Single-molecule counting of biomolecules with femtoliter dropret chamber array

We attempted to develop a novel digital counting method to measure the number of target biomolecules at a single molecule level by use of an array of one million femtoliter water-in-oil (W/O) droplet. Target molecules were first captured on antibody-modified microbeads after labeled with reporter enzyme. After beads were entrapped individually in micron-sized w/o droplets of which volume are femtoliter scale, fluorogenic enzyme assay was carried out to count the number of captured target molecules. As a demonstration, enzyme-linked immunosorbent (ELISA) assay of prostate specific antigen was conducted. The limit of detection of prostate specific antigen reached 2 aM, ten million times enhanced compared with conventional ELISA.