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A novel microfluidic solid-phase extraction (μSPE) device efficiently isolates cell-free DNA (cfDNA) for cancer mutation detection. This low-cost, high-recovery method simplifies liquid biopsy workflows for improved diagnostics.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Microfluidics

Background:

  • Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) are vital liquid biopsy markers for disease detection, particularly in oncology.
  • Accurate cfDNA analysis requires high-quality input DNA, necessitating efficient extraction methods with high recovery and minimal interference.
  • Current methods often face challenges in recovering the full cfDNA size range and avoiding genomic DNA co-extraction.

Purpose of the Study:

  • To develop and validate a novel, low-cost microfluidic solid-phase extraction (μSPE) device for cfDNA isolation.
  • To achieve high cfDNA recovery across a broad size spectrum, including short fragments, with minimal genomic DNA contamination.
  • To demonstrate the clinical utility of the μSPE device in cancer patient samples for mutation detection.

Main Methods:

  • Fabrication of a UV/O3-activated plastic microfluidic chip with surface-confined carboxylic acid functionalities.
  • Utilized a novel immobilization buffer (IB) containing polyethylene glycol and salts to condense cfDNA onto the activated surface.
  • Incorporated micropillars within the μSPE device to enhance extraction capacity and scalability.

Main Results:

  • Achieved >90% recovery of model cfDNA fragments (100-700 bp) and >70% recovery of short cfDNA fragments (50 bp).
  • Demonstrated significant reduction in co-extracted genomic DNA interference through optimized IB composition.
  • Successfully quantified cfDNA levels in healthy donors and cancer patients (non-small-cell lung, colorectal) and detected KRAS mutations.

Conclusions:

  • The novel μSPE device offers a simple, low-cost, and highly efficient method for cfDNA extraction from plasma.
  • The μSPE platform provides high cfDNA recovery, including short fragments, with minimal genomic DNA contamination, crucial for liquid biopsy applications.
  • This technology shows significant potential for routine clinical diagnostics, enabling sensitive detection of actionable mutations in cancer patients.