Polyethylene Glycol-Mediated Directional Conjugation of Biological Molecules for Enhanced Immunoassays at the Point-of-Care

Rapid and reliable point-of-care (POC) diagnostic tests can have a significant impact on global health. One of the most common approaches for developing POC systems is the use of target-specific biomolecules. However, the conjugation of biomolecules can result in decreased activity, which may compromise the analytical performance and accuracy of the developed systems. To overcome this challenge, we present a polymer-based cross-linking protocol for controlled and directed conjugation of biological molecules. Our protocol utilizes a bifunctional thiol-polyethylene glycol (PEG)-hydrazide polymer to enable site-directed conjugation of IgG antibodies to the surface of screen-printed metal electrodes. The metal surface of the electrodes is first modified with thiolated PEG molecules, leaving the hydrazide groups available to react with the aldehyde group in the Fc fragments of the oxidized IgG antibodies. Using anti-Klebsiella pneumoniae carbapenemase-2 (KPC-2) antibody as a model antibody used for antimicrobial resistance (AMR) testing, our results demonstrate a ~10-fold increase in antibody coupling compared with the standard N-hydroxysuccinimide (NHS)-based conjugation chemistry and effective capture (>94%) of the target KPC-2 enzyme antigen on the surface of modified electrodes. This straightforward and easy-to-perform strategy of site-directed antibody conjugation can be engineered for coupling other protein- and non-protein-based biological molecules commonly used in POC testing and development, thus enhancing the potential for improved diagnostic accuracy and performance.