Research For Electrochemical Biosensing Analysis Of DNA Methylation Based On Tetrahedral Nanostructure

Objective: DNA methylation and its related factors are of great significance in the diagnosis,treatment,and prognosis of various diseases.Traditional methods of DNA methylation measurement usually rely on intricate apparatuses or cumbersome procedures,while electrochemical biosensing detection technology,which is featured by high-sensitivity,low-cost and easy-to-operate,is now becoming the hotspot of this field.The aim of this study is to design an electrochemical biosensor for ultrasensitive DNA methylation detection based on multiple signal amplifying strategies such as the Aurum nanoparticle(AuNP)electrodeposition,hybridization chain reaction(HCR)and enzymatic reaction,hoping to offer a precise and sensitive method for the diagnosis and treatment of DNA methylation-related disease.Methods: Partially complementary sequences were designed and the DNA tetrahedral nanostructure probe(TSP)was synthesized through the denaturation and renaturation of DNA.The TSP was characterized by the atomic force microscopy(AFM)and the polyacrylamide gel electrophoresis(PAGE),and then immobilized on the surface of a gold electrode modified with AuNPs.After proceeding the target DNA hybridization,HpaII endonuclease digestion,HCR amplification,and streptavidin-horseradish peroxidase(S-HRP)conjugation,the electrode was immersed into the tetramethylbenzidine(TMB)solution.The electron transfer on the electrode surface was measured via electrochemical detection methods,which could indicate the methylation information of the target DNA sequence.After optimizing the experimental conditions such as TSP immobilization time,target DNA hybridization time and HpaII endonuclease digestion time,the performance of the electrochemical biosensor,such as the linear range,detection limit,storage stability and specificity,were investigated under the optimal conditions.Results: The results of the AFM and PAGE showed that the TSP was synthesized effectively with a high yield and the tetrahedral nanostructure was successfully formed.Electrochemical biosensing detection results showed that each modification step of the electrochemical biosensor was effectively achieved.Under the optimal experimental conditions,the electrochemical biosensor exhibited a linear range between 1 amol/L to 1 pmol/L,and the detection limit reached about 0.93 amol/L.The base-mismatched target sequence hybridization experiment and the human serum recovery experiment demonstrated that the electrochemical biosensor was of good specificity,and the 30-days storage stability was also verified.Conclusion: Our electrochemical biosensor was capable for the detection of the methylated DNA target sequences with ultrahigh sensitivity,ideal specificity and stability,which could offer a precise and sensitive method for the early diagnosis and treatment of DNA methylation-related diseases.