J.I.B. Wilson

Robust Fluorescent Detection of Protein Fatty-Acylation with Chemical Reporters

Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

Analysis of chemical vapour deposited diamond films by X-ray photoelectron spectroscopy

Abstract X-ray photoelectron spectroscopy (XPS) for elemental and phase analysis of diamond surfaces is reviewed, including both single crystal natural diamond and polycrystalline chemical vapour deposition (CVD) films. The core C1s peaks, plasmon losses and valence band spectra of sp2 graphite are compared with those of sp3 diamond, and briefly with mixed sp2/sp3 hard carbon films. Surface reconstructions are reviewed for diamond in the presence of hydrogen or following thermal annealing. Attention is drawn to the destructive effects of argon ion bombardment ‘cleaning’. The synthesis of CVD diamond, including the initial nucleation process, is described and the significance of hydrogen is explained. Chemical modification of the surface of diamond films is discussed, with relevance to sensors and field electron emission. XPS analysis of (111) polycrystalline films treated by atom beams of N, O, H, Cl is described and the influence of unwanted oxygen is emphasised. For both atom beam and furnace oxidation, the oxygen is incorporated into the (111) diamond surface as bridging C–O–C.

The role of C2 in nanocrystalline diamond growth

This paper presents findings from a study of nanocrystalline diamond (NCD) growth in a microwave plasma chemical vapor deposition reactor. NCD films were grown using Ar∕H2∕CH4 and He∕H2∕CH4 gas compositions. The resulting films were characterized using Raman spectroscopy, scanning electron microscopy, and atomic force microscopy. Analysis revealed an estimated grain size of the order of 50nm, growth rates in the range 0.01–0.3μm∕h, and sp3- and sp2-bonded carbon content consistent with that expected for NCD. The C2 Swan band (dΠg3↔aΠu3) was probed using cavity ring-down spectroscopy to measure the absolute C2(a) number density in the plasma during diamond film growth. The number density in the Ar∕H2∕CH4 plasmas was in the range from 2to4×1012cm−3, but found to be present in quantities too low to measure in the He∕H2∕CH4 plasmas. Optical emission spectrometry was employed to determine the relative densities of the C2 excited state (d) in the plasma. The fact that similar NCD material was grown whether using Ar or He as the carrier gas suggests that C2 does not play a major role in the growth of nanocrystalline diamond.This paper presents findings from a study of nanocrystalline diamond (NCD) growth in a microwave plasma chemical vapor deposition reactor. NCD films were grown using Ar∕H2∕CH4 and He∕H2∕CH4 gas compositions. The resulting films were characterized using Raman spectroscopy, scanning electron microscopy, and atomic force microscopy. Analysis revealed an estimated grain size of the order of 50nm, growth rates in the range 0.01–0.3μm∕h, and sp3- and sp2-bonded carbon content consistent with that expected for NCD. The C2 Swan band (dΠg3↔aΠu3) was probed using cavity ring-down spectroscopy to measure the absolute C2(a) number density in the plasma during diamond film growth. The number density in the Ar∕H2∕CH4 plasmas was in the range from 2to4×1012cm−3, but found to be present in quantities too low to measure in the He∕H2∕CH4 plasmas. Optical emission spectrometry was employed to determine the relative densities of the C2 excited state (d) in the plasma. The fact that similar NCD material was grown whether usin...

Silicon‐silicon dioxide interface: An infrared study

We have investigated, using differential infrared spectroscopy, the transition region between single‐crystal silicon and its natural thermally grown oxide. By monitoring the thickness‐dependent behavior of the stretching mode of the Si–O bond near 1075 cm−1, we isolate bulk oxide characteristics and features arising from interface constraints, finding that films up to 100 A are still affected by interface effects. For thicker films we observe a consistent degree of asymmetry in the Si‐O absorption band of about 9 cm−1, which does not exhibit a strong dependence on thickness. By contrast, the peak position, width, and degree of symmetry are found to be sensitively dependent upon film thickness below 100 A, providing evidence for structurally distinct phases of silicon dioxide. Our initial interpretation suggests that the infrared spectra of such thin layers may be significantly affected by strain originating at the Si‐oxide interface, rather than oxygen deficiency or overstoichiometry.

