How antibody surface coverage on nanoparticles determines the activity and kinetics of antigen capturing for biosensing

The antigen-capturing activity of antibody-coated nanoparticles is very important for affinity-based bioanalytical tools. In this paper, a comprehensive study is reported of the antigen-capturing activity of antibodies that are nondirectionally immobilized on a nanoparticle surface. Superparamagnetic nanoparticles (500 nm) were covalently functionalized with different quantities of monoclonal antibodies against cardiac troponin I (cTnI). At a low antibody surface coverage, up to 4% of the immobilized antibodies could capture antigen molecules from solution. At high antibody coverage (=50 × 10(2) antibodies per nanoparticle, i.e., = 64 × 10(2) antibodies per µm(2)), the fraction of antigen-capturing antibodies drops well below 4% and the number of active antibodies saturates at about 120 per nanoparticle. The fraction of active antibodies is small, yet surprisingly their dissociation constants (Kd) are low, between 10 and 200 pM. In addition, the surface-binding activity of the antibody-coated nanoparticles was analyzed in an optomagnetic sandwich immunoassay biosensor, measuring cTnI in undiluted blood plasma. The data show that the immunoassay response scales with the number of active antibodies, increasing initially and saturating at higher antibody densities. The observations are summarized in a molecular sketch of the attachment, ordering, and functionality of antibodies on the nanoparticle surface.