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We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs. ∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an …