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Published on: December 23, 2022
A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small
Yi Li1, Fei Deng1, Ewa M Goldys1
1Graduate School of Biomedical Engineering and ARC Centre of Excellence in Nanoscale Biophotonics, University of New South Wales, Sydney, 2052, Australia; Australian Centre for NanoMedicine, University of New South Wales, Sydney, 2052, Australia.
CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE) technology enhances immunoassays for detecting non-nucleic acid targets. This innovation achieves attomolar sensitivity for small proteins, improving upon existing methods.
Area of Science:
- Biotechnology
- Biosensing
- Molecular Diagnostics
Background:
- CRISPR/Cas biosensing excels in nucleic acid detection but faces limitations for non-nucleic acid targets.
- Conventional immunoassays often lack the sensitivity required for detecting low-abundance analytes.
- There is a need for broadly applicable methods to enhance immunoassay sensitivity.
Purpose of the Study:
- To develop a universally applicable signal amplification strategy for immunoassays using CRISPR/Cas12a.
- To demonstrate attomolar detection limits for small proteins by integrating CRISPR/Cas12a with antibody-based assays.
- To create a simple and user-friendly approach to overcome sensitivity limitations in immunoassays.
Main Methods:
- Synthesized a hybrid conjugate of antibody and single-stranded DNA oligonucleotide.
- Integrated CRISPR/Cas12a-based signal amplification into various immunoassay formats (primary/secondary antibody).
- Validated the system using cytokine detection (IFN-γ, EGFR) in buffer and human plasma.
Main Results:
- Achieved attomolar (1 fg/mL) sensitivity for small protein detection.
- Demonstrated a 6-log linear dynamic range for analytes like IFN-γ and EGFR.
- Showcased a 1000-fold increase in sensitivity compared to a commercial ELISA kit for IFN-γ.
- Successfully detected IFN-γ in human plasma samples with high sensitivity and broad linear range.
Conclusions:
- CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE) effectively enhances immunoassay sensitivity for non-nucleic acid targets.
- CRUISE offers a simple, user-friendly, and broadly applicable strategy to achieve attomolar detection limits.
- This technology provides a valuable alternative for researchers needing improved sensitivity and detection ranges in immunoassays.
