Contributions of Microelectrochemical Scanning Techniques for the Efficient Detection of Localized Corrosion Processes at the Cut Edges of Polymer-Coated Galvanized Steel.

Spatially resolved information on corrosion reactions operating at the cut edges of coated metals can be obtained using microelectrochemical scanning techniques using a suitable selection of operation modes and scanning probes. The scanning vibrating electrode technique (SVET) provides current density maps with a spatial resolution of the order of the dimensions of the sample, which allows the temporal evolution of the corrosion reactions to be followed over time. This leads to the identification and localization of cathodic and anodic sites, although the technique lacks chemical specificity for the unequivocal identification of the reactive species. The application of scanning electrochemical microscopy (SECM) was previously limited to image cathodic reaction sites, either due to oxygen consumption in the amperometric operation or by the alkalinisation of the electrolyte in potentiometric operation. However, it is shown that anodic sites can be effectively monitored using an ion-selective microelectrode (ISME) as a probe. The ISME probes detected differences in the local concentrations of Zn2+ and OH- ions from the cut edges of a complete coil coating system compared to the same system after the polymeric layers were removed. In this way, it has been shown that the inhibitor loading in the polymer layers effectively contributes to reducing the corrosion rates at the cut edge, thus helping to extend the useful life of the sacrificial galvanized layer bonded directly to the steel matrix. Additionally, these two probe configurations can be integrated into a multi-electrode tip for potentiometric operation to simultaneously monitor localized changes in pH values and metal ion dissolution in a single scan. Spatial and temporal distributions were further investigated using different rastering procedures, and the potential of constructing pseudomaps for 2D-imaging is described.

Thermotropic and structural effects of poly(malic acid) on fully hydrated multilamellar DPPC-water systems.

The thermotropic and structural effects of low molecular weight poly(malic acid) (PMLA) on fully hydrated multilamellar dipalmitoylphosphatidylcholine (DPPC)-water systems were investigated using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and freeze-fracture transmission electron microscopy (FFTEM). Systems of 20wt% DPPC concentration and 1 and 5wt% PMLA to lipid ratios were studied. The PMLA derivatives changed the thermal behavior of DPPC significantly and caused a drastic loss in correlation between lamellae in the three characteristic thermotropic states (i.e., in the gel, rippled gel and liquid crystalline phases). In the presence of PBS or NaCl, the perturbation was more moderate. The structural behavior on the atomic level was revealed by FTIR spectroscopy. The molecular interactions between DPPC and PMLA were simulated via modeling its measured infrared spectra, and their peculiar spectral features were interpreted. Through this interpretation, the poly(malic acid) is inferred to attach to the headgroups of the phospholipids through hydrogen bonds between the free hydroxil groups of PMLA and the phosphodiester groups of DPPC.

Corrosion Protection and Heat Resistance of Paints for Outdoor Use.

Innovative heat- and corrosion-resistant coating approaches, applicable in indirect-food-contact outdoor environments, have been developed. Two systems, a direct-to-metal single-layer, polysiloxane-based, oven-dried system and a bilayer, zinc phosphate active pigment-containing, ambient-cured system were developed to overcome the shortcomings of the traditional bilayer, zinc-rich primer-based heat-resistant surface-protective solutions for outdoor cooking equipment, such as barbecue grills. This case study aims to optimize the application conditions, measure and evaluate the impact of surface preparation and compare thermo-resistant and anticorrosive properties of different coating systems focusing on eco-efficiency. The anticorrosion efficiency of the coatings was characterized using salt-spray chamber corrosion tests and electrochemical impedance spectroscopy. The thermo-resistant character of the coatings was tested by cyclic and constant heat treatment, after which the physical integrity of the coatings was evaluated by optical microscopy. In the overall performance of the coatings, the roughening of the steel substrate surface and the thickness of the coatings were also considered as influential parameters. The study revealed that the newly developed coatings have superior anticorrosion performance to the usually applied Zn-rich coating. The Single-layered Coating has excellent corrosion resistance under certain conditions and has the advantage of fast layer application. The Bilayered Coating showed excellent heat- and corrosion-resistance properties even on a surface without sand-blasting.

Silica@zirconia Core@shell Nanoparticles for Nucleic Acid Building Block Sorption.

