Co-reactant confined and vertically ordered silica nanochannel regulated electrochemiluminescence for homogeneous detection of miRNA

Electrochemiluminescence (ECL) arises from the collisional annihilation between the electro-excited luminophore and co-reactant. Most ECL-based bioassays require extensive free co-reactants that collide with luminophores randomly. Herein, the N,N-dimethylaniline (DMA) group was electro-grafted on the ITO electrode surface as the bottom co-reactant layer, and the vertically ordered mesoporous silica film (VMSF) was prepared as the upper layer to regulate the accessibility of Ru(bpy)(3)(2+). Thus, the dual-layer functionalized VMSF/N-ITO was newly built for the controllable ECL study. Relevant conditions, including the DMA electro-grafting, VMSF thickness, buffer concentration and pH value, were discussed to improve the ECL efficiency. Based on the VMSF/N-ITO platform, a homogeneous ECL bioassay was developed for miRNA-21 detection. The target nucleotide sequence induced the hybridization chain reaction (HCR) of probes H1 and H2 to absorb free Ru(bpy)(3)(2+) as much as possible. Residual Ru(bpy)(3)(2+) permeated the VMSF nanochannels to collide with the DMA co-reactant in producing an ECL response. There was a linear relationship between triangle ECL intensity and logarithmic concentration of miRNA-21 in the 1 fM similar to 100 pM range, and the detection limit was 0.25 fM. The developed approach was probes immobilization-free, highly sensitive, and anti-interference, opening a new avenue for ECL-based bioassays.