Pulmonary toxicity and translocation of gallium phosphide nanowires to secondary organs following pulmonary exposure in mice.

BACKGROUND: III-V semiconductor nanowires are envisioned as being integrated in optoelectronic devices in the near future. However, the perspective of mass production of these nanowires raises concern for human safety due to their asbestos- and carbon nanotube-like properties, including their high aspect ratio shape. Indeed, III-V nanowires have similar dimensions as Mitsui-7 multi-walled carbon nanotubes, which induce lung cancer by inhalation in rats. It is therefore urgent to investigate the toxicological effects following lung exposure to III-V nanowires prior to their use in industrial production, which entails risk of human exposure. Here, female C57BL/6J mice were exposed to 2, 6, and 18 µg (0.12, 0.35 and 1.1 mg/kg bw) of gallium phosphide (III-V) nanowires (99 nm diameter, 3.7 µm length) by intratracheal instillation and the toxicity was investigated 1, 3, 28 days and 3 months after exposure. Mitsui-7 multi-walled carbon nanotubes and carbon black Printex 90 nanoparticles were used as benchmark nanomaterials. RESULTS: Gallium phosphide nanowires induced genotoxicity in bronchoalveolar lavage cells and acute inflammation with eosinophilia observable both in bronchoalveolar lavage and lung tissue (1 and 3 days post-exposure). The inflammatory response was comparable to the response following exposure to Mitsui-7 multi-walled carbon nanotubes at similar dose levels. The nanowires underwent partial dissolution in the lung resulting in thinner nanowires, with an estimated in vivo half-life of 3 months. Despite the partial dissolution, nanowires were detected in lung, liver, spleen, kidney, uterus and brain 3 months after exposure. CONCLUSION: Pulmonary exposure to gallium phosphide nanowires caused similar toxicological effects as the multi-walled carbon nanotube Mitsui-7.

Use of dielectrophoresis for directing T cells to microwells before nanostraw transfection: modelling and experiments.

Nanostraw substrates have great potential for achieving minimally invasive cell transfection. Cells located on the nanostraw substrate are subjected to mild DC electric pulses applied across the nanostraw substrate, which open pores in the cell membrane on top of the nanostraws and drives charged cargo through these pores via electrophoresis. However, with this method, the current may leak through uncovered nanostraws, thereby decreasing the desired effect in the cell-covered nanostraws. A minimization of the number of uncovered nanostraws could be achieved by high cell coverage, but this is challenging when working with small cell populations. Nanostraw substrates of smaller area could be covered by smaller cell populations but are hard to integrate into fluidics systems. Here, we use simulations and experiments to show that this issue can be addressed by covering the nanostraw substrate with an insulating layer containing pores of similar size to cells. The pores act as traps into which cells can be guided using dielectrophoresis, ensuring a high degree of occupancy while maintaining a high cell viability, even if the total number of cells is low.

Uptake of nanowires by human lung adenocarcinoma cells.

Semiconductor nanowires are increasingly used in optoelectronic devices. However, their effects on human health have not been assessed fully. Here, we investigate the effects of gallium phosphide nanowires on human lung adenocarcinoma cells. Four different geometries of nanowires were suspended in the cell culture for 48 hours. We show that cells internalize the nanowires and that the nanowires have no effect on cell proliferation rate, motility, viability and intracellular ROS levels. By blocking specific internalization pathways, we demonstrate that the nanowire uptake is the result of a combination of processes, requiring dynamin and actin polymerization, which suggests an internalization through macropinocytosis and phagocytosis.

Biosensing using arrays of vertical semiconductor nanowires: mechanosensing and biomarker detection.

Due to their high aspect ratio and increased surface-to-foot-print area, arrays of vertical semiconductor nanowires are used in numerous biological applications, such as cell transfection and biosensing. Here we focus on two specific valuable biosensing approaches that, so far, have received relatively limited attention in terms of their potential capabilities: cellular mechanosensing and lightguiding-induced enhanced fluorescence detection. Although proposed a decade ago, these two applications for using vertical nanowire arrays have only very recently achieved significant breakthroughs, both in terms of understanding their fundamental phenomena, and in the ease of their implementation. We review the status of the field in these areas and describe significant findings and potential future directions.

