Investigation of the effect of phytohormone on the expression of microRNA-159a in Arabidopsis thaliana seedlings based on mimic enzyme catalysis systematic electrochemical biosensor

摘要

MicroRNAs (miRNAs) play very important roles in plant growth and development as well as phyto- hormones. More importantly, microRNAs were recently found to be a new growth regulator involved in plant hormone signaling. Therefore, for investigating the expression change of microRNAs in plants exposed to phytohormones and understanding the effect of phytohormones on microRNAs expression, we developed a simple, sensitive, and label-free method for microRNAs biosensing based on mimic enzyme catalysis signal amplification, where carboxylic graphene-hemin hybrid nanosheets was synthesized and used to catalyze the oxidation reaction of hydroquinone in the presence of H_2O_2 due to the intrinsic peroxidase-like activity of hemin on the carboxylic graphene surface. The electrochemical reduction current of the oxidative product of benzoquinone was depended on the hybridization amount of microRNAs and used to monitor the microRNAs hybridization event. Under optimal detection conditions, the current response was proportional to the logarithm concentration of microRNA-159a from 0.5 pM to 1.0 nM with the detection limit of 0.17 pM (S/N=3). The fabricated biosensor showed highly reproducible (Relative standard deviation (RSD) was 3.53% for 10 biosensors fabricated indepen- dently) and detection selectivity (Even discriminating single-base mismatched microRNA sequence). We also found that abscisic acid, a kind of phytohormone, had greatly influence on microRNA-159a expression in Arabidopsis thaliana seedlings. With increasing abscisic acid concentration and prolonging incubation time, both the expression level of microRNA-159a increased. This graphene-hemin-based approach provides a novel avenue to detect microRNA with high sensitivity and selectivity while avoiding laborious label, disadvantages of bio-enzymes and complex operations for microRNAs separa- tion and enrichment, which might be attractive for genetic analysis and clinic biomedical application.