Double hairpin-assembled probe-mediating catalytic hairpin assembly and primer exchange reaction for sensitive and label-free miRNA analysis in gastric carcinoma

Abstract

Abstract The aberrant expression of microRNA (miRNA) is closely associated with various pathological processes, such as the development of gastric cancer. High-efficiency quantification of miRNAs is significant for the diagnosis, prognosis, and treatment of cancers. However, the sensitive and reliable detection of miRNA remains a huge challenge. We depict here a novel fluorescent approach for sensitive and label-free miRNA detection by exploiting a designed detection scaffold to integrate the catalytic hairpin assembly and primer exchange reaction (PER). In this method, the detection scaffold that is constructed based on the hybridization between two hairpin structure probes (H1 probe and H2 probe), is capable of specifically recognizing target miRNA and activating signal amplification, and the PER process transcribes numerous G-rich sequences to induce ThT-based label-free signal generation. Based on the efficient signal amplification strategy and label-free signal generation mode, the method exhibits a wide detection range of 7 orders of magnitude and a high repeatability (coefficient of variation, 2.76%), implying that the proposed approach will be a robust tool in quantification of miRNA and early diagnosis of disease. Graphical abstract

Keywords

• microRNA
• Catalytic hairpin assembly (CHA)
• Primer exchange reaction (PER)
• Thioflavin T (ThT)