Simultaneous EGFR mutation detection using a sensitive dual-target biosensor based on 3D hollow carbon nanosphere synergy with circular bipedal DNA walkers.

A sensitive dual-target electrochemical biosensor was developed integrating circular bipedal DNA walkers with a three-dimensional hollow carbon nanospheres-tetrametallic nanoparticle (3D HCNs-TT NPs) modified platform, enabling concurrent detection of two critical epidermal growth factor receptor (EGFR) exon 19 mutations: E746_A750 deletion (Mut DNA A) and L747_S752delinsS (Mut DNA B). The circular bipedal DNA walkers address the limitations of low amplification efficiency in traditional single-legged DNA walkers and mitigate the sensitivity reduction caused by excessively long walking strands. Additionally, we used the 3D HCNs-TT NPs nanocomposite materials to modify the electrodes, which significantly increased the electron transfer rate and provided more active sites for molecular probe loading, thereby further enhancing the performance of the detection platform. This biosensor demonstrated good performance, with detection limits as low as 3.4 fM (Mut DNA A) and 2 fM (Mut DNA B) (S/N = 3). The designed sensor was applied for detection using clinical samples, including cells with gene mutations and diluted human serum, and these results confirm the sensor's potential for clinical translation, offering a potential tool for early non-small cell lung cancer (NSCLC) diagnosis and personalized tyrosine kinase inhibitors (TKI) therapy guidance.