Application of Tyrosinase-Mediated Oxidative Coupling to Site-Selective Surface Bioconjugation

Bioconjugation is key to enabling the diverse applications that biomolecules are capable of mediating. While strategies for solution-phase bioconjugation are of critical importance, many applications of biomolecules require surface immobilization. Thus, efficient surface bioconjugation strategies are required in order to enable the full potential of biomolecules, particularly proteins. Many current immobilization methods are not site-specific. Although non-site-specific methods are sometimes effective, such as the new approach for tyrosinase immobilization provided herein, site-specificity is often critical to maintaining protein activity once immobilized. This work focuses on a new method for surface bioconjugation that employs an enzymatic oxidative coupling strategy. The method was optimized on gold nanoparticles, and it was demonstrated that phenol functional groups on gold nanoparticle surfaces can be directly oxidized by the enzyme tyrosinase to enable subsequent site-selective bioconjugation of proteins, including multivalent protein assemblies, and DNA. Cellulose was also explored as a solid support, and small molecule oxidative coupling is described. Appropriate optimization may allow the oxidative coupling approach to be efficient for biomolecule immobilization on cellulose substrates. Progress was also made toward oxidative coupling of proteins on gold chips, with the goal of generating reversible Surface Plasmon Resonance sensor chips to study biomolecule interactions. Overall, the work herein offers a new route for protein immobilization on a range of surfaces, which expands the toolbox of surface bioconjugation and may improve or enable a variety of devices, diagnostics, and other systems that require efficient protein immobilization strategies