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Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF- α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production

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ABSTRACT

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

No MeSH data available.


Endotoxin detection in GBMs using the endpoint chromogenic LAL assay.GBMs (50 μg/ml) were incubated with LAL (containing enzyme), or substrate, or both LAL and substrate. After the incubation period, the absorbance of the substrate was measured. The data showed significant LPS contamination in GO-D and FLG, while assay interference was observed in the case of GO-A and GO-B.
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pone.0166816.g002: Endotoxin detection in GBMs using the endpoint chromogenic LAL assay.GBMs (50 μg/ml) were incubated with LAL (containing enzyme), or substrate, or both LAL and substrate. After the incubation period, the absorbance of the substrate was measured. The data showed significant LPS contamination in GO-D and FLG, while assay interference was observed in the case of GO-A and GO-B.

Mentions: Following synthesis/procurement and characterization of the GBM samples, their endotoxin levels were measured using standard methods, i.e., different formats of the LAL assay (see Methods). The endpoint chromogenic LAL assay results showed similar absorbance in cases of GO-A and GO-B with or without the presence of LAL and substrate (Fig 2). This indicated false positive results for these GO samples, due to optical interference with the LAL assay at the detection wavelength. GO-C displayed very low endotoxin levels in the endpoint chromogenic LAL assay (Fig 2) and it could therefore not be concluded whether or not there is any interference with the assay. On the other hand, GO-D and FLG displayed high levels of endotoxin in this assay, i.e., 1.5 EU/ml and 1.4 EU/ml, respectively, and low intrinsic material absorbance at the wavelength used (405 nm).


Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF- α Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production
Endotoxin detection in GBMs using the endpoint chromogenic LAL assay.GBMs (50 μg/ml) were incubated with LAL (containing enzyme), or substrate, or both LAL and substrate. After the incubation period, the absorbance of the substrate was measured. The data showed significant LPS contamination in GO-D and FLG, while assay interference was observed in the case of GO-A and GO-B.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120825&req=5

pone.0166816.g002: Endotoxin detection in GBMs using the endpoint chromogenic LAL assay.GBMs (50 μg/ml) were incubated with LAL (containing enzyme), or substrate, or both LAL and substrate. After the incubation period, the absorbance of the substrate was measured. The data showed significant LPS contamination in GO-D and FLG, while assay interference was observed in the case of GO-A and GO-B.
Mentions: Following synthesis/procurement and characterization of the GBM samples, their endotoxin levels were measured using standard methods, i.e., different formats of the LAL assay (see Methods). The endpoint chromogenic LAL assay results showed similar absorbance in cases of GO-A and GO-B with or without the presence of LAL and substrate (Fig 2). This indicated false positive results for these GO samples, due to optical interference with the LAL assay at the detection wavelength. GO-C displayed very low endotoxin levels in the endpoint chromogenic LAL assay (Fig 2) and it could therefore not be concluded whether or not there is any interference with the assay. On the other hand, GO-D and FLG displayed high levels of endotoxin in this assay, i.e., 1.5 EU/ml and 1.4 EU/ml, respectively, and low intrinsic material absorbance at the wavelength used (405 nm).

View Article: PubMed Central - PubMed

ABSTRACT

Nanomaterials may be contaminated with bacterial endotoxin during production and handling, which may confound toxicological testing of these materials, not least when assessing for immunotoxicity. In the present study, we evaluated the conventional Limulus amebocyte lysate (LAL) assay for endotoxin detection in graphene based material (GBM) samples, including graphene oxide (GO) and few-layered graphene (FLG). Our results showed that some GO samples interfered with various formats of the LAL assay. To overcome this problem, we developed a TNF-α expression test (TET) using primary human monocyte-derived macrophages incubated in the presence or absence of the endotoxin inhibitor, polymyxin B sulfate, and found that this assay, performed with non-cytotoxic doses of the GBM samples, enabled unequivocal detection of endotoxin with a sensitivity that is comparable to the LAL assay. FLG also triggered TNF-α production in the presence of the LPS inhibitor, pointing to an intrinsic pro-inflammatory effect. Finally, we present guidelines for the preparation of endotoxin-free GO, validated by using the TET.

No MeSH data available.