Abstract
The ability to detect double-stranded DNA (dsDNA) as a biomarker without denaturing it to single-stranded DNA (ss-DNA) continues to be a major challenge. In this work, we report a sandwich biosensor for the detection of the ds-methylated MGMT gene, a potential biomarker for brain tumors and breast cancer. The purpose of this biosensor is to achieve simultaneous recognition of the gene sequence, as well as the presence of methylation. The biosensor is based on reduced graphene oxide (rGO) electrodes decorated with gold nanoparticles (AuNPs) and uses Peptide Nucleic Acid (PNA) that binds to the ds-MGMT gene. The reduction of GO was performed in two ways: electrochemically (ErGO) and thermally (TrGO). XPS and Raman spectroscopy, as well as voltammetry techniques, showed that the ErGO was more efficiently reduced, had a higher C/O ratio, showed a smaller crystallite size of the sp2 lattice, and was more stable during measurement. It was also revealed that the electro-deposition of the AuNPs was more successful on the ErGO surface due to the higher At% of Au on the ErGO electrode. Therefore, the ErGO/AuNPs electrode was used to develop biosensors to detect the ds-MGMT gene. PNA, which acts as a bio-recognition element, was used to form a self-assembled monolayer (SAM) on the ErGO/AuNPs surface via the amine-AuNPs interaction, recognizing the ds-MGMT gene sequence by its invasion of the double-stranded DNA and the formation of a triple helix. The methylation was then detected using biotinylated-anti-5mC, which was then measured using the amperometric technique. The selectivity study showed that the proposed biosensor was able to distinguish between blank, non-methylated, non-complementary, and target dsDNA spiked in mouse plasma. The LOD was calculated to be 0.86 pM with a wide linear range of 1 pM to 50 µM. To the best of our knowledge, this is the first report on using PNA to detect ds-methylated DNA. This sandwich design can be modified to detect other methylated genes, making it a promising platform to detect ds-methylated biomarkers.
Highlights
DNA methylation is the most exhaustively characterized epigenetic alteration of DNA in which methyl groups (CH3) are covalently bound to DNA
A biosensor for the detection of the ds-MGMT gene has been developed in this work using an ErGO/AuNPs-modified electrode
A high C/O ratio was achieved using electrochemical reduction of graphene oxide solution (GO) without using any harmful reductants and in a shorter period of time compared to the techniques reported in other papers
Summary
DNA methylation is the most exhaustively characterized epigenetic alteration of DNA in which methyl groups (CH3) are covalently bound to DNA. Chen et al [16] developed an electrochemical biosensor for DNA methylation detection using tetrahedron DNA probes which were anchored to a AuNPs-coated gold electrode with avidin-HRP as the label This biosensor showed a dynamic range of 1 aM to 1 pM, with the LOD of 0.93 aM. The LOD was calculated to be 0.86 pM with a wide linear range, and the developed biosensor showed a high sensitivity in mouse plasma This biosensor is the first report for the detection of the double-stranded methylated gene, is bisulfite- and PCR-free, and can be tailor-made to detect other methylated genes, which can be beneficial in point-of-care (POC) programs as an inexpensive platform to detect methylated DNA biomarkers. PBS tablets pH 7.4 were obtained from Fisher Scientific (Loughborough, UK) and the PBS buffer solution was prepared in Milli-Q water
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