Plasma polymer and biomolecule modification of 3-D scaffolds for tissue engineering

Plasma polymerization was used to coat a melt electrospun polycaprolactone scaffold to improve cell attachment and organization. Plasma polymerization was performed using an amine containing monomer, allylamine, which then allowed for the subsequent immobilization of biomolecules i.e. heparin and fibroblast growth factor-2. The stability of the plasma polymerized amine-coating was demonstrated by X-ray photoelectron spectroscopy analysis and imaging time-of-flight secondary ion mass spectrometry revealed that a uniform plasma amine-coating was deposited throughout the scaffold. Based upon comparison with controls it was evident that the combination scaffold aided cell ingress and the formation of distinct fibroblast and keratinocyte layers.

Preservation of native conformation during aluminium-induced aggregation of tau protein

ALUMINIUM exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27(Al) NMR spectroscopy. The CD studies revealed a five-fold increase in the observed ellipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. Al-27 NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits,the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for tills process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.

GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Polypyrrole based amperometric glucose biosensors

Biosensors have gained immense acceptance in the field of medical diagnostics, besides environmental, food safety and biodefence applications due to its attributes of real-time and rapid response. This synergistic combination of biotechnology and microelectronics comprises a biological recognition element coupled with a compatible transducer device. Diabetes is a disease of major concern since the ratio of world population suffering from it is increasing at an alarming rate and therefore the need for development of accurate and stable glucose biosensors is evident. There are many commercial glucose biosensors available yet some limitations need attention. This review presents a detailed account of the polypyrrole based amperometric glucose biosensors. The polymer polypyrrole is used extensively as a matrix for immobilization of glucose oxidase enzyme owing to its favourable features such as stability under ambient conditions, conductivity that allows it to be used as an electron relay, ability to be polymerized under neutral and aqueous mild conditions, and more. The simple one-step electrodeposition on the electrode surface allows easy entrapment of the enzyme. The review is structured into three categories (a) the first-stage biosensors: which report the studies from the inception of use of polypyrrole in glucose biosensors during which time the role of the polymer and the use of mediators was established. This period saw extensive work by two separate groups of Schuhmann and Koopal who contributed a great deal in understanding the electron transfer pathways in polypyrrole based glucose biosensors, (b) the second-stage biosensors: which highlight the shift of polypyrrole from a conventional matrix to composite matrices with extensive use of mediators focused at improving the selectivity of response, and (c) third-stage biosensors: the remarkable properties of nanoparticles and carbon nanotubes and their outstanding ability to mediate electrontransfers have seen their indispensable use in conjugation with polypyrrole for development of glucose biosensors with improved sensitivity and stability characteristics which is accounted in the review, which thus traces the evolution of polypyrrole from a conventional matrix, to composites and thence to the form of nanotube arrays, with the objective of addressing the vital issue of diabetes management through the development of stable and reliable glucose biosensors.

Electrochemical Functionalization of a Gold Electrode with Redox-Active Self-Assembled Monolayer for Electroanalytical Application

The electrochemical functionalization of a Au electrode with a redox-active monolayer and the electroanalytical applications of the functionalized electrode are described. Reaction of the electrochemically derived o-quinone on the self-assembled monolayer (SAM) of 6-mercaptopurine (MPU) on a Au electrode gives a redox-active 4-(6-mercapto-purin-9-yl)benzene-1,2-diol (MPBD) self-assembly under optimized conditions. Electrochemical quartz crystal microbalance technique has been employed to follow the functionalization of the electrode in real time. Electrochemically derived o-quinone reacts at the N(9) position of the self-assembled MPU in neutral pH. Raman spectral measurement confirms the reaction of o-quinone on MPU self-assembly. MPBD shows a well-defined reversible redox response, characteristic of a surface-confined redox mediator at 0.21 V in neutral pH. The anodic peak potential (Epa) of MPBD shifts by −60 mV while changing the solution pH by 1 unit, indicating that the redox reaction involves two electrons and two protons. The surface coverage (Γ) of MPBD was 7.2 ± 0.3 × 10-12 mol/cm2. The apparent heterogeneous rate constant (ksapp) for MPBD was 268 ± 6 s-1. MPBD efficiently mediates the oxidation of nicotinamide adenine dinucleotide (NADH) and ascorbate (AA). A large decrease in the overpotential and significant increase in the peak current with respect to the unmodified electrode has been observed. Surface-confined MPBD has been successfully used for the amperometric sensing of NADH and AA in neutral pH at the nanomolar level.

