Polyethyleneimine/polyethylene glycol-conjugated gold nanoparticles as nanoscale positive/negative controls in nanotoxicology: testing in frog embryo teratogenesis assay-Xenopus and mammalian tissue culture system.

Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis optimization or colloidal stability under physiological conditions until a group at the National Institute of Standards and Technology recently succeeded in producing remarkably stable polyethyleneimine (PEI)-coated AuNPs (Au-PEI). This improved version of Au-PEI (Au-PEI25kB) has increased the demand for toxicity and teratogenicity information for applications in nanomedicine and nanotoxicology. In vitro assays for Au-PEI25kB in various cell lines showed substantial active cytotoxicity. For advanced toxicity research, the frog embryo teratogenesis assay-Xenopus (FETAX) method was employed in this study. We observed that positively-charged Au-PEI25kB exhibited significant toxicity and teratogenicity, whereas polyethylene glycol conjugated AuNPs (Au-PEG) used as comparable negative controls did not. There is a characteristic avidity of Au-PEI25kB for the jelly coat, the chorionic envelope (also known as vitelline membrane) and the cytoplasmic membrane, as well as a barrier effect of the chorionic envelope observed with Au-PEG. To circumvent these characteristics, an injection-mediated FETAX approach was utilized. Like treatment with the FETAX method, the injection of Au-PEI25kB severely impaired embryo development. Notably, the survival/concentration curve that was steep when the standard FETAX approach was employed became gradual in the injection-mediated FETAX. These results suggest that Au-PEI25kB may be a good candidate as a nanoscale positive control material for nanoparticle analysis in toxicology and teratology.

Crystallize It before It Diffuses: Kinetic Stabilization of Thin-Film Phosphorus-Rich Semiconductor CuP2.

Numerous phosphorus-rich metal phosphides containing both P-P bonds and metal-P bonds are known from the solid-state chemistry literature. A method to grow these materials in thin-film form would be desirable, as thin films are required in many applications and they are an ideal platform for high-throughput studies. In addition, the high density and smooth surfaces achievable in thin films are a significant advantage for characterization of transport and optical properties. Despite these benefits, there is hardly any published work on even the simplest binary phosphorus-rich phosphide films. Here, we demonstrate growth of single-phase CuP2 films by a two-step process involving reactive sputtering of amorphous CuP2+x and rapid annealing in an inert atmosphere. At the crystallization temperature, CuP2 is thermodynamically unstable with respect to Cu3P and P4. However, CuP2 can be stabilized if the amorphous precursors are mixed on the atomic scale and are sufficiently close to the desired composition (neither too P poor nor too P rich). Fast formation of polycrystalline CuP2, combined with a short annealing time, makes it possible to bypass the diffusion processes responsible for decomposition. We find that thin-film CuP2 is a 1.5 eV band gap semiconductor with interesting properties, such as a high optical absorption coefficient (above 105 cm-1), low thermal conductivity (1.1 W/(K m)), and composition-insensitive electrical conductivity (around 1 S/cm). We anticipate that our processing route can be extended to other phosphorus-rich phosphides that are still awaiting thin-film synthesis and will lead to a more complete understanding of these materials and of their potential applications.

Growth and Decomposition of Pt Surface Oxides.

The formation and thermal stability of Pt surface oxides on a Pt thin film were studied in situ using ambient-pressure X-ray photoelectron spectroscopy. At an oxygen pressure of 73 Pa (550 mTorr), the surface Pt oxide was gradually formed, evidenced by the O 1s peak at 529.5 eV as the Pt film was heated. The Pt oxide peak reached a maximum between 217 and 317 °C and then decreased as the sample temperature was further increased. A similar response was seen on cooling from 480 to 23 °C; the intensity of the Pt oxide peak first increased and then decreased. The remaining Pt surface oxides partially decomposed during ultra-high-vacuum (UHV) pumping and completely decomposed during heating in UHV, which highlights the challenge of characterizing these surfaces with UHV instruments. These results have important implications for the understanding of the surface states of platinum films in different environments and the roles of different catalytic mechanisms.

Practical Guide to the Design, Fabrication, and Calibration of NIST Nanocalorimeters.

We report here on the design, fabrication, and calibration of nanocalorimeter sensors used in the National Institute of Standards and Technology (NIST) Nanocalorimetry Measurements Project. These small-scale thermal analysis instruments are produced using silicon microfabrication approaches. A single platinum line serves as both the heater and temperature sensor, and it is made from a 500 µm wide, 100 nm thick platinum trace, suspended on a 100 nm thick silicon nitride membrane for thermal isolation. Supplemental materials to this article (available online) include drawing files and LabVIEW code used in the fabrication and calibration process.

