A toehold-mediated strand displacement cascade-based DNA assay method via flow cytometry and magnetic separation

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• EGFR T790M mutations are critical biomarkers in non-small-cell lung cancer (NSCLC).
• Accurate detection of T790M is essential for guiding clinical treatment and monitoring therapeutic efficacy.
• Current detection methods face challenges in sensitivity and applicability to complex biological matrices.

Purpose of the Study:

• To develop a novel, sensitive flow cytometry-based assay for detecting EGFR T790M circulating tumor DNA.
• To evaluate the assay's performance in distinguishing single-nucleotide polymorphisms and its tolerance to blood serum matrix.

Main Methods:

• A flow cytometry sensing strategy utilizing a toehold-initiated hybridization chain reaction (HCR).
• Biotin-labeled initiator sequences and helper hairpin sequences facilitate target recycling and signal amplification.
• Products of HCR are captured by magnetic beads and analyzed via flow cytometry.

Main Results:

• The proposed method demonstrates sensitive detection of the EGFR T790M mutation.
• The assay successfully distinguishes single-nucleotide polymorphisms, indicating high specificity.
• The method shows tolerance to complex matrices, such as blood serum, highlighting its potential for clinical application.

Conclusions:

• The novel flow cytometry-based assay offers a sensitive and specific approach for T790M detection.
• This method holds significant promise for application in liquid biopsies for NSCLC patient management.
• The assay's robustness in complex matrices supports its potential for routine clinical use.