications, one which could be applied to the realization of minimally invasive sensing technology. In this work, the encapsulation and functional characterization of a bienzymatic coupled catalytic system within polyelectrolyte microcapsules is described. The two components, glucose oxidase (GOx) and horseradish peroxidase (HRP), were coprecipitated with calcium carbonate microspheres, followed by layer-by-layer assembly to form ultrathin polymer film coatings that act as capsule walls after removal of the sacrificial carbonate cores. Encapsulated concentrations of GOx and HRP were determined to be 19.7 ( 1.0 and 29.4 ( 3.6 mg/mL, respectively. An 85% decrease in the rate of glucose consumption relative to GOx and HRP in free solution was observed, which is attributed to substrate diffusion limitations. To further understand the temporal and spatial dynamics of the two-step reaction, a technique for monitoring microscale glucose consumption was developed using confocal imaging techniques. Time-based acquisition of capsule/Amplex Red suspensions was performed, from which it was observed that the high concentration of enzyme immobilized within the capsule walls resulted in a greater rate and quantity of glucose consumption at the capsule periphery when compared to glucose consumption within the capsule interior. These findings demonstrate the function of a bienzymatic catalytic system within the controlled environment of polyelectrolyte microspheres and a novel approach to analysis of the internal reactions using confocal imaging that will allow direct comparison with reaction-diffusion modeling and further explorations to optimize the distribution and activity of the encapsulated species. Introduction The ability to sequester molecules within a controlled volume or compartment is of considerable interest to the design of biological sensors, in particular, protein-based sensors compris- ing components that may be cytotoxic and/or immunogenic. 1 By compartmentalizing the sensing chemistry within a semi- permeable capsule, allowing transport of the analyte of interest across the compartment barrier, the molecular recognition and specific interaction with the analyte can occur without com- promising the surrounding environment. Microscale carriers offer easy introduction into to the sample of interest, via injection or infusion. 2,3 While traditional methods of localizing sensing components include component immobilization within hydrogel, 4 silica, 5 and polymer 6 matrixes, recent developments using carriers fabricated by nanoassembly techniques offer promise for biological sensing applications. 7-12 In particular, developments involving enzyme immobilization within meso- porous particles, 13 precipitated crystals, 11 enzyme-doped ultrathin films, 14,15 and calcium alginate microspheres in combination with self-assembled multilayers 16,17 provide several immobiliza- tion options from which to select. Layer-by-layer (LbL) self-assembly, a technique based on the adsorption of charged molecules or particles onto an oppositely charged substrate, 18 has been demonstrated for preparation of ultrathin films with precisely controlled properties such as thickness, composition, and permeability. 15,19-22 Additionally, methods to simply and efficiently produce polymeric capsules have been developed utilizing this technique through LbL film deposition of charged polyelectrolytes onto sacrificial templates, which are subsequently dissolved. 23 Recently, microcapsule fabrication has transitioned from polymer templates (e.g., melamine formaldehyde) to inorganic particles, such as man- ganese, cadmium, and calcium carbonate microparticles, 24 which can be dissolved with mild core dissolution conditions that favor complete template dissolution and retention of enzymatic activity. 24,25 Whereas traditional encapsulation methods based on techniques involving alterations of pH 26,27 and solvent polarity 23 along with initial-layer decomplexation 28 have been shown to encapsulate molecules, exposure to harsh conditions can reduce biological viability of the molecules, which could potentially limiting these methods for enzymatic-based sensor fabrication. An additional advantage of calcium carbonate (CaCO 3 ) particles is the characteristic mesoporous structure, such that, during LbL film deposition, polyelectrolyte complexes adsorb not only to the particle surface but also within the interpenetrat- ing pores. Following core dissolution, a polyelectrolyte capsule containing a polyelectrolyte matrix that spans the capsule interior remains. 29 Three methods of macromolecular encapsulation have been previously demonstrated using polyelectrolyte micro- capsules fabricated from CaCO 3 microparticle templates. 29-31 These involve loading of preformed capsules, 29 loading porous CaCO 3 microparticles prior to multilayer film assembly, 31 and * To whom correspondence should be addressed. Phone: 318-257-5100. Fax: 318-257-5104. E-mail: mcshane@latech.edu. ² Max-Planck Institute of Colloids and Interfaces. ‡ Louisiana Tech University. § Current address: Interdisciplinary Research Centre in Biomedical Materials, Queen Mary University, E1 4NS, London, United Kingdom. 710 Biomacromolecules 2006, 7, 710-719 10.1021/bm050304j CCC: $33.50 © 2006 American Chemical Society Published on Web 02/18/2006