High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity

Abstract

A major challenge in many clinical diagnostic applications is the measurement of low-abundance proteins and other biomolecules in biological fluids. Digital technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional protein detection methods. However, current digital ELISA technologies still possess insufficient sensitivities for many rare protein biomarkers and require specialized instrumentation or time-consuming workflows that have limited their widespread implementation. To address these challenges, we have developed a more sensitive and streamlined digital ELISA platform, Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), which attains low attomolar limits of detection, with an order of magnitude enhancement in sensitivity over these other methods. MOSAIC uses a rapid, automatable flow cytometric readout that vastly increases throughput and is easily integrated into existing laboratory infrastructure. As MOSAIC provides high sampling efficiencies for rare target molecules, assay bead number can readily be tuned to enhance signal-to-background with high measurement precision. Furthermore, the solution-based signal readout of MOSAIC expands the number of analytes that can simultaneously be measured for higher-order multiplexing with femtomolar sensitivities or below, compared with microwell- or droplet-based digital methods. As a proof of principle, we apply MOSAIC toward improving the detectability of low-abundance cytokines in saliva and ultrasensitive multiplexed measurements of eight protein analytes in plasma and saliva. The attomolar sensitivity, high throughput, and broad multiplexing abilities of MOSAIC provide highly accessible and versatile ultrasensitive capabilities that can potentially accelerate protein biomarker discovery and diagnostic testing for diverse disease applications.

Keywords: biomarkers; diagnostics; digital; immunoassay; single molecule; ultrasensitive protein detection.

Figure 1.

Molecular On-bead Signal Amplification for Individual Counting (MOSAIC). Single target molecules are first captured with an excess number of antibody-coated paramagnetic beads, such that each bead carries either zero or one target molecule according to a Poisson distribution. Upon formation of single immunocomplex sandwiches with a biotinylated detector antibody and labeling with a streptavidin-DNA conjugate, rolling circle amplification (RCA) is carried out to generate a long DNA concatemer attached to each immunocomplex. Fluorescently labeled DNA probes are hybridized to the RCA product, allowing “on” and “off” beads to be counted via flow cytometry.

Figure 2.

Analytical sensitivities of MOSAIC assays across different analytes and assay bead numbers. (A) Calibration curves for IL-10 MOSAIC assays using different assay bead numbers. The signal readout is denoted by average molecules per bead (AMB). (B) Effect of MOSAIC assay bead number on the limit of detection (LOD) and signal-to-background for IL-10 detection. LOD values were calculated as three standard deviations above the background AMB. Relative signal-to-background for each assay bead number was determined as the signal-to-background of a specific calibrator normalized to the signal-to-background of the same calibrator using 100 000 assay beads. (C) Calibration curve for the corresponding IL-10 Simoa assay, using 100 000 assay beads and 400 000 helper beads. (D) Effects of MOSAIC assay bead number on LOD and relative signal-to-background across additional analytes. All error bars represent the standard deviations of three replicates, with six replicates performed for the blank.

Figure 3.

Increasing sampling efficiency improves analytical sensitivity and precision. (A,B) Limits of detection for calibration curves generated from subsets of varying bead numbers randomly sampled from MOSAIC calibration curves for IL-10 (A) and IFN-γ (B). Each color denotes the starting total assay bead number. Each point represents the median of 100 randomly sampled subsets, with error bars representing the interquartile range. Open circles denote values for which the upper quartile had a positive infinity value due to very high measurement coefficients of variation (CVs) at very low bead subset sizes. (C,D) Measurement CVs of the background signal for randomly sampled bead subsets of varying bead numbers for IL-10 (C) and IFN-γ (D).

Figure 4.

Measurements of IFN-γ concentrations in human plasma and saliva using MOSAIC and Simoa. (A−D) Measured IFN-γ concentrations in the 17 plasma (A,B) and 26 saliva (C,D) samples using MOSAIC and Simoa. Concentrations shown represent the measured concentration values in the 4-fold diluted plasma samples. The Pearson correlation coefficients were 0.80 and 0.31 for the plasma and saliva (among detectable values) samples, respectively. The low correlation coefficient in saliva may be attributed to the small fraction of detectable samples using Simoa as well as some samples with IFN-γ levels near the LOD of Simoa or MOSAIC. Red dashed lines denote assay LODs, which were calculated as three standard deviations above the AMB of the background (buffer only). Samples with measurements below the assay LOD were assigned a value equal to the LOD. Samples for which measured values were below the LOD of either or both assays are denoted by an open circle. Error bars represent the standard deviations of two replicates.

Figure 5.

Multiplexing with MOSAIC technology. (A) Schematic of multiplexing with MOSAIC. Beads coated with antibodies to different target analytes are encoded by using different fluorescent dyes with different wavelengths, intensities, and/or using multiple bead sizes. Upon capture of the single analyte molecules on each bead type, formation of single immunocomplex sandwiches, and labeling with streptavidin-DNA, rolling circle amplification is carried out and the mixture of beads is analyzed by flow cytometry. Beads are differentiated by a series of gates in different fluorescence channels, and the average molecules per bead for each bead type is then determined from the intensities in the fluorescence channel corresponding to the probe color. (B,C) Measured concentrations of eight protein analytes in human plasma (top) and saliva (bottom) using an eight-plex MOSAIC assay (B) and two four-plex Simoa assays (C). Concentrations shown are the measured concentration values in the 16-fold and 8-fold diluted plasma and saliva samples, respectively. Measurements below the assay LOD are assigned a value equal to the LOD and denoted by open symbols.