Improving the Sensitivity of a Multiplexed Digital Immunoassay Based on Extremely High Bead Analysis Efficiency.

The development of a detection methodology with high sensitivity, stability, and user-friendliness for quantification of proteins at subfemtogram levels is essential for clinical applications such as early screening, disease diagnosis, and monitoring disease progression. A traditional micropartition-based digital enzyme-linked immunosorbent assay (dELISA) results in significant bead loss due to intricate partitioning based on Poisson distribution, multistep reaction operations, nonglobal signal recognition, and reading modes, which have not yet achieved the ultimate detection sensitivity. This study introduces an ultrasensitive multiplexed digital immunoassay with extremely high bead analysis efficiency (HiBeA) through integrating the bead transfer strategy in multistep immunoreaction processing and flow cytometry detection mode.