Porous nanozymes: the peroxidase-mimetic activity of mesoporous iron oxide for the colorimetric and electrochemical detection of global DNA methylation

Nanomaterials (nanozymes) with peroxidase-mimetic activity have been widely used in biosensing platforms as low-cost, relatively stable and prevailing alternatives to natural enzymes. Herein, we report on the synthesis and application of the peroxidase-mimetic activity of mesoporous iron oxide (MIO) for the detection of global DNA methylation in colorectal cancer cell lines. The target DNA was extracted and denatured to get ssDNA followed by direct adsorption onto the surface of a bare screen-printed gold electrode (SPGE). A 5-methylcytosine antibody (5mC) functionalized nanomaterial (MIO-5mC) was then used to recognise the methylcytosine groups present on the SPGE. The MIO-5mC conjugates catalyse the TMB solution in the presence of hydrogen peroxide to give the colorimetric (i.e., naked-eye observation) and electrochemical detection of DNA methylation. The assay could successfully detect as low as 10% difference in the global DNA methylation level in synthetic samples and cell lines with good reproducibility and specificity (%RSD = <5%, for n = 3). This strategy avoids the use of natural enzyme horseradish peroxidase (HRP), traditional PCR based amplification and bisulfite treatment steps that are generally used in many conventional DNA methylation assays. We envisage that our assay could be a low-cost platform with great potential for genome-wide DNA methylation analysis in point-of-care applications.