Multiparameter single-particle motion analysis for homogeneous digital immunoassay.

Digital homogeneous non-enzymatic immunosorbent assay (digital Ho-Non ELISA) is a new class of digital immunoassay that enables highly sensitive quantification of biomolecules using a simple protocol. In digital Ho-Non ELISA, nanoparticles are tethered onto the surface of femtoliter reactors via captured target molecules. The tethered particles capturing target molecules are identified as those showing a confined Brownian motion with root-mean-square displacement (RMSD) values in a defined range. The present work aims to improve the specificity to discriminate tethered particles via single-target molecules from non-specifically immobilized particles by analyzing two nanoparticle parameters. First, in order to suppress the broadening of RMSD due to the heterogeneity of bead size, we corrected the RMSD with the fluorescence intensity of the beads. Second, focusing on the shape of Brownian motion in the x-y trajectory, we classified motion patterns by aspect ratio of the trajectory. By using multiparameter single-particle motion analysis with corrected RMSD and aspect ratio, a 3.9-fold enhanced sensitivity in PSA assay was achieved compared to the conventional RMSD analysis approach. This new strategy would increase the potential of digital immunoassays.

Multiplexed homogeneous digital immunoassay based on single-particle motion analysis.

Homogeneous digital immunoassay is a powerful analytical method for highly sensitive protein biomarker detection with a simple protocol. However, it has not been multiplexed yet. In this study, we developed a multiplexed homogeneous digital immunoassay based on single-particle motion analysis (digital homogeneous non-enzyme-linked immunosorbent assay, digital Ho-Non ELISA). In this assay, multiple target antigen molecules react with the optical subpopulation of magnetic nanobeads labeled with fluorescent dyes and capture antigen-specific antibodies. Then, these beads are magnetically pulled into femtoliter-sized reactors. The surface of these reactors is modified with multiple detection antibodies specific to each antigen by molecular tethers. Each antigen on the particles reacts with the detection antibodies anchored to the surface of the reactors. Magnetic force enhances the efficiency of bead encapsulation in the reactors, and subsequent physical compartmentalization of beads enhances the binding efficiency of the antigen-antibody reaction. The tethered beads show characteristic Brownian motion distinct from free diffusion or non-specific binding of the antigen-free beads. The color of the beads is attributed to target-identification, and the number of tethered beads is attributed to the concentration of the specific target. We measured two biomarkers (PSA and IL6) as model targets by multiplexed digital Ho-Non ELISA. Our method showed higher sensitivity compared to previous digital Ho-Non ELISA and could detect multiple targets simultaneously with the same performance as in single-plex detection. This new strategy has the potential to open a new avenue for next-generation multiplexed immunoassays in in vitro diagnostics.

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.