Simultaneous Isolation of Circulating Nucleic Acids and EV-Associated Protein Biomarkers From Unprocessed Plasma...

Area of Science:

• Biomarker Discovery
• Molecular Diagnostics
• Personalized Medicine

Background:

• Personalized medicine relies on understanding cellular and molecular disease mechanisms.
• Accessing multiple biomarker classes (DNA, RNA, proteins) in unprocessed states is crucial for accurate analysis.
• Current methods often require extensive sample processing, potentially altering biomarker integrity.

Purpose of the Study:

• To characterize a novel platform for simultaneous isolation and on-chip detection of cfDNA, EV-RNA, and EV-associated proteins.
• To evaluate the platform's ability to process unprocessed biological fluids using AC Electrokinetics (ACE).
• To demonstrate the compatibility of the isolated biomarkers with downstream analytical techniques.

Main Methods:

• Utilized AC Electrokinetics (ACE) on a microelectrode array (ACE chip) for biomarker capture from unprocessed human biofluids.
• Performed simultaneous on-chip visualization of cfDNA, EV-RNA, and EV-associated proteins using specific dyes and antibodies.
• Eluted captured biomarkers for downstream analysis via PCR, RT-PCR, NGS, and nanoparticle characterization.

Main Results:

• Successfully isolated and detected cfDNA, EV-RNA, and EV-associated proteins directly on the ACE chip.
• Identified tumor-specific variants and housekeeping gene mRNA (PGK1) in isolated cfDNA and RNA.
• Confirmed the presence of extracellular vesicles (EVs) with characteristics of exosomes through protein and size analysis.

Conclusions:

• The ACE platform enables simultaneous isolation and analysis of unprocessed cfDNA, EV-RNA, and EV-associated proteins.
• The platform yields high-quality biomarkers compatible with established downstream technologies like PCR and NGS.
• This technology holds potential as a sample preparation method for advanced biomarker analysis in personalized medicine.