Tetrazine Ligation on Semiconductor Quantum Dots Requires Specific Surface Coating and Tagging Architectures

The tetrazine ligation, an inverse electron-demand Diels-Alder reaction between tetrazine and a dienophile such as norbornene (Nb) or <i>trans</i>-cyclooctene (TCO), has characteristics that make it a potentially ideal chemistry for the preparation of nanoparticle bioconjugates. Although this click chemistry has been used for this purpose, relatively little is known about how to optimize nanoparticle surface chemistry and tagging with reactive functional groups to maximize the chances of successful conjugation. Here, we addressed this open question with quantum dots (QDs) by preparing and testing a panel of coatings for tetrazine ligation with a small-molecule fluorescent dye and an immunoglobulin G antibody. These coatings included Nb-appended dithiol-anchored ligands based on a small-molecule design, a poly(ethylene glycol) (PEG) oligomer, and multiple dextran variants. Additional coatings included a PEGylated amphiphilic polymer and a PEGylated dithiol-anchored coordinating polymer, both of which were modified with TCO, Nb, or tetrazine groups. The Nb-appended ligands lost their reactivity between bulk solution and binding to the QDs, whereas both types of polymer-coated QD were reactive toward a small-molecule fluorescent dye, regardless of the click-reactive group tagged on the PEG. However, the efficiency of the ligation of both polymer coatings with antibodies was highly dependent on whether the dienophile was Nb or TCO, whether this group was tagged on the QDs or the antibodies, and if the polymer was amphiphilic or coordinating. Immunofluorescent labeling of a cancer cell line revealed that the greater versatility of the coordinating polymer coating for ligation came at the expense of substantial nonspecific binding to cells, which was avoided by the amphiphilic polymer coating. These observations and trends are discussed to arrive at recommendations for maximizing the probability of successful tetrazine ligation to form bioconjugates from QDs and other colloidal nanoparticles.