Abstract
Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.