Clinical diagnosis of EML4–ALK mutation in NSCLC by a gold nanoparticle beacon

Abstract

The existence of EML4–ALK, a mutated fusion gene in non-small-cell lung cancer (NSCLC), is an important consideration in the decision of the treatment options for NSCLC patients. However, there has been no standardized methods for EML4–ALK fusion gene detection up to now, which makes the development of novel detection probes particularly appealing. A molecular beacon is effective in detecting the level of a particular mRNA in living cells with easy signal visualization and convenient application. In this study, a molecular beacon composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences was synthesized and characterized. The presence of the EML4–ALK fusion gene will specifically open the hairpin structure, leading to the recovery of FITC fluorescence. This molecular beacon could precisely distinguish NSCLC cell lines with different levels of EML4–ALK mRNA expression, as indicated by obvious fluorescence in the EML4–ALK positive H2228 cell line and a negligible signal in the negative A549 cell line. The sensitivity and specificity of the molecular beacon were further verified in H2228 cells treated with small interfering RNAs (siRNAs) against ALK, which demonstrated decreased fluorescence after gene silencing. These results were simultaneously confirmed by flow cytometry and Q-PCR. More importantly, our molecular beacon successfully identified the EML4–ALK fusion gene in tissue specimens from 2 of 23 NSCLC patients. The results were consistent with Q-PCR quantification. Therefore, this molecular beacon holds great potential in clinical diagnosis of the EML4–ALK fusion gene with remarkable sensitivity and convenience.