Multiple Loci DNA Methylation Profiling for Rapid Colorectal Cancer Diagnosis Based on a Smartphone-Integrated Microfluidic Biosensing Platform

Methylation level of circulating tumor DNA (ctDNA) is widely recognized as a pivotal biomarker in precision oncology for tumor characterization and risk assessment. However, current point-of-care testing platforms for methylation analysis are constrained in achieving sufficient detection specificity due to their limitations in single-locus detection and their dependence on complex instrumentation. In this study, to overcome the above limitations, a smartphone-integrated, microfluidic chip technology-based biosensing platform was engineered for simultaneous analysis of multiple methylation loci in ctDNA. In this platform, whole blood samples from suspected individuals underwent GlaI methylation-sensitive enzyme digestion and target-specific primer hybridization, followed by microfluidic chip loading for on-chip colorimetric reactions. Methylation levels at specific loci were quantified through chamber-specific absorbance measurements by using the developed portable biosensing platform. The biosensor achieved simultaneous detection of three specific methylation loci (CpG sites) on the Septin 9 gene, which are associated with colorectal cancer. The detection limits (LODs) for the individual methylation loci are 0.25, 0.4, and 0.525 fM, respectively. This platform demonstrates high accuracy in detecting methylation signatures within blood samples from colorectal cancer patients, showing a significant correlation with conventional diagnostic methods. With its low cost and portable features, the developed biosensing platform may provide an economical solution for early-stage screening of cancer for the healthcare system in remote and under-resourced regions.