Sensitive and specific detection of ctDNA using Copper-Free click chemistry and magnetic bead Technology

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies, enabling earlier detection and real-time assessment of disease progression. However, a significant hurdle in its widespread adoption is the extremely low concentration of ctDNA in biological samples, especially during the early stages of cancer, making sensitive and specific detection challenging. This work addresses the critical problem of developing a highly sensitive and specific method for low abundance ctDNA detection. We developed a novel, highly sensitive, and specific method for ctDNA analysis, employing copper-free click chemistry (strain-promoted azide-alkyne cycloaddition, SPAAC) for enzyme-free amplification, coupled with magnetic bead-assisted fluorometric detection. This enzyme-free approach significantly enhanced specificity and reduced background noise. We meticulously optimized parameters, including primer length and annealing temperature, finding that 30-base primers and a 50 °C annealing temperature yielded optimal amplification efficiency. Our method successfully detected ctDNA at concentrations as low as 10 pM (15 bp primer). Agarose gel electrophoresis confirmed highly specific amplification with minimal non-specific products, and the assay demonstrated excellent allelic discrimination, accurately distinguishing single-nucleotide mutations. Importantly, the method proved robust in complex human serum samples, demonstrating its practical applicability. This innovative, cost-effective, and enzyme-free platform overcomes many limitations of current ctDNA detection technologies. By enabling highly sensitive and specific detection of low abundance ctDNA, this methodology represents a significant leap forward for non-invasive cancer diagnostics, paving the way for earlier disease detection, improved treatment monitoring, and the broader implementation of personalized medicine.