Highly-selective detection of EGFR mutation gene in lung cancer based on surface enhanced Raman spectroscopy and asymmetric PCR

Abstract

The evaluation of EGFR mutation genes in circulating tumor DNA (ctDNA) in blood sample is key for patients with lung cancer. Surface-enhanced Raman scattering (SERS) has potential for trace detection of DNA or RNA. The detection rate offered by current methods can not meet clinical demand. By combining asymmetric polymerase chain reaction (PCR) and SERS, a highly-selective detection for EGFR mutation genes in lung cancer was developed. Sea-urchin like Au nanoclusters (AuNCs) were synthesized via Ag seed-mediated growth. AuNCs with a diameter of 120 nm were covered with 79 nanopricks (20 nm). Then, EGFR mutation specific molecular beacons (MBs) labeled with Cy3 were coated on the surface of AuNCs. The loading amount of MBs was calculated as 5720 ± 740 on one AuNCs. These AuNCs probes had good efficiency (equilibrium time: 20 minutes) with high sensitivity (detection limit: 5.8 nM), high specificity (capable of single-base mismatch recognition) and good stability against nucleases. Following this, asymmetric PCR was performed to obtain large numbers of single-stranded DNA (ssDNA, E746-A750del). The ssDNA was incubated with the AuNCs probes and tested quantitatively based on the SERS signals of the AuNCs probes. This combined asymmetric PCR-SERS method had a very high detection threshold (4.24 fM). The asymmetric PCR-SERS method was shown to have an overall sensitivity of 75% and specificity of 100% in a further 15 clinical blood samples. This method is proved to be promising for non-invasive and sensitive detection of EGFR mutations in ctDNA.