Polarization-based immunoassay in aqueous solution using Au nanoparticle-labeled antibody

Here we describe an ultrasensitive antigen-antibody immunoassay using gold nanoparticles (AuNPs). Polarization microscopy is used to discriminate individual AuNP dimers from isolated single AuNPs by means of their Brownian motion in aqueous solution. The optical anisotropy and rotational diffusion time were measured to provide accurate and robust discrimination. Since the size of a naked antibody is comparable to that of an AuNP, the distance between two AuNPs (inter-dimer distance) is rather large, and therefore the optical anisotropy is seriously degraded. To address this problem, we digested the antibody with the protease pepsin to reduce the distance. Autocorrelation analysis allowed discrimination of the difference in optical anisotropy and rotational diffusion time. Setting an appropriate threshold for the measurement enabled sufficient accuracy in the discrimination.