Gene therapy approaches for obesity-induced adipose neuropathy: Device-targeted AAV-mediated neurotrophic factor delivery to adipocytes in subcutaneous adipose.

Maintaining functional adipose innervation is critical for metabolic health. We found that subcutaneous white adipose tissue (scWAT) undergoes peripheral neuropathy (PN) with obesity, diabetes, and aging (reduced small-fiber innervation and nerve/synaptic/growth-cone/vesicle markers, altered nerve activity). Unlike with nerve injuries, peripheral nerves do not regenerate with PN, and therefore new therapies are needed for treatment of this condition affecting 20-30 million Americans. Here, we validated a gene therapy approach using an adipocyte-tropic adeno-associated virus (AAV; serotype Rec2) to deliver neurotrophic factors (brain-derived neurotrophic factor [BDNF] and nerve growth factor [NGF]) directly to scWAT to improve tissue-specific PN as a proof-of-concept approach. AAVRec2-BDNF intra-adipose delivery improved tissue innervation in obese/diabetic mice with PN, but after longer periods of dietary obesity there was reduced efficacy, revealing a key time window for therapies. AAVRec2-NGF also increased scWAT innervation in obese mice and was more effective than BDNF, likely because Rec2 targeted adipocytes, the tissue's endogenous NGF source. AAVRec2-NGF also worked well even after 25 weeks of dietary obesity, unlike BDNF, which likely needs a vector that targets its physiological cellular source (stromal vascular fraction cells). Given the differing effects of AAVs carrying NGF versus BDNF, a combined therapy may be ideal for PN.

A Microfabricated, Flow-Driven Grinding Mill for Mechanical Cell Lysing.

This paper presents the design, microfabrication, and demonstration of a novel microfluidic grinding mill for the lysis of the dinoflagellate, Alexandrium, a neurotoxin-producing genus of algae that is responsible for red tide and paralytic shellfish poisoning. The mill consists of a high-speed, hydrodynamically driven microrotor coupled to a micro grinding mill that lyses robust algal cells by mechanical abrasion with single-pass efficiencies as high as 97%. These efficiencies are comparable to, or better than, current mechanical and chemical lysing methods without adding complications associated with harsh chemical additives that can interfere with subsequent downstream bioanalysis. Release of cytoplasm from lysed algae was confirmed using polymerase chain reaction (PCR) amplification of Alexandrium DNA using dinoflagellate primers.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin.

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

Directing the spatial patterning of motor neuron differentiation in engineered microenvironments.

Embryonic development of the spinal cord proceeds through a carefully orchestrated temporal and spatial sequence of chemical cues to provide precise patterning of adult cell types. Recreating this complex microenvironment in a standard cell culture dish is difficult, if not impossible. In this paper, a microfluidic device is used to recapitulate, in vitro, the graded patterning events which occur during early spinal cord development. The microdevice design is developed using COMSOL modeling, with which the spatiotemporal profiles of multiple, diffusible morphogens are simulated. Four independently addressed source/sinks are employed to generate two overlapping orthogonal gradients within a cell culture chamber, mimicking the dorsoventral and anteroposterior axes of the developing embryo. Mouse embryonic stem cells are directed therein to differentiate into motor neurons in a spatially organized manner, reminiscent of a neural tube.

A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA.

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA-RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

Fabrication and characterization of a solid-state nanopore with self-aligned carbon nanoelectrodes for molecular detection.

Stochastic molecular sensors based on resistive pulse nanopore modalities are envisioned as facile DNA sequencers. However, recent advances in nanotechnology fabrication have highlighted promising alternative detection mechanisms with higher sensitivity and potential single-base resolution. In this paper we present the novel self-aligned fabrication of a solid-state nanopore device with integrated transverse graphene-like carbon nanoelectrodes for polyelectrolyte molecular detection. The electrochemical transduction mechanism is characterized and found to result primarily from thermionic emission between the two transverse electrodes. Response of the nanopore to Lambda dsDNA and short (16-mer) ssDNA is demonstrated and distinguished.

The applications of in situ electron energy loss spectroscopy to the study of electron beam nanofabrication.

An in situ electron energy loss spectroscopy (EELS) technique has been developed to investigate the dynamic processes associated with electron-beam nanofabrication on thin membranes. In this article, practical applications germane to e-beam nanofabrication are illustrated with a case study of the drilling of nanometer-sized pores in silicon nitride membranes. This technique involves successive acquisitions of the plasmon-loss and the core-level ionization-loss spectra in real time, both of which provide the information regarding the hole-drilling kinetics, including two respective rates for total mass loss, individual nitrogen and silicon element depletion, and the change of the atomic bonding environment. In addition, the in situ EELS also provides an alternative method for endpoint detection with a potentially higher time resolution than by imaging. On the basis of the time evolution of in situ EELS spectra, a qualitative working model combining knock-on sputtering, irradiation-induced mass transport, and phase separation can be proposed.

