Characterization of a reversible thermally-actuated polymer-valve: A potential dynamic treatment for congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a fetal defect comprising an incomplete diaphragm and the herniation of abdominal organs into the chest cavity that interfere with fetal pulmonary development. Though the most promising treatment for CDH is via interventional fetoscopic tracheal occlusion (TO) surgery in-utero, it has produced mixed results due to the static nature of the inserted occlusion. We hypothesize that a suitable noninvasively-actuatable, cyclic-release tracheal occlusion device can be developed to enable dynamic tracheal occlusion (dTO) implementation. OBJECTIVE: To conduct an in-vitro proof-of-concept investigation of the construction of thermo-responsive polymer valves designed for targeted activation within a physiologically realizable temperature range as a first step towards potential development of a noninvasively-actuatable implantable device to facilitate dynamic tracheal occlusion (dTO) therapy. METHODS: Six thermo-responsive polymer valves, with a critical solution temperature slightly higher than normal physiological body temperature of 37°C, were fabricated using a copolymer of n-isopropylacrylamide (NIPAM) and dimethylacrylamide (DMAA). Three of the valves underwent ethylene oxide (EtO) sterilization while the other three served as controls for EtO-processing compatibility testing. Thermal response actuation of the valves and their steady-state flow performances were evaluated using water and caprine amniotic fluid. RESULTS: All six valves consisting of 0.3-mole fraction of DMAA were tested for thermal actuation of caprine amniotic fluid flow at temperatures ranging from 30-44°C. They all exhibited initiation of valve actuation opening at ~40°C with full completion at ~44°C. The overall average coefficient of variation (CV) for the day-to-day flow performance of the valves tested was less than 12%. Based on a Student t-test, there was no significant difference in the operational characteristics for the EtO processed versus the non-EtO processed valves tested. CONCLUSIONS: We successfully fabricated and demonstrated physiological realizable temperature range operation of thermo-responsive polymer valves in-vitro and their suitability for standard EtO sterilization processing, a prerequisite for future in-vivo surgical implantation testing.

Carbon Nanofiber Arrays: A Novel Tool for Microdelivery of Biomolecules to Plants.

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, a common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5-500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants.

Transfer of vertically aligned carbon nanofibers to polydimethylsiloxane (PDMS) while maintaining their alignment and impalefection functionality.

Vertically aligned carbon nanofibers (VACNFs) are synthesized on Al 3003 alloy substrates by direct current plasma-enhanced chemical vapor deposition. Chemically synthesized Ni nanoparticles were used as the catalyst for growth. The Si-containing coating (SiN(x)) typically created when VACNFs are grown on silicon was produced by adding Si microparticles prior to growth. The fiber arrays were transferred to PDMS by spin coating a layer on the grown substrates, curing the PDMS, and etching away the Al in KOH. The fiber arrays contain many fibers over 15 µm (long enough to protrude from the PDMS film and penetrate cell membranes) and SiN(x) coatings as observed by SEM, EDX, and fluorescence microscopy. The free-standing array in PDMS was loaded with pVENUS-C1 plasmid and human brain microcapillary endothelial (HBMEC) cells and was successfully impalefected.

Weakly charged cationic nanoparticles induce DNA bending and strand separation.

Weakly charged cationic nanoparticles cause structural changes including local denaturing and compaction to DNA under mild conditions. The charged ligands bind to the phosphate backbone of DNA and the uncharged ligands penetrate the helix and disrupt base pairing. Mobility shifts in electrophoresis, molecular dynamics, and UV-vis spectrophotometry give clues to the details of the interactions.

Vertically aligned carbon nanofiber as nano-neuron interface for monitoring neural function.

