Sequential enzymatic reactions and stability of biomolecules immobilized onto phospholipid polymer nanoparticles

Polymer nanoparticles for sequential enzymatic reactions were prepared by combining a phospholipid polymer shell with a polystyrene core. The active ester groups for the bioconjugation and phospholipid polar groups were incorporated into the phospholipid polymer backbone using a novel active ester monomer and 2-methacryloyloxyethyl phosphorylcholine. For the sequential enzymatic reactions, acetylcholinesterase, choline oxidase, and horseradish peroxidase-labeled IgG were immobilized onto the nanoparticles. As substrates, acetylcholine chloride, choline chloride, and tetramethylbenzidine were added to the nanoparticle suspension, the acetylcholine chloride was converted to choline chloride, the choline chloride was oxidized by choline oxidase, and hydrogen peroxide was then formed as an enzymatic degradation product. The hydrogen peroxide was used for the next enzymatic reaction (oxidized by peroxidase) with tetramethylbenzidine. The sequential enzymatic reactions on the nanoparticles via degradation products (hydrogen peroxide) were significantly higher than that of the enzyme mixture. This result indicated that the diffusion pathway of the enzymatic products and the localization of the immobilized enzyme were important for these reactions. These nanoparticles were capable of facilitating sequential enzymatic reactions.