Sensitive,Enzyme-free And Label-free Electrochemical Sensor For K-ras G12D Point Mutation Detection

Objective:K-ras gene is the most frequently mutated gene in the epidermal growth factor receptor（EGFR）signaling pathway and the most common type of mutation is K-ras G12D point mutation（K-ras G12DM）.The mutation status of K-ras gene is considered to be a key indicator for the early diagnosis of colorectal cancer patients and the evaluation of the efficacy of targeted drugs.Considering some shortcomings of the existing detection methods,such as limited sensitivity and specificity,complicated operating procedures,and expensive equipment,this study aims to construct a sensitive,simple,and efficient electrochemical sensor for the detection of low abundance mutation markers,providing a powerful tool for the diagnosis and treatment of related diseases.Methods:1.Surface modification of gold electrode:The TCEP-reduced capture probe（H₂）was modified on the electrode surface,and then the non-specific sites on the electrode surface were covered with a blocking agent（MCH）.2.Establishment of a liquid-phase cyclic amplification system:First,the reaction strands（S₁,S₂）were mixed with the target DNA to form a complete DNAzyme active structure.Subsequently,the substrate chain（H₁）of DNAzyme and the hybridization reaction chains（H₃,H₄）were successively added to the above-mentioned mixed solution for the amplification reaction.The DNAzyme cleavage cycle was verified by agarose gel electrophoresis.3.Construction and analysis of the electrochemical sensor:The liquid-phase amplification system was transferred to the surface of modified gold electrode to activate hybridization chain reaction.Methylene blue（MB）was used as an electroactive indicator to generate electrochemical signal.The process of sensor construction was verified by electrochemical impedance spectroscopy（EIS）and cyclic voltammetry（CV）.After optimizing the experimental conditions,differential pulse voltammetry（DPV）was used to electrochemically detect K-ras G12DM with different concentrations,and a standard curve were drawn.4.Performance evaluation of sensor:including sensitivity,specificity,stability,and recovery experiments.Results:1.The catalytic cleavage process of DNAzyme was verified by agarose gel electrophoresis.2.The layer-by-layer assembly process of the sensor was verified EIS and CV,indicating that the electrochemical sensor has been successfully constructed.3.Under optimal conditions,the electrochemical signal was linearly related to the logarithm of the concentration of target DNA.The detection range was 0.5 fM-50 nM,and the linear equation is I（μA）=2.75928 lg c（pM）+14.2228（R²=0.99348）.4.The sensor had good analytical performance,detection limit as low as 0.5 fM,good specificity and repeatability,and satisfactory recovery results.Conclusion:By integrating two signal amplification mechanisms,DNAzyme and HCR,a sensitive and efficient electrochemical sensing strategy was successfully fabricated for the detection of low-concentration K-ras G12D point mutation,with satisfactory repeatability,specificity,and recovery.In addition,the sensing strategy required no protease,no labeling,low cost,which had a high clinical application potential.