Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

The detection of single protein molecules1,2 in blood could help identify many new diagnostic protein markers. We report an approach for detecting hundreds to thousands of individual protein molecules simultaneously that enables the detection of very low concentrations of proteins. Proteins are captured on microscopic beads and labeled with an enzyme, such that each bead has either one or zero enzyme-labeled proteins. By isolating these beads in arrays of 50-femtoliter reaction chambers, single proteins can be detected by fluorescence imaging. By singulating molecules in these arrays, ~10–20 enzymes can be detected in 100 μL (~10−19 M). Single molecule enzyme-linked immunosorbent assays (digital ELISA) based on singulation of enzyme labels enabled the detection of clinically-relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA3-5. Digital ELISA detected prostate specific antigen in all tested sera from patients who had undergone radical prostatectomy, down to 14 fg/mL (0.4 fM). The clinical use of protein biomarkers for the differentiation of healthy and disease states, and for monitoring disease progression, requires the measurement of low concentrations of proteins in complex samples. Current immunoassays measure proteins at concentration