Epifluorescent single-molecule counting with Streptavidin-Phycoerythrin conjugates.

Abstract

Single-molecule methods, specifically single-molecule counting, convey high sensitivity in research applications. However, single-molecule counting experiments require specialized equipment or consumables to perform. Here, we utilize commercially available Streptavidin-Phycoerythrin (SA-PE) conjugates for single-molecule detection and counting on a standard epifluorescence microscope. We show that SA-PE conjugates can be used for single-molecule counting on glass surfaces, similarly to TIRF microscopy experiments. Additionally, we show that direct fluorescent imaging of SA-PE on magnetic microparticles has applications for single-molecule diagnostics, with analyte detection sensitivities in the low femtomolar range. We use this approach for detecting Hepatitis B surface antigen (HBsAg) and achieve improved sensitivity in comparison to ensemble measurements. Lastly, we also demonstrate the capability for single-molecule counting on the surface of fixed human cells. We anticipate that utilization of commercial bright conjugates will have broad appeal for researchers interested in single-molecule counting assays.