Comparison of Gold Nanoparticle and Fluorophore-conjugated Antibodies for Labeling Epitopes in Permeabilized Cells

Immunocytochemistry (IHC) and immunofluorescence (IF) offer crucial diagnostic insights in clinical settings. Most IF assays are performed using fluorophore-conjugated antibodies, but these fluorophores can be subject to issues, such as photobleaching and autofluorescence, that result in lower signal-to-noise ratios (SNR). Gold nanoparticles provide greater signal stability and scatter light well, making them easily separable from the background of biological tissues and improving SNR. In this study, we sought to determine the labeling efficiency, signal quality, and image artifacts of 2.2, 10, and 40 nm diameter gold nanoparticle probes conjugated to antibodies in IF applications on fixed, permeabilized cells in comparison to traditional fluorophores. Overall, micrographs of nanoparticle labels had higher SNR due to lower background signal, and punctate appearances as compared with the continuously distributed signal of the immunofluorescent label. Signal-to-noise ratios varied with nanoparticle diameter, and signal fidelity was worse for keratin versus epithelial growth factor receptor (EGFR). Labeling of EGFR was successful using both extracellular and intracellular epitopes, while poor labeling of keratin 19 with 10 nm diameter nanoparticles was improved by pretreatment with heat and sonication, suggesting hindrance of nanoparticle labels within the fixed, permeabilized cell.