The development of delivery systems for the immobilization of nucleic acid cargo molecules is of prime importance due to the need for safe administration of DNA or RNA type of antigens and adjuvants in vaccines. Nanoparticles (NP) in the size range of 20-200 nm have attractive properties as vaccine carriers because they achieve passive targeting of immune cells and can enhance the immune response of a weakly immunogenic antigen via their size. We prepared high capacity 50 nm diameter silica@zirconia NPs with monoclinic/cubic zirconia shell by a green, cheap and up-scalable sol-gel method. We studied the behavior of the particles upon water dialysis and found that the ageing of the zirconia shell is a major determinant of the colloidal stability after transfer into the water due to physisorption of the zirconia starting material on the surface. We determined the optimum conditions for adsorption of DNA building blocks, deoxynucleoside monophosphates (dNMP), the colloidal stability of the resulting NPs and its time dependence. The ligand adsorption was favored by acidic pH, while colloidal stability required neutral-alkaline pH; thus, the optimal pH for the preparation of nucleic acid-modified particles is between 7.0-7.5. The developed silica@zirconia NPs bind as high as 207 mg dNMPs on 1 g of nanocarrier at neutral-physiological pH while maintaining good colloidal stability. We studied the influence of biological buffers and found that while phosphate buffers decrease the loading dramatically, other commonly used buffers, such as HEPES, are compatible with the nanoplatform. We propose the prepared silica@zirconia NPs as promising carriers for nucleic acid-type drug cargos.

In Situ Investigation of the Cytotoxic and Interfacial Characteristics of Titanium When Galvanically Coupled with Magnesium Using Scanning Electrochemical Microscopy.

Recently, the cytotoxic properties of galvanically coupled Ti-Mg particles have been shown in different cells. This cytotoxic effect has been attributed mainly to Mg due to its tendency to undergo activation when coupled with Ti, forming a galvanic cell consisting of an anode (Mg) and a cathode (Ti). However, the role of the Ti cathode has been ignored in explaining the cytotoxic effect of Ti-Mg particles due to its high resistance to corrosion. In this work, the role of titanium (Ti) in the cytotoxic mechanism of galvanically coupled Ti-Mg particles was examined. A model galvanic cell (MGC) was prepared to simulate the Mg-Ti particles. The electrochemical reactivity of the Ti sample and the pH change in it due to galvanic coupling with Mg were investigated using scanning electrochemical microscopy (SECM). It was observed that the Ti surface changed from passive to electrochemically active when coupled with Mg. Furthermore, after only 15 min of galvanic coupling with Mg, the pH in the electrolyte volume adjacent to the Ti surface increased to an alkaline pH value. The effects of the galvanic coupling of Ti and Mg, as well as those of the alkaline pH environment, on the viability of Hs27 fibroblast cells were investigated. It was shown that the viability of Hs27 cells significantly diminished when Mg and Ti were galvanically coupled compared to when the two metals were electrically disconnected. Thus, although Ti usually exhibited high corrosion resistance when exposed to physiological environments, an electrochemically active surface was observed when galvanically coupled with Mg, and this surface may participate in electron transfer reactions with chemical species in the neighboring environment; this participation resulted in the increased pH values above its surface and enhanced generation of reactive oxygen species. These features contributed to the development of cytotoxic effects by galvanically coupled Ti-Mg particles.

Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography.

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.

Electrochemical time of flight method for determination of diffusion coefficients of glucose in solutions and gels.

The diffusion coefficient of glucose in different media is an important parameter in life sciences, as well as in biotechnology and microbiology. In this work a simple, fast method is proposed that is based on the electrochemical time of flight principle. In most of the earlier time of flight experiments performed, a constant flight distance was applied. In the present work a scanning electrochemical microscope (SECM) was applied as a measuring tool. With use of the SECM, the flying distance could be changed with high precision, making measurements with several flight distances more accurate and reliable values could be obtained for solutions as well as for gels. The conventional voltammetric methods are not applicable for glucose detection. In our work electrocatalytic copper oxide coated copper microelectrodes and micro-sized amperometric enzyme sensors were used as detectors, while microdroplet-ejecting pneumatically driven micropipettes were used as a source.