Nanowires for Biosensing: Lightguiding of Fluorescence as a Function of Diameter and Wavelength.

Semiconductor nanowires can act as nanoscaled optical fibers, enabling them to guide and concentrate light emitted by surface-bound fluorophores, potentially enhancing the sensitivity of optical biosensing. While parameters such as the nanowire geometry and the fluorophore wavelength can be expected to strongly influence this lightguiding effect, no detailed description of their effect on in-coupling of fluorescent emission is available to date. Here, we use confocal imaging to quantify the lightguiding effect in GaP nanowires as a function of nanowire geometry and light wavelength. Using a combination of finite-difference time-domain simulations and analytical approaches, we identify the role of multiple waveguide modes for the observed lightguiding. The normalized frequency parameter, based on the step-index approximation, predicts the lightguiding ability of the nanowires as a function of diameter and fluorophore wavelength, providing a useful guide for the design of optical biosensors based on nanowires.

Single cell analysis of proliferation and movement of cancer and normal-like cells on nanowire array substrates.

Nanowires are presently investigated in the context of various biological and medical applications. In general, these studies are population-based, which results in sub-populations being overlooked. Here, we present a single cell analysis of cell cycle and cell movement parameters of cells seeded on nanowires using digital holographic microscopy for time-lapse imaging. MCF10A normal-like human breast epithelial cells and JIMT-1 breast cancer cells were seeded on glass, flat gallium phosphide (GaP), and on vertical GaP nanowire arrays. The cells were monitored individually using digital holographic microscopy for 48 h. The data show that cell division is affected in cells seeded on flat GaP and nanowires compared to glass, with much fewer cells dividing on the former two substrates compared to the latter. However, MCF10 cells that are dividing on glass and flat GaP substrates have similar cell cycle time, suggesting that distinct cell subpopulations are affected differently by the substrates. Altogether, the data highlight the importance of performing single cell analysis to increase our understanding of the versatility of cell behavior on different substrates, which is relevant in the design of nanowire applications.

Parallel computation with molecular-motor-propelled agents in nanofabricated networks.

The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.

Surface nanostructures for fluorescence probing of supported lipid bilayers on reflective substrates.

The fluorescence interference contrast (FLIC) effect prevents the use of fluorescence techniques to probe the continuity and fluidity of supported lipid bilayers on reflective materials due to a lack of detectable fluorescence. Here we show that adding nanostructures onto reflective surfaces to locally confer a certain distance between the deposited fluorophores and the reflecting surface enables fluorescence detection on the nanostuctures. The nanostructures consist of either deposited nanoparticles or epitaxial nanowires directly grown on the substrate and are designed such that they can support a lipid bilayer. This simple method increases the fluorescence signal sufficiently to enable bilayer fluorescence detection and to observe the recovery of fluorescence after photobleaching in order to assess lipid bilayer formation on any reflective surface.

Nanowire-imposed geometrical control in studies of actomyosin motor function.

Recently, molecular motor gliding assays with actin and myosin from muscle have been realized on semiconductor nanowires coated with Al2O3. This opens for unique nanotechnological applications and novel fundamental studies of actomyosin motor function. Here, we provide a comparison of myosin-driven actin filament motility on Al2O3 to both nitrocellulose and trimethylchlorosilane derivatized surfaces. We also show that actomyosin motility on the less than 200 nm wide tips of arrays of Al2O3-coated nanowires can be used to control the number, and density, of myosin-actin attachment points. Results obtained using nanowire arrays with different inter-wire spacing are consistent with the idea that the actin filament sliding velocity is determined both by the total number and the average density of attached myosin heads along the actin filament. Further, the results are consistent with buckling of long myosin-free segments of the filaments as a factor underlying reduced velocity. On the other hand, the findings do not support a mechanistic role in decreasing velocity, of increased nearest neighbor distance between available myosin heads. Our results open up for more advanced studies that may use nanowire-based structures for fundamental investigations of molecular motors, including the possibility to create a nanowire-templated bottom-up assembly of 3D, muscle-like structures.

