Sensitive colorimetric biosensing for methylation analysis of p16/CDKN2 promoter with hyperbranched rolling circle amplification

A simple, fast and sensitive colorimetric biosensing method for DNA methylation analysis was developed by combining methylation-sensitive endonuclease based digestion with hyperbranched rolling circle amplification (HRCA) induced signal enhancement. The assay was carried out on a DNA capture probe modified 96-cell microplate with four sequential steps of target recognition, endonuclease-based digestion, isothermal HRCA, and enzyme-catalyzed colorimetric readout within 3 h. The semi-quantitative and precise analysis of methylated DNA could be easily achieved by naked eye and absorbance measurements, respectively. The strategy exhibited excellent detection specificity and accuracy with a log-linear response to methylated DNA from 100 fM to 10 nM. As a proof of concept, the assay was applied to investigate the methylation status of p16/CDKN2 promoter of breast cancer patients. The methylated p16 concentration was not significantly associated with the clinical parameters. The proposed method allowed efficient methylation detection with simplicity, rapidness, low cost and high sensitivity, showing great promise for application in early diagnosis of methylation-related diseases.