Improving theSensitivity of a Multiplexed DigitalImmunoassay 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. Typically, a bead analysis ratio over 95% was achieved, ensuring high sensitivity, efficiency, and stability of the established HiBeA using as few as only 5,000 beads. As a proof of concept, HiBeA was utilized for the multiplexed detection of IL-10 and IL-6, achieving detection limits of 5.9 and 8.8 fg/mL, respectively. This signifies a 3- to 4-fold enhancement in detection sensitivity under the same reaction time while using only 1% of the assay bead number compared to the commercial single-molecule array (SiMoA) system. HiBeA presents ultrasensitivity, robust detection stability based on tailored, multistep operation of immune-reaction, and the ability to perform multiplexed detection, thereby offering substantial prospects for the advancement of ultrasensitive clinical diagnostics.