Sensitive, enzyme-free and label-free electrochemical sensor for K-ras G12D point mutation detection based on double cascade amplification reaction

The levels of K-ras gene mutation was thought to be closely related to the early diagnosis of colorectal cancer and the evaluation of the efficacy of targeted drugs. Here, a high-efficiency, enzyme- and label-free electrochemical sensor for K-ras G12D point mutation (K-ras G12DM) determination was developed by concatenation of split Mg2+-dependent DNAzyme and hybridization chain reaction (HCR). The presence of target induced split DNAzyme to assemble into an intact and activated conformation, catalyzing the cyclic cleavage of substrates to produce a mass of intermediates. Subsequently, the intermediate products triggered downstream HCR events to form long notched dsDNA, which would couple a large amount of methylene blue (MB) through π − π stacking interactions to generate a significant electrochemical signal. Under optimal conditions, this sensor platform achieved a highly sensitive detection of the target, with the lowest detection limit of 0.5 f. and a good linear relationship in the range of 0.5 f. to 50 nM. Furthermore, the biosensor not only easily distinguished target from high concentrations of mismatched target analogs, but also detected rare mutations with mutation frequencies as low as 0.001%. Due to its low cost, high sensitivity and high specificity, this strategy has broad prospects for other low-abundance targets and point mutations.