Use of PtC Nanotips for Low-Voltage Quantum Tunneling Applications.

The use of focused ion and focused electron beam (FIB/FEB) technology permits the fabrication of micro- and nanometer scale geometries. Therefore, FIB/FEB technology is a favorable technique for preparing TEM lamellae, nanocontacts, or nanowires and repairing electronic circuits. This work investigates FIB/FEB technology as a tool for nanotip fabrication and quantum mechanical tunneling applications at a low tunneling voltage. Using a gas injection system (GIS), the Ga-FIB and FEB technology allows both additive and subtractive fabrication of arbitrary structures. Using energy dispersive X-ray spectroscopy (EDX), resistance measurement (RM), and scanning tunneling microscope (STM)/spectroscopy (STS) methods, the tunneling suitability of the utilized metal-organic material-platinum carbon (PtC) is investigated. Thus, to create electrode tips with radii down to 15 nm, a stable and reproducible process has to be developed. The metal-organic microstructure analysis shows suitable FIB parameters for the tunneling effect at high aperture currents (260 pA, 30 kV). These are required to ensure the suitability of the electrodes for the tunneling effect by an increased platinum content (EDX), a low resistivity (RM), and a small band gap (STM). The STM application allows the imaging of highly oriented pyrolytic graphite (HOPG) layers and demonstrates the tunneling suitability of PtC electrodes based on high FIB aperture currents and a low tunneling voltage.

Development and Proof of Concept of a Miniaturized MEMS Quantum Tunneling Accelerometer Based on PtC Tips by Focused Ion Beam 3D Nano-Patterning.

Most accelerometers today are based on the capacitive principle. However, further miniaturization for micro integration of those sensors leads to a poorer signal-to-noise ratio due to a small total area of the capacitor plates. Thus, other transducer principles should be taken into account to develop smaller sensors. This paper presents the development and realization of a miniaturized accelerometer based on the tunneling effect, whereas its highly sensitive effect regarding the tunneling distance is used to detect small deflections in the range of sub-nm. The spring-mass-system is manufactured by a surface micro-machining foundry process. The area of the shown polysilicon (PolySi) sensor structures has a size smaller than 100 µm × 50 µm (L × W). The tunneling electrodes are placed and patterned by a focused ion beam (FIB) and gas injection system (GIS) with MeCpPtMe3 as a precursor. A dual-beam system enables maximum flexibility for post-processing of the spring-mass-system and patterning of sharp tips with radii in the range of a few nm and initial distances between the electrodes of about 30-300 nm. The use of metal-organic precursor material platinum carbon (PtC) limits the tunneling currents to about 150 pA due to the high inherent resistance. The measuring range is set to 20 g. The sensitivity of the sensor signal, which depends exponentially on the electrode distance due to the tunneling effect, ranges from 0.4 pA/g at 0 g in the sensor operational point up to 20.9 pA/g at 20 g. The acceleration-equivalent thermal noise amplitude is calculated to be 2.4-3.4 mg/Hz. Electrostatic actuators are used to lead the electrodes in distances where direct quantum tunneling occurs.

CLC-b-mediated NO-3/H+ exchange across the tonoplast of Arabidopsis vacuoles.

Nitrate is frequently the major nitrogen source for plants and is generally assimilated during the day. In the absence of light, nitrate can transiently accumulate in the vacuolar lumen via tonoplast transporters. CLC-a, a member of the CLC family of anion transporters, is critically involved in this nitrate storage in the vacuole, while other CLC family members apparently have different roles in diverse cell organelles. Here, CLC-b, a close relative of CLC-a, was functionally expressed in oocytes and analyzed. CLC-b conducted strongly outwardly rectifying anionic currents that were largest in the presence of nitrate. Fluorescence ratio changes of oocytes loaded with a pH-dependent fluorescent dye suggested that NO(-)(3) transport is associated with H(+) exchange. CLC-b was localized at the tonoplast, as was CLC-c, when tagged with the green fluorescent protein. CLC-b expression was strongest in young roots, hypocotyl and cotyledons. The physiological role of CLC-b was analyzed using two independent knock-out alleles. Both lines grew as the wild type in various conditions. The total chloride and nitrate content was identical in clcb lines and the wild type, potentially suggesting that mutants were able to compensate the loss of CLC-b.

H-independent glutamine transport in plant root tips.