Role of oligo(malic acid) on the formation of unilamellar vesicles.

Stable unilamellar dipalmitoylphosphatidylcholine vesicles were produced by using oligo(malic acid) and cholesterol. Detailed physico-chemical characterization prove that by using oligo(malic acid) the substitution of PEGylated lipids for sterically stabilization comes possible. The polymer molecules cover the outer surface of spherical-shaped vesicles, and an asymmetrical composition occurs in the two leaflets of the phospholipid bilayer. The oligo(malic-acid) and cholesterol are enriched in the outer side assuring the stabilization of vesicles. Cholesterol plays an important role in the self-assembly of components as it makes the entering of oligomers possible deep into the polar head-region of lipids. The presence of oligo(malic acid) molecules does not induce degradation by hydrolysis of lipid molecules but the vesicle system turns into a sensitive form giving a possibility for pH sensitive targeting. Preliminary investigation on the investigated oligo(malic acid)-stabilized vesicles do not show any toxic effect promising their applicability in the field of liposomal drug delivery.

An approach to in situ detection of hydrogen peroxide: application of a commercial needle-type electrode.

The reactive oxygen species are thought to play major roles in developing different physiological disorders. A commercial, needle-type amperometric glucose enzyme sensor manufactured for human patients was investigated. This sensor measures glucose by detecting hydrogen peroxide evolved in the enzymatic reaction of glucose. In the experiments, the immobilized enzyme layer of the sensor was inactivated. The applicability of this 'inhibited' glucose sensor for detecting hydrogen peroxide was tested. The simple battery powered, single purpose electronic unit was replaced by an advanced electrochemical workstation. The sensitivity, selectivity and lower limit of detection of the hydrogen peroxide measurements were investigated. Voltammetric measurements were carried out in intensively stirred buffered aqueous media, in plasma samples as well as in subcutan areas of anesthetized Wistar rats. Preliminary measurements carried out with the amperometric and periodically interrupted amperometric technique predicted that the human clinical sensor, after our enzyme inhibition step, can be used for checking the elevation of the hydrogen peroxide level in different subcutan areas of human subjects.

Preparation and 68Ga-radiolabeling of porous zirconia nanoparticle platform for PET/CT-imaging guided drug delivery.

This paper describes the preparation of gallium-68 (68Ga) isotope labeled porous zirconia (ZrO2) nanoparticle (NP) platform of nearly 100nm diameter and its first pharmacokinetic and biodistribution evaluation accomplished with a microPET/CT (µPet/CT) imaging system. Objectives of the investigations were to provide a nanoparticle platform which can be suitable for specific delivery of various therapeutic drugs using surface attached specific molecules as triggering agents, and at the same time, suitable for positron emission tomography (PET) tracing of the prospective drug delivery process. Radiolabeling was accomplished using DOTA bifunctional chelator. DOTA was successfully adsorbed onto the surface of nanoparticles, while the 68Ga-radiolabeling method proved to be simple and effective. In the course of biodistribution studies, the 68Ga-labeled DOTA-ZrNPs showed proper radiolabeling stability in their original suspension and in blood serum. µPet/CT imaging studies confirmed a RES-biodistribution profile indicating stable nano-sized labeled particles in vivo. Results proved that the new method offers the opportunity to examine further specifically targeted and drug payload carrier variants of zirconia NP systems using PET/CT imaging.

Direct immobilization of manganese chelates on silica nanospheres for MRI applications.

The development of tissue specific magnetic resonance imaging (MRI) contrast agents (CAs) is very desirable to achieve high contrast ratio combined with excellent anatomical details. To this end, we introduce a highly effective manganese(II) containing silica material, with the aim to shorten the longitudinal (T1) relaxation time. The microporous silica nanospheres (MSNSs) with enlarged porosity and specific surface area were prepared by a surfactant assisted aqueous method. Subsequently, the surface silanol groups were amino-functionalized, reacted with diethylenetriaminepentaacetic (DTPA) dianhydride and finally deposited with Mn2+. After comprehensive characterization, the MRI properties of functionalized MSNSs were investigated. The resulting nanospheres demonstrated substantial contrast enhancement during the in vitro MRI investigations, which was also evidenced by significant contrast enhancement on T1-weighted MR images in vivo. Moreover, in vitro cytotoxicity assay of functionalized MSNSs on hepatocyte mono- and hepatocyte-Kuppfer cell co-cultures showed no significant decrease in cell viability. Our findings confirmed our hypothesis, that Mn2+-chelating MSNSs are appropriate candidates for liver-specific T1-weighted MRI CAs with high relaxivities (r1=7.18mM-1s-1).

