(599bs) An Integrated Approach for Enhanced Protein Conjugation and Capture with Viral Nanotemplates and Hydrogel Microparticle Platforms Via Rapid Bioorthogonal Reactions

Protein sensing platforms with high performance are highly desired in various applications such as medical diagnostics, bioprocess monitoring and bioterrorism detection. While planar platforms including enzyme-linked immunosorbent assay (ELISA) and protein microarrays have been typically utilized for target protein quantification, these platforms suffer from low loading capacity of probe biomolecules (i.e. antibodies) and nonspecific adsorption of target proteins.  3D structured platforms with non-fouling properties (e.g. polymer brush- and hydrogel-based platforms) have emerged to improve antibody loading capacity and to suppress nonspecific adsorption.  However, there still exist challenges in these 3D platforms; (1) limited enhancement in antibody loading and target protein capture capacity due to diffusion-limited 3D structures, and (2) potential damage to antibodies during fabrication processes.

In this work, we exploited an integrated approach for protein sensing platforms with enhanced antibody loading and target protein capture capacity by utilizing tobacco mosaic virus (TMV) as rigid nanotubular templates assembled with hydrogel microparticles and rapid bioorthogonal tetrazine (Tz)–trans-cyclooctene (TCO) cycloaddition reaction.  Protein conjugation results with a red fluorescent protein (R-Phycoerythrin, R-PE) as a model protein showed significantly enhanced protein conjugation capacity of TMV-assembled particles (TMV-particles) over hydrogel particles without TMV templates.  Further in-depth comparison of protein conjugation kinetics with slower conjugation reaction (strain-promoted alkyne–azide cycloaddition (SPAAC) reaction) showed less diffusion-limited structure of the TMV-particles for protein conjugation.  We also examined target protein capture capacity of the TMV-particles by utilizing an anti-R-PE antibody (R-Ab)–R-PE pair as a model system, whose results showed considerably improved target protein capture capacity over R-Ab conjugated hydrogel particles.  Finally, we demonstrate controllable protein and antibody conjugation capacity by simply varying TMV concentrations without any negative effect of high TMV assembly density on protein conjugation and target protein capture capacity.  Overall, these results illustrate a facile post-fabrication protein conjugation approach with viral nanotemplates assembled onto hydrogel microparticles for enhanced protein conjugation and sensing that can be readily enlisted in a wide range of protein sensing applications.