Ultrasensitive, Colorimetric Detection of MicroRNAs based on Isothermal Exponential Amplification Reaction-Assisted Gold Nanoparticle Amplification

• 1、State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237

Abstract：: MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are linked to a large number of human diseases and disorders. In this work, we developed a colorimetric method for rapid, ultrasensitive miRNA detection via isothermal exponential amplification reaction (EXPAR)-assisted gold nanoparticle (AuNP) amplification. The sensing probe designed with a tandem phosphorothioate modification in the backbone of the polyadenines at the 5' terminus was employed to directly assemble onto the surface of AuNP with high adsorption affinity. The recognition domain at the 3' terminus of the sensing probe hybridizes with target miRNAs to trigger EXPAR with exponential signal amplification. With the amplification reaction with the action of DNA polymerase, the sensing probe gradually detaches from the AuNP, resulting in the aggregation of bare AuNPs in the high-salt reaction environment due to lack of DNA protection. The presence of AuNP aggregation is conveniently measured by UV-vis spectroscopy. Our proposed method could provide a linear detection range from 50 fM to 10 nM with a detection limit of 46 fM within 60 min, and also discriminate a single-nucleotide difference between homologous miRNAs.

Keywords： MicroRNA detection Colorimetric method Isothermal exponential amplification reaction Gold nanoparticle Phosphorothioate modification