Multi-environment Nanocalorimeter with Electrical Contacts for Use in the Scanning Electron Microscope.

We have developed a versatile nanocalorimeter sensor which allows imaging and electrical measurements of samples under different gaseous environments using the scanning electron microscope (SEM) and can simultaneously measure the sample temperature and associated heat of reaction. This new sensor consists of four independent heating/sensing elements for nanocalorimetry and eight electrodes for electrical measurements, all mounted on a 50 nm thick, 250 µm × 250 µm suspended silicon nitride membrane. This membrane is highly electron transparent and mechanically robust enabling in situ SEM observation under realistic temperatures, environmental conditions and pressures up to one atmosphere. To demonstrate this new capability, we report here on 1) in situ SEM-nanocalorimetry study of melting and solidification of polyethylene oxide, 2) the temperature dependence of conductivity of a nanowire; 3) the electron beam induced current measurements (EBID) of a nanowire in vacuum and air. Furthermore, the sensor is easily adaptable to operate in liquid environment and is compatible with most existing SEM. This versatile platform couples nanocalorimetry with in situ SEM imaging under various gaseous and liquid environments and is applicable to materials research, nanotechnology, energy, catalysis and biomedical applications.

Nanocalorimetry-coupled time-of-flight mass spectrometry: identifying evolved species during high-rate thermal measurements.

We report on measurements integrating a nanocalorimeter sensor into a time-of-flight mass spectrometer (TOFMS) for simultaneous thermal and speciation measurements at high heating rates. The nanocalorimeter sensor was incorporated into the extraction region of the TOFMS system to provide sample heating and thermal information essentially simultaneously with the evolved species identification. This approach can be used to measure chemical reactions and evolved species for a variety of materials. Furthermore, since the calorimetry is conducted within the same proximal volume as ionization and ion extraction, evolved species detected are in a collision-free environment, and thus, the possibility exists to interrogate intermediate and radical species. We present measurements showing the decomposition of ammonium perchlorate, copper oxide nanoparticles, and sodium azotetrazolate. The rapid, controlled, and quantifiable heating rate capabilities of the nanocalorimeter coupled with the 0.1 ms temporal resolution of the TOFMS provides a new measurement capability and insight into high-rate reactions, such as those seen with reactive and energetic materials, and adsorption\desorption measurements, critical for understanding surface chemistry and accelerating catalyst selection.

Hydrated/dehydrated lipid phase transitions measured using nanocalorimetry.

The phase transition evolution with hydration of a model system, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), was investigated with a fast nanocalorimetry system. Using nanocalorimetry, it is possible to measure the gel to liquid phase transitions that occur on millisecond to second time scales and quantify the time to recover the hydrated state. The results show the phase transition occurring in a few milliseconds and the relaxation or recovery time from the dehydrated state back to original hydrated state occurring with times dependent on the local humidity. With relative humidity (RH) of 43% or higher, the recovery time can be less than a few seconds. With RH of 11% or lower, the recovery time is extended to greater than a minute. The recovery process is controlled by mechanisms that depend on the lipid molecular repacking and water transport from the environment. Nanocalorimetry provides a powerful method to investigate the kinetics of such transformations in lipids and other biological and pharmaceutical moieties.

Combining nanocalorimetry and dynamic transmission electron microscopy for in situ characterization of materials processes under rapid heating and cooling.

Nanocalorimetry is a chip-based thermal analysis technique capable of analyzing endothermic and exothermic reactions at very high heating and cooling rates. Here, we couple a nanocalorimeter with an extremely fast in situ microstructural characterization tool to identify the physical origin of rapid enthalpic signals. More specifically, we describe the development of a system to enable in situ nanocalorimetry experiments in the dynamic transmission electron microscope (DTEM), a time-resolved TEM capable of generating images and electron diffraction patterns with exposure times of 30 ns-500 ns. The full experimental system consists of a modified nanocalorimeter sensor, a custom-built in situ nanocalorimetry holder, a data acquisition system, and the DTEM itself, and is capable of thermodynamic and microstructural characterization of reactions over a range of heating rates (10(2) K/s-10(5) K/s) accessible by conventional (DC) nanocalorimetry. To establish its ability to capture synchronized calorimetric and microstructural data during rapid transformations, this work describes measurements on the melting of an aluminum thin film. We were able to identify the phase transformation in both the nanocalorimetry traces and in electron diffraction patterns taken by the DTEM. Potential applications for the newly developed system are described and future system improvements are discussed.

Calcium signaling is gated by a mechanical threshold in three-dimensional environments.

