Highly Sensitive 3D-Nanoplasmonic-Based Epidermal Growth Factor Receptor Mutation Multiplex Assay Chip for Liquid Biopsy

Economical mutation detection method with high analytical and clinical sensitivity is necessary for early cancer diagnosis and screening. In this study, a novel 3D-nanoplasmonic-based multiplex mutation assay chip is developed to detect epidermal growth factor receptor (EGFR) mutations. This assay kit comprises a 3D-nanoplasmonic substrate immobilized with capture probes and primer–probe sets for recombinase polymerase amplification, wild-type inhibition, and fluorescence detection, enabling multiplex detection of EGFR exon 19 deletions, exon 20 insertions, and exon 21 L858R point mutations. The strategy facilitates the detection of all deletions and insertions within the target region with extremely high analytical sensitivity, detecting as low as 1 × 10⁻⁹% mutation frequency, implying three copies/reactions and 100 zM. The synergistic effects of plasmon-enhanced fluorescence from the 3D-nanoplasmonic substrate and wild-type inhibitor contribute to this high analytical sensitivity. Moreover, the developed chip exhibits 100% accuracy in the clinical testing of plasma samples from normal individuals and patients with benign lung tumor and malignant lung tumor. With high sensitivity and multiplexing capabilities, this assay operates at a low reaction temperature (around 37 °C) and requires a short processing time, ≈70 min post-cell-free DNA extraction. These features make the chip a valuable tool for easy and widespread cancer screening.