Characterization of a Single-Molecule Sensitive Digital Flow Cytometer for Amplification-Free Digital Assays

Digital assays such as digital PCR for nucleic acids and digital ELISA for proteins provide absolute quantitation and greater accuracy, sensitivity, and reproducibility than their analogue counterparts (real-time PCR and standard ELISA), but current digital assays involve amplification (e.g., DNA amplification in digital PCR and signal amplification in digital ELISA), which makes high multiplexing difficult, often requires complex and expensive sample compartmentalization, and adds reaction steps. We have developed a single-molecule sensitive flow cytometer, which we termed a digital flow cytometer (dFC). dFC optimizes the sensitivity and efficiency of single-molecule detection by using smaller, planar microfluidic channels, a smaller probe volume, and a shorter working distance/higher numerical aperture objective than used in current commercial high-sensitivity flow cytometers, allowing digital assays via direct single-molecule counting. This paper describes our characterization of the analytical performance of this system when detecting antibody-dye conjugates and demonstrates absolute concentration measurements of commercial antibody-dye conjugates. The dFC exhibited a single-molecule detection efficiency with which over 98% for antibodies conjugated with 18 different small-molecule, phycobiliprotein, and semiconducting polymer dyes were separated from noise, a low false-positive rate, a stable baseline signal, and accurate concentration measurements with a dynamic range spanning 4 orders of magnitude. This system can be used for authenticating antibody-dye conjugates used in flow cytometry and tissue imaging studies and in the development of multiplexed, amplification-free digital assays for nucleic acids and proteins.