Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid...

Area of Science:

• Biomedical Engineering
• Molecular Diagnostics
• Oncology

Background:

• Accurate screening of cancer point mutations is crucial for effective patient management and treatment selection.
• Existing methods like next-generation sequencing and digital PCR face limitations in speed, simplicity, and cost-effectiveness for detecting low-concentration mutations.
• There is a significant need for rapid, sensitive, and affordable diagnostic tools for cancer mutation detection.

Purpose of the Study:

• To develop and validate a novel method combining allele-specific PCR (AS-PCR) with a quartz crystal microbalance (QCM) biosensor for sensitive cancer point mutation detection.
• To assess the sensitivity and accuracy of the AS-PCR/QCM approach for detecting clinically relevant mutations like BRAF V600E and KRAS G12D.
• To evaluate the applicability of this methodology for analyzing clinical tissue and plasma samples in oncology.

Main Methods:

• Integration of allele-specific PCR (AS-PCR) for targeted amplification of cancer point mutations.
• Utilization of a high fundamental frequency quartz crystal microbalance (QCM) array as a biosensor for ultrasensitive detection.
• Detection mechanism based on energy dissipation measurement of acoustically 'lossy' liposomes binding to surface-anchored dsDNA targets.

Main Results:

• The AS-PCR/QCM method successfully detected 1 mutant copy in 10^4 wild-type molecules (0.01% mutant allele frequency, MAF) in spiked samples.
• Validation studies on patient-derived melanoma, colorectal, and lung cancer samples (tissue and plasma) showed excellent agreement with Sanger sequencing and ddPCR.
• The methodology demonstrated high efficiency in detecting mutations below 1% MAF in real clinical samples, confirming its practical utility.

Conclusions:

• The combined AS-PCR and QCM biosensor approach offers high sensitivity and specificity for detecting cancer point mutations.
• This method is cost-effective, compatible with routine workflows, and capable of multiplexed analysis (24-array biochip), making it suitable for clinical implementation.
• The developed technology represents a promising tool for routine molecular screening in clinical oncology laboratories, supporting both tissue and liquid biopsies.