ECL-CRISPR array for multiplexed detection of miRNAs

We describe here an electrochemiluminescent (ECL) array for individually detecting 3 miRNAs utilizing CRISPR/Cas13a. Detection involves binding a target miRNA to Cas 13a protein that includes the RNA complement to the target, This activated Cas13a then cleaves a poly-RNA rich in r-Guanosine to produce electrochemiluminescent (ECL) activators that increases ECL output proportional to target miRNA concentration. Specifically, poly-r-guanosine (poly-r-G) is cleaved by the collateral RNase activity of Cas13a to generate small poly-r-G fragments that are efficient in activating ECL of (bis-2,2'-bipyridyl) ruthenium polyvinylpyridine ([Ru(bpy)₂PVP₁₀] (ClO₄)₂) (RuPVP) films on sensor electrodes at +1.1 V vs. Ag/AgCl. The 3D-printed array was used to detect three Alzheimer's disease (ALZ) miRNA biomarkers (30e-5p, 34c-3p and 200c-5p). ECL is generated in the 3D-printed array designed with reference, counter and four separate RuPVP sensor electrodes. Detection limits for miRNAs were 7.4 fg/mL to 7 pg/mL with high sensitivities in linear dynamic ranges from 70 pg/mL to 70 μg/mL. Limits of detection (LOD) were 42 pg/mL, 0.074 fg/mL, and 0.15 fg/mL for miR30e-5p, miR34c-5p, and miR200c-3p, respectively. Spike recovery studies and patient plasma analyses after RNA extraction gave high accuracy and specificity, and excellent correlation with a referee CRISPR fluorescence method.