An electrochemical biosensing platform initiated simultaneously from multi-directions with programmable enzyme-free strategy for DNA variant detection.

Single-nucleotide variations (SNVs) represent vital clinical and biological information in the onset and progression of many cancers, but lacking of cost-effective, high-sensitive and reliable SNVs detection method. In this study, we propose a programmable electrochemical biosensing strategy initiated simultaneously from multi-directions by enzyme-free amplifying circuit for high-sensitivity SNVs detection. Through elaborate design, we utilized the power of conventional enzyme-free catalytic reaction to activate a multidirectional initiation self-assembly process, enabling multiple amplification. This innovative cascade strategy significantly improved the amplification performance and detection sensitivity. Subsequently, KRAS gene of cancer cells was used as proof-of concept model for SNVs recognition to demonstrate the capability. With the help of cascade design, the single-base differences between SNV sequence and wild-type sequence (WT) could be differentiated and amplified effectively. Consequently, abundant Y-shaped DNA structure efficiently was induced by DNA variant to generate on the electrode surface, facilitating the incorporation of methylene blue (MB) redox indicator. Therefore, a "signal-on" electrochemical biosensing platform was constructed. Our enzyme-free biosensor achieved a low detection limit of 36 aM and a broader linear range spanning from 100 aM to 1 nM under optimal experimental conditions. The capability of proposed cascaded DNA network to detect DNA variants in complex cancer cells and serum samples indicated the potential applicability in real sample analysis.