Boronate affinity-assisted oriented antibody conjugation on quantum dot nanobeads for improved detection performance in lateral flow immunoassay

Abstract The biological activity of immunoprobes is one of the most factors to determine the analytical performance of lateral flow immunoassay. However, the immunoprobes prepared using the carbodiimide method experience some limitations, such as random and disorder antibody orientation, thus reducing their binding affinity against the target antigen. Herein, we reported a facile approach for one-pot fabrication of boronate-tagged quantum dot nanobeads (QBs), and the boronate affinity reaction was introduced to assist the antibody-oriented attachment on the surface of QBs by the specific reaction of the boric acid group on QB surface and the carbohydrate residues containing cis-diol on the Fc fragment of antibodies. The fabricated immunoprobes were then used for the LFIA of hepatitis B surface antigen at the optimal conditions. In comparison with the carbodiimide method, the proposed conjugation strategy showed better antibody orientation, lower usage of labeled antibody (25 µg antibodies per mg boronate-tagged QBs vs. 350 µg antibodies per mg carboxyl modified QBs), shorter coupling time (5 min vs. 90 min), and higher antibody biological activity (10.8-fold higher than that of carboxyl-modified QBs). Furthermore, the developed boronate-tagged QB-based LFIA exhibited a low detection limit of 0.062 ng mL−1 and a wide dynamic linear range from 0.1 ng mL−1 to 1600 ng mL−1 for the hepatitis B surface antigen quantitative determination. In addition, the proposed oriented antibody strategy also showed the general applicability for detecting other four biomarkers by simply replacing the corresponding antibodies. In brief, this method is promising as a universal fabrication technology for the preparation of high-performance nanoparticle assemblies-based immunoprobes with directional antibody conjugation.