A portable and quantitative detection of microRNA-21 based on cascade enzymatic reactions with dual signal outputs

MicroRNAs (miRNAs) are physiological status-related molecules which can be used as biomarkers for diseases, such as cancers. The point-of-care testing (POCT) of miRNAs has great application potential in early diagnosis and process monitoring of diseases. In this paper, a fast and dual signal outputs detection for microRNA-21 (miRNA-21) was established by using both personal glucose meter (PGM) and fluorescence spectrometer. In such an assay protocol, a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate (AMP). The hairpin structure can be specifically degraded by exonuclease T (Exo T) after hybridization with the target miRNA-21, releasing a large amount of AMP as the reporter. Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions. The machinery includes two parts: an adenosine triphosphate (ATP) generation system and a glucose consumption/NADPH production system. The produced AMP in the former step triggers the production of ATP, and subsequently the consumption of glucose and the production of NADPH. The changes of both glucose and NADPH are proportional to the concentration of miRNA-21, and can be determined by PGM and fluorescence spectrometer, respectively. Besides, the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions. Under the optimal experimental conditions, the PGM signal is linearly correlated with the concentration of miRNA-21 in the range from 5 to 150 nM, with the limit of detection (LOD) of 3.65 nM. The LOD of fluorescence detection mode is even lowered to 0.03 nM. The miRNA-21-spiked serum samples, as well as the actual serum samples from cancer patients, have been successfully detected by this detection strategy. Thus the established assay provides a POCT solution for cancer diagnosis and prognosis.