A Unified Multiplex Digital PCR-based Platform for Multi-class Biomarker Quantification from Low-volume Blood

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biomarker Discovery

Background:

• Current multi-analyte blood tests face limitations in multiplexing capacity, workflow complexity, and data integration.
• Accurate quantification of diverse biomarkers is crucial for advancing diagnostic capabilities.

Purpose of the Study:

• To introduce FluoMag-dCoDe, a unified platform for simultaneous quantification of multiple biomarker classes from blood.
• To overcome the limitations of existing methods for high-dimensional biomarker analysis.

Main Methods:

• Developed FluoMag-dCoDe, a platform converting analytes into fluorescence-coded DNA for digital PCR (dPCR).
• Utilized a universal primer and TaqMan probe set for multiplexed quantification in a single dPCR reaction.
• Demonstrated co-measurement of eight mRNA and three protein immune-response biomarkers.

Main Results:

• Achieved high quantitative fidelity with minimal cross-interference between analytes.
• Showed strong concordance with RT-qPCR for mRNA quantification.
• Exhibited superior analytical sensitivity to ELISA for protein detection, including TRAIL, from small blood volumes (∼35 µL).

Conclusions:

• FluoMag-dCoDe unifies mRNA and protein quantification into a single, standardized readout.
• The platform offers robust performance from low blood volumes, enabling high-dimensional biomarker analysis for precision diagnostics.
• Established a scalable approach for advanced biomarker analysis in clinical settings.