Bioluminescent enzyme inhibition-based assay to predict the potential toxicity of carbon nanomaterials

Abstract

A bioluminescent enzyme inhibition-based assay was applied to predict the potential toxicity of carbon nanomaterials (CNM) presented by single- and multi-walled nanotubes (SWCNT and MWCNT) and aqueous solutions of hydrated fullerene С₆₀ (C₆₀HyFn). This assay specifically detects the influence of substances on parameters of the soluble or immobilised coupled enzyme system of luminescent bacteria: NAD(P)Н:FMN-oxidoreductase+luciferase (Red+Luc). A protocol based on the optical properties of CNM for correcting the results of the bioluminescent assay was also developed. It was shown that the inhibitory activity of CNM on Red+Luc decreased in the following order: MWCNT>SWCNT>C₆₀HyFn. The soluble enzyme system Red+Luc had high sensitivity to MWCNT and SWCNT, with values of the inhibition parameter IC₅₀ equal to 0.012 and 0.16mg/L, respectively. The immobilised enzyme system was more vulnerable to C₆₀HyFn than its soluble form, with an IC₅₀ equal to 1.4mg/L. Due to its technical simplicity, rapid response time and high sensitivity, this bioluminescent method has the potential to be developed as a general enzyme inhibition-based assay for a wide variety of nanomaterials.

Keywords: Bioluminescence; Enzyme inhibition-based assay; Luciferase; Nanomaterials; Nanotoxicity; Nanotubes.