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Stensberg et al. Page 9 useful for guiding future regulations, as there are no rules currently in place to control the commercialization of Ag NP enhanced products [112]. However, efforts to obtain this knowledge is limited by available technologies for monitoring physiological changes during Ag NP exposure. While semiquantitative physiological assessment methods are widely employed and still very useful, one major drawback is that they are destructive (i.e., organisms need to be sacrificed). This imposes significant limits on the amount and quality of information that can be obtained from an individual specimen during experimental trials. Over the last few decades the use of electrochemical microsensors and nanosensors has become more prominent for biological research applications [113]. Used in both intracellular and extracellular applications, these sensors have allowed us to measure analytes related to metabolism, stress and cell communication/signaling in ways which were not previously conceivable. Traditional use of nano/microsensors has involved penetration into cells/tissues or extracellular measurements along the surface of cells/ tissues. Intracellular micro- and nano-sensors have been shown to cause membrane damage and cytotoxicity, respectively [114]. While recent developments in intracellular nanosensors have allowed them to be used in minimally invasive formats [115], most extracellular micro/ nanosensors are still used invasively due to low signal-to-noise ratio and a lack of multidimensional spatial resolution. One extracellular technique that has alleviated these problems is the self-referencing microsensor technique. This sensor modality significantly improves signal-to-noise ratio and provides direct measurement of dynamic analyte flux, increasing spatial resolution with minimal sacrifice of temporal resolution [116]. While the use of microsensors in self- referencing modality has been around for decades [117], its potential for real-time physiological sensing has yet to be fully realized. The operation of sensors in self- referencing mode involves the oscillation of a single microsensor between two points, separated by a constant distance via computer-controlled stepper motors. Flux information can be obtained in real time by measuring Ag concentrations at each point, based on Fick’s first law of diffusion. The combination of a dc-coupled amplification scheme and oscillatory movement of a single electrode produces significant noise filtration and an increase in signal-to-noise ratio [116]. While there are currently no microsensors for the direct detection of Ag NPs, other sensors can be employed for monitoring physiological responses to Ag NPs. For instance, Ag+ can affect the uptake of H+ and various ions (e.g., Na+ and K+), simple sugars (e.g., glucose) and metabolic analytes necessary for cellular growth and development [118]. Self-referencing micro sensors can be used to monitor the real-time flux of glucose [119,120], glutamate [121], indoleacetic acid [122] and hydrogen peroxide. The latter case has been used to detect an increase in H2O2 efflux after exposing an excised, murine spinal cord to citrate-stabilized Ag NPs at 1 ppm (Figure 2). This release of H2O2 was most likely the result of oxidative stress induced by Ag NPs, in agreement with previous experiments [123,124]. While others have noted net increases in H2O2 efflux due to Ag NP exposure, the self-referencing microsensor technique provides a higher resolution of temporal and spatial data not obtainable with previous techniques. This high degree of resolution enables the use of metrics such as time to response, duration of response, peak efflux and total efflux, m easured as integrated flux (Table 2) [122]. Amperometric sensors have also been used with enzymes that produce electroactive species generated from redox reactions. For example, glucose oxidase converts glucose into gluconic acid and H2O2, which can be measured by amperometry [125]. Enzyme-based biosensors can also be used in the self-referencing modality near cell/tissue surfaces and measure the effect of Ag NPs on physiological transport of various analytes. 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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