Designing a fluorescence padlock probe-based biosensor and colorimetric assay for the detection of G12D KRAS mutation

Aim: Cell-free DNA in the plasma is known to be a potential biomarker for noninvasive diagnosis of oncogenic mutations. The authors aimed to design an optimized padlock probe-based hyperbranched rolling circle amplification biosensor to detect the KRAS G12D mutation using fluorescence and colorimetric methods. Methods: Single-factor experiments, Plackett-Burman design and response surface methodology were applied to optimize the padlock probe-based hyperbranched rolling circle amplification reaction. Results: The maximum fluorescence intensity was achieved at a padlock probe concentration of 1.5 pM and target concentration of 9 pM at 38 degrees C ligation temperature. The proposed biosensor has a low detection limit of 60 fM of target DNA and a linear response in the concentration range of 60 fM to 0.2 pM. Conclusion: The results indicated the power of these assays to detect KRAS point mutations in liquid state reactions.

Automated summary

The study aimed to design an optimized biosensor to detect the KRAS G12D mutation in plasma using padlock probe-based hyperbranched rolling circle amplification. The results showed the biosensor has a low detection limit and linear response in detecting KRAS point mutations in liquid state reactions.