Rapid detection of NPY methylation in colorectal cancer using a singlet oxygen-based photoelectrochemical ratiometric...

Aberrant methylation of Neuropeptide Y (NPY) gene has emerged as a promising biomarker for monitoring metastatic colorectal cancer and predicting its early recurrence. Current standard approaches for the detection of NPY hypermethylation levels rely on the use of enzymes and are associated with high costs, long times of analysis and interferences from PCR inhibitors, which limits their applicability in point-of-care or low-resource environments. Therefore, the need for an affordable, rapid, and sensitive technology for NPY methylation detection is clear.

Herein we present a novel, rapid, sensitive, highly selective, and enzyme-free photoelectrochemical ratiometric bio-platform for the detection and quantification of NPY gene hypermethylation. At the core of this method lies the use of magnetic microbeads and singlet oxygen (¹O₂)-based photoelectrochemical detection. The use of bisulfite conversion and hybridization temperature allowed to achieve a 99 % discrimination efficiency of the methylated NPY target from the unmethylated one. Detection of 18 pM (18 fmol) of 94-nucleotide methylated NPY target sequence with 6 methylation sites in hybridization buffer and 45 pM (45 fmol) against the background of genomic DNA was achieved without any amplification. Ultimately, ratiometric quantitative sensing of methylated NPY gene over reference albumin gene was carried out and an LOD of 2 % methylation level was obtained.

Overall, this technique demonstrated immense potential in overcoming the complexity, high costs, and need for enzymes of current strategies for hypermethylation detection and thus represents a great innovation in (photo)electrochemical sensing of aberrant DNA methylation. It, therefore, holds great promise for establishing in future a clinically relevant point-of-care testing device.