Cells interpret their mechanical environment using diverse signaling pathways that affect complex phenotypes. These pathways often interact with ubiquitous 2(nd)-messengers such as calcium. Understanding mechanically-induced calcium signaling is especially important in fibroblasts, cells that exist in three-dimensional fibrous matrices, sense their mechanical environment, and remodel tissue morphology. Here, we examined calcium signaling in fibroblasts using a minimal-profile, three-dimensional (MP3D) mechanical assay system, and compared responses to those elicited by conventional, two-dimensional magnetic tensile cytometry and substratum stretching. Using the MP3D system, we observed robust mechanically-induced calcium responses that could not be recreated using either two-dimensional technique. Furthermore, we used the MP3D system to identify a critical displacement threshold governing an all-or-nothing mechanically-induced calcium response. We believe these findings significantly increase our understanding of the critical role of calcium signaling in cells in three-dimensional environments with broad implications in development and disease.

Synthetic protocells interact with viral nanomachinery and inactivate pathogenic human virus.

We present a new antiviral strategy and research tool that could be applied to a wide range of enveloped viruses that infect human beings via membrane fusion. We test this strategy on two emerging zoonotic henipaviruses that cause fatal encephalitis in humans, Nipah (NiV) and Hendra (HeV) viruses. In the new approach, artificial cell-like particles (protocells) presenting membrane receptors in a biomimetic manner were developed and found to attract and inactivate henipavirus envelope glycoprotein pseudovirus particles, preventing infection. The protocells do not accumulate virus during the inactivation process. The use of protocells that interact with, but do not accumulate, viruses may provide significant advantages over current antiviral drugs, and this general approach may have wide potential for antiviral development.

Spaceflight-related suboptimal conditions can accentuate the altered gravity response of Drosophila transcriptome.

Genome-wide transcriptional profiling shows that reducing gravity levels during Drosophila metamorphosis in the International Space Station (ISS) causes important alterations in gene expression: a large set of differentially expressed genes (DEGs) are observed compared to 1g controls. However, the preparation procedures for spaceflight and the nonideal environmental conditions on board the ISS subject the organisms to additional environmental stresses that demonstrably affect gene expression. Simulated microgravity experiments performed on the ground, under ideal conditions for the flies, using the random position machine (RPM), show much more subtle effects on gene expression. However, when the ground experiments are repeated under conditions designed to reproduce the additional environmental stresses imposed by spaceflight procedures, 79% of the DEGs detected in the ISS are reproduced by the RPM experiment. Gene ontology analysis of them shows they are genes that affect respiratory activity, developmental processes and stress-related changes. Here, we analyse the effects of microgravity on gene expression in relation to the environmental stresses imposed by spaceflight. Analysis using 'gene expression dynamics inspector' (GEDI) self-organizing maps reveals a subtle response of the transcriptome to microgravity. Remarkably, hypergravity simulation induces similar response of the transcriptome, but in the opposite direction, i.e. the genes promoted under microgravity are usually suppressed under hypergravity. These results suggest that the transcriptome is finely tuned to normal gravity and that microgravity, together with environmental constraints associated with space experiments, can have profound effects on gene expression.

Synthetic protocells to mimic and test cell function.

Synthetic protocells provide a new means to probe, mimic and deconstruct cell behavior; they are a powerful tool to quantify cell behavior and a useful platform to explore nanomedicine. Protocells are not simple particles; they mimic cell design and typically consist of a stabilized lipid bilayer with membrane proteins. With a finite number of well characterized components, protocells can be designed to maximize useful outputs. Energy conversion in cells is an intriguing output; many natural cells convert transmembrane ion gradients into electricity by membrane-protein regulated ion transport. Here, a synthetic cell system comprising two droplets separated by a lipid bilayer is described that functions as a biological battery. The factors that affect its electrogenic performance are explained and predicted by coupling equations of the electrodes, transport proteins and membrane behavior. We show that the output of such biological batteries can reach an energy density of 6.9 x 10(6) J m(-3), which is approximately 5% of the volumetric energy density of a lead-acid battery. The configuration with maximum power density has an energy conversion efficiency of 10%.

Self-assembled gold nanoparticle molecular probes for detecting proteolytic activity in vivo.

Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface-defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5- to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model.

Three-dimensional conductive constructs for nerve regeneration.

