Revolutionizing the capture efficiency of ultrasensitive digital ELISA via an antibody oriented-immobilization strategy.

Bead-based digital ELISA, the most sensitive protein quantification method, has drawn much attention to exploring ultra-low abundance biomarkers in the life sciences and clinical applications. However, its major challenge refers to the low antigen capture efficiency in the immunoreaction process due to the low probability of collision between the deficient concentration of the analytes and the captured antibody-immobilized on the beads. Here, we achieved significantly improved reaction efficiency in the digital signal formation by fixing the orientation of antibodies and revealed the kinetic mechanism for the first time. A facile and fast antibody conjugation strategy that formed boronate ester complexes was designed to retain the uniform orientation of antibodies with controllable antibody density. Remarkably, the oriented immobilized antibody exhibited stronger antigen-binding capacity and faster antigen-binding speed compared to randomly immobilized antibodies, with capture efficiency increasing approximately 14-fold at 15 μg of antibody per 1 mg microbeads (0.035 antibody nm -2 ) under 0.5 h incubation. Combined with theoretical analysis, we verified that the improved capture efficiency of the oriented antibodies mainly originated from the considerable rise in the binding rate constant ( k on ) rather than the increase in antigen-binding sites, which further prominently decreased the limit of detection (LoD) in a shorter incubation time compared with the randomly immobilized antibody. In conclusion, the antibody oriented conjugation method effectively overcomes the low capture efficiency challenge of bead-based digital ELISA. It paves a promising way for further improving the digital immunoassay performance and promotes the early diagnosis of diseases by recognizing more ultra-low abundance significant biomarkers.