Robert Northcutt

Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Polypyrrole membranes as scaffolds for biomolecule immobilization

Enzymes have evolved over hundreds of years through changes in ecosystems (climate, atmosphere, hydrology, etc). The evolutionary changes driven by the need to survive has led to enzymes with diverse functionality such as reduction of carbon dioxide and methane to other forms of carbon, fixation of nitrogen, and high temperature biochemical processes. While these enzymes have useful properties, engineering a scalable cell-free system with these enzymes will be useful for stable production of desired products without involving the vagaries of cellular metabolism. This article presents various approaches to incorporate ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a conducting polymer (polypyrrole (PPy)) doped with a bulky anion (dodecylbenzenesulfonate (DBS)) in an effort to create functional devices for the conversion of carbon dioxide into precursors for high-value chemicals. We demonstrate that the tailored device creates an environment where the enzyme can retain its function while being protected from denaturing conditions. It is envisioned that the 3-PGA produced by RuBisCO will be converted into value-added products.

Fabrication and characterization of an integrated ionic device from suspended polypyrrole and alamethicin-reconstituted lipid bilayer membranes

Conducting polymers are electroactive materials that undergo conformal relaxation of the polymer backbone in the presence of an electrical field through ion exchange with solid or aqueous electrolytes. This conformal relaxation and the associated morphological changes make conducting polymers highly suitable for actuation and sensing applications. Among smart materials, bioderived active materials also use ion transport for sensing and actuation functions via selective ion transport. The transporter proteins extracted from biological cell membranes and reconstituted into a bilayer lipid membrane in bioderived active materials regulate ion transport for engineering functions. The protein transporter reconstituted in the bilayer lipid membrane is referred to as the bioderived membrane and serves as the active component in bioderived active materials. Inspired by the similarities in the physics of transduction in conducting polymers and bioderived active materials, an integrated ionic device is formed from the bioderived membrane and the conducting polymer membrane. This ionic device is fabricated into a laminated thin-film membrane and a common ion that can be processed by the bioderived and the conducting polymer membranes couple the ionic function of these two membranes. An integrated ionic device, fabricated from polypyrrole (PPy) doped with sodium dodecylbenzenesulfonate (NaDBS) and an alamethicin-reconstituted DPhPC bilayer lipid membrane, is presented in this paper. A voltage-gated sodium current regulates the electrochemical response in the PPy(DBS) layer. The integrated device is fabricated on silicon-based substrates through microfabrication, electropolymerization, and vesicle fusion, and ionic activity is characterized through electrochemical measurements.

Mechanoelectrochemistry of soft electroactive materials via surface-tracked scanning electrochemical microscopy

Real time measurement of time-correlated ion transport and volumetric changes in electroactive materials is necessary to understand and model mechanoelectrochemistry. Reversible reduction and oxidation of soft electroactive materials such as conducting polymers result in the deformation of the material due to ion transport into and out of the polymer backbone. In cells, ion transport and volumetric expansion are collectively responsible for homeostasis that is essential for life functions and hence, mechanoelectrochemistry of cells is essential to understand cell and developmental biology. The characterization methods required to investigate mechanoelectrochemistry require nanoscale spatial resolution for the imaging of a redox active site in a polymer or a small group of transmembrane proteins in a single cell. Towards this goal, we present an imaging technique using scanning electrochemical microscopy (SECM) hardware with shear-force (SF) feedback for high bandwidth mechanoelectrochemistry characterization. In this proceedings article, we demonstrate this technique referred to as surface-tracked scanning electrochemical microscopy technique (ST-SECM) that is realized by measuring the structural feedback of the glass electrode to position the electrode in 10s of nanometers above the surface of a polypyrrole membrane doped with dodecylbenzenesulfonate (PPy(DBS)). Two ultra-microelectrodes of controlled dimensions (of 20 μm and 30 μm glass diameter) were fabricated using a hydrofluoric acid etching technique and were used to generate a spatially correlated ion storage map of PPy(DBS). We compare the developed technique to a three-dimensional discrete scan over the surface and show that a ST-SECM technique produces a higher resolution and takes approximately 200 fewer minutes as compared to the conventional technique.

An Investigation of Morphology Dependent Charge Storage in Polypyrrole Membranes

PPy-based membranes exchange ions with electrolyte through reversible redox processes and hence are best suited as electrodes for batteries and super capacitors. The energy density of batteries and super capacitors are dependent on the specific capacitance of the conducting polymer and can be represented through a mechanistic model for ion transport. Through this model, the specific capacitance of polypyrrole-based membranes is shown to be dependent on the number of accessible redox sites at the electrolyte-polymer interface. The accessibility of redox sites at the electrolyte-polymer interface can be increased by controlling the morphological properties and distribution of dopant in the polymer backbone. Thus, by nanostructuring and by controlling the distribution of the dopant in the polymer, we have shown that the capacitance of PPy-based membranes can be increased to 490 F.g−1 for a 50 mV.sec−1 scan rate and 0.6 g.cm−2 specific mass. Despite this value of specific capacitance being the highest reported for PPy-based membranes to date, it is estimated that only 69% of active redox sites are used for ion storage and hence can be increased further. Maximizing specific capacitance requires an understanding of spatial distribution of redox sites in the polymer backbone and its corresponding chemoelectrical activity. In order to generate a spatial map of ion storage in PPy-based membranes, this article presents for the first time a shear-force (SF) based topography imaging and scanning electrochemical microscopy (SECM) imaging of the PPy(DBS) under reduced and oxidized conditions. From a correlated topography and chemoelectrical activity of PPy-based membrane, the data shows the availability of redox sites in the polymer and it is projected that this result will enhance the design and nanostructuring of PPy-based membranes and distribution of dopant in the backbone.Copyright

