Ferrocenylnaphthalene Diimide-Based Electrochemical Detection of Aberrant Methylation in hTERT Gene

Abstract

Since aberrant methylation at CpG sites is linked to the silencing of tumor suppressor genes, DNA methylation analysis is important for cancer diagnosis. We developed ferrocenylnaphthalene diimide (FND), which has two ferrocenyl moieties at the substituent termini, as an electrochemical indicator for hybridized DNA duplexes. In this study, we attempted to detect aberrant methylation of human telomerase reverse transcriptase gene (hTERT), an efficient cancer marker, using FND-based hybridization coupled with electrochemical detection via a multi-electrode chip.

Oral Cancer Screening Based on Methylation Frequency Detection in hTERT Gene Using Electrochemical Hybridization Assay via a Multi‐electrode Chip Coupled with Ferrocenylnaphthalene Diimide

“…Clinical specimens were tissue, local exfoliated oral cells (EOC‐E) and exfoliated oral cells from entire oral cavity (EOC‐L). Methylation specific PCR (MSP) products from clinical samples were treated by using DNeasy Blood and Tissue Kit, BisulFlash DNA Modification Kit and PCR with ZymoTaq Premix according to a procedure described previously .…”

“…Probe DNAs (Table ) were immobilized to a multi‐electrode chip according to the pattern of probe DNA by the previously reported method . Reaction mixture, 0.25 ng/μL MSP products, 5×SSC and 50% Formamide (Deionized)) was incubated for 15 minutes at 95°C, after that, it was stored at 4 °C until use.…”

“…This simple technique is based on fact that FND (Figure B) is concentrated double‐stranded DNA specifically by threading intercalation . We applied the electrochemical hybridization assay for PCR products from synthetic oligonucleotide, genome DNA from one oral squamous cell carcinoma (OSCC) patient, and one healthy volunteer as template using multi‐electrode chip to detect methylation frequency of CpG sites of hTERT promoter regions . The obtained results showed that methylation frequency of sample DNAs was realized by the electrochemical hybridization assay with DNA probes carrying different amounts of methylation on the chip and also showed the good correlation between cancer development and methylation frequency .…”

Electrochemical Aberrant Methylation Detection Based on Ferrocenyl Naphthalene Diimide Carrying β‐Cyclodextrin, FNC

“…Electrochemical techniques have an advantage in DNA detection, which was pioneered by Palecek [8] and its application in the detection of methylated cytosine has been reported; for example, methods based on the different redox potential of methylated cytosine and other nucleic acid bases [8][9][10], the enzymatic reaction of HRPmodified antibody for methylated cytosine coupled with H 2 O 2 /hydroxyquinone [11], or the electrochemical hybridization assay of MSP product using electroactive DNA ligands [12][13][14][15][16][17].…”

“…We have been developing an electrochemical methylation detection device using ferrocenyl naphthalene diimide (FND) derivatives as hybridization indicators for p53 [18], p16 [13], CDH4 [14], and hTERT genes [15,16] in combination with the MSP product after bisulfite treatment of the methylation-specific DNA probe immobilized on the gold electrode.…”