CRISPR/Cas13a Enabled Fluorescent and Electrochemiluminescent Strategies for Multiplex Detection of Alzheimer’s Associated Biomarkers

We have developed two CRISPR-Cas13a biosensing platforms for multiplexed detection of microRNAs (miRNAs). Both utilize Cas13a’s collateral RNase activity, activated upon target recognition, to cleave reporters/ activators and generate optical signals. A 3D-printed electrochemiluminescent (ECL) array with RuPVP films enables Cas13a mediated cleavage of poly-r-guanosine into short rG fragments, which act as efficient activators to enhance ECL output. We detected Alzheimer’s (ALZ) disease biomarkers miR-30e-5p, miR-34c-5p, and miR-200c-3p with limits of detection (LOD) at low or sub fg/mL across broad dynamic ranges, and offer portable, rapid, cost-effective approach for multiplex analysis. A fluorescence-based assay was also developed in a 3D-printed 96-well plate using immobilized Cas13a/crRNA complexes in hydrogel wells. Upon target recognition, cleavage of a quenched reporter generates fluorescence, yielding low fg/mL LODs with dynamic ranges up to 1800 pg/mL. Both assays are amplification-free, compatible with plasma, and distinguish ALZ-positive from ALZ-negative patients. Using these methods, we are actively developing CRISPR strategies to detect phosphorylated tau-proteins (pTau-181, pTau -217) that are excellent emerging ALZ biomarkers, alongside the miRNAs, creating a powerful diagnostic panel. These CRISPR platforms provide sensitive, amplification-free, and practical tools for biomarker quantification.