One-step Circulating Tumor DNA Microfluidic Sensing Based on Toehold-mediated Entropy-driven Bipedal DNA Walker and Dendritic Hybridization Chain Reaction
Circulating tumor DNA (ctDNA) in blood,present at low abundance,serves as a critical biomarker for cancer. Precise detection of ctDNA is of great significance for early diagnosis,disease monitoring,and prognosis evaluation. In this study,a microfluidic chip-based entropy-driven bipedal DNA walker combined with dendritic hybridisation chain reaction was designed as a cascade signal amplification strategy for detection of ctDNA in blood in a microfluidic chip. In the presence of the target,toehold A interacted with target and triggerd a bidirectional strand displacement reaction with the aid of fuel strand F,thereby releasing the target and the 8-17 DNAzyme active centre. Among them,the released target was recycled in a new round of entropy-driven chain replacement reaction. Meanwhile,8-17 DNAzyme active center activated by Pb2+would act on the cleavage site of the substrate hairpin at the microbead surface,exposing the capture probes on the surface of the microbead. The numerous capture probes induced a dendritic hybridization chain reaction,which resulted in fluorescent signals being concentrated on the surface of the microbeads. With the breast cancer mutant gene PIK3CAE545K as the target model,under the optimal experimental conditions,the linear range of sensor was 50-10000 fmol/L,the detection limit was 0.94 fmol/L (LOD,3σ),and the regression equation was y=31.65 lgCT+474.08 (CT is the concentration mutant gene PIK3CAE545K sequence,fmol/L). This method showed spiked recoveries between 98.8％ and 106.5％ when applied to detection of mutant gene PIK3CAE545K in human serum. Characterized by its sensitivity,specificity,anti-interference capability,high throughput,and one-step operation,this method was ideally suited for the rapid analysis of complex samples.
DNA walkerEntropy-driven strand displacement reactionMicrofluidic chip8-17 DNAzymeDendritic hybridization chain reaction
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维普