Neural chips, which are capable of simultaneous multisite neural recording and stimulation, have been used to detect and modulate neural activity for almost thirty years. As neural interfaces, neural chips provide dynamic functional information for neural decoding and neural control. By improving sensitivity and spatial resolution, nano-scale electrodes may revolutionize neural detection and modulation at cellular and molecular levels as nano-neuron interfaces. We developed a carbon-nanofiber neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes and demonstrated its capability of both stimulating and monitoring electrophysiological signals from brain tissues in vitro and monitoring dynamic information of neuroplasticity. This novel nano-neuron interface may potentially serve as a precise, informative, biocompatible, and dual-mode neural interface for monitoring of both neuroelectrical and neurochemical activity at the single-cell level and even inside the cell. FROM THE CLINICAL EDITOR: The authors demonstrate the utility of a neural chip with lithographically defined arrays of vertically aligned carbon nanofiber electrodes. The new device can be used to stimulate and/or monitor signals from brain tissue in vitro and for monitoring dynamic information of neuroplasticity both intracellularly and at the single cell level including neuroelectrical and neurochemical activities.

Role of ion flux on alignment of carbon nanofibers synthesized by DC plasma on transparent insulating substrates.

A key factor to the implementation of devices with vertically aligned carbon nanofibers (VACNFs) is fundamental understanding of how to control fluctuations in the growth direction of the fibers. Here we demonstrate synthesis of VACNF on transparent and insulating substrates by continuous direct current (DC) plasma for realization of cellular interface suitable for transmission optical microscopy. To maintain continuous glow discharge above the substrate, a metal grid electrode layer (Cr) was deposited over silica with windows of exposed silica ranging in size from 200 µm to 1 mm. This electrode geometry allows for synthesis of VACNFs even within an insulating window. This observation and the observed trends in the alignment of nanofibers in the vicinity of grid electrodes have indicated that the alignment does not correspond to the direction of the electric field at the substrate level, contrary to previously proposed alignment mechanism. Computational modeling of the plasma with this grid cathode geometry has shown that nanofiber alignment trends follow calculated ion flux direction rather than electrical field. The new proposed alignment mechanism is that ion sputtering of the carbon film on a catalyst particle defines the growth direction of the nanofibers. With this development, fiber growth direction can be better manipulated through changes in ionic flux direction, opening the possibility for growth of nanofibers on substrates with unique geometries.

Effects of ultramicroelectrode dimensions on the electropolymerization of polypyrrole.

Anode geometry can significantly affect the electrochemical synthesis of conductive polymers. Here, the effects of anode dimensions on the electropolymerization of pyrrole are investigated. Band microelectrodes were prepared with widths ranging from 2 to 500 mum. The anode dimension has a significant effect on the resulting thickness of polymer film. The electropolymerization process deviates significantly from that predicted by simple mass transfer considerations when electrode dimensions are less than approximately 20 mum. Polymer film thickness is thinner than expected when electrode dimensions become less than approximately 10 mum. A simple mathematical model was derived to explain the observed effects of anode dimensions on the polymerization process. Simulation results confirm that diffusive loss of reaction intermediates accounts for the observed experimental trends. The described simulation facilitates understanding of the electropolymerization processes and approaches to the controlled deposition of polypyrrole, particularly at the submicron scale, for microelectromechanical systems and biomedical applications.

Immobilization and release strategies for DNA delivery using carbon nanofiber arrays and self-assembled monolayers.

We report a strategy for immobilizing dsDNA (double-stranded DNA) onto vertically aligned carbon nanofibers and subsequently releasing this dsDNA following penetration and residence of these high aspect ratio structures within cells. Gold-coated nanofiber arrays were modified with self-assembled monolayers (SAM) to which reporter dsDNA was covalently and end-specifically bound with or without a cleavable linker. The DNA-modified nanofiber arrays were then used to impale, and thereby transfect, Chinese hamster lung epithelial cells. This mechanical approach enables the transport of bound ligands directly into the cell nucleus and consequently bypasses extracellular and cytosolic degradation. Statistically significant differences were observed between the expression levels from immobilized and releasable DNA, and these are discussed in relation to the distinct accessibility and mode of action of glutathione, an intracellular reducing agent responsible for releasing the bound dsDNA. These results prove for the first time that an end-specifically and covalently SAM-bound DNA can be expressed in cells. They further demonstrate how the choice of immobilization and release methods can impact expression of nanoparticle delivered DNA.

