Integration of T7 exonuclease-triggered amplification and cationic conjugated polymer biosensing for highly sensitive detection of microRNA

Abstract

A novel and highly sensitive method for detection of microRNA (miRNA) was developed by integration of T7 exonuclease-triggered amplification and cationic conjugated polymer (CCP) biosensing. First, a fluorescein-labeled probe was designed with the complementary sequence to the target miRNA. When target miRNA was absent in the solution, the fluorescence probe interacted with CCP through the strong electrostatic interactions, leading to the highly efficient fluorescence resonance energy transfer (FRET) from CCP to fluorescein. In the presence of target miRNA, the probe hybridized with the miRNA to form DNA/miRNA duplex hybrids. Then, T7 exonuclease digested cyclically the fluorescence probes in hybrids and triggered the enzyme amplification reaction, generating a large number of single nucleotides. Owing to the weak electrostatic interaction between CCP and the single nucleotide, the FRET from CCP to fluorescein would not take place, which effectively reduced the background and significantly enhanced the sensitivity and the dynamic range of miRNA detection. The linear range of the assay was 0.2-100 pM and the detection limit 0.08 pM was 58 times lower than that of the endonuclease-based assay. The method is simple, cost-effective, and with no need for the sophisticated instrument, and has broad application prospects for miRNA detection and early diagnosis.

Keywords: Cationic conjugated polymer; Fluorescence resonance energy transfer; MicroRNA; T7 exonuclease.