Work

The Specific Low-Interference dsDNA Copper Nanoclusters for Visual Fluorescent Detection and Quantification of the EGFR L858R Point Mutation in Whole Single-Tube Magnetic Purification System

Year: 2026

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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.

Source: Analytical Chemistry

DOI: 10.1021/acs.analchem.6c00089

Authors: Ke-Peng Lai, Ravery Sebuyoya, Kung-Hung Lin, Hwang-Shang Kou, Chun-Chi Wang

Institutions: Kaohsiung Medical University, Zuoying Armed Forces General Hospital, Kaohsiung Medical University Chung-Ho Memorial Hospital

Language: English

Topic: Advanced biosensing and bioanalysis techniques

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: hybrid

Estimated APC paid: $4,500