The unique electrochemical properties of conductive polymers can be utilized to form stand-alone polymeric tubes and arrays of tubes that are suitable for guides to promote peripheral nerve regeneration. Noncomposite, polypyrrole (PPy) tubes ranging in inner diameter from 25 microm to 1.6 mm as well as multichannel tubes were fabricated by electrodeposition. While oxidation of the pyrrole monomer causes growth of the film, brief subsequent reduction allowed mechanical dissociation from the electrode mold, creating a stand-alone, conductive PPy tube. Conductive polymer nerve guides made in this manner were placed in transected rat sciatic nerves and shown to support nerve regeneration over an 8-week time period.

Designing artificial cells to harness the biological ion concentration gradient.

Cell membranes contain numerous nanoscale conductors in the form of ion channels and ion pumps that work together to form ion concentration gradients across the membrane to trigger the release of an action potential. It seems natural to ask if artificial cells can be built to use ion transport as effectively as natural cells. Here we report a mathematical calculation of the conversion of ion concentration gradients into action potentials across different nanoscale conductors in a model electrogenic cell (electrocyte) of an electric eel. Using the parameters extracted from the numerical model, we designed an artificial cell based on an optimized selection of conductors. The resulting cell is similar to the electrocyte but has higher power output density and greater energy conversion efficiency. We suggest methods for producing these artificial cells that could potentially be used to power medical implants and other tiny devices.

Poly(glycerol sebacate) nanofiber scaffolds by core/shell electrospinning.

The novel biomaterial poly(glycerol sebacate) (PGS) holds great promise for tissue engineering and regenerative medicine. PGS is a rubbery, degradable polymer much like elastin; however, it has been limited to cast structures. This work reports on the formation of PGS nanofibers in random non-woven mats for use as tissue engineering scaffolds by coaxial core/shell electrospinning. PGS nanofibers are an inexpensive and synthetic material that mimics the chemical and mechanical environment provided by elastin fibers. Poly(lactide) was used as the shell material to constrain the PGS during the curing process and was removed before cell seeding. Human microvascular endothelial cells from skin (HDMEC) were used to evaluate the in-vitro cellular compatibility of the PGS nanofiber scaffolds. [Figure: see text].

Label-free immunodetection with CMOS-compatible semiconducting nanowires.

Semiconducting nanowires have the potential to function as highly sensitive and selective sensors for the label-free detection of low concentrations of pathogenic microorganisms. Successful solution-phase nanowire sensing has been demonstrated for ions, small molecules, proteins, DNA and viruses; however, 'bottom-up' nanowires (or similarly configured carbon nanotubes) used for these demonstrations require hybrid fabrication schemes, which result in severe integration issues that have hindered widespread application. Alternative 'top-down' fabrication methods of nanowire-like devices produce disappointing performance because of process-induced material and device degradation. Here we report an approach that uses complementary metal oxide semiconductor (CMOS) field effect transistor compatible technology and hence demonstrate the specific label-free detection of below 100 femtomolar concentrations of antibodies as well as real-time monitoring of the cellular immune response. This approach eliminates the need for hybrid methods and enables system-scale integration of these sensors with signal processing and information systems. Additionally, the ability to monitor antibody binding and sense the cellular immune response in real time with readily available technology should facilitate widespread diagnostic applications.

Electropolymerization on microelectrodes: functionalization technique for selective protein and DNA conjugation.

A critical shortcoming of current surface functionalization schemes is their inability to selectively coat patterned substrates at micrometer and nanometer scales. This limitation prevents localized deposition of macromolecules at high densities, thereby restricting the versatility of the surface. A new approach for functionalizing lithographically patterned substrates that eliminates the need for alignment and, thus, is scalable to any dimension is reported. We show, for the first time, that electropolymerization of derivatized phenols can functionalize patterned surfaces with amine, aldehyde, and carboxylic acid groups and demonstrate that these derivatized groups can covalently bind molecular targets, including proteins and DNA. With this approach, electrically conducting and semiconducting materials in any lithographically realizable geometry can be selectively functionalized, allowing for the sequential deposition of a myriad of chemical or biochemical species of interest at high density to a surface with minimal cross-contamination.

Approaches for biological and biomimetic energy conversion.

This article highlights areas of research at the interface of nanotechnology, the physical sciences, and biology that are related to energy conversion: specifically, those related to photovoltaic applications. Although much ongoing work is seeking to understand basic processes of photosynthesis and chemical conversion, such as light harvesting, electron transfer, and ion transport, application of this knowledge to the development of fully synthetic and/or hybrid devices is still in its infancy. To develop systems that produce energy in an efficient manner, it is important both to understand the biological mechanisms of energy flow for optimization of primary structure and to appreciate the roles of architecture and assembly. Whether devices are completely synthetic and mimic biological processes or devices use natural biomolecules, much of the research for future power systems will happen at the intersection of disciplines.