Analytical Chemistry

Bioconjugation of Electron-Probe Au25 Nanocluster to Monoclonal Antibody

Authors

Abstract

We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody without protein engineering, in a step toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the tryptophan (Trp)-selective bioconjugation step by using easy-to-analyze hydroxylamine (ABNOH) reagents in a pH-neutral buffer, instead of using N-oxyl radicals (ABNO) under acidic conditions as previously developed. This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure, utilizing first Trp-selective bioconjugation for homogeneous introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach bicyclononyne (BCN)-presenting, redox-sensitive Au25 nanocluster, was key to successful immunogold synthesis. This procedure is scalable. The covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates. In comparison with a non-homogenous variant prepared by lysine-selective bio-conjugation, Trp-selective conjugates exhibited both satisfactory gold cluster modification and minimal loss of antigen-binding ability.

Content

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Supplementary material

Thumbnail image of SI_Biconjugation of Au25 to trastuzumab.pdf
Supporting Information
Detailed experimental conditions and methods, Figure, and NMR charts of new compounds (PDF)