Measuring endotoxin contamination in NP preparations. As preliminary step, evaluation of the interference of Au NPs on the pNA readings at 405 nm was performed. Different concentrations of Au NPs (4, 13, and 20 nm diameter) could significantly increase the readout at OD405 of 125 μM pNA (round symbols) (p < 0.05 for all concentrations of NPs 4 nm, for the three highest of NPs 13 nm, and for the highest of NPs 20 nm) (upper left panel). The selected pNA concentration corresponds to that developed by 0.6 EU of endotoxin in the Endpoint Chromogenic LAL assay (lower left panel). The increase caused by NPs could therefore be misinterpreted as a significant increase in the presence of endotoxin. For this reason, endotoxin evaluation was then performed only on NP dilutions that did not cause significant interference with the pNA readings (typically, ≤ 1 μg/ml for Au NPs 4 nm, ≤ 4 μg/ml for Au NPs 13 nm, and ≤ 12.5 μg/ml for Au NPs 20 nm). Five separate batches of Au NPs 4 nm and their solvents were tested. For dry NPs, the solvent was endotoxin-free PBS. Batch-to-batch variability in the endotoxin contamination was evident (upper right panel). The importance of avoiding such contamination is shown by the powerful effect of minute amounts of endotoxin in activating IL-1β gene expression in human monocytes (lower right) (p < 0.05 for all endotoxin concentrations vs. control; square symbols).