Periodically interrupted amperometry. A way of improving analytical performance of membrane coated electrodes.

Amperometry is a powerful voltammetric measuring method. Its application is specially advantageous when used in combination with a separation step or with some other sample treatment method providing selectivity. The selectivity is often achieved by coating the amperometric working electrode surface with a membrane of special character. Size exclusion membrane, immobilized enzyme containing reaction layer, protecting dialysis membrane, perm selective ion exchange film etc can be mentioned here. In conventional amperometry the measuring potential is continuously applied, therefore in case of membrane coated electrodes the electrode process depletes the diffusion layer. In this work the performance of a new periodically interrupted amperometric (PIA) measuring program has been investigated in case of glucose enzyme sensor. The measuring program allowing time for reloading the diffusion layer provided higher current and therefore improved sensitivity and lower limit of detection.

Electrochemical behavior of ferrocene in ionic liquid media.

Chemistry and applicability of ionic liquids (IL), - organic salts with low melting point - are in the focus of interest today. The ILs with melting point below room temperature are expected to be good solvents. Their applicability in organic synthetic work, in separation processes as well as in electrochemistry is very promising. In the work reported here the voltammetric behavior of ferrocene in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM+ PF6-) ionic liquid has been investigated. Conventional size and micro platinum and carbon electrodes were employed in CV and in chronoamperometric measurements. Karl Fischer method was used for the determination of water content of the solvent. Voltammetric measurements without addition of background electrolyte could be carried out in (BMIM+ PF6-) ionic liquid. A broad potential window could be used. Concentration dependence of the electrochemically determined diffusion coefficient of the ferrocene was observed.

Electropolymerized molecular imprinting on glassy carbon electrode for voltammetric detection of dopamine in biological samples.

A simple and reliable method for preparing a selective dopamine (DA) sensor based on a molecularly imprinted polymer of ethacridine was proposed. The molecularly imprinted polymer electrode was prepared through electrodepositing polyethacridine-dopamine film on the glassy carbon electrode and then removing DA from the film via chemical induced elution. The molecular imprinted sensor was tested by cyclic voltammetry as well as by differential pulse voltammetry (DPV) to verify the changes in oxidative currents of DA. In optimized DPV conditions the oxidation peak current was well-proportional to the concentration of DA in the range from 2.0×10(-8)M up to 1×10(-6)M. The limit of detection (3σ) of DA was found to be as low as 4.4nM, by the proposed sensor that could be considered a sensitive marker of DA depletion in Parkinson's disease. Good reproducibility with relative standard deviation of 1.4% and long term stability within two weeks were also observed. The modified sensor was validated for the analysis of DA in deproteinized human serum samples using differential pulse voltammetric technique.

Capsaicin-Sensitive Sensory Nerves Mediate the Cellular and Microvascular Effects of H2S via TRPA1 Receptor Activation and Neuropeptide Release.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

Lu-177-Labeled Zirconia Particles for Radiation Synovectomy.

The present article describes the preparation of ß-emitter lutetium-177-labeled zirconia colloid and its preliminary physicochemical and biological evaluation of suitability for local radionuclide therapy. The new (177)Lu-labeled therapeutic radiopharmaceutical candidate was based on the synthesis mode of a previously described zirconia nanoparticle system. The size and shape of the developed radiopharmaceutical compound were observed through a scanning electron microscope and dynamic light scattering methods. The radiocolloid had a 1.7 µm mean diameter and showed high in vitro radiochemical and colloid size stability at room temperature and during the blood sera stability test. After the in vitro characterizations, the product was investigated in the course of the treatment of a spontaneously diseased dog veterinary patient's hock joint completed with single-photon emission computed tomography (SPECT) imaging follow-up measurements and a dual-isotope SPECT imaging tests with conventional (99m)Tc-methanediphosphonic acid bone scintigraphy. In the treated dog, no clinical side-effects or signs of histopathological changes of the joints were recorded during the treatment. SPECT follow-up studies clearly and conspicuously showed the localization of the (177)Lu-labeled colloid in the hock joint as well as detectable but negligible leakages of the radiocolloid in the nearest lymph node. On the basis of biological follow-up tests, the orthopedic team assumed that the (177)Lu-labeled zirconia colloid-based local radionuclide therapy resulted in a significant and long-term improvement in clinical signs of the patient without any remarkable side-effects.

