Expand detection windows for identifying single nucleotide polymorphisms using a competitive toehold-mediated strand displacement ratiometric sensing platform

The subtle free energy difference introduced by a single nucleotide mutation results in poor specificity of almost all DNA hybridization probe-based single nucleotide polymorphism (SNP) detection techniques. The development of SNP biosensing strategies with both specificity and sensitivity is a hot and difficult issue in the current field. In this study, we creatively constructed a competitive toehold-mediated strand displacement sensing platform (CTMSD) based on the traditional TMSD reaction, which increased the energy barrier through the intrinsic competition mechanism and expanded the detection window of SNPs. Furthermore, based on the characteristics of the CTMSD platform, the dual-signal detection mode was introduced to change the function model of the detection curve through reporting internal reference ratio signal. The new detection curve model not only compensated for sensitivity, significantly enhanced the discrimination factor, but also greatly expanded the detection window with infinite robustness factor over the detection range. The expansion of the detection window and the improvement of specificity of CTMSD for SNP recognition based on the ratiometric signal output model were verified by computer simulations and experiments. In addition, as a deformation of the strand displacement reaction, the CTMSD was readily adaptable to commonly used signal amplification techniques, such as catalytic hairpin assembly (CHA). Through the CTMSD-CHA performance analysis and real testing of cell genomic samples, the practical application value of CTMSD with the ratiometric signal output model was confirmed. This study provides an important reference for the design and improvement of SNP biosensors and even for all nucleic acid biosensors.