Inducible RNA interference-mediated gene silencing using nanostructured gene delivery arrays.

RNA interference (RNAi) has become a powerful biological tool over the past decade. In this study, a tetracycline-inducible small hairpin RNA (shRNA) vector system was designed for silencing cyan fluorescent protein (CFP) expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% +/- 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene-silencing effects using RNAi in single cells and cell populations.

Actuatable membranes based on polypyrrole-coated vertically aligned carbon nanofibers.

Nanoporous membranes are applicable to a variety of research fields due to their ability to selectively separate molecules with high efficiency. Of particular interest are methods for controlling membrane selectivity through externally applied stimuli and integrating such membrane structures within multiscale systems. Membranes comprised of deterministically grown, vertically aligned carbon nanofibers (VACNFs) are compatible with these needs. VACNF membranes can regulate molecular transport by physically selecting species as they pass between the fibers. Defined interfiber spacing allows for nanoscale control of membrane pore structure and resultant size selectivity. Subsequent physical or chemical modification of VACNF structures enables the tuning of physical pore size and chemical specificity allowing further control of membrane permeability. In this work, the dynamic physical modulation of membrane permeability that results when VACNFs are coated with an electrically actuatable polymer, polypyrrole, is demonstrated. Electrochemical reduction of polypyrrole on the VACNFs results in controlled swelling of the diameter of the nanofibers that in turn decreases the pore size. Dynamic control of membrane pore size enables selective transport and gating of nanoscale pores.

End-specific strategies of attachment of long double stranded DNA onto gold-coated nanofiber arrays.

We report the effective and site-specific binding of long double stranded (ds)DNA to high aspect ratio carbon nanofiber arrays. The carbon nanofibers were first coated with a thin gold layer to provide anchorage for two controllable binding methods. One method was based on the direct binding of thiol end-labeled dsDNA. The second and enhanced method used amine end-labeled dsDNA bound with crosslinkers to a carboxyl-terminated self-assembled monolayer. The bound dsDNA was first visualized with a fluorescent, dsDNA-intercalating dye. The specific binding onto the carbon nanofiber was verified by a high resolution detection method using scanning electron microscopy in combination with the binding of neutravidin-coated fluorescent microspheres to the immobilized and biotinylated dsDNA. Functional activity of thiol end-labeled dsDNA on gold-coated nanofiber arrays was verified with a transcriptional assay, whereby Chinese hamster lung cells (V79) were impaled upon the DNA-modified nanofibers and scored for transgene expression of the tethered template. Thiol end-labeled dsDNA demonstrated significantly higher expression levels than nanofibers prepared with control dsDNA that lacked a gold-binding end-label. Employing these site-specific and robust techniques of immobilization of dsDNA onto nanodevices can be of advantage for the study of DNA/protein interactions and for gene delivery applications.

Vertically aligned carbon nanofiber arrays record electrophysiological signals from hippocampal slices.

Vertically aligned carbon nanofiber (VACNF) electrode arrays were tested for their potential application in recording neuro-electrophysiological activity. We report, for the first time, stimulation and extracellular recording of spontaneous and evoked neuroelectrical activity in organotypic hippocampal slice cultures with ultramicroelectrode VACNF arrays. Because the electrodes are carbon-based, these arrays have potential advantages over metal electrodes and could enable a variety of future applications as precise, informative, and biocompatible neural interfaces.

Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays.

Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

Controlling the Dimensions of Carbon Nanofiber Structures through the Electropolymerization of Pyrrole.

