Copper-Free Click Chemistry-Mediated Cyclic Ligation Amplification for Highly Sensitive and Non-Label Electrochemical Detection of Gene Mutation

Herein, a new signal amplification strategy on the basis of copper-free click chemistry-mediated cyclic ligation of DNA for detecting cancer-related mutant p53 (mp53) gene with high sensitivity is described. The complementary sandwich hybridization of the target mp53 gene with the azide-modified capture probes and the dibenzocyclooctyne-conjugated and G-quadruplex containing signal probes pulls the azide and dibenzocyclooctyne moieties into close proximity, leading to copper-free click ligation of the signal probes on the sensor surface. Subsequent thermal recycling of the target gene results in the attachment of lots of signal probes on the electrode. The many G-quadruplex segments in the signal probes then bind and confine hemin within the vicinity of the electrode surface, thereby yielding substantially amplified reduction current response for detecting the mutant target mp53 sequences with a low detection limit of 7.7 fM. This sensor can also discriminate mutant and wild gene with single base mutation and can realize target gene detection in diluted human serums. Distinct from conventional copper-mediated click chemistry and ligase-based DNA ligation, our approach is copper- and enzyme-free, making our method a simple and robust signal amplification sensing means for detecting low levels of different DNA biomarkers.