High-Fidelity Single Molecule Quantification in a Flow Cytometer Using Multiparametric Optical Analysis

Microfluidic techniques are widely used for high-throughput quantification and discrete analysis of micron-scale objects like cells, but are difficult to apply to molecular-scale targets. Instead, single-molecule methods primarily rely on low-throughput imaging of immobilized molecules using high-resolution microscopy. Here we report that commercial-grade flow cytometers can detect single nucleic acid targets following enzymatic extension and dense labeling with multiple distinct fluorophores. We focus on microRNAs, short nucleic acids that can be extended by rolling circle amplification and labeled with multicolor fluorophores to generate repetitive nucleic acid products with sub-micron sizes and tunable multispectral profiles. By cross-correlating the multiparametric optical features, signal-to-background ratios were amplified 1600-fold to allow single-molecule detection across 4 orders of magnitude of concentration. The limit of detection was measured to be 47 femtomolar, which is 100-fold better than ...