Analytical Chemistry ARTICLE Figure 1. TEM images of (a) maghemite and (b) magnetite. Figure 2. Eﬀect of dUSPION exposure on (a) MTS absorbance read- ings in a cell free system and (b) on absorbance in the absence of the MTS reagent, n = 3 signiﬁcantly (*p < 0.05) diﬀerent to untreated control. in background absorbance in an acellular system (p < 0.05) (Figure 2a). For investigating whether the optical properties of dUSPION have a direct eﬀect on absorbance readings, the absorbance of dUSPION alone at the wavelength required for the MTS assay was investigated. Interestingly, the results showed that 100 μg/mL of dUSPION are capable of signiﬁcantly increasing the absorbance readings compared to the control level in the absence of the MTS reagent (p < 0.05) (Figure 2b). The MTS assay is a simple and sensitive colorimetric method that has been used in the past to quantitate NM induced cyto- toxicity, including zinc oxide, titanium dioxide, and silica- and alkoxy silane coated iron oxide NP.35,36 However, in the current study it is clearly demonstrated that dUSPION induce a substantial increase in absorbance at the wavelengths required for the MTS assay, thereby severely confounding the sensitivity and speciﬁcity of the assay for quantifying cell viability in response to dUSPION exposure. This could potentially lead to misinterpretations of Figure 3. Eﬀect of dUSPION exposure on calcein ﬂuorescence in a cell free system, n = 3 signiﬁcantly (*p < 0.05) diﬀerent to untreated control. biological response. This suggests that MTS can be used to evaluate viability of cells treated with dUSPION only if it is taken into consideration that higher concentrations of dUSPION might aﬀect background signal. The present study is not alone in demonstrat- ing NM-induced tetrazolium-based assay interference. Studies have also shown that carbon nanotubes can adsorb another common tetrazolium compound, MTT, used for cytotoxicity studies, onto their surface leading to a quenching and an alteration in absorbance.11,12 However, the eﬀect in the case of dUSPION depends on the type of probe and the oxidation state of the dUSPION used. Incubation of dUSPION with the ﬂuorescent probe calcein (also frequently used to quantify cell viability), resulted in a dose- dependent decrease in the intensity of the resultant ﬂuorescent signal at 520 nm with 1 10 2 μg/mL maghemite, reaching sig- niﬁcance at 10 μg/mL (p < 0.05; Figure 3). A similar proﬁle was also observed with magnetite, but only the highest concentration (100 μg/mL) signiﬁcantly reduced the calcein ﬂuorescent signal in a cell free system (p < 0.05). The reduction in ﬂuorescence intensity observed at the higher dUSPION concentrations suggests quench- ing is induced by the NP that is independent of their oxidative status. The precise mechanism involved in the assay interferences observed is not well understood. It is evident that dUSPION alters the optical properties of the assay probes. It is known that carbon NM such as single walled carbon nanotubes (SWCNT) can adsorb dyes onto their surface, likely through van der Waals forces which subsequently quench or alter their absorbance or ﬂuorescent properties.11,34 It is not known if dUSPION adsorb colorimetric and ﬂuorometric dyes in the same manner as SWNCT. According to Worle-Knirsch et al. in 2006 and later veriﬁed by Casey et al. in 2007, SWNCT interact with insoluble MTT-formazan crystals that are formed after MTT reduction by cellular enzymes.11,34 However the present study was in a cell free system, thus dUSPION are unlikely to interact with MTS in exactly the same way as SWNCT interact with MTT. However, it is possible that dUSPION somehow adsorb calcein and MTS onto their surface leading to quenching of ﬂuorescence in the case of calcein and enhancement of absorbance readings in the case of MTS. Interestingly, a dUSPION solution alone examined without the MTS assay components signiﬁcantly increased absorbance readings at 490 nm to levels similar to that seen with the MTS assay components. This suggests that the increased MTS response is mostly due to the contribution of dUSPION’s own optical properties (Figure 2b). Potentially, factors such as surface chemistry, fabrication process, or types of surfactants used to disperse the NM (in this study, dextran) may also play a role in governing the interactions and degree of interference with 3781 dx.doi.org/10.1021/ac200103x |Anal. Chem. 2011, 83, 3778–3785