A compact nanobody-DNAzyme conjugate enables antigen detection and signal amplification.

Hemin-utilizing G-quadruplex DNAzymes with peroxidase-like (POX) activity are widely used as signal reporters in biosensing technology. However, their application to protein detection has been mostly limited to sandwich-type assays involving streptavidin or nanoparticles as indirect bridging platforms between DNAzymes and antibodies. Herein, we describe the generation of a compact, covalently DNAzyme-labeled nanobody which was successfully tested in a direct enzyme-linked immunosorbent assay (ELISA). The conjugation approach was based on the self-labeling protein tag mVirD2, a truncated bacterial relaxase able to covalently bind DNA with 1:1 stoichiometry at a specific amino acid residue. The hybrid molecule combined the nanobody antigen binding affinity and specificity with the DNAzyme catalytic capability to oxidize peroxidase substrates (e.g. ABTS, H2O2). The proposed strategy is simple and cost-effective, enables development into multiplex formats and provides reagents with hitherto unmet reproducibility in terms of POX activity instrumental for both colorimetric and electrochemical reactions. As a proof-of-concept, it was demonstrated that DNAzyme-nanobody conjugates are convenient immunoreagents for rapid and specific detection of the toxic alga Alexandrium minutum.