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.

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.

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.

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.

Airbrushed nickel nanoparticles for large-area growth of vertically aligned carbon nanofibers on metal (Al, Cu, Ti) surfaces.

Vertically aligned carbon nanofibers (VACNFs) were grown by plasma-enhanced chemical vapor deposition (PECVD) using Ni nanoparticle (NP) catalysts that were deposited by airbrushing onto Si, Al, Cu, and Ti substrates. Airbrushing is a simple method for depositing catalyst NPs over large areas that is compatible with roll-to-roll processing. The distribution and morphology of VACNFs are affected by the airbrushing parameters and the composition of the metal foil. Highly concentrated Ni NPs in heptane give more uniform distributions than pentane and hexanes, resulting in more uniform coverage of VACNFs. For VACNF growth on metal foils, Si micropowder was added as a precursor for Si-enriched coatings formed in situ on the VACNFs that impart mechanical rigidity. Interactions between the catalyst NPs and the metal substrates impart control over the VACNF morphology. Growth of carbon nanostructures on Cu is particularly noteworthy because the miscibility of Ni with Cu poses challenges for VACNF growth, and carbon nanostructures anchored to Cu substrates are desired as anode materials for Li-ion batteries and for thermal interface materials.

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.

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 ligand monolayers on catalytic nickel nanoparticles for synthesizing vertically aligned carbon nanofibers.

Vertically aligned carbon nanofibers (VACNFs) were synthesized using ligand-stabilized Ni nanoparticle (NP) catalysts and plasma-enhanced chemical vapor deposition. Using chemically synthesized Ni NPs enables facile preparation of VACNF arrays with monodisperse diameters below the size limit of thin film lithography. During pregrowth heating, the ligands catalytically convert into graphitic shells that prevent the catalyst NPs from agglomerating and coalescing, resulting in a monodisperse VACNF size distribution. In comparison, significant agglomeration occurs when the ligands are removed before VACNF growth, giving a broad distribution of VACNF sizes. The ligand shells are also promising for patterning the NPs and synthesizing complex VACNF arrays.

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.

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.

Reversible electrowetting of vertically aligned superhydrophobic carbon nanofibers.

Reversible electrostatically induced wetting (electrowetting) of vertically aligned superhydrophobic carbon nanofibers has been investigated. Carbon nanofibers on a 5 x 5 microm pitch were grown on Si substrates, electrically insulated with a conformal dielectric, and hydrophobized with fluoropolymer. This nanostructured scaffold exhibited superhydrophobic behavior for saline (theta approximately 160 degrees). Electrowetting induced a contact angle reduction to theta approximately 100 degrees. Competitive two-liquid (dodecane/saline) electrowetting exhibited reversibility on the same nanostructured scaffold. Without applied bias, ultra-fine-point tip (approximately 25 nm radius) nanofibers result in effectively zero capacitance with the overlying saline layer. Complete electrowetting of the substrate is confirmed as capacitance values increase by several orders of magnitude with increased wetting. These results demonstrate the applicability of reversible electrowetting on nanostructured scaffolds and use of nanofabricated structures that can be integrated with various micro- and nanoelectronic technologies.

Surface patterning of silica nanostructures using bio-inspired templates and directed synthesis.

Natural systems excel in directing the synthesis of inorganic materials for various functional purposes. One of the best-studied systems is silica synthesis, as occurs in diatoms and marine sponges. Various biological and synthetic polymers have been shown to template and catalyze silica formation from silicic acid precursors. Here, we describe the use of poly-L-lysine to promote the synthesis of silica in neutral, aqueous solution and when immobilized onto a silicon support structure under similar conditions. Either reagent jetting or conventional photolithography techniques can be used to pattern the templating polymer. Spots created by reagent jetting led to the creation of silica structures in the shape of a ring that may be a result of the spotting process. Photolithographically defined poly-L-lysine spots led to thin laminate structures after exposure to a dilute aqueous silicic acid solution. The laminate structures were nanostructured and highly interconnected. Photolithographic patterning of (3-aminopropyl)trimethoxysilane, a reagent that mimics the lysine functional group, led to similar silica coatings even though low-molecular-weight materials do not rapidly promote silica synthesis in solution. This result highlights the importance of functional-group arrangement for templating and promoting the synthesis of inorganic materials. The described surface-patterning techniques offer a route to integrate conventional silicon-patterning technologies with biologically based material synthesis. Such combined fabrication techniques enable controlled assembly over multiple length scales and an approach to understanding interfacial silica synthesis, as occurs in natural systems.