Highly Sensitive, Multiplexed, and Accessible Digital Protein Measurement with MagDroplex

Introduction: Highly sensitive and multiplexed protein detection is crucial across various biological contexts. The most advanced, digital enzyme-linked immunosorbent assay (dELISA), has enabled 1000-fold increase in sensitivity over traditional immunoassays but involves complex devices or microfluidic control for single bead trapping, has limited multiplexing capability, and lacks broad accessibility. To address these challenges, we present MagDroplex, an innovative approach that simplifies protein detection at single-molecule level while significantly enhancing multiplexing capability and accessibility. MagDroplex synergistically integrates magnetic proximity extension and digital polymerase chain reaction (dPCR) to transform the detection of proteins into the quantification of single DNA molecules. By detecting DNA rather than enzyme labels, our approach eliminates precise bead control as required in dELISA, facilitates concurrent detection of multiple targets through various PCR multiplexing methodologies, and leverages the widespread adoption of dPCR technology for broader implementation.

Materials and

Methods: In MagDroplex, we first perform multiplexed proximity extension, using multiple magnetic beads and antibody-oligo conjugates (PEA probes) with unique fluorescence codes to concurrently capture and recognize various protein targets. Fine-tuned parameters such as oligo-to-antibody ratios and bead quantity, coupled with stringent assay conditions facilitated by triple antibody bindings and bead-enabled washings, ensure optimal protein-to-DNA conversion with minimal background noises. For precise DNA quantification and preventing interferences of the beads during PCR, we employ heat denaturation to release extended DNA from beads by disrupting the antigen-antibody binding. Next, we employ widely-used Bio-Rad QX200 droplet dPCR (ddPCR) platform for DNA quantification and multiplexed fluorescence-coding signal generation, where each droplet acts as an isolated PCR reaction chamber, capable of generating a unique fluorescence signature corresponding to the specific DNA present, leading to the formation of multiple clusters for accurate identification and quantification of the corresponding protein targets (Figure 1).

Results, Conclusions, and Discussions: As a demonstration, we applied MagDroplex towards an eight-plex detection targeting IL-8, GM-CSF, IL-1β, IL-2, IL-6, TNF-α, IFN-γ, and IL-4. The oligos on PEA probes for each target were assigned with distinct fluorescence codes consisting of varying number of FAM and HEX TaqMan probe binding sites, which, upon protein-to-DNA conversion, DNA release, and ddPCR, generated unique fluorescence signatures resulting from different intensities at FAM and HEX channels. Our results revealed distinct droplet populations, each characterized by unique fluorescence signatures that precisely matches the pre-designed codes for each target. Using a custom Python-based data analysis pipeline utilizing Gaussian Mixture Model, we effectively identified and quantified eight distinct droplet clusters (Figure 2). Next, we assessed the detection sensitivities of each target in MagDroplex by testing serially diluted protein mixtures spiked in fetal bovine serum at equal concentrations. We achieved sub-femtomolar to attomolar detection limits for each target, demonstrating an overall performance on par with each singleplex dELISA (Figure 3). Moreover, the multiplexing capability of MagDroplex can be easily expanded by incorporating fluorescence codes across more channels and using more droplets, and our approach is readily available for any microfluidic dPCR devices.

Overall, with its high sensitivity, expandable multiplexing capability, and broad accessibility, MagDroplex is poised to significantly advance biomarker research, diagnostics, and precision medicine in the future.

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