Analytical Chemistry There are several examples in the literature of the ﬂuorescent probe DCFH-DA being used for the quantiﬁcation of oxidative stress induced by NM, as this is one of the primary mechanisms associated with adverse cellular responses to NM. Some exam- ples include iron oxide NP exposed to mesenchymal stem cells and HeLa (human cervival carcinoma) cells, SWCNT-induced ROS in HaCaT (human keratinocyte) cells, ambient ultraﬁne particles, cationic polystyrene nanospheres, TiO2, fullerol NP and carbon black in RAW 264.7 phagocytic cells, and silver nano- particles in human hepatoma and skin keratinocytes.4,39 42 It is not clear from these studies whether the possibility of confound- ing factors, which we have found to be associated with NM, have been taken into consideration, and whether the appropriate controls have been included. The distinct diﬀerence in the oxidative potential of maghemite and magnetite may be related to the oxidative state of the iron ions in the complex. In maghemite (Fe2O3) iron ions are mostly Fe3þ, while in magnetite (Fe3O4) they are a mixture of Fe3þ and Fe2þ with a Fe2þ/Fe3þ ratio of 0.43543 (Table 1). It is possible that Fe2þ surface ions undergo Fenton reaction by reacting with any H2O2 that may be available within the aqueous environment of the assay, to produce a hydroxyl radical. H2O2 can also react in a Fenton-like reaction with Fe3þ to generate [FeIIIOOH]2þ which can go on to generate OOH 3 , OH 3 , or OH , it has also been suggested that reaction of Fe3þ with H2O2 generates super- oxide. It is known that Fe2þ is more reactive than Fe3þ; however, in the present study it seems that the Fenton-like reaction in- volving Fe3þ is more potent. One possible explanation for this is the size of dUSPION agglomerates. Particle sizing using DLS suggests that in the buﬀer used for the DCFH-DA assay (Hepes- buﬀered (20 mM) Hanks balanced salt solution with glucose (5 mM)), magnetite forms bigger agglomerates than maghemite, thus magnetite would have less exposed surface area and poten- tially less ions available to react (Table 1). It is also possible that maghemite is a more stable molecule than magnetite and con- sequently does not release as many iron ions to react with the assay components. Fenton and Fenton-like reactions are known to generate diﬀerent ROS and intermediate species (see below).44 Thus, another possible explanation for the diﬀerences observed when using the DCFH-DA assay is that DCFH-DA may not be as sensitive in detecting ROS produced by magnetite as compared to ROS produced by maghemite. Fenton reaction Fe2þ Fe2þ þ H2O2 f Fe3þ þ OH þ OH 3 Fe2þ þ H2O2 f ½FeIV OH 2þ þ H2O Fenton-like reaction Fe3þ or or Fe3þ þ H2O2 f ½FeIIIOOH 2þ þ Hþ ½FeIIIOOH 2þ f Fe2þ þ OOH 3 ½FeIIIOOH 2þ f ½FeIV O 2þ þ OH 3 ARTICLE Figure 7. Eﬀect of dUSPION exposure on APF ﬂuorescence response in an acellular system, n = 3, signiﬁcantly (*p < 0.05) diﬀerent to untreated control. or ½FeIIIOOH 2þ f ½FeV O 3þ þ OH Similar to DCFH-DA, APF is a ﬂuorometric probe used for the detection of oxidative species. APF is oxidized by free radicals to yield a highly ﬂuorescent product which can be detected at 520 nm. Concentrations of maghemite and magnetite between 1 10 3 and 1 μg/mL did not have a signiﬁcant eﬀect on the resultant intensity of the APF ﬂuorescence signal. However, 10 and 100 μg/mL maghemite and 100 μg/mL magnetite caused a signiﬁcant decrease in background APF ﬂuorescence signal (Figure 7), which could be due to adsorption onto the surface of the NPs, thus quenching the ﬂuorescence response. This is in contrast to the results seen when using DCFH with maghemite, which reported increased oxidative stress at 1 100 μg/mL (Figure 6). This may be because APF is oxidized by fewer free radical species than DCFH as it is much more selective, only detecting the hydroxyl radical and peroxynitrite anion. Thus, APF may not be detecting the speciﬁc ROS produced by maghemite, which are possibly ROS products of Fenton-like reactions. The contrasting results obtained from using these two diﬀerent oxidative stress assays again highlights the diﬃculties and complexity of testing the safety of NM. Although this study sheds light on NP-USPION interactions in an acellular environment, one important point is that the undesirable interactions demonstrated in the present study may or may not be mimicked exactly in a cellular milieu. It is quite likely that these interactions will still occur in the culture media when the cells are exposed to NP and the test reagent as demon- strated by Zhang and colleagues, who have shown that the brown color of the USPION led to higher cell viability readings.45 Additionally factors in a cellular system, such as media compo- nents or cell debris, may modulate the interactions observed in the acellular system. Alternatively, intracellular masking of NP with proteins and other metabolites following cellular uptake could result in a dampened interaction. ’ CONCLUSIONS In conclusion, not all standard biological assays are compatible with dUSPION and this holds great signiﬁcance because these NM are routinely used in various biomedical applications sub- sequent to toxicity testing that utilizes colorimetric/ﬂuorometric probes such as those used in the current study. The present study shows that colorimetric assays such as MTS, and ﬂuorometric probes including calcein, DCFH-DA, and APF can interact with dUSPION especially at higher doses. Therefore, control 3783 dx.doi.org/10.1021/ac200103x |Anal. Chem. 2011, 83, 3778–3785