Multiplexed and streamlined DNA methylation detection of colorectal cancer-related genes using graphically encoded hydrogel microparticles and rolling circle amplification.

Detection of DNA methylation in multiple genes can contribute to the prediction, diagnosis, and prognosis of diverse diseases. However, current DNA methylation detection technologies, such as PCR and microarrays, suffer from limited multiplexing capabilities or complicated assay procedures that require pre-amplification. To circumvent these limitations, we present a graphically encoded hydrogel microparticle-based multiplexed DNA methylation process mediated by rolling circle amplification (RCA) for signal enhancement. Unlike typical DNA methylation detection technologies, the developed process does not depend on pre-amplification and has a large multiplexing capacity based on graphic-based encoding. By the streamlined integration of bisulfite-based sequence conversion, ligation of padlock DNA, and hydrogel-based RCA, colorectal cancer-related genes (VIM and SDC2) can be detected with sensitivities of 23.3 fM and 54.1 fM. Furthermore, methylated DNA in a mixture of methylated and unmethylated DNA can be differentiated at levels as low as 0.1 %. The robust multiplexing capability of VIM and SDC2 was also confirmed by the negligible non-specific signal and consistency of the detection signal with the singleplex assay (81.9 % and 94.7 % recovery rates for VIM and SDC2, respectively). Finally, the practical applicability of the assay was validated by analyzing the methylation levels of VIM and SDC2 in cellular DNA extracted from normal and colorectal cancer cell lines. Given its multiplexing capability, streamlined workflow, robust sensitivity, and specificity, the developed assay can contribute to various biomedical and omics fields, including cancer diagnosis.