A novel electrochemical biosensor was constructed for specific and ultrasensitive detection of PIK3CAH1047R gene mutation based on NsbI restriction enzyme-mediated strand displacement amplification (NsbI-SDA) and four-way DNA junction for the first time. In this biosensor, the NsbI restriction enzyme combined with strand displacement amplification (SDA) was able to specifically distinguish PIK3CAH1047R gene mutation and increase the number of DNA copies to improve electrochemical response. In the presence of target mutation gene, DNA fragments produced by the cleavage event of NsbI restriction enzyme could trigger the SDA reaction to generate massive linker chains. When the linker chains were captured on the electrode, the four-way DNA junction was then attached at the end of linker chain. By integrating electroactive molecules of methylene blue (MB) into four-way DNA junction, this sandwich-like electrochemical biosensor was able to determine the specific distinction of target mutation gene with a low detection limit of 0.001%. Finally, this strategy could be used to analyze mutation gene spiked into human serum samples, indicating the potential application in genetic analysis and clinical disease diagnosis.
Keywords: Electrochemical biosensor; Four-way DNA junction; Methylene blue; NsbI-SDA; PIK3CA gene mutation.
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