Electrically conductive polymers, such as polypyrrole (PPy), show promise for modifying the dimensions and properties of micro- and nanoscale structures. Mechanisms for controlling the formation of PPy films of nanoscale thickness were evaluated by electrochemically synthesizing and examining PPy films on planar gold electrodes under a variety of growth conditions. Tunable PPy coatings were then deposited by electropolymerization on the sidewalls of individual, electrically addressable carbon nanofibers (CNFs). The ability to modify the physical size of specific nanofibers in controllable fashion is demonstrated. The biocompatibility, potential for chemical functionalization, and ability to effect volume changes of this nanocomposite can lead to advanced functionality, such as specific, nanoscale valving of materials and morphological control at the nanoscale.

Resident neuroelectrochemical interfacing using carbon nanofiber arrays.

Carbon nanofiber electrode architectures are used to provide for long-term, neuroelectroanalytical measurements of the dynamic processes of intercellular communication between excitable cells. Individually addressed, vertically aligned carbon nanofibers are incorporated into multielement electrode arrays upon which excitable cell matrixes of both neuronal-like derived cell lines (rat pheochromocytoma, PC-12) and primary cells (dissociated cells from embryonic rat hippocampus) are cultured over extended periods (days to weeks). Electrode arrays are characterized with respect to their response to easily oxidized neurotransmitters, including dopamine, norepinephrine, and 5-hydroxytyramide. Electroanalysis at discrete electrodes following long-term cell culture demonstrates that this platform remains responsive for the detection of easily oxidized species generated by the cultured cells. Preliminary data also suggests that quantal release of easily oxidized transmitters can be observed at nanofiber electrodes following direct culture and differentiation on the arrays for periods of at least 16 days.

Synthetic nanoscale elements for delivery of materials into viable cells.

Arrays of vertically aligned carbon nanofibers (VACNFs) provide structures that are well suited for the direct integration and manipulation of molecular-scale phenomena within intact, live cells. VACNFs are fabricated via a combination of microfabrication techniques and catalytic plasma-enhanced chemical vapor deposition. In this chapter, we discuss the synthesis of VACNFs and detail the methods for introducing these arrays into the intracellular domain of mammalian cells for the purpose of delivering large macromolecules, specifically plasmid DNA, on a massively parallel basis.

A magnetocaloric pump for microfluidic applications.

A magnetocaloric pump provides a simple means of pumping fluid using only external thermal and magnetic fields. The principle, which can be traced back to the early work of Rosensweig, is straightforward. Magnetic materials tend to lose their magnetization as the temperature approaches the material's Curie point. Exposing a column of magnetic fluid to a uniform magnetic field coincident with a temperature gradient produces a pressure gradient in the magnetic fluid. As the fluid heats up, it loses its attraction to the magnetic field and is displaced by cooler fluid. The impact of such a phenomenon is obvious: fluid propulsion with no moving mechanical parts. Until recently, limitations in the magnetic and thermal properties of conventional materials severely limited practical operating pressure gradients. However, recent advancements in the design of metal substituted magnetite enable fine control over both the magnetic and thermal properties of magnetic nanoparticles, a key element in colloidal-based magnetic fluids (ferrofluids). This paper begins with a basic description of the process and previous limitations due to material properties. This is followed by a review of existing methods of synthesizing magnetic nanoparticles as well as an introduction to a new approach based on thermophilic metal-reducing bacteria. We compare two compounds and show, experimentally, significant variation in specific magnetic and thermal properties. We develop the constitutive thermal, magnetic, and fluid dynamic equations associated with a magnetocaloric pump and validate our finite-element model with a series of experiments. Preliminary results show a good match between the model and experiment as well as approximately an order of magnitude increase in the fluid flow rate over conventional magnetite-based ferrofluids operating below 80 degrees C. Finally, as a practical demonstration, we describe a novel application of this technology: pumping fluids at the "lab-on-a-chip" microfluidic scale.