(61b) An Ultrasensitive Flow-Based Digital ELISA for Detection of Attomolar Protein Concentrations

Measurements of very low levels of biomolecules, including proteins and nucleic acids, are essential for many clinical diagnostic applications, but remain a critical challenge due to insufficient analytical sensitivity. While digital measurement methods such as digital enzyme-linked immunosorbent assay (ELISA) have dramatically improved measurement sensitivities by up to 1000-fold over traditional bulk analytical methods, there remain many potential biomarkers of interest that exist in accessible biofluids at concentrations below the detection limits of current digital ELISA methods. To address this challenge, we have developed a more sensitive, streamlined digital ELISA platform, Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), that achieves low- to mid-attomolar sensitivities for protein detection. Based on nucleic acid signal amplification and a high-throughput flow cytometric signal readout, MOSAIC enhances analytical sensitivity by over an order of magnitude compared to the current state-of-the-art digital ELISA method. Importantly, the simplified digital readout process provides a cost-effective, readily accessible platform that can be executed with common laboratory instrumentation. Furthermore, due to its improved sampling efficiencies and versatile detection platform, the MOSAIC technology can enable high-order multiplexing, which will have significant utility in applications involving precious low-volume samples or biomarker screening.