A Single Microfluidic Device Approach to Direct Isolation, Purification, and Amplification of cfDNA from Undiluted...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Bioengineering

Background:

• Circulating cell-free DNA (cfDNA) serves as a crucial biomarker for disease detection and monitoring.
• Current cfDNA analysis requires extensive laboratory processing, limiting accessibility and real-time application.

Purpose of the Study:

• To develop an integrated microfluidic system for point-of-care cfDNA processing.
• To simplify and accelerate the diagnostic screening and monitoring of diseases using cfDNA.

Main Methods:

• A microfluidic device was engineered to combine cfDNA collection, purification, and on-chip amplification.
• Dielectrophoretic (DEP) forces were utilized for cfDNA collection from undiluted plasma.
• On-chip polymerase chain reaction (PCR) was implemented using an integrated thermal cycling system.

Main Results:

• The integrated system successfully consolidated cfDNA collection, purification, and amplification steps.
• On-chip PCR demonstrated a 3-fold improvement in the lower limit of detection and a 5-fold increase in DNA yield compared to off-chip methods.
• The system successfully detected KRAS mutations in a pancreatic cancer patient's plasma, showcasing clinical diagnostic potential.

Conclusions:

• The developed microfluidic system offers a streamlined approach for cfDNA analysis at the point-of-care.
• This technology has the potential to significantly enhance diagnostic screening accessibility and enable real-time patient monitoring.
• Further development could lead to a fully self-contained system for rapid cfDNA mutation detection.