Highly sensitive mutation quantification by high-dynamic-range capillary-array electrophoresis (HiDy CE).

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Accurate quantification of rare cancer mutations is crucial for effective treatment monitoring.
• Existing methods for detecting EGFR mutations can be time-consuming and costly.
• There is a need for rapid, sensitive, and cost-effective diagnostic tools.

Purpose of the Study:

• To develop a simple, robust, and ultra-small four-color-fluorescence detection system.
• To apply this system to high-dynamic-range capillary-array electrophoresis (HiDy CE) for quantifying rare EGFR mutants.
• To assess the sensitivity, dynamic range, and clinical potential of the developed system.

Main Methods:

• Integration of a four-capillary array, four-lens array, four-dichroic-mirror array, and CMOS sensor into a single device.
• Application of the system to high-dynamic-range capillary-array electrophoresis (HiDy CE).
• Co-amplification of serially diluted EGFR mutant (MT) and constant-concentration wild type (WT) using competitive PCR, followed by HiDy CE analysis.

Main Results:

• Quantification of a rare EGFR exon 19 deletion mutant (MT) in the presence of excess wild type (WT).
• Achieved high sensitivity with a limit of detection (LOD) of 0.004% MT/WT.
• Demonstrated a four-orders-of-magnitude dynamic range (0.01-100% MT/WT).

Conclusions:

• The developed ultra-small fluorescence detection system integrated into HiDy CE provides highly sensitive and accurate quantification of rare EGFR mutations.
• HiDy CE offers significant advantages over existing methods, including higher speed, increased sample throughput, and lower cost per sample.
• The HiDy CE system shows great potential for routine clinical practice in molecular diagnostics.