Wash- and Amplification-Free Digital Immunoassay Based on Single-Particle Motion Analysis

Digital enzyme-linked immunosorbent assay (ELISA) is a powerful analytical method for highly sensitive protein biomarker detection. The current protocol of digital ELISA requires multiple washing steps and signal amplification using an enzyme, which could be the potential drawback in in vitro diagnosis. In this study, we propose a digital immunoassay method, which we call "Digital HoNon-ELISA" (digital homogeneous non-enzymatic immunosorbent assay) for highly sensitive detection without washing and signal amplification. Target antigen molecules react with antibody-coated magnetic nanoparticles, which are then magnetically pulled into femtoliter-sized reactors. The antigens on the particles are captured by antibodies anchored on the bottom surface of the reactor via molecular tethers. Magnetic force enhances the efficiency of particle encapsulation in the reactors. Subsequent physical compartmentalization of the particles enhances the binding efficiency of antigen-carrying particles to the antibodies. The tethered particles show characteristic Brownian motion within a limited space by the molecular tethering, which is distinct from free diffusion or nonspecific binding of antigen-free particles. The number of tethered particles directly correlates with the concentration of the target antigen. Digital HoNon-ELISA was used with a prostate-specific antigen to achieve a detection of 0.093 pg/mL, which is over 9.0-fold the sensitivity of commercialized highly sensitive ELISA (0.84 pg/mL) and comparable to digital ELISA (0.055 pg/mL). This digital immunoassay strategy has sensitivity similar to digital ELISA with simplicity similar to homogeneous assay. Such similarity allows for potential application in rapid and simple digital diagnostic tests without the need for washing and enzymatic amplification.