An electron microscopy investigation of the structure of porous silicon by oxide replication.

The morphology of porous silicon is studied by scanning electron microscopy (SEM) by making an oxide replica of the pore structure. Highly branched n-type porous silicon samples were prepared and a replica was formed by oxidation of the pores followed by selective removal of the silicon substrate to expose the oxide pores. Scanning and transmission electron microscopy images confirmed many previously held assumptions about porous silicon formation, including the fractal structure and crystallographic propagation; they also provided a clearer understanding of the details of pore formation. The replica procedure also provides a platform for a more facile and comprehensive analysis of the porous silicon morphology.

Nanopore with Transverse Nanoelectrodes for Electrical Characterization and Sequencing of DNA.

A DNA sequencing device which integrates transverse conducting electrodes for the measurement of electrode currents during DNA translocation through a nanopore has been nanofabricated and characterized. A focused electron beam (FEB) milling technique, capable of creating features on the order of 1 nm in diameter, was used to create the nanopore. The device was characterized electrically using gold nanoparticles as an artificial analyte with both DC and AC measurement methods. Single nanoparticle/electrode interaction events were recorded. A low-noise, high-speed transimpedance current amplifier for the detection of nano to picoampere currents at microsecond time scales was designed, fabricated and tested for future integration with the nanopore device.

Frequency dependence of gold nanoparticle superassembly by dielectrophoresis.

Dielectrophoresis is an effective method for capturing nanoparticles and assembling them into nanostructures. The frequency of the dielectrophoretic alternating current (ac) electric field greatly influences the morphology of resultant nanoparticle assemblies. In this study, frequency regimes associated with specific gold nanoparticle assembly morphologies were identified. Gold nanoparticles suspended in water were captured by microelectrodes at different electric field frequencies onto thin silicon nitride membranes. The resultant assemblies were examined by transmission electron microscopy. For this system, the major frequency-dependent influence on morphology appears to arise not from the Clausius-Mossotti factor of the dielectrophoretic force itself, but instead from ac electroosmotic fluid flow and the influence of the electrical double layer at the electrode-solution interface. Frequency regimes of technological interest include those forming one-dimensional nanoparticle chains, microwires, combinations of microwires and nanoparticle chains suitable for nanogap electrode formation, and dense three-dimensional assemblies with very high surface area.

Solid-phase direct write (SPDW) of carbon via scanning force microscopy.

The fabrication of carbon nanostructures by direct writing with a scanning force microscope is described. A conductive atomic force tip is used to collect carbon from a glassy carbon substrate and redeposit it onto a gold thin film under voltage control. The resulting patterns are examined with atomic force microscopy and Auger electron spectrometry. Writing of carbon lines with widths as small as 40 nm is demonstrated.

Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement.

A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).

Dielectrophoretic manipulation of finite sized species and the importance of the quadrupolar contribution.

Dielectrophoresis (DEP) is the movement of polarizable species in a nonuniform electric field. DEP is used to attract (positive DEP) to or repel from (negative DEP) regions of high field intensity and is useful for manipulating species, including biological species. Current theoretical and numerical approaches used to predict the response to DEP forces assume that the target species is a point particle; however, in practice, the target species is of finite size, e.g., macromolecules, spores and assay beads. To elucidate the importance of target species size effects, higher order terms in the DEP force multipole expansion must be considered [Electrophoresis 23, 1973 (2002)]]. In this paper, we used the method of Green's function to derive and explore the importance of the quadrupolar contribution to the DEP forces acting on finite-sized species produced by a planar, interdigitated array of electrodes. Based on the analysis, it was found, for example, that at a fixed height of 20 mum in an interdigitated DEP array with an electrode width and spacing of 20 mum energized by a 10 Vp p, 1.0 MHz ac signal, the quadrupolar contribution to the total DEP force was 5% for a latex bead with 4.2 mum in radius and 10% for the one with 6 mum in radius. For a fixed, fractional quadrupolar contribution, beta , both the exact calculation and the scaling estimate elucidate that the critical size of particle increase linearly with the electrode width (and spacing) at a fixed height, while the critical particle radius increases with a square-root dependence on the width height above the electrode in the electrode array.

Micromachined, silicon filament light source for spectrophotometric microsystems.

A miniature broadband light source is a critical element in a spectrophotometric microsystem. The design, fabrication, and characterization of a highly stable, miniature broadband light source that comprises filaments of single-crystal silicon are presented. Electrical current versus voltage and radiant emittance spectra under constant voltage bias are measured and related to filament dimensions. A maximum stable operating temperature for these filaments is estimated to be 1200 K. Resistance drift is demonstrated to be less than 0.5% over a 10-h period of continuous operation with visible incandescence. Emittance spectra of a multifilament array, measured at three different electrical biases, are presented and shown to compare well with theoretical blackbody radiation spectra. A continuous, total radiated power of 10.7 mW was achieved with a 1 mm x 1 mm filament array with peak emittance at lambda=2.7 micrometers.