Electrogenerated Chemiluminescence Biosensor with a Tripod Probe for the Highly Sensitive Detection of MicroRNA

A novel probe for the highly sensitive detection of microRNA with enhanced helix accessibility and good assembling without backfilling was developed using a tripod structure fabricated by triplex DNA. A layer of triplex DNA assembled on electrodeposited reduced graphene oxide was used as the capture probe, and a subsequent hybridization chain reaction that promoted the efficient intercalation of the electrogenerated chemiluminescence (ECL) emitter [Ru(bpy)₂(dppz)]²⁺ (bpy refers to 2,2'-bipyridine, and dppz refers to dipyrido[3,2- a:2',3'- c]phenazine) was used as an analytical-signal amplifier. The fabricated biosensor was examined with an anodic ECL mode using tri- n-propyl amine as the coreactant. The construction of the biosensor was systematically characterized with various techniques including atomic-force microscopy, gel electrophoresis, cyclic voltammetry, and electrochemical-impedance spectroscopy, and its performance was optimized under a variety of experimental conditions, especially the concentration of each reagent as well as the incubation time. Under the optimal experimental conditions, the reported biosensor showed a very low limit of detection of 0.10 fM (S/N = 3) and a wide linear dynamic range covering 0.50 fM to 100 pM toward microRNA-155 with excellent specificity, stability, and reproducibility. Finally, the biosensor was successfully applied to the detection of microRNA-155 extracted from the colon-cancer cell line DLD1, demonstrating its potential application in the sensitive detection of biological samples in the early diagnosis of diseases.