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

Abstract

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.

Keywords: RCA; amplification; biomarker; diagnostics; miRNA; microRNA; multiplexing.

Figure 1.

Schematic diagram depicting fluorescent labeling and fluorescence detection of single nucleic acid molecules using a flow cytometer. In this example, ~22-mer microRNA is extended by rolling circle amplification using a circular DNA template and DNA polymerase to generate large molecules with repeating sequence motifs. The products are then labeled with a mixture of multicolor intercalating dyes and dye-DNA molecules that bind through hybridization. The products are then analyzed as individual events by multispectral fluorescence and scattering in a flow cytometer. The schematics illustrate molecular mechanisms but are not drawn to scale. The specific dye-DNA probes applied determine the density of labeling and the fraction of amplicon that is double-stranded.

Figure 2.

Characterization of extended and fluorescently labeled microRNA at the single molecule level. (a) Fluorescence micrographs of Cy3-conjugated dye-DNA probes (left) and miR-375 amplicons labeled with the same dye-DNA probes (right). Scale bars represent 5 μm. Intensity histograms are shown below for probes alone (blue) and labeled microRNA products (red). (b) Temporal fluorescence intensity in a diffraction limited confocal spot for dye-DNA probes in solution (top) and labeled microRNA products (bottom).

Figure 3.

Detection of fluorescently labeled and extended microRNA in a flow cytometer. (a,b) Scatter plots show events detected for microRNA amplicons labeled with both dye-DNA probes and SYBR Green. (a) Correlation between side scattering and SYBR Green fluorescence intensity. Black lines indicate gates. (b) Same measurement as (a) showing Cy5 and Cy3 intensity channels for each event. (c) Correlation between molecular counts and microRNA concentration with all other conditions held constant. Red line indicates the linear dynamic range. (d) microRNA quantification through reverse transcription polymerase chain reaction. Plot shows the measured cycle threshold for each microRNA concentration using TaqMan qRT-PCR reagents and a real-time PCR instrument. All data points and error bars represent the mean and standard deviation, respectively, of three technical replicates.

Figure 4.

Multispectral labeling of extended microRNA through hybridization with multicolor dye-DNA. (a) Fluorophore absorption (A, red) and photoluminescence (P.L., blue) spectra for tested dyes are shown with normalized intensities. Gray highlight indicates emission bandpass filters. (b) Representative data for event counts in biparametric color channels using the dye combinations indicated at top. Axes show logarithmically scaled fluorescence intensity for indicated fluorophores. (c) Ratiometric labeling of miR amplicons with Cy3/Cy5 dye pairs shows discrete bands with narrow intensity profiles. Data were collected for 10 pM microRNA concentrations and >79 000 counts.

Figure 5.

Multiplexed detection and quantification of microRNA in a flow cytometer. (a,b) Schematic diagrams depict fluorescent labeling and detection of (a) miR-375 using Cy3-DNA and Cy5-DNA probes or (b) miR-30a-5p using A430-DNA and A594-DNA probes with each amplicon labeled with SYBR Green. All templates and hybridization probes are mixed together to perform the multiplexed assay. The schematics illustrate molecular mechanisms but are not drawn to scale. (c,e) Scatter plots show events detected in the miR-375-specific Cy3/Cy5 biparametric color channel or the (d,f) Alexa-430/Alexa-594 color channel for amplicons generated through RCA reactions containing (c,d) 10 pM miR-375 alone or (e,f) 10 pM miR-30a-5p alone. Events were filtered by SYBR Green and side-scatter prior to gating in dye-DNA channels. (g) Correlation between Cy3/Cy5 counts and miR-375 concentration in the presence and absence of 10 pM miR-30a-5p. Trend lines represent the linear dynamic range. (h) Correlation between Alexa-430/Alexa-594 counts and miR-30a-5p concentration in the presence and absence of 10 pM miR-375. Trend lines represent the linear dynamic range.

Figure 6.

Optical differentiation and barcoding of microRNAs. (a) Schematic depiction of multicolor labeling based on sequence-specific dye-DNA hybridization. (b) Heat map shows false positive count rates for the biparametric color channels from Figure 4b. Fluorophore pairs are plotted on the y-axis, and optical channels are plotted on the x-axis. Channels and fluorophores corresponding to numerical labels are provided in SI Table S2. (c) Heat map of empirical false positive count rates between ratiometric channels from Figure 4c. Fluorophore pairs are plotted on the y-axis, and optical channels are plotted on the x-axis. Channels and fluorophores corresponding to numerical labels are provided in SI Table S3. (d) Calculated optical barcode number for indicated color code number and ratiometric code number. Colors indicate three (green), five (red), or seven (blue), color combinations. Solid curves show the total code numbers, whereas dashed and dotted lines indicate code numbers with maximum false positive count rates of 10⁻³ and 10⁻⁶, respectively.