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Stensberg et al. Page 10 Another type of microsensor that can be used in a self-referencing modality is the ion- selective electrode (ISE), which can detect a vast array of biologically relevant cations [125]. With regard to Ag NP and Ag+ toxicity, self-referencing ISEs have been used to evaluate toxicity based on the aberrant flux of Na+ and H+ [118]. For example, H+ efflux was measured from Daphnia magna embryos dosed with silver nitrate (Figure 3a) and Ag NPs (Figure 3b). Increases in time to response, peak response and total integrated flux were observed for Ag NP exposure when compared with embryos exposed to Ag+ (Table 3). A micro-ISE for Ag+ has been recently developed and demonstrated in self-referencing mode, with detection limits below 100 nm [126]. The sensor can monitor the rate of Ag+ uptake without interference from Ag NPs (Figure 4) and is thus particularly useful for separating the physiological effects of Ag+ from those of Ag NPs. In particular, any physiological response that does not correlate directly with Ag+ influx can be attributed specifically to Ag NP toxicity. The ability to noninvasively segregate the effects of Ag NPs and Ag+ on physiological transport will be crucial for establishing a mode of action for Ag NPs. Finally, optical microsensors have been used in a self-referencing modality [127,128]. These have some advantages over electrochemical sensors, as they are relatively unaffected by electrical or mechanical noise. The self-referencing optical microsensors have been used to measure real-time O2 flux as a metric for physiological stress in fathead minnows, exposed to several environmental contaminants [129]. However, preliminary data on Daphnia magna embryos exposed to Ag NPs indicate little or no effect on O2 consumption (Figure 5 & Table 4). Biological imaging with Ag NPs Silver nanoparticles exhibit a strong optical activity due to plasmon resonance, an electrodynamic phenomenon based on the excitation of conduction electrons at specific frequencies of light [12]. These plasmon resonances enhance the detection and tracking of Ag NPs by a number of optical imaging methods, from simple light scattering to multiphoton luminescence, surface-enhanced Raman scattering (SERS) and in vivo biomedical imaging modalities. With major exception to the darkfield imaging studies, most of these cases can be considered exploratory in their use of Ag NPs, but also demonstrate their potential utility in the design of in vitro and in vivo studies that address downstream toxicological effects. Ultimately, the accumulation of toxicological data will determine the scope and limitations in developing Ag NPs as imaging agents for clinical use. On the other hand, these novel imaging tools present opportunities for tracing the various pathways and fates of Ag NPs in biological systems. Ag NPs in optical darkfield microscopy Colloidal Ag NPs are widely recognized as optical labels for biosensing and imaging applications based on light scattering [12]. Ag NPs below 50 nm typically support strong extinctions between 400 and 500 nm (blue–green region of the visible spectrum), although individual NPs as small as 2.6 nm can be detected under optimal conditions [130]. The plasmon resonances can shift toward longer wavelengths if the particles are larger than 50 nm or are anisotropic in shape [131]. This wavelength-dependent scattering enables Ag NPs to be distinguished according to their physical characteristics allowing independent tracking of NP uptake rate as a function of size or shape. Xu and coworkers have investigated the uptake of Ag-coated gold NPs by several different organisms using darkfield optical imaging [132]. In one such study involving Pseudomonas aeruginosa, an opportunistic bacterial pathogen, the active uptake and efflux of single NPs as large as 80 nm in diameter were monitored with transport times ranging from minutes to hours, depending on the Nanomedicine (Lond). Author manuscript; available in PMC 2012 May 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptPDF Image | Toxicological studies on silver nanoparticles
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