Quadratic Nonlinearity of One- and Two-Electron Oxidized Metalloporphyrins and Their Switching in Solution

We report the quadratic nonlinearity of one- and two-electron oxidation products of the first series of transition metal complexes of meso-tetraphenylporphyrin (TPP). Among many MTPP complexes, only CuTPP and ZnTPP show reversible oxidation/reduction cycles as seen from cyclic voltammetry experiments. While centrosymmetric neutral metalloporphyrins have zero first hyperpolarizability, β, as expected, the cation radicals and dications of CuTPP and ZnTPP have very high β values. The one- and two-electron oxidation of the MTPPs leads to symmetry-breaking of the metal−porphyrin core, resulting in a large β value that is perhaps aided in part by contributions from the two-photon resonance enhancement. The calculated static first hyperpolarizabilities, β0, which are evaluated in the framework of density functional theory by a coupled perturbed Hartree−Fock method, support the experimental trend. The switching of optical nonlinearity has been achieved between the neutral and the one-electron oxidation products but not between the one- and the two-electron oxidation products since dications that are electrochemically reversible are unstable due to the formation of stable isoporphyrins in the presence of nucleophiles such as halides.

Direct Synthesis of Terminally ``Clickable'' Linear and Hyperbranched Polyesters

1,3-Dipolar cycloaddition of an organic azide and an acetylenic unit,often referred to as the ``click reaction'', has become an important ligation tool both in the context of materials chemistry and biology. Thus, development of simple approaches to directly generate polymers that bear either an azide or an alkyne unit has gained considerable importance. We describe here a straightforward approach to directly prepare linear and hyperbranched polyesters that carry terminal propargyl groups. To achieve the former, we designed an AB-type monomer that carries a hydroxyl group and a propargyl ester, which upon self-condensation under standard transesterification conditions yielded a polyester that carries a single propargyl group at one of its chain-ends. Similarly, an AB(2) type monomer that carries one hydroxyl group and two propargyl ester groups, when polymerized under the same conditions yielded a hyperbranched polymer with numerous clickable'' propargyl groups at its molecular periphery. These propargyl groups can be readily clicked with different organic azides, such as benzyl azide, omega-azido heptaethyleneglycol monomethylether or 9-azidomethyl anthracene. When an anthracene chromophore is clicked, the molecular weight of the linear polyester could be readily estimated using both UV-visible and fluorescence spectroscopic measurements. Furthermore, the reactive propargyl end group could also provide an opportunity to prepare block copolymers in the case of linear polyesters and to generate nanodimensional scaffolds to anchor variety of functional units, in the case of the hyperbranched polymer. (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3200-3208, 2010.

A low-cost lead-acid battery with high specific-energy

Lightweight grids for lead-acid battery grids have been prepared from acrylonitrile. butadiene styrene (ABS) copolymer followed by coating with lead. Subsequently, the grids have been electrochemically coated with a conductive and corrosion-resistant layer of polyaniline. These grids are about 75% lighter than those employed in conventional lead-acid batteries. Commercial-grade 6V/3.5 Ah (C-20-rate) lead-acid batteries have been assembled and characterized employing positive and negative plates constituting these grids. The specific energy of such a lead-acid battery is about 50 Wh/kg. The batteries can withstand fast charge-discharge duty cycles.

A hybrid electrochemical-thermal method for the preparation of large ZnO nanoparticles

A simple and efficient two-step hybrid electrochemical-thermal route was developed for the synthesis of large quantity of ZnO nanoparticles using aqueous sodium bicarbonate electrolyte and sacrificial Zn anode and cathode in an undivided cell under galvanostatic mode at room temperature. The bath concentration and current density were varied from 30 to 120 mmol and 0.05 to 1.5 A/dm(2). The electrochemically generated precursor was calcined for an hour at different range of temperature from 140 to 600 A degrees C. The calcined samples were characterized by XRD, SEM/EDX, TEM, TG-DTA, FT-IR, and UV-Vis spectral methods. Rietveld refinement of X-ray data indicates that the calcined compound exhibits hexagonal (Wurtzite) structure with space group of P63mc (No. 186). The crystallite sizes were in the range of 22-75 nm based on Debye-Scherrer equation. The TEM results reveal that the particle sizes were in the order of 30-40 nm. The blue shift was noticed in UV-Vis absorption spectra, the band gaps were found to be 5.40-5.11 eV. Scanning electron micrographs suggest that all the samples were randomly oriented granular morphology.

Nonlinear Transport in Hybrid Polypyrrole-Gold Nanostructures

We have studied charge transport in nanometer scale films of polypyrrole (PPy) that were grown electrochemically onto discontinuous ultrathin films of gold. The gold films consisted of 100 nm size islands, separated from each other by nanometer-size gaps. The thickness of PPy can be varied from 30 to 200 nm. The I-V characteristics of these hybrid PPy-Au nanostructures show strong non-linearity at low temperatures, and in particular for the more insulating samples. The hopping transport is further verified from the log / versus V-1/4 plots. Furthermore, the I-V data follow an empirical relation dlog//dV(1/4) similar to T-1/2.

Effect of surface roughness on diffusion-limited charge transfer

A theory is developed for diffusion-limited charge transfer on a non-fractally rough electrode. The perturbation expressions are obtained for concentration, current density and measured diffusion-limited current for arbitrary one- and two-dimensional surface profiles. The random surface model is employed for a rough electrode\electrolyte interface. In this model the gross geometrical property of an electrochemically active rough surface - the surface structure factor-is related to the average electrode current, current density and concentration. Under short and long time regimes, various morphological features of the rough electrodes, i.e. excess area (related to roughness slope), curvature, correlation length, etc. are related to the (average) current transients. A two-point Pade approximant is used to develop an all time average current expression in terms of partial morphological features of the rough surface. The inverse problem of predicting the surface structure factor from the observed transients is also described. Finally, the effect of surface roughness is studied for specific surface statistics, namely a Gaussian correlation function. It is shown how the surface roughness enhances the overall diffusion-limited charge transfer current.

Probing Single and Bilayer Graphene Field Effect Transistors By Raman Spectroscopy

This article is a review of our work related to Raman studies of single layer and bilayer graphenes as a function Fermi level shift achieved by electrochemically top gating a field effect transistor. Combining the transport and in situ Raman studies of the field effect devices, a quantitative understanding is obtained of the phonon renormalization due to doping of graphene. Results are discussed in the light of time dependent perturbation theory, with electron phonon coupling parameter as an input from the density functional theory. It is seen that phonons near and Gamma and K points of the Brillouin zone are renormalized very differently by doping. Further, Gamma-phonon renormalization is different in bilayer graphene as compared to single layer, originating from their different electronic band structures near the zone boundary K-point. Thus Raman spectroscopy is not only a powerful probe to characterize the number of layers and their quality in a graphene sample, but also to quantitatively evaluate electron phonon coupling required to understand the performance of graphene devices.

Electrochemical co-deposition of bimetallic Pt-Ru nanoclusters dispersed on poly(3,4-ethylenedioxythiophene) and electrocatalytic behavior for methanol oxidation

Nanoclusters of bimetallic Pt-Ru are electrochemically deposited on conductive polymer, poly(3,4-ethylenedioxythiophene)(PEDOT), which is also electrochemically deposited on a carbon paper substrate. The bimetallic deposition is carried out in an acidic electrolyte consisting of chloroplatinic acid and ruthenium chloride at 0.0 V versus saturated calomel electrode (SCE) on PEDOT coated carbon paper. A thin layer PEDOT on a carbon paper substrate facilitates the formation of uniform, well-dispersed, nano clusters of Pt-Ru of mean diameter of 123 nm, which consist of nanosize particles. In the absence of PEDOT, the size of the clusters is about 251 nm, which are unevenly distributed on carbon paper substrate. Cyclic voltammetry studies suggest that peak currents of methanol oxidation are several times greater on PtRu-PEDOT electrode than on Pt-Ru electrode in the absence of PEDOT. (C) 2011 Elsevier B.V. All rights reserved.

A new polymeric flame retardant additive for vinyl polymers

Diethyl allyl phosphate (DEAP) monomer has been synthesized, and characterized, using H-1 NMR and direct ionization mass spectrometric (DI-MS) techniques. It was free-radically polymerized to yield the poly(diethyl allyl phosphate) (PDEAP). The direct pyrolysis-mass spectrometric (DP-MS) analysis of the PDEAP revealed that it undergoes thermal degradation to yield mainly the monomer. Utility of PDEAP as a potent flame-retardant additive in polystyrene (PS) and poly(methyl methacrylate) (PMMA) has also been established.