The article describes a novel approach called Single Molecule Enzyme-Linked Immunosorbent Assay (SiMoA) that enables the detection of single protein molecules at sub-femtomolar concentrations in serum. The method involves capturing proteins on microscopic beads, labeling them with enzymes, and isolating these beads in arrays of 50-femtoliter reaction chambers. By imaging these arrays, single protein molecules can be detected due to the high local concentration of fluorescent product molecules. The sensitivity of SiMoA is significantly higher than conventional ELISA, with a detection limit of ~10–20 enzymes in 100 μL, corresponding to an LOD of ~200 aM (6 fg/mL) for prostate-specific antigen (PSA) in whole serum. The authors demonstrate the clinical utility of this method by successfully detecting PSA in serum samples from patients who had undergone radical prostatectomy, with concentrations ranging from 14 fg/mL to 9.4 pg/mL. This approach has the potential to facilitate early diagnosis and treatment of diseases by detecting very low concentrations of proteins in blood.The article describes a novel approach called Single Molecule Enzyme-Linked Immunosorbent Assay (SiMoA) that enables the detection of single protein molecules at sub-femtomolar concentrations in serum. The method involves capturing proteins on microscopic beads, labeling them with enzymes, and isolating these beads in arrays of 50-femtoliter reaction chambers. By imaging these arrays, single protein molecules can be detected due to the high local concentration of fluorescent product molecules. The sensitivity of SiMoA is significantly higher than conventional ELISA, with a detection limit of ~10–20 enzymes in 100 μL, corresponding to an LOD of ~200 aM (6 fg/mL) for prostate-specific antigen (PSA) in whole serum. The authors demonstrate the clinical utility of this method by successfully detecting PSA in serum samples from patients who had undergone radical prostatectomy, with concentrations ranging from 14 fg/mL to 9.4 pg/mL. This approach has the potential to facilitate early diagnosis and treatment of diseases by detecting very low concentrations of proteins in blood.