Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging

Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide, which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the nontargeted covalent labeling of bovine serum albumin using a fluorescently labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface-bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.