Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity

Abstract

This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin–magnetic microbeads (Strep–MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (−0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA–polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MGMT tumor suppressor gene. Amplification factors of 43.6 and 55.2 were achieved using ProtA–polyHRP80 or Histostar, respectively, compared to the conventional secondary antibody labeling. This amplification was crucial to detect methylation events at single-nucleotide resolution achieving limits of detection (LODs) of 23.0 and 13.2 pM, respectively, without any target DNA amplification. The ProtA–polyHRP80-based bioplatform, selected as a compromise between sensitivity and cost per determination, exhibited full discrimination toward the target 5-hmC against the closely related 5-mC. In addition, the bioplatform detected 5-hmC at the regional level (MGMT promoter region) in just 10 ng of genomic DNA (gDNA, ∼2700 genomes) extracted from cancer cells and tissues from colorectal cancer (CRC) patients within 60 min.

Analyses of DNA methylation involved in the activation of nuclear karyopherin alpha 2 leading to the progression and prognostic significance across human breast cancer

“…DNA methylation is an early event in the process of tumorigenesis and gradually enhances during tumor progression [35]. Therefore, DNA methylation of precancerous lesions or early tumors is the most promising method for early diagnosis and prognosis of cancer.…”

Electrochemical Biosensors for Determination of Colorectal Tumor Biomarkers

“…DNA methyltransferases catalyze the covalent binding of the methyl group at the fifth carbon of the cytosine ring 5-methylcytosine (5-mC). After, 5-mC can be oxidized to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) methylcytosine dioxygenase oxidase [115]. The O 6 -methylguanine-DNA methyltransferase (MGMT) gene, whose functional product removes alkylating groups from O 6 -guanine, is an important example of this epigenetic mechanism [94,116].…”

“…Povedano and co-authors [115] developed the first electrochemical bioplatform to detect methylation events at localized sites with single-base sensitivity. The strategy is based on streptavidin-modified magnetic beads with a biotinylated-DNA capture probe, followed by hybridization with a synthetic target DNA sequence with a single 5-hydroxymethylcytosine (5-hmC) in the promoter region of the MGMT gene, the recognition of the methylation event by a specific antibody (anti-5-hmC), and further conjugation with one bioreagent for the signal amplification.…”

<p>Analyses of DNA Methylation Involved in the Activation of Nuclear Karyopherin Alpha 2 Leading to Identify the Progression and Prognostic Significance Across Human Breast Cancer</p>

“…DNA methylation is an early event in the process of tumorigenesis and gradually enhances during tumor progression. 36 Therefore, DNA methylation of precancerous lesions or early tumors is the most promising method for early diagnosis and prognosis of cancer. By examining its DNA methylation status through Figure 9 KPNA2 expression is upregulated by hypomethylation in breast tumors.…”