Polyion Complex-Templated Synthesis of Cross-Linked Single-Enzyme Nanoparticles

Single-enzyme nanoparticles (SENs), which encapsulate individual enzymes in a thin polymer network, offer exciting possibilities for stabilizing enzymes and tuning their activity, but most approaches to date rely on covalent modification of the enzyme. We introduce here a new approach to their synthesis in which a pre-prepared polymer synthesized by reversible addition-fragmentation chain-transfer polymerization is first tethered to the surface via electrostatic interactions. Isothermal titration calorimetry and asymmetric flow field-flow fraction (AF4) in combination with multiangle light scattering (MALS) reveal weak binding of 2-5 chains/enzymes. The strength of binding can be tuned based on the charge density of the bound polymer. AF4-MALS and small-angle X-ray scattering confirm the formation of a thin cross-linked shell around the enzyme following chain extension of this polymer in the presence of a bis-functional monomer. The mild conditions of this method of SEN formation, which avoids any covalent modification of the enzyme, result in no loss in activity on our model enzyme (glucose oxidase) and 4-fold increase in thermal stability. It offers much greater control over the chemistry of the SEN, which we demonstrate by incorporation of trehalose between the enzyme and cross-linked shell.