BACKGROUND: Glutamine is one of the primary amino acids in nitrogen assimilation and often the most abundant amino acid in plant roots. To monitor this important metabolite, a novel genetically encoded fluorescent FRET-reporter was constructed and expressed in Arabidopsis thaliana. As a candidate for the glutamine fluxes, the root tip localized, putative amino acid transporter CAT8 was analyzed and heterologously expressed in yeast and oocytes. PRINCIPAL FINDINGS: Rapid and reversible in vivo fluorescence changes were observed in reporter-expressing root tips upon exposure and removal of glutamine. FRET changes were detected at acid and neutral pH and in the presence of a protonophore, suggesting that part of the glutamine fluxes were independent of the pH. The putative amino acid transporter CAT8 transported glutamine, had a half maximal activity at approximately 100 microM and the transport was independent of external pH. CAT8 localized not only to the plasma membrane, but additionally to the tonoplast, when tagged with GFP. Ultrastructural analysis confirmed this dual localization and additionally identified CAT8 in membranes of autophagosomes. Loss-of function of CAT8 did not affect growth in various conditions, but over-expressor plants had increased sensitivity to a structural substrate analog, the glutamine synthetase inhibitor L-methionine sulfoximine. CONCLUSIONS: The combined data suggest that proton-independent glutamine facilitators exist in root tips.

Visualization of arginine influx into plant cells using a specific FRET-sensor.

Amino acids are not only the building blocks of proteins, but are the metabolic precursors of a variety of primary and secondary metabolites. In order to detect and visualize how plants transport, sense, and store amino acids with sub-cellular specificity, chimeric fluorescent proteins that respond to changes in amino acid concentrations were constructed. The reporter element of these sensors consists of a periplasmic bacterial protein that undergoes large, non-enzymatic conformational changes upon binding of its substrate. The receptor protein was attached to ECFP and an environmentally insensitive YFP derivative at opposite ends. Fluorescence resonance energy transfer changes were specifically observed after addition of arginine and to a lesser extent ornithine. The recombinant sensor showed a concentration-dependent increase in the fluorescence ratio with an apparent in vitro affinity for arginine of approximately 2 mM. A mutation in the binding pocket lowered the affinity and decreased the specificity. When expressed in E. coli, an increase in the fluorescence ratio was specifically detected after exposure to arginine and ornithine. Transient expression of the sensor in plant cell protoplasts and stable expression in Arabidopsis roots revealed specific fluorescence changes upon addition of arginine. The analysis suggests that fluorescent amino acid sensors may be versatile tools for studying the in vivo dynamics of metabolism and compartmentalization in plants.

Function of the anion transporter AtCLC-d in the trans-Golgi network.

Anion transporting proteins of the CLC type are involved in anion homeostasis in a variety of organisms. CLCs from Arabidopsis have been shown to participate in nitrate accumulation and storage. In this study, the physiological role of the functional chloride transporter AtCLC-d from Arabidopsis was investigated. AtCLC-d is weakly expressed in various tissues, including the root. When transiently expressed as a GFP fusion in protoplasts, it co-localized with the VHA-a1 subunit of the proton-transporting V-type ATPase in the trans-Golgi network (TGN). Stable expression in plants showed that it co-localized with the endocytic tracer dye FM4-64 in a brefeldin A-sensitive compartment. Immunogold electron microscopy confirmed the localization of AtCLC-d to the TGN. Disruption of the AtCLC-d gene by a T-DNA insertion did not affect the nitrate and chloride contents. The overall morphology of these clcd-1 plants was similar to that of the wild-type, but root growth on synthetic medium was impaired. Moreover, the sensitivity of hypocotyl elongation to treatment with concanamycin A, a blocker of the V-ATPase, was stronger in the clcd-1 mutant. These phenotypes could be complemented by overexpression of AtCLC-d in the mutant background. The results suggest that the luminal pH in the trans-Golgi network is adjusted by AtCLC-d-mediated transport of a counter anion such as Cl(-) or NO(3)(-).

Active site structure and catalytic mechanisms of human peroxidases.

Myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase are heme-containing oxidoreductases (EC 1.7.1.11), which bind ligands and/or undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties, and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II, and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-)catalases. Based on the published crystal structures of free MPO and its complexes with cyanide, bromide and thiocyanate as well as on sequence analysis and modeling, we critically discuss structure-function relationships. This analysis highlights similarities and distinguishing features within the mammalian peroxidases and intents to provide the molecular and enzymatic basis to understand the prominent role of these heme enzymes in host defense against infection, hormone biosynthesis, and pathogenesis.