A novel nest hybridization chain reaction based electrochemical assay for sensitive detection of circulating tumor DNA

As an ideal biomarker candidate, circulating tumor DNA (ctDNA) plays a vital role in noninvasive diagnosis of cancer. However, most traditional approaches for quantifying ctDNA are cumbersome and expensive. In the present work, a novel electrochemical biosensor based on nest hybridization chain reaction was proposed for the sensitive and specific detection of PIK3CA E545K ctDNA with a simple process. The nest hybridization chain reaction was initiated by the hybridization of two dumbbell-shaped DNA units which were assembled by two classes of well-designed DNA probes respectively, leading to the formation of a complex DNA structure. In the presence of target ctDNA, the amplified hybridization chain reaction products were captured by target ctDNA, resulting in a significant increase of electrochemical signal. Under the optimal conditions, the developed biosensor exhibited good analytical performance for the detection of target ctDNA with the linear range from 5 pM to 0.5 nM and the detection limit of 3 pM. Furthermore, this assay was successfully applied to the detection of ctDNA in spiked-in samples, pleural effusion and serum samples of malignant tumor patients. This simple and cost-effective sensing system holds great potentials for ctDNA detection and cancer diagnosis.