Enzyme Immobilization within a Hyaluronic Acid Matrix for Biosensor Applications

Enzymes make up an integral system in the success and efficiency of biological reactions; their specific activities have been transferred from biological to synthetic environments and applied within the chemical, environmental, and biomedical industries to increase processing efficiency. There are, however, limitations to enzyme-based technologies including fragility of the biocatalyst, lack in user-controllability of its reaction, ephemeral shelf-life, and low capacity for large-scale usage. To approach such limitations, a technique involving enzyme immobilization within biocompatible and biodegradable hyaluronic acid (HA)-based hydrogels of varying pore-sizes has been explored. Hydrogels are self-assembled upon the respective crosslinking of hydrophobic dodecylamine, hexylamine, and octadecylamine with hydrophilic HA via 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide reaction chemistry. With synthesis conducted at 1:10, 1:1, and 10:1 molar ratios of amine to HA, user-controllability of hydrogel surface morphology can be warranted, offering increased applicability of immobilization with a range of enzyme sizes, attributes, and modes of implementation. Upon the production of these amphiphilic matrices, biocatalysts are entrapped within the net-like gel samples for enablement of protection from environment-induced denaturation and degradation while maintaining substrate access to the protein active site and product release from the system, respectively. Diversification of pore sizes in synthesized gels provide trial opportunity for maximization of both concentration of immobilized enzyme as well as activity of such enzyme. Immobilization emphasis is placed on glucose oxidase (GOx), one of the model enzymes used in biosensors and the pharmaceutical industry, before this proof-of-concept immobilization platform can be extended to other enzymes and other applications. Following physical adsorption of GOx within the HA hydrogels, it is envisioned that activity, stability, and sensitivity of the entrapped enzyme can be optimized through manipulation of the platform’s pore sizing, as controlled through amine-HA pairing and proportion. Further, attachment of the formulated hydrogels onto functionalized gold electrodes creates opportunity in the development of a biosensor with continuous, real-time tracking capabilities. Such analyses could therefore enhance the body of knowledge and realm of implementation of enzyme immobilization, particularly in the medical sector through applications such as biosensing, drug delivery, and tissue engineering.