Direct single-molecule counting for immunoassay applications

Single-molecule methods offer specificity in studying complex systems and dynamics, but they also offer high sensitivity for basic enumeration. We apply single-molecule TIRF to immunoassays by counting the number of target molecules captured on a streptavidin surface. We demonstrate the utility of using single-molecule counting on eluted detection conjugate, following the capture and sandwich formation portions of the assay having been completed on microparticles. This approach is simple and effective, and creates the opportunity for a universal detection platform that can be used to perform a variety of diagnostic and blood screening assays. We take advantage of the low volume requirements of single-molecule detection and apply a sample reloading approach to concentrate sample onto the detection surface. Due to the high affinity of the streptavidin-biotin reaction, concentration through reloading is both quick and robust. These findings are demonstrated on a model system and in an HIV p24 antigen assay. Single-molecule detection techniques do not need to be complex to exhibit power and flexibility, and so can become valuable in the field of immunoassay diagnostics. •Super-sensitive quantitative methods are important for the new generation of virus and disease marker detection.•Implementation of reliable single-molecule counting/reloading minimizes uncertainties.•Direct single-molecule counting can be used in diagnostic immunoassays.