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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.