Label-free sensing platform for miRNA-146a based on chromo-fluorogenic pyrophosphate recognition

In this study we applied the dual-responsive chromo-fluorescent Cu²⁺ chelate 1C for the recognition of miRNA-146a through a pyrophosphate (PPi) sensing strategy in a rolling circle amplification (RCA) process. This approach for the recognition of miRNA-146a was highly robust, selective, and sensitive down to the attomolar (fluorogenic) and sub-micromolar (chromogenic) ranges under modified biochemical conditions at elevated temperature. Probe 1 selectively recognized Cu²⁺ and PPi ions in a sequential manner, as evidenced by colorless→pink→colorless transitions; the fluorescence emissions centered at 480 nm underwent a corresponding on–off–on sequence in the bluish-green region. We attribute this reversible switching upon the addition of Cu²⁺/PPi ions to effective chelation-induced ligand-to-metal charge/electron transfer that resulted in opening of the lactam ring upon complexation and closing of the lactam ring upon decomplexation. We also report a label-free approach for monitoring miRNA-146a amplification in an RCA process under modified T4 ligase and ϕ₂₉ buffer conditions, using the Cu²⁺ ensemble 1C at pH 7.0 (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid: HEPES, 10 mM MgCl₂); the time required to perform this process (40–50 min) was relatively shorter than conventional RCA process. This ensemble 1C could recognize miRNA-146a colorimetrically (from pink to colorless) and fluorimetrically (“turn-on” mode) at concentrations within the highly sensitive atto-/nanomolar range under physiological conditions. This cost-effective label-free sensing strategy appears to be a universal method for detecting miRNAs according to the specified length of the template.