The oxidation of (100) textured diamond

The thermal oxidation of highly textured (100) chemical vapour deposited (CVD) diamond has been investigated using a combination of high resolution X-ray photoelectron spectroscopy. The diamond samples were oxidised in dry O in a vacuum 2 furnace at temperatures up to 800 8C. The kinetics of oxidation of well-defined crystal facets of (100) diamond have been studied using in-situ laser interferometry and thermogravimetric analysis. Concomitant scanning probe microscopic examination of individual facets after oxidation revealed negligible changes to the surface roughness. Oxygen containing functional groups such as ether (COC) and carbonyl ()C_O) have been observed using X-ray photoelectron spectroscopy at all the surface coverages investigated. Even at higher coverages the formation of higher oxidation states such as carboxylic acid groupings was negligible (-2.5%). Angle resolved X-ray photoelectron spectroscopy measurements have been used to discriminate between surface and sub-surface oxygen. 2002 Elsevier Science B.V. All rights reserved.

Diamond-based glucose sensors

Abstract A series of diamond-based glucose sensors, based on the interaction of glucose with the enzyme glucose oxidase (GOD), has been produced. For each sensor, the sensitivity to glucose was assessed and, with some devices, the range of glucose concentrations over which the sensor showed a linear response was determined. The sensing electrode formed one electrode of an electrochemical cell, and a tungsten counter electrode formed the other. All the sensors were diamond-based, with the nature of the working electrode being the distinguishing feature. The first device was a diamond platinum-GOD sensor. However, this device was prone to interference from other electroactive chemicals in the blood such as vitamin C and acetaminophen. To minimise the metal content of the sensors, two further sensors were produced using heavily boron doped diamond as the conducting electrode in place of the platinum. In the first case, the GOD was immobilised on to the surface of the diamond by electrochemical deposition, and in the second, the GOD was “wired” directly to the electrode by covalent bonding to the electrode surface.

STRUCTURE OF ULTRATHIN SILICON DIOXIDE FILMS

Differential infrared spectroscopy has been used to study the silicon‐oxygen stretching band of thin silicon dioxide films thermally grown on single‐crystal silicon. We consistently observe an asymmetry in the spectra of films thicker than about 100 A, of about 9 cm−1. The peak position, width, and degree of asymmetry are also found to be sensitively dependent upon film thickness below 100–150 A, while above this level these features are only very weakly dependent upon film thickness, indicating the presence of a thin layer of different structural or bonding properties. Our interpretation suggests that the infrared spectra of layers up to 100 A thick are significantly affected by strain originating at the silicon‐oxide interface, in agreement with recent observations.

Absorption of infrared radiation in silicon

The absorption of 9–11 μm radiation by thin wafers of lightly doped, n‐type Si has been measured at several lattice temperatures from 300 to 800 K. The temperature dependence of the absorption coefficient at λ=10.6 μm is extracted from the data and compared with previous measurements and also with recent theoretical models. A novel processing technique is described in which coupling of the CO2 laser radiation to the Si lattice is significantly enhanced by the simultaneous absorption of radiation from an argon laser.

Microfluidics based on ZnO/nanocrystalline diamond surface acoustic wave devices

Surface acoustic wave (SAW) devices with 64 μm wavelength were fabricated on a zinc oxide (ZnO) film deposited on top of an ultra-smooth nanocrystalline diamond (UNCD) layer. The smooth surface of the UNCD film allowed the growth of the ZnO film with excellent c-axis orientation and low surface roughness, suitable for SAW fabrication, and could restrain the wave from significantly dissipating into the substrate. The frequency response of the fabricated devices was characterized and a Rayleigh mode was observed at ∼65.4 MHz. This mode was utilised to demonstrate that the ZnO/UNCD SAW device can be successfully used for microfluidic applications. Streaming, pumping, and jetting using microdroplets of 0.5 and 20 μl were