A clamp-improved universal amplified system for ratiometric fluorescent detection of single-nucleotide polymorphisms coupled with a novel dual-emissive silver nanocluster

Rapid, sensitive and precise detection of single-nucleotide polymorphisms (SNPs) with risk association is of great significance but still remains challenging in resource-limited clinical settings. Herein, a convenient, low-cost and label-free ratiometric fluorescent sensing strategy is developed by coupling peptide nucleic acid (PNA) clamp-improved catalyzed hairpin assembly (CHA) reaction with a novel hairpin DNA-templated dual-emissive silver nanocluster (AgNC). In the presence of targets containing single-base mismatches, the sensing platform initiates the amplification and enlarges the distance between the Ag emitter pair in AgNC, resulting in a decrease of red emission accompanied by a significant optical readout. On the contrary, the PNA clamp is capable of specifically blocking the perfectly homologous counterpart in DNA sequence and thereby impeding unwanted amplification. In this study, the SNP of oncogenic Kirsten ras (KRAS) gene has been accurately and effectively genotyped with a detection limit of 9.6 pM. Moreover, this nanosensor can effectively discriminate a series of SNPs with vivid color change and its universality can be simply achieved by a rational design of the relative sequences. This platform performs well for both target DNA spiked cell lysate samples and PCR amplicons from standard cell lines, implying its great practical application prospect for bioanalysis and biosensors.