Research On A New Method Of Electrochemical Detection Of Circulating Tumor DNA Based On Walkers

Circulating tumor DNA(ctDNA)is a single-stranded or doublestranded gene fragments originating from tumor or circulating tumor cells,and subsequently released with apoptosis and necrosis directly,or released from macrophages to body fluids such as peripheral blood and cerebrospinal fluid with the absorption of necrotic cells.studies have shown that ctDNA has many cancer-related molecular characteristics,such as single nucleotide variation,methylation change and so on.As a potential tumor biomarker,ctDNA has gained increasing attention in term of cancer assessment.Thus,quantitative analysis of ctDNA plays an vital role in early diagnosis,treatment guidance,prognosis evaluation of cancer.However,the biggest challenge for quantitative detection of ctDNA is that its contents in body fluid are very low,and the existing detection methods can not achieve highly sensitive detection.Therefore,an electrochemical biosensor based on targetdriven rolling walker and DOX@TDN-Au signal tag is designed for the ultrasensitive detection of ctDNA.In this study,a multiple-legged and highly integrated DNA rolling walker based electrochemical biosensor was developed for ultrasensitive ctDNA analysis through rolling circle amplification(RCA)with doxorubicin@tetrahedron-Au(DOX@TDN-Au)as electrochemical indicator.Upon target-driven RCA,the multiple-legged walker could move along with the predesigned track by strand displacement reactions,resulting in numbers of legs binding irreversibly to iStep probes.The binding of massive legs to iStep probes could effectively impede DOX@TDN-Au tags binding on the surface of sensor and then reached a “signal off” state.Benefiting from the highly amplified efficiency of rolling walker machine and DOX@TDN-Au tags,the established biosensor performed high sensitivity for target detection with a low limit of detection down to 0.29 fM.Moreover,the target ctDNA could hybridize with the ring and capture probe simultaneously,greatly enhancing the specificity of the developed biosensing method.Thus,this biosensing method is a promising tool for detection of ctDNA in the field of clinical diagnostic and tumor progression assessment.