Introduction to Digital Immunoassay

Single molecule counting is an ultra-sensitive immunoassay technology that allow detection of proteins and nucleic acids at lowest possible levels. It’s a simple, flexible, robust, and sensitive multiplex immunoassay platform for measuring biomarkers in most common sample types.

Detection and quantification of molecular biomarkers are critical to disease diagnosis and progression monitoring. Various approaches have been developed, but the most well-established technology is ELISA (enzyme-linked immunosorbent assay), which amplifies antibody-biomarker binding via enzymatic reactions and converts reaction products into an optical signal (e.g., color changes), Although ELISA is widely used in clinical and research labs, its detection limit, total test time (sample to result) and dynamic range are often insufficient for clinical applications. Recent technological advances have made it possible to detect single molecules, which have been used to measure the binding of a single biomarker molecule to a capture antibody with improved detection limit. This single-molecule approach has been referred to as digital immunoassay.

Digital ELISA

Digital ELISA was developed approximately 10 years ago as an ultra-sensitive detection technique (see image). When the enzymatic reaction is achieved in femtoliter-sized micro-wells, the accumulated product is detectable at very low concentrations of the target, thereby achieving single molecule detection. Using a digital ELISA, a femtomolar concentration of recombinant nucleoprotein for influenza A virus was detected. A digital ELISA using bifunctional fluorescence magnetic nano-spheres with gold nanoparticles (AuNPs) was applied to detect avian influenza virus. The LOD was 7.8 fg/mL, which was promising for single bio-molecular detection. Many improvements have been described for digital ELISA.

The left panel shows that antibody-conjugated magnetic beads are first used to capture single molecules of the target protein (i.e., antigen), and the protein-bead complexes are labeled with a second antibody-conjugated enzyme. The right panel shows a case of low antigen concentration and a case of high antigen concentration. In the tube, first, antibody-conjugated magnetic beads, an antigen, and a second antibody-conjugated enzyme are sequentially mixed. Then, the bead complexes are assessed using a precision-fabricated, femtoliter-volume microwell array capable of capturing one bead per well. After adding the substrate as a fluorophore, fluorescence images of a small section of the well array are obtained. The concentration of protein in bulk solution is correlated with the percentage of beads that carry a protein molecule.