A Multiplex Digital Polymerase Chain Reaction-Based Platform with a Unified Readout for Multiclass Biomarker Quantification from Low-Volume Blood.

Abstract

Simultaneous quantification of multiple biomarker classes from blood is essential for improving diagnostic accuracy, yet current methods remain limited by low multiplexing capacity, reliance on separate instruments and workflows, and inconsistent signal readouts that hinder data integration. Here, we present fluorescence-coded, magnetic bead-enhanced digital co-detection of multianalytes (FluoMag-dCoDe), a platform that converts diverse analytes into distinct fluorescence-coded DNA templates for multiplexed quantification in a single digital polymerase chain reaction (dPCR) using a universal primer and TaqMan probe set. We demonstrate comeasurement of eight mRNA and three protein immune-response biomarkers in a two-color dPCR format. The platform achieves high quantitative fidelity with minimal cross-interference, showing strong concordance with single-plex RT-qPCR for mRNAs and superior analytical sensitivity to ELISA for proteins from ∼35 μL fingerstick-compatible blood volumes. By unifying mRNA and protein quantification in a single standardized readout while sustaining robust performance from low blood volumes, FluoMag-dCoDe establishes a scalable approach for high-dimensional biomarker analysis in precision diagnostics.