Target-triggered autocatalytic sequence recycling for sensitive and simultaneous detection of microRNA and mRNA via multi-donor iFRET signal amplification

Abstract Simultaneous monitoring of different biomarker molecules can potentially offer high accuracy for disease diagnosis. In this work, by using a new biological auto-cycling proximity recording (APR) approach and multi-donor-induced fluorescence resonance energy transfer (iFRET), we establish a multiplexed and sensitive fluorescent method for simultaneous detection of microRNA-155 and osteopontin mRNA for accurate alcoholic liver disease diagnosis. The presence of the two target sequences triggers the APR process via two toehold-mediated strand displacement reactions for the generation of many dsDNAs with the ROX and Cy5 dyes linked to their termini. Subsequent excitation of the SYBR Green I dye intercalated into the dsDNA strands exhibits amplified fluorescence at distinct wavelengths via iFRET for sensitive and simultaneous detection of microRNA-155 and osteopontin mRNA. With the synergistic signal amplification by APR and iFRET, our method shows sub-femtomolar detection limits for the two RNA sequences (e.g., 0.5 and 0.3 fM for microRNA-155 and osteopontin mRNA, respectively). In addition, such a method can also realize high selectivity and the detection of the two RNA sequences in diluted serum samples, indicating its potential application for accurate diagnosis of other diseases.