Immunoassay Detection using Direct Single-Molecule Counting

Abstract

Single-molecule methods have a great deal of specificity for studying complex systems and dynamics, but they also offer high sensitivity for basic enumeration. We apply single-molecule TIRF to immunoassays by simply and literally counting the number of target molecules captured on a streptavidin surface. We demonstrate that utility of using single-molecule counting on eluted detection conjugate, the capture and sandwich formation portions of the assay having completed on microparticles or other systems. This approach is simple and effective, and creates the opportunity for a universal detection platform that can be used to perform a variety of 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 both model and HIV p24 antigen assay systems. Single-molecule detection techniques do not need to be complex to exhibit both power and flexibility, and so can become valuable in the field of immunoassay diagnostics.