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 but are difficult to apply to molecular-scale targets. Instead, single-molecule methods primarily rely on low-throughput microscopic imaging of immobilized molecules. 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 (RCA). We labeled RCA-extended microRNAs with multicolor fluorophores to generate repetitive nucleic acid products with submicron 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 fM, which is 100-fold better than gold-standard methods based on polymerase chain reaction. Furthermore, multiparametric analysis allowed discrimination of different microRNA sequences in the same solution using distinguishable optical barcodes. Barcodes can apply both ratiometric and colorimetric signatures, which could facilitate high-dimensional multiplexing. Because of the wide availability of flow cytometers, we anticipate that this technology can provide immediate access to high-throughput multiparametric single-molecule measurements and can further be adapted to the diverse range of molecular amplification methods that are continually emerging.