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We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

Simultaneous Detection of Single Molecules and Singulated Ensembles of Molecules Enables Immunoassays with Broad Dynamic Range