Probing for DNA methylation with a voltammetric DNA detector

Abstract

A label-free electrochemical detection of DNA hybridization is used for probing synthetic methylated ssDNA 27-mer or 33-mer targets from the GSTP1-gene. The method is based on electrostatic modulation of the anion-exchange kinetics of a polypyrrole bilayer film deposited on platinum-microelectrodes to which a synthetic single-stranded 15-mer GSTP-1 promoter probe DNA has been attached (DNA detector). The effect of the contact of this DNA-detector with non-methylated and methylated complementary DNA sequences in Tris-buffer is compared using cyclic voltammetry (CV). The DNA-hybridization taking place at the electrode surface leads to a significant decrease of the CV area recorded after exposure to complementary target DNA in comparison to the CV change recorded for non-complementary DNA target. The performance of this miniaturized DNA detector was optimized with respect to hybridization time, temperature, and concentration of the target. It was also evaluated with respect to selectivity, sensitivity, and reproducibility. These results are significant for their possible use as a screening test for hypermethylated DNA sequences. Label-free electrochemical method for the detection of prostate cancer using a DNA biomarker.

Voltammetric Label‐free Detection of DNA Hypermethylation Using Polypyrrole‐modified Microelectrode Array

“…In methylated DNA, the 5-methylcytosine residues are left unchanged during the bisulfite modification process Table 2. For optimal efficiency of conversion, as recommended by Epigentek's protocol, 5 μL of the 0.1 μM target DNA samples were used [26]. After the bisulfite treatment, 20 μL of each DNA target sample was collected and diluted with 330 μL of TrisÀ HCl buffer to final concentration of 1.12 × 10 À 2 μg/ml These samples were stored in a refrigerator until time of use.…”

Electrochemical Sensing of Cancer‐related Global and Locus‐specific DNA Methylation Events

“…Saheb et al. proposed an electrochemical method for DNA hybridization detection of synthetic methylated 27‐mer or 33‐mer ssDNA targets from the GSTP1 ‐gene (biomarker of prostate cancer). This method employed previous bisulfite treatment and electrostatic modulation of the anion‐exchange kinetics of a polymerized polypyrrole and poly [2,5‐dithienyl‐(N‐3‐phosphorylpropyl)pyrrole] bilayer film deposited on platinum‐microelectrodes to which a synthetic 15‐mer GSTP‐1 promoter ss‐DNA probe was attached.…”

“…The electrochemical strategies reported so far for cancer‐related DNA methylation are based on the use of: the different affinity interaction of DNA bases onto Au‐SPE and graphene ; selective PNA and DNA , probes; anion‐exchange kinetics ; 5‐mC specific antibodies ; electrochemical indicators (FDN , [Ru(NH 3 ) 6 ] 3+ and [Fe(CN) 6 ] 3− ); DNAzyme and enzyme [35, 42]‐based catalysis and quantum dots and nanocomposites as tracing tags. The reported strategies employ a wide variety of electrodes (GEC , gold , SPCE , platinum microelectrodes , Au‐SPE , gold microelectrodes , gold electrodes array , BiFE ) and electrochemical techniques (DPV , CV , SWV and (chrono)amperometry ) to perform the transduction. It is worth to mention also that some of the developed PCR‐free strategies use nanomaterials as electrode modifiers or advanced labels to achieve the required sensitivity.…”

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

“…In this context, electrochemical biosensors offer unique advantages to overcome the aforementioned limitations for the detection of DNA methylation status . However, the electrochemical biosensors reported so far for this purpose, using both PCR and PCR independent approaches, have been mostly limited to synthesized DNA targets and scarcely explored in clinical samples. Moreover, most of the developed PCR‐free electrochemical biosensors require multiple reagents and complex and long protocols involving bisulfite treatment and amplification strategies using nanomaterials to comply with the required sensitivity.…”

“…However, the electrochemical biosensors reported so far for this purpose, using both PCR and PCR independent approaches, have been mostly limited to synthesized DNA targets and scarcely explored in clinical samples. Moreover, most of the developed PCR‐free electrochemical biosensors require multiple reagents and complex and long protocols involving bisulfite treatment and amplification strategies using nanomaterials to comply with the required sensitivity. Therefore, novel electrochemical biosensing strategies to perform reliable quantification of DNA methylation in clinically relevant and minimally treated samples with simple and rapid protocols, are in high demand.…”