Evaluation of Dibenzocyclooctyne and Bicyclononyne Click Reaction on Azido-Functionalized Antifouling Polymer Brushes via Microspotting

Strain-promoted alkyne-azide cycloaddition (SPAAC) has become an indispensable tool in bioorthogonal conjugation and surface immobilization. While numerous studies have focused on enhancing the reactivity of cyclooctynes, a facile method to evaluate the binding efficiency for cyclooctyne-azide-based immobilization without any sophisticated facilities is still missing. In the present work, different derivatives of dibenzocyclooctyne/bicyclononyne (DBCO/BCN) linked to either a fluorophore or a biotin-moiety are patterned on ultra-low fouling polymer brushes, which can avoid unspecific protein contamination without any prior blocking steps. The polymer brushes are composed of an antifouling bottom block and azide-terminated top block. The assessment of binding efficiency is conducted on ordered arrays spotted by microchannel cantilever spotting (mu CS) with a normal fluorescent microscope. Both cyclooctynes demonstrate reliable binding performance with azide-bearing diblock polymer brushes via mu CS, but DBCO shows a higher surface density of molecular immobilization according to the protein binding assays. This work provides a reference for choosing appropriate cyclooctyne to couple with azides and can be useful for the design of biosensors or bio-platforms for analyte detection, cell capture, and other biological applications.

Automated summary

This study evaluates the binding efficiency between cyclooctyne and azide-based immobilization by patterning different derivatives of azide-functionalized dibenzocyclooctyne/bicyclononyne (DBCO/BCN) on ultra-low fouling polymer brushes. The results provide a reference for choosing appropriate cyclooctynes to couple with azides.