Plasmon hybridization in pyramidal metamaterials: a route towards ultra-broadband absorption.

Pyramidal metamaterials are currently developed for ultra-broadband absorbers. They consist of periodic arrays of alternating metal/dielectric layers forming truncated square-based pyramids. The metallic layers of increasing lengths play the role of vertically and, to a less extent, laterally coupled plasmonic resonators. Based on detailed numerical simulations, we demonstrate that plasmon hybridization between such resonators helps in achieving ultra-broadband absorption. The dipolar modes of individual resonators are shown to be prominent in the electromagnetic coupling mechanism. Lateral coupling between adjacent pyramids and vertical coupling between alternating layers are proven to be key parameters for tuning of plasmon hybridization. Following optimization, the operational bandwidth of Au/Ge pyramids, i.e. the bandwidth within which absorption is higher than 90%, extends over a 0.2-5.8 µm wavelength range, i.e. from UV-visible to mid-infrared, and total absorption (integrated over the operational bandwidth) amounts to 98.0%. The omni-directional and polarization-independent high-absorption properties of the device are verified. Moreover, we show that the choice of the dielectric layer material (Si versus Ge) is not critical for achieving ultra-broadband characteristics, which confers versatility for both design and fabrication. Realistic fabrication scenarios are briefly discussed. This plasmon hybridization route could be useful in developing photothermal devices, thermal emitters or shielding devices that dissimulate objects from near infrared detectors.

Molecular motor transport through hollow nanowires.

Biomolecular motors offer self-propelled, directed transport in designed microscale networks and can potentially replace pump-driven nanofluidics. However, in existing systems, transportation is limited to the two-dimensional plane. Here we demonstrate fully one-dimensional (1D) myosin-driven motion of fluorescent probes (actin filaments) through 80 nm wide, Al2O3 hollow nanowires of micrometer length. The motor-driven transport is orders of magnitude faster than would be possible by passive diffusion. The system represents a necessary element for advanced devices based on gliding assays, for example, in lab-on-a-chip systems with channel crossings and in pumpless nanosyringes. It may also serve as a scaffold for bottom-up assembly of muscle proteins into ordered contractile units, mimicking the muscle sarcomere.

Molecular motor propelled filaments reveal light-guiding in nanowire arrays for enhanced biosensing.

Semiconductor nanowire arrays offer significant potential for biosensing applications with optical read-out due to their high surface area and due to the unique optical properties of one-dimensional materials. A challenge for optical read-out of analyte-binding to the nanowires is the need to efficiently collect and detect light from a three-dimensional volume. Here we show that light from fluorophores attached along several µm long vertical Al2O3 coated gallium phosphide nanowires couples into the wires, is guided along them and emitted at the tip. This enables effective collection of light emitted by fluorescent analytes located at different focal planes along the nanowire. We unequivocally demonstrate the light-guiding effect using a novel method whereby the changes in emitted fluorescence intensity are observed when fluorescent cytoskeletal filaments are propelled by molecular motors along the wires. The findings are discussed in relation to nanobiosensor developments, other nanotechnological applications, and fundamental studies of motor function.

Ultrafast molecular motor driven nanoseparation and biosensing.

Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 µm², with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.

Tracking actomyosin at fluorescence check points.

