Entropy-Driven Circuit Integrated with Ligases toRegulate DNA-AuNP Network Disintegration for Colorimetric Detectionof Single Nucleotide Polymorphisms

In recent years, entropy-driven circuit (EDC) dynamic DNA networks have garnered significant attention in nucleic acid detection owing to their simplicity, efficiency, and flexible design. Nevertheless, conventional EDC reactions face a constraint in achieving optimal signal amplification due to a solitary and feeble driving force. To overcome this limitation, we innovatively devised a gold nanoparticle (AuNP) dispersion-enhanced EDC (Au-EDC) approach, pioneering a novel colorimetric signal amplification and output system. The system was harmoniously integrated with the ligase chain reaction (LCR) for precise single nucleotide polymorphism (SNP) genotyping. Specifically, LCR was selectively executed solely on the positive strand of the mutant target (MT), facilitating precise point-to-strand information transduction. Subsequently, the LCR product triggered the Au-EDC cycling reaction, causing the DNA-AuNPs network to progressively disintegrate and release a pronounced colorimetric signal. This strategic design ingeniously harnessed the entropy increase that occurs as AuNPs undergo a transition from aggregated to dispersed states, offering a supplemental impetus for the EDC cycle. The integrated LCR-Au-EDC system excelled in detecting MT at concentrations as low as 320 fM and differentiating pooled samples with mutation frequencies as low as 0.1%. Moreover, the system accurately performed SNP genotyping on the real genomes derived from soybean leaves. Consequently, this study not only develops a colorimetric signal amplification and output sensing system based on EDC reactions but also provides a cost-effective and efficient SNP genotyping tool.