DNA methylation detection by a novel fluorimetric nanobiosensor for early cancer diagnosis.
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(-13)M with detection limit of 1.2 × 10(-16)M and on the other hand, fluorescence intensity declined linearly with concentration of 3.2 × 10(-15)-8.0 × 10(-13)M methylated DNA and detection limit was 3.1 × 10(-16)M. 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.
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran.
M
M
Dadmehr
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran. Electronic address: smhosseini@khayam.ut.ac.ir.
M
M
Hosseini
Department of Biochemistry, Tarbiat Modares University, Tehran, Iran.
S
S
Hosseinkhani
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran; Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran.
M R
MR
Ganjali
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran.
M
M
Khoobi
Department of Chemistry, Kharazmi University, Karaj, Tehran, Iran.
H
H
Behzadi
Department of Molecular Biology, Tofigh Daru Co., Tehran, Iran.
M
M
Hamedani
Department of Molecular Biology, Tofigh Daru Co., Tehran, Iran.
R
R
Sheikhnejad
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D055088:Early Detection of Cancer / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005470:Fluorometry / Q000295:instrumentation*; D006801:Humans; D036103:Nanotechnology / Q000295:instrumentation*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation
D004273:DNA, Neoplasm
DNA methylation; Dipyridamole; Fe@Au nanoparticle; Fluorescence; Nanobiosensor