Characterization of Electrochemical Capacity of a Biotemplated Polypyrrole Membrane

Recent studies of polypyrrole (PPy) electrodes have been increasing the interfacial surface area in order to increase electrochemical performance. We present a novel method of electropolymerizing PPy doped with dodecylbenzenesulfonate (DBS) referred to as biotemplating. A biotemplated conducting polymer utilizes phospholipid vesicles in order to form a three dimensional structure with a sponge-like shape. The vesicles, measuring 1–2 μm in diameter, are added in situ with the polymerization solution. They become enveloped while maintaining their structure during electropolymerization of PPy(DBS). The result of this structure is a significant increase in surface area compared to current techniques. There are several advantages in using biotemplated conducting polymers as battery electrodes. Compared to a planar PPy(DBS) membrane, biotemplated PPy(DBS) membranes have a roughly 50% increased storage capacity. There is an expected reduction in volumetric expansion during ion ingress/egress into the polymer backbone. This reduction would result in decreased fatigue loading and improving cyclability. Further, biotemplated PPy(DBS) membranes can be fabricated into thin structures with increased flexibility, allowing them to be rolled into various packaging sizes. In this article, the charge density of a biotemplated PPy(DBS) membrane as a function of charging and discharging currents is compared to a planar PPy(DBS) membrane. The structural enhancement offers systemic advantages by providing higher volumetric energy density and decreased fatigue loading for applications involving conducting polymer electrodes.Copyright

Dynamics of ion transport in a bio-derived ionic transistor

Biological processes and electromechanical function in ionic polymers share ion transport as the fundamental processes for sensing, actuation and energy harvesting. Inspired by the similarity in protein-bound cell membranes and polypyrrole membrane (an ionic polymer), our group is developing a hybrid device that provides the template for integrating biology and electronics. The integrated device, referred to as a bio-derived ionic transistor (BIT), consists of a bilayer lipid membrane (BLM) formed on a polypyrrole membrane and has two inputs that regulates the output of the device. This proceedings article will discuss the constructional features of proposed actuator, fabrication procedure of a prototype actuator and discuss a modeling framework for analyzing the dynamics of the ionic response.

Electrochemical Analysis of Alamethicin Reconstituted Planar Bilayer Lipid Membranes Supported on Polypyrrole Membranes

Conducting polymers possess similarity in ion transport function to cell membranes and perform electro-chemo-mechanical energy conversion. In an in vitro setup, protein-reconstituted bilayer lipid membranes (bioderived membranes)perform similar energy conversion and behave like cell membranes. Inspired by the similarity in ionic function between a conducting polymer membrane and cell membrane, this article presents a thin-film laminated membrane in which alamethicin-reconstituted lipid bilayer membrane is supported on a polypyrrole membrane. Owing to the synthetic and bioderived nature of the components of the membrane, we refer to the laminated membrane as a hybrid bioderived membrane. In this article, we describe the fabrication steps and electrochemical characterization of the hybrid membrane. The fabrication steps include electropolymerization of pyrrole and vesicle fusion to result in a hybrid membrane; and the characterization involves electrical impedance spectroscopy, chronoamperometry and cyclic voltammetry. The resistance and capacitance of BLM have the magnitude of 4.6×109 Ω-cm2 and 1.6×10−8 F/cm2 .The conductance of alamethicin has the magnitude of 6.4×10−8 S/cm2 . The change in ionic conductance of the bioderived membrane is due to the electrical field applied across alamethicin, a voltage-gated protein and produces a measurable change in the ionic concentration of the conducting polymer substrate.Copyright

Polypyrrole Bridge as a Support for Alamethicin-Reconstituted Planar Bilayer Lipid Membranes

Conducting polymer actuators and sensors utilize electrochemical reactions and associated ion transport at the polymer-electrolyte interface for their engineering function. Similarly, a bioderived active material utilizes ion transport through a protein and across a bilayer lipid membrane for sensing and actuation functions. Inspired by the similarity in ion transport process in a bilayer lipid membrane (BLM) and conducting polymers, we propose to build an integrated ionic device in which the ion transport through the protein in the bilayer lipid membrane regulates the electrolytic and mechanical properties of the conducting polymer. This article demonstrates the fabrication and characterization of a DPhPC planar BLM reconstituted with alamethicin and supported on a polypyrrole bridge measuring 100 μm × 500 μm and formed across micro-fabricated gold pads. The assembly is supported on silicon dioxide coated wafers and packaged into an electronic-ionic package for electrochemical characterization. The various ionic components in the integrated ionic device are characterized using electrical impedance spectroscopy (EIS), cyclic voltammetry (CV), and chronoamperometry (CA) measurements. The results from our experimental studies demonstrate the procedure to fabricate a rugged electro active polymer supported BLM that will serve as a platform for chemical, bioelectrical sensing and VOC detection.Copyright