Dual-signal enrichment enhanced entropy-driven circuits integrated ligases for highly sensitive genotyping detection

Entropy driver circuits (EDCs) are favored in the field of nucleic acid detection due to their fastness, simplicity, and high compatibility. However, traditional EDC reactions have weak driving forces and single signal outputs, leading to unsatisfactory signal amplification effects. Here, we rationally utilized the DNA loading function and light quenching properties of gold nanoparticles (AuNPs) to develop a dual-signal enrichment enhanced EDC strategy, which was harmoniously integrated with the ligase chain reaction (LCR) for specific detection of single nucleotide polymorphisms. First, LCR transduces the single-base mutation information within mutant targets (MT) into single-stranded DNA information, simultaneously achieving preliminary amplification of the biological signal. Subsequently, the enhanced EDC cycling reaction is activated by the LCR products, accompanied by the detachment of a large number of FAM-modified probes from the surface of AuNPs to output fluorescent signals. The enhanced EDC strategy effectively addresses the issue of inadequate signal amplification in traditional EDC methods. Ultimately, the integrated system with dual-signal amplification capability is proven to accurately detect MT at concentrations as low as 30.3 fM. In addition, 0.01 % of MT in the heterozygous sample pool can be specifically identified by the detection system. Additionally, we verified the practical application potential of the sensing system using real soybean genome samples. Therefore, this study not only proposes an effective EDC enhancement strategy and develops an efficient and practical SNP detection tool, but also provides an important reference idea for the rational construction of the detection platform based on EDC reaction.