The Specific Low-Interference dsDNA Copper Nanoclusters for Visual Fluorescent Detection and Quantification of the...

Existing fluorescence-based techniques for single-nucleotide variation detection are limited by nonspecific fluorescence interference and complex analytical workflows. To address these challenges, we developed a single-tube fluorescence detection strategy integrating restriction fragment length polymorphism with poly-AAT-templated copper nanoclusters. Owing to the significantly higher synthesis efficiency of copper nanoclusters on AT-rich sequences than on random DNA templates, fluorescence interference from residual genomic DNA and nonspecific amplification products was effectively suppressed. As a result, strong fluorescence emission was generated predominantly from double-stranded poly-AAT, enabling reliable visual discrimination of single-nucleotide variation ratios under UV illumination. Biotin-labeled primers combined with streptavidin-coated magnetic beads enabled efficient separation of digested DNA fragments, eliminating the need for electrophoresis and further simplifying the workflow. The entire analytical procedure─including PCR amplification, enzymatic digestion, magnetic separation, and fluorescence measurement─was completed within a single tube, highlighting its suitability for automation and integration into microfluidic platforms. The method was validated for detection of the EGFR L858R mutation in patients with nonsmall cell lung cancer, yielding an excellent linear calibration curve (r = 0.9981), recovery rates of 95-110%, and a detection limit of 2.33%. These results demonstrate sensitivity comparable to that of commercial qPCR and next-generation sequencing, while offering advantages in instrument cost, simplicity, and analytical specificity.