A dual-trigger entropy drive circuit based on competitive hybridization for high-specific enzyme-free detection of single nucleotide polymorphisms

Abstract

Single nucleotide polymorphisms (SNPs) play a pivotal role in the detection of major diseases and the breeding of molecular designs. However, current SNP detection methods often rely heavily on expensive proteases, or alternatively, enzyme-free detection methods grapple with limited specificity. Addressing this issue, our study presents an enzyme-free, highly specific, simple, and efficient detection platform. First, we introduced additional base mismatches into the traditional entropy-driven circuit (EDC) reaction to establish a foundational distinction between mutant (MT) and wild-type (WT) sequences. On this basis, we introduced the concept of competitive hybridization and developed a dual-trigger EDC reaction (DEDC) platform, which responded to both wild-type target (WT) and mutant target (MT). By strategically leveraging the signals from both WT and MT, we constructed a ratiometric signal output mode, substantially enhancing the discrimination factor between WT and MT and maximizing the specificity of the detection system. Within the DEDC reaction system, the driving force solely consists of the increase in the system's entropy, with no enzymes involved throughout the entire process, thereby achieving simple and efficient specific detection of SNPs. Notably, MT, previously considered an interference in assays, is repurposed as a trigger signal, making DEDC particularly suitable for the identification of heterozygous samples with low mutational abundances. By analyzing the performance of this platform and using it for genotyping detection of soybean real genome samples, the practical application potential of the CTMSD platform was verified. The CTMSD platform based on EDC reactions has the potential to become a universal biosensing paradigm for future biochemical applications.