Emerging concepts for on-chip biotechnologies aim to replace microfluidic flow by active, molecular-motor driven transport of cytoskeletal filaments, including applications in bio-simulation, biocomputation, diagnostics, and drug screening. Many of these applications require reliable detection, with minimal data acquisition, of filaments at many, local checkpoints in a device consisting of a potentially complex network of channels that guide filament motion. Here we develop such a detection system using actomyosin motility. Detection points consist of pairs of gold lines running perpendicular to nanochannels that guide motion of fluorescent actin filaments. Fluorescence interference contrast (FLIC) is used to locally enhance the signal at the gold lines. A cross-correlation method is used to suppress errors, allowing reliable detection of single or multiple filaments. Optimal device design parameters are discussed. The results open for automatic read-out of filament count and velocity in high-throughput motility assays, helping establish the viability of active, motor-driven on-chip applications.

Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

Food chain transport of nanoparticles affects behaviour and fat metabolism in fish.

Nano-sized (10(-9)-10(-7) m) particles offer many technical and biomedical advances over the bulk material. The use of nanoparticles in cosmetics, detergents, food and other commercial products is rapidly increasing despite little knowledge of their effect on organism metabolism. We show here that commercially manufactured polystyrene nanoparticles, transported through an aquatic food chain from algae, through zooplankton to fish, affect lipid metabolism and behaviour of the top consumer. At least three independent metabolic parameters differed between control and test fish: the weight loss, the triglycerides∶cholesterol ratio in blood serum, and the distribution of cholesterol between muscle and liver. Moreover, we demonstrate that nanoparticles bind to apolipoprotein A-I in fish serum in-vitro, thereby restraining them from properly utilising their fat reserves if absorbed through ingestion. In addition to the metabolic effects, we show that consumption of nanoparticle-containing zooplankton affects the feeding behaviour of the fish. The time it took the fish to consume 95% of the food presented to them was more than doubled for nanoparticle-exposed compared to control fish. Since many nano-sized products will, through the sewage system, end up in freshwater and marine habitats, our study provides a potential bioassay for testing new nano-sized material before manufacturing. In conclusion, our study shows that from knowledge of the molecular composition of the protein corona around nanoparticles it is possible to make a testable molecular hypothesis and bioassay of the potential biological risks of a defined nanoparticle at the organism and ecosystem level.

Tracking the small with the smallest--using nanotechnology in tracking zooplankton.

A major problem when studying behavior and migration of small organisms is that many of the questions addressed for larger animals are not possible to formulate due to constraints on tracking smaller animals. In aquatic ecosystems, this problem is particularly problematic for zoo- and phytoplankton, since tracking devices are too heavy to allow the organism to act naturally. However, recent advances in nanotechnology have made it possible to track individual animals and thereby to focus on important and urgent questions which previously have not been possible to address. Here we report on a novel approach to track movement and migratory behavior of millimeter sized aquatic animals, particularly Daphnia magna, using the commercially available nanometer sized fluorescent probes known as quantum dots. Experimental trials with and without quantum dots showed that they did not affect behavior, reproduction or mortality of the tested animals. Compared to previously used methods to label small animals, the nano-labeling method presented here offers considerable improvements including: 24 h fluorescence, studies in both light and darkness, much improved optical properties, potential to study large volumes and even track animals in semi-natural conditions. Hence, the suggested method, developed in close cooperation between biologists, chemists and physicists, offers new opportunities to routinely study zooplankton responses to light, food and predation, opening up advancements within research areas such as diel vertical/horizontal migration, partial migration and other differences in intra- and interspecific movements and migration.

Fluorescence resonance energy transfer between phenanthrene and PAMAM dendrimers.

We describe herein an adsorption-induced energy transfer between phenanthrene, a major environmental pollutant, and a fluorescently labeled dendrimer acting as a host molecule. We find experimentally that such energy transfer is the most efficient at a solvent pH of 8 and for a phenanthrene:dendrimer molar ratio of 1:2. Using molecular dynamics simulations we show that the strongest binding interactions occur between phenanthrene and the primary amines of the dendrimer. The simulations provide evidence that at low pH, phenanthrene-phenanthrene interactions are favorable and compete with phenanthrene-dendrimer binding. This study offers a new scheme for detecting dendrimer molecular assembly and a physical basis for exploiting dendrimer nanotechnologies for water purification and environmental remediation.