Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing

Abstract

The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery

“…First, potential nanoparticle-dye interferences must be considered to avoid false-positive and false-negative results. [26,27] No interference was observed measuring luminescence of ATP standards in the presence of liposomes ( Figure S1). Figure 1 Representative confocal-multiphoton images are shown in Figure 3a and results of image analysis of the sequestered z-stacks are given in Figure 3b.…”

Cytotoxicity of Group 5 Transition Metal Ditellurides (MTe2; M=V, Nb, Ta)

“…[39][40][41] As such, control experiments were performed in the absence of cells to check for possible sources of particle-induced interferences,t od etermine the suitability of the assays. Therefore, we set out to determinep ossible forms of particle-inducedi nterferences for two commonly used assays which have similar working mechanisms:W ST-8 and methyl thiazolyldiphenyl-tetrazoliumbromide (MTT) assays.…”

“…[10,35] The remaining cell viability of the A549 cellsa fter 24 hi ncubation with variousM Te 2 concentrations was measured and analysed using the water-soluble tetrazolium salt (WST-8) assay.F urthermore, controle xperiments were also conducted in the absence of cells to check for possible sources of particle-induced interferences between MTe 2 with cell viability assaym arkersw hichc ould cause false positive or false negative resultstob eproduced. [39][40][41] Results and Discussion…”

Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo

“…While false-negative results by the inhibition of LDH activity by Si, Au, and CdSe NPs 96 was avoided, the addition of BSA caused false-negative effects in protein detection via the Bradford reagent. 97 For the identification of potential assay interference, the incubation of NPs with the assay compounds alone (in the absence of cells) and with cells alone (in the absence of assay compounds) can be used. These controls are, however, only useful when the NPs interact with assay compounds and with the readout; assay interactions by Au NPs, which increased the detected amount of dead cells by shuttling the indicator dye, propidium iodide, into the cells, would not have been revealed.…”