Dual-targets binding protection mediated rolling circle transcription with tandem fluorescent RNA aptamers for label-free detection of liver cancer biomarkers

As one of the malignant tumors with rising incidence, early diagnosis and accurate treatment of liver cancer play a crucial role in improving the cure rate of patients. Alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are two important tumor markers in the diagnosis of liver cancer. Although antibody analysis methods such as immunoassay have been developed, they are still challenging considering the sensitivity and operating cost. Here, we describe a fluorescent RNA aptamer-based rolling circle transcription (FRA-RCT) test, which can ultra-sensitively and accurately detect the presence of both AFP and CEA target proteins at the same time. The dual-targets sensor integrating the aptamers of AFP and CEA can be protectively bound to the target proteins in advance, while the remaining complements are bound by the trigger chains. Through the in vitro transcription process of FRA-RCT, the amount of the targets is successfully converted to the amount of the remaining triggers and thus amplified into two fluorescent RNA aptamer products. This collection of modular components greatly improves the signal gain and specificity of the FRA-RCT technology. In addition, we have successfully distinguished the positive serum samples of liver cancer patients from the negative healthy controls, and realized the point of care testing (POCT) of different targets by using portable chips. The differential fluorescence output proved its effectiveness and practicability. Therefore, this dual-targets aptamer sensor has a promising prospect in simultaneous cancer diagnosis at low cost and high efficiency.