A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS...

Area of Science:

• Biotechnology
• Molecular Biology
• Oncology

Background:

• The Kirsten rat sarcoma virus (KRAS) gene is frequently mutated in human cancers, driving tumor cell proliferation.
• Targeting KRAS mutations is challenging due to limited effective drugs.
• Accurate detection of KRAS mutations is essential for personalized cancer treatment.

Purpose of the Study:

• To develop an innovative microfluidic chip for efficient KRAS gene sample microsegmentation.
• To create a sensitive and specific system for KRAS gene mutation detection.
• To evaluate the chip's performance in digital polymerase chain reaction (dPCR) applications.

Main Methods:

• A microfluidic chip with parallel fluid channels and cylindrical microchambers was designed and fabricated using a "Glass-PDMS-glass" sandwich structure.
• Automated platforms were used to verify the chip's liquid separation performance.
• A four-color fluorescence detection system integrated with the chip and a flat-panel instrument for thermal cycling was employed for KRAS mutation analysis.

Main Results:

• The microfluidic chip demonstrated stable performance with a dynamic detection range of four orders of magnitude.
• The system achieved a gene mutation detection limit of 0.2%.
• The four-color fluorescence system successfully distinguished three different KRAS gene mutation types simultaneously on a single chip.

Conclusions:

• The developed microfluidic chip shows significant potential for digital polymerase chain reaction (dPCR) applications in KRAS mutation detection.
• This technology offers a promising platform for sensitive and multiplexed cancer mutation analysis.
• The system facilitates improved diagnostic capabilities for KRAS-driven cancers.