Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles

Engineered nanoparticles (NP) are generally contaminated by bacterial endotoxin, a ubiquitous bacterial molecule with significant toxic and inflammatory effects. The presence of endotoxin, if not recognised, can be responsible for many of the in vitro and in vivo effects attributed to NPs. The Limulus Amoebocyte Lysate (LAL) assay, the test requested by regulatory authorities for assessing endotoxin contamination in products for human use, is not immediately applicable for testing endotoxin in NP preparations, mainly due to the possible interference of NPs with the assay readouts and components. In this study, we have compared different commercially available LAL assays for detecting endotoxin in gold, silver and iron oxide NPs. Different NP chemistry, concentrations and surface coatings could differently interfere with the LAL assays' results. After accurate testing of the possible interaction/interference of NPs with the various assay components, the modified chromogenic LAL assay proved the most suitable assay for measuring endotoxin in NP samples, provided the appropriate controls are performed. Thus, endotoxin determination can be performed in NP preparation with commercial LAL assays only after assay validation, i.e. once possible interference of NPs with the assay components and readouts has been excluded.