Bi-functional antibody-CRISPR/Cas12a ribonucleoprotein conjugate for improved immunoassay performance

Fei Deng; Yi Li; Tim Hall; Graham Vesey; Ewa M Goldys

doi:10.1016/j.aca.2023.341211

Bi-functional antibody-CRISPR/Cas12a ribonucleoprotein conjugate for improved immunoassay performance

Anal Chim Acta. 2023 Jun 8:1259:341211. doi: 10.1016/j.aca.2023.341211. Epub 2023 Apr 11.

Authors

Fei Deng¹, Yi Li², Tim Hall³, Graham Vesey³, Ewa M Goldys⁴

Affiliations

¹ Graduate School of Biomedical Engineering, University of New South Wales, Sydney, 2052, Australia; ARC Centre of Excellence in Nanoscale Biophotonics, University of New South Wales, Sydney, 2052, Australia. Electronic address: fei.deng@unsw.edu.au.
² Graduate School of Biomedical Engineering, University of New South Wales, Sydney, 2052, Australia; ARC Centre of Excellence in Nanoscale Biophotonics, University of New South Wales, Sydney, 2052, Australia. Electronic address: yi.li6@unsw.edu.au.
³ Graduate School of Biomedical Engineering, University of New South Wales, Sydney, 2052, Australia.
⁴ Graduate School of Biomedical Engineering, University of New South Wales, Sydney, 2052, Australia; ARC Centre of Excellence in Nanoscale Biophotonics, University of New South Wales, Sydney, 2052, Australia.

PMID: 37100476
DOI: 10.1016/j.aca.2023.341211

Abstract

Protein conjugates are commonly used in biochemistry, including diagnostic platforms such as antibody-based immunoassays. Antibodies can be bound to a variety of molecules creating conjugates with desirable functions, particularly for imaging and signal amplification. Cas12a is a recently discovered programable nuclease with the remarkable capability to amplify assay signals due to its trans-cleavage property. In this study, we directly conjugated antibody with Cas12a/gRNA ribonucleoprotein without the loss of function in either constituent. The conjugated antibody was suitable for immunoassays and the conjugated Cas12a was capable of amplifying the signal produced in an immunosensor without the need to change the original assay protocol. We applied the bi-functional antibody-Cas12a/gRNA conjugate to successfully detect two different types of targets, a whole pathogenic microorganism, Cryptosporidium, and a small protein, cytokine IFN-γ, with sensitivity reaching one single microorganism per sample and 10 fg/mL for IFN-γ, respectively. With simple substitution of the antibody conjugated with the Cas12a/gRNA RNP, this approach can potentially be applied to increase sensitivity of a variety of immunoassays for a broad range of different analytes.

Keywords: CRISPR/Cas12a biosensing; Immunoassay; Protein conjugation; User-friendly.

MeSH terms

Antibodies
Biosensing Techniques*
CRISPR-Cas Systems
Cryptosporidiosis*
Cryptosporidium*
Humans
Immunoassay
Immunoconjugates*
Ribonucleoproteins

Substances

Immunoconjugates
Antibodies
Ribonucleoproteins