A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor.

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)6]³â»/4⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, a biosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limit and a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.