Multiplexed detection of miRNA and protein biomarkers for Alzheimer’s disease using CRISPR
Monday, March 3, 2025 10:00 AM to 12:00 PM · 2 hr. (America/New_York)
Expo Floor
Poster
Bioanalytical & Life Science
Information
Alzheimer’s Disease (AD) is a prevalent neurodegenerative disease without a permanent cure. Demand for fast, dependable diagnostics calls for mediating strategies. In response, we are developing low-cost, 3D printed point of care diagnostic arrays for AD, featuring multiplexity for a combination of miRNAs and protein biomarkers to be detected simultaneously. Our approach uses innovative CRISPR/ cas13a technology coupled to electro chemiluminescent (ECL) and fluorescent (FL) detection techniques. The unique ability of CRISPR RNA to bind target RNAs and activate an indiscriminate collateral cleavage of an RNA substrate is employed to detect target miRNAs 30e-5p, 34c-5p and 200c-3p. For ECL detection, poly-r-guanine (poly-rG) or poly-r-guanine-r-adenine (poly-rGA) is added to the sample, and CRISPR activity cleaves it into small RNA fragments that act as efficient activators for ECL signal, generated by a bis-2,2’-bipyridyl ruthenium polyvinyl pyridine dye coated in multiple layers on pyrolytic graphite electrodes. A FL probe with a fluorophore coupled to a quencher via a short RNA strand provides detection for the FL assay. Target protein (e.g. pTau-181) detection employs secondary antibodies (Ab2) and miRNA mimics attached to polymer coated Silica nanoparticles (SiNP). pTau-181 in patient samples bind to Ab2 resulting in a miRNA-SiNP-Ab2-pTau-181 immunocomplex which is separated by a subsequent incubation with capture/ primary antibody (Ab1) on magnetic beads (MB). miRNA-SiNP-Ab2-pTau-181-Ab1-MB complex along with miRNAs in the sample are the analytes for ECL and FL assays. ECL signals are captured by a CDC camera in a dark box and a plate reader measure signals for the FL assay to generate calibration plots for subsequent sample analyses. Spike recovery studies verify the validity, accuracy and efficiency of the assay while specificity and clinical relevance will be verified by analyzing patient plasma samples and comparing results to referee PCR and immunoassays.
Day of Week
Monday
Poster Format
SEAC Poster Abstract
Session Number
PS-S05
Application
Bioanalytical
Methodology
Fluorescence and Luminescence
Primary Focus
Methodology
Morning or Afternoon
Morning
Poster Co-Authors
Co-Authors
Rachelle Canete - Department of Chemistry, University of Connecticut, James F. Rusling - Department of Chemistry, University of Connecticut, Institute of Materials Science, University of Connecticut, School of Chemistry, University of Galway, Department of Surgery and Neag Cancer Center, UConn Health, Sherif M. Shawky - Department of Chemistry, University of Connecticut, P.I. Thilini De Silva - Department of Chemistry, University of Connecticut, Oscar Clement - Department of Chemistry, University of Connecticut, Palash Vora - Department of Chemistry, University of Connecticut, Jessica L. Rouge - Department of Chemistry, University of Connecticut, Keshani Hiniduma - Department of Chemistry, University of Connecticut
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