Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.

Most of the dynamic DNA devices are rationally constructed by utilizing toehold-mediated DNA strand displacement reactions. However, such approaches have been mainly limited to the operation with double-stranded hybridization and lack the versatility of DNA scaffold responses for additional levels of controlling DNA strand displacement reactions. Herein, we propose a toehold activation strategy based on the DNA tetraplex (G-quadruplex or i-motif), where the toehold domain is designed by attaching a complementary single-stranded segment (CS) to a G-rich/C-rich segment. Modulating G-quartet/C·C(+) numbers and/or the CS lengths can easily tune the strand displacement kinetics. This scheme allows fine control of DNA strand displacement rates over 2 orders of magnitude by adjusting the concentration of various environmental stimuli. This strategy expands the rule set of designing dynamic DNA devices and will be useful in building diverse environmental stimuli-fuelled molecular devices.

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