DNA methylation detection by a novel fluorimetric nanobiosensor for early cancer diagnosis

Abstract

A very sensitive and convenient fluorescence nanobiosensor for rapid detection of DNA methylation based on Fe3O4/Au core/shell nanoparticles has been developed. Specific site of CpG islands of adenomatous polyposis coli (APC), a well studied tumor suppressor gene, was used as the detection target DNA sequence. The characteristics of nanoparticles were determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), UV–visible spectroscopy and X-ray diffraction (XRD) spectroscopy. Fe@Au nanoparticles functionalized by bounding of single stranded DNA (ssDNA) probe through sulfhydryl group at the 5′ phosphate end. Then unmethylated and methylated complementary target ssDNA were hybridized with the immobilized ssDNA probe. Dipyridamole, a pharmaceutical agent used for the first time as a fluorescence probe which significantly interacted with hybridized unmethylated and methylated DNA. Upon the addition of the target unmethylated and methylated ssDNA, the fluorescence intensity increased in linear range by concentration of unmethylated ssDNA from 1.6×10−15 to 6.6×10−13M with detection limit of 1.2×10−16M and on the other hand, fluorescence intensity declined linearly with concentration of 3.2×10−15–8.0×10−13M methylated DNA and detection limit was 3.1×10−16M. We have also shown that nanobiosensor could distinguish ratio of methylation in series of partially methylated DNA targets with identical sequences. A density functional theory (DFT) calculation was also performed to investigate the interaction between Dipyridamole with unmethylated and methylated cytosine. Finally real sample analysis suggested that nanobiosensor could have practical application for methylation detection in human plasma sample.