The bioorthogonal cycloaddition reaction between tetrazine and trans-cyclooctene (TCO) is rapidly growing in use for molecular imaging and cell-based diagnostics. We have surprisingly uncovered that the majority of TCOs conjugated to monoclonal antibodies using standard amine-coupling procedures are non-reactive. We show that antibody-bound TCOs are not inactivated by trans-cis isomerization and that the bulky cycloaddition reaction is not sterically hindered. Instead, TCOs are likely masked by hydrophobic interactions with the antibody. We show that introducing TCO via hydrophilic polyethyleneglycol (PEG) linkers can fully preserve reactivity, resulting in >5-fold enhancement in functional density without affecting antibody binding. This is accomplished using a novel dual bioorthogonal approach in which heterobifunctional dibenzylcyclooctyne (DBCO)-PEG-TCO molecules are reacted with azido-antibodies. Improved imaging capabilities are demonstrated for different cancer biomarkers using tetrazine-modified fluorophore and quantum dot probes. We believe that the PEG linkers prevent TCOs from burying within the antibody during conjugation, which could be relevant to other bioorthogonal tags and biomolecules. We expect the improved TCO reactivity obtained using the reported methods will significantly advance bioorthogonal pretargeting applications.

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Single cell secretion studies have provided valuable insight into population heterogeneity, but low detection sensitivity requires cells to secrete thousands of molecules. We employed semiconductor quantum dots and a new single particle imaging approach to improve detection sensitivity to only a few secreted molecules per cell.