Visualization test strip reading of urine miRNA via cascade selective recognition reactions for non-invasive diagnosis of bladder cancer

This study proposed a homogeneous enzyme-free and nucleic acid-based catalytic hairpin assembly and cascade recognition reactions strategy that used calcein as the signal reporter for dual visualization (color and length) and fluorescence signal detection of bladder cancer marker miRNA-126 in clinical urine. This strategy was based on the ability of calcein and CuS nanoparticles (NPs) to recognize C-Ag+-C structure/Ag+/Cu2+/CuS NPs and C-Ag+-C structure/Ag+, respectively. Due to the integration of cascade amplification and material cation exchange reaction (CER), the sensitivity of homogeneous enzyme-free miRNA analysis at room temperature was significantly improved. This study achieved the limits of detections (LODs) as low as 0.25 aM of miRNA-126 and single-cell level of T24 cells with fluorescence mode. And the reading mode of color and length changes could detect miRNA and cells with concentrations as low as 5 aM and 1 cell/mL without instruments, respectively. And less reduction of fluorescence signal was observed when target miRNA was replaced with these base-mismatched RNAs. Then miRNA-126 in 66 clinical urine samples was quantified. The experimental results were consistent with the polymerase chain reaction (PCR) kit, computed tomography (CT), and pathological findings. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.954, and the assay showed 100% specificity and 90% sensitivity.