Analytical Chemistry ARTICLE Figure 4. Eﬀect of dUSPION exposure on CyQUANT ﬂuorescence in an acellular system (n = 3). Figure 5. Eﬀect of dUSPION exposure on EthD-1 ﬂuorescence emis- sion in a cell free system (n = 3). colorimetric and ﬂuorometric dyes. However, in the present study, dextran alone without the MTS assay components did not increase absorbance levels at 490 nm (data not shown). An alternative cell viability test system, the CyQUANT assay is a sensitive technique used for the determination of cell num- bers in culture, and unlike MTT or MTS, this assay does not depend on cellular metabolic activity. This assay is based on the CyQUANT GR dye ﬂuorescing only when bound to cellular nucleic acids (DNA and/or RNA) in lysed cells. The current study shows that in a cell- free system increasing concentrations of maghemite or magnetite did not interfere with the assay (up to 100 μg/mL; Figure 4). Thus, this assay could be used as an alternative to the MTT or MTS assays. Similarly, neither form of dUSPION used in this study interfered with EthD-1 ﬂuorescence in a cell free system (Figure 5). Interestingly, as opposed to the other probes tested in this study, the function of which is reliant on chemical reactions, both CyQUANT and EthD-1 work through enhancing ﬂuorescence intensity upon binding to nucleic acids. While dUSPION appear to present limited interference when the assay is dependent on a physical change, such as binding to nucleic acids, it appears that if the assay relies on a chemical reaction, the dUSPION may be interacting with the assay components directly or may be interfering at some step in the chemical reaction. Fluorescence-based dyes are also key reporters for oxidative stress, for example the ﬂuorometric probe DCFH-DA is widely used for the detection of intracellular ROS.37,38 As illustrated in Figure 6a, there was a dose-dependent decrease in ﬂuorescence intensity of DCFH with increasing concentrations of magnetite, which was signiﬁcant at concentrations of 1, 10, and 100 μg/mL (p < 0.05; Figure 6a). TBHP and increasing concentrations of magnetite demonstrated a synergistic eﬀect with a dose dependent increase in ﬂuorescence, reaching signiﬁcance at 100 μg/mL of magnetite (p < 0.05; Figure 6a). Our results suggest that this Figure 6. Eﬀect of dUSPION on DCF ﬂuorescence response in an acellular system: (a) magnetite and (b) maghemite in the absence or presence of 2 mM NAC. Signiﬁcantly (*p < 0.05) diﬀerent relative to zero dUSPION control and signiﬁcantly (**p < 0.05) diﬀerent relative to zero dUSPION control with TBHP treatment. Signiﬁcantly (*p < 0.05) diﬀerent relative to zero dUSPION control, and signiﬁcantly (**p < 0.05) diﬀerent when comparing each dose of dUSPION treatment plus NAC to its relevant zero NAC control. n = 3 for each experiment. decrease in DCF signal is not due to any antioxidant eﬀect that magnetite may have, since magnetite did not reduce TBHP- induced increases in DCF ﬂuorescence. Thus, it is likely that in a cell free system magnetite quenches the ﬂuorescence response at higher doses, possibly through adsorption of the probe onto their surface. Somehow, in the presence of a ROS-inducer such as TBHP, this eﬀect is reversed. The present results again demon- strate the unpredictability of responses when using such assays to measure NM safety and the importance of performing preliminary tests to check for assay NM interactions. Interestingly, maghemite presented the opposite response, causing an increase in ﬂuorescence intensity from 1 μg/mL. At subsequent concentrations, the increase in ﬂuorescence signal was dramatic, equating to a 60-fold elevation at 10 μg/mL and a 40- fold increase in background ﬂuorescence when 100 μg/mL maghemite was used (Figure 6b). To determine whether this in- crease in signal was indeed caused by oxidative stress, the experi- ment was repeated in the presence of the antioxidant NAC. The maghemite-induced increase in ﬂuorescence signal at 1, 10, and 100 μg/mL was indeed found to be signiﬁcantly reduced (p < 0.05). Thus, demonstrating that maghemite induces oxidative stress in the acellular system (as opposed to interacting with the dye itself) which can be substantially reduced using NAC (Figure 6b). This suggests that maghemite has a much higher oxidative potential than magnetite. It is possible that in the same manner as magnetite, maghemite is able to quench ﬂuorescence response at low concentrations. However, due to its higher oxidative potential, at higher concentrations the massive increase in oxidative species production masks any ﬂuorescence quenching eﬀect that the maghemite may have, resulting in an overall increase in ﬂuorescence response 3782 dx.doi.org/10.1021/ac200103x |Anal. Chem. 2011, 83, 3778–3785