Encapsulation of organophosphorus acid anhydrolase (OPAA) in nanostructured materials for the detection and decontamination of chemical warfare agents
Organophosphorus acid anhydrolase (OPAA) was successfully encapsulated in situ into silica and organically-modified silica materials using the acid-catalyzed hydrolysis and co-condensation of tetramethylorthosilicate and organosiloxanes via the non-surfactant templated sol-gel process. To begin, studies with guanidinium hydrochloride and urea indicated the enzyme was susceptible to denaturation (i.e., unfolding) and renaturation. The urea study followed the simple two-stage folding model with an estimated Gibbs free energy value of 1.5 kcal/mol. A systematic approach was developed to prepare the mesoporous materials to encapsulate enzymes. The enzyme was found to be damaged by methanol that was generated as a by-product of sol-gel reactions. This setback was alleviated by a simple low volume-shrinkage sol-gel approach where methanol was evacuated before addition of enzyme. Various samples were prepared using either D-fructose or poly(ethylene glycol) as the pore forming template and characterized in the presence of various amounts of OPAA. By varying the template concentration or the concentration of the starting materials, the pore parameters can be tuned to have high surface area of 500-800 m2/g, large pore volume ranging from 0.2-0.8 cm3/g, and designable pore diameter ranging from 2-6 nm. As a result, the enzyme remained active in the encapsulated form in both aqueous and mixed aqueous-organic solvents. The immobilization of OPAA in mesoporous materials significantly increased the stability of OPAA against the denaturation by organic solvents. The organically-modified gel sample in 20 % acetone showed a significant retainment of enzyme activity up to approximately 90 % in comparison with aqueous solution. By employing a simple regeneration procedure involving buffer wash, the encapsulated samples retained activity after several reuses with little or no indications of enzyme leakage. Gold-doped mesoporous materials were also prepared for the detection of cyanide using surface enhanced Raman spectroscopy (SERS). The materials were SERS active and out-performed the detection limit of conventional colloidal gold. Though the materials were not optimized, the detection limit of cyanide was 1 ppm. These results clearly indicated that biomolecules, such as OPAA, can be entrapped into mesoporous materials via the nonsurfactant templated pathway. Unlike many other encapsulation methods, this pathway is simple to perform, easily modified (hence the introduction of the low volume shrinkage approach), and produces a material that remains active and reusable.