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Biofilm detachment often has detrimental effects such as pipe obstruction and infection, yet the detachment mechanisms underlying dispersal remain largely unknown. In this study, a stress response mechanism known as glutathione-gated potassium efflux (GGKE) was evaluated as an active detachment mechanism in the dispersal of Pseudomonas aeruginosa biofilms. N-ethylmaleimide (NEM) was used to activate potassium efflux proteins (Kef) associated with the GGKE pathway. This stress response mechanism was hypothesized to lead to altered cation concentration, which can potentially affect polymer bridging in biofilms, and ultimately cause biofilm detachment. Results showed the activation of GGKE by NEM exposure caused biofilm detachment without inducing a measurable change in viability, and detached biomass concentration and composition were dependent on NEM concentration. More detached biomass was observed with higher concentrations of NEM, with a trend of increasing polymer detachment. The detachment was likely resulting from a weakened biofilm structural integrity induced by bridge denaturing from GGKE activation. This study is important in understanding biofilm detachment from engineered systems such as membrane aerated bioreactors.

Silver has many diverse applications, including use as a biocide for biomedical devices and potable water systems. Increased use of silver has led to a demand for technologies which can measure dissolved species in situ. In addition to the development of tools such as electrodes for measuring dissolved silver concentration, techniques are needed which can quantify spatially and temporally dynamic transport of silver. For the first time, we demonstrate the use of a self-referencing Ag+-selective microelectrode for non-invasively quantifying Ag+ flux in biomedical and environmental applications. Characterization of sensor performance included detailed analysis of microelectrode sensitivity, selectivity, response time, limit of detection, and linear range. Liquid membrane microelectrode performance was similar to modern solid state electrodes, and in self-referencing modality differential signal acquisition was sufficient for use in a wide range of applications. Self-referencing microelectrodes were used to profile concentration boundary layer transport, providing a “snapshot” of microgradients in the unstirred boundary layer. In addition, time resolved, non-invasive measurements were used to quantify Ag+ release rate (efflux) from two common antimicrobial biomedical devices, and Ag+ uptake rate (influx) to a mature Pseudomonas aeruginosa biofilm during disinfection. Use of this technique will increase our understanding of silver transport in engineered antimicrobial devices and systems. In addition, the technique may be used to better our understanding of Ag+ release from nanoparticles.

Embryos, unlike adults, are typically sessile, which allows for an increase in the available metrics that can be used to assess chemical toxicity. We investigate Daphnia magna development rate and oxygen consumption as toxicity metrics and compare them to arrested embryo development using four different techniques with potassium cyanide (KCN) as a common toxicant. The EC50 (95 % CI) for arrested development was 2,535 (1,747–3,677) μg/L KCN. Using pixel intensity changes, recorded with difference imaging, we semi-quantitatively assessed a decrease in development rate at 200 μg/L KCN, threefold lower than the arrested development lowest observed effect concentration (LOEC). Respirometry and self-referencing (SR) microsensors were two unique techniques used to assess oxygen consumption. Using respirometry, an increase in oxygen consumption was found in the 5 μg/L KCN treatment and a decrease for 148 μg/L, but no change was found for the 78 μg/L KCN treatment. Whereas, with SR microsensors, we were able to detect significant changes in oxygen consumption for all three treatments: 5, 78, and 148 μg/L KCN. While SR offered the highest sensitivity, the respirometry platform developed for this study was much easier to use to measure the same endpoint. Oxygen consumption may be subject to change during the development process, meaning consumption assessment techniques may only be useful only for short-term experiments. Development rate was a more sensitive endpoint though was only reliable four of the six embryonic developmental stages examined. Despite being the least sensitive endpoint, arrested embryo development was the only technique capable of assessing the embryos throughout all developmental stages. In conclusion, each metric has advantages and limitations, but because all are non-invasive, it is possible to use any combination of the three.

Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9–15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.

Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag(+) and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO3 and Ag NPs (130-650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag(+). This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag(+) was more efficient than Ag NPs at displacing Na(+) within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.

A novel method for spatially controlled layer-by-layer assembly of enzyme-based amperometric biosensors was developed for multi-analyte sensing with high spatial and temporal resolution. Stereo-electrodes with 75 μm tip separation and 1–3 μm tip size were functionalized using a simple three-step electrodeposition scheme to produce biosensors for simultaneous detection of glucose and lactate. Layers of nanostructured platinum, enzyme-doped conductive polymer poly(3,4-ethylenedioxythiophene) [PEDOT] and non-conductive polymer poly(o-aminophenol) [PoAP] were electrodeposited to achieve increased electroactive surface area, fine control over site and quantity of enzyme, and reduced interference. The resulting dual-electrode sensors showed good linear range, fast response times and negligible crosstalk. The proposed scheme can be easily expanded to multi-electrode systems and is adaptable to lab-on-chip platforms for a wide range of analytes.

Silver nanoparticles (Ag NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products may lead to an increase in toxic levels of environmental silver, but regulatory control over the use or disposal of such products is lagging due to insufficient assessment on the toxicology of Ag NPs and their rate of release into the environment. In this article we discuss recent research on the transport, activity and fate of Ag NPs at the cellular and organismic level, in conjunction with traditional and recently established methods of nanoparticle characterization. We include several proposed mechanisms of cytotoxicity based on such studies, as well as new opportunities for investigating the uptake and fate of Ag NPs in living systems.

Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental (zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily applied to any field of science concerned with bridging the gap between form and function.