Total synthesis of isotopically enriched Si-29 silica NPs as potential spikes for isotope dilution quantification of natural silica NPs.

A new method was developed for the preparation of highly monodisperse isotopically enriched Si-29 silica nanoparticles ((29)Si-silica NPs) with the purpose of using them as spikes for isotope dilution mass spectrometry (IDMS) quantification of silica NPs with natural isotopic distribution. Si-29 tetraethyl orthosilicate ((29)Si-TEOS), the silica precursor was prepared in two steps starting from elementary silicon-29 pellets. In the first step Si-29 silicon tetrachloride ((29)SiCl4) was prepared by heating elementary silicon-29 in chlorine gas stream. By using a multistep cooling system and the dilution of the volatile and moisture-sensitive (29)SiCl4 in carbon tetrachloride as inert medium we managed to reduce product loss caused by evaporation. (29)Si-TEOS was obtained by treating (29)SiCl4 with absolute ethanol. Structural characterisation of (29)Si-TEOS was performed by using (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy and Fourier-transform infrared (FTIR) spectroscopy. For the NP preparation, a basic amino acid catalysis route was used and the resulting NPs were analysed using transmission electron microscopy (TEM), small angle X-ray scattering (SAXS), dynamic light scattering (DLS) and zeta potential measurements. Finally, the feasibility of using enriched NPs for on-line field-flow fractionation coupled with multi-angle light scattering and inductively coupled plasma mass spectrometry (FFF/MALS/ICP-MS) has been demonstrated.

Development and study of an amperometric biosensor for the in vitro measurement of low concentration of putrescine in blood.

An amperometric biosensor was developed for the in vitro determination of putrescine in blood samples because elevated level of putrescine in blood can be a diagnostic indicator of certain kinds of cancer. The electrochemical transducer consisted of a flat form, three electrode amperometric micro-cell fabricated with thin film photolithography on flexible Kapton substrate. An immobilized putrescine oxidase (PUO) layer provided the biocatalytic oxidation of the putrescine, while the generated hydrogen peroxide was detected on the platinum-working electrode. An electropolymerized poly(m-phenylenediamine) (pPDA) size-exclusion layer was used to protect the working electrode from fouling and to prevent signal generation by common electroactive interferents present in blood. The preparation of the biocatalytic enzyme- and outer protective layers was optimized for improved sensitivity and response time. A detection limit of 50 nM was achieved in pH-adjusted whole blood samples, which is below pathological levels.

Preparation, purification, and characterization of aminopropyl-functionalized silica sol.

A new, simple, and "green" method was developed for the surface modification of 20 nm diameter Stöber silica particles with 3-aminopropyl(diethoxy)methylsilane in ethanol. The bulk polycondensation of the reagent was inhibited and the stability of the sol preserved by adding a small amount of glacial acetic acid after appropriate reaction time. Centrifugation, ultrafiltration, and dialysis were compared in order to choose a convenient purification technique that allows the separation of unreacted silylating agent from the nanoparticles without destabilizing the sol. The exchange of the solvent to acidic water during the purification yielded a stable colloid, as well. Structural and morphological analysis of the obtained aminopropyl silica was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements, Fourier-transform infrared (FTIR), (13)C and (29)Si MAS nuclear magnetic resonance (NMR) spectroscopies, as well as small angle X-ray scattering (SAXS). Our investigations revealed that the silica nanoparticle surfaces were partially covered with aminopropyl groups, and multilayer adsorption followed by polycondensation of the silylating reagent was successfully avoided. The resulting stable aminopropyl silica sol (ethanolic or aqueous) is suitable for biomedical uses due to its purity.

Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe.

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 µM. Treatment with 0, 250, 500 or 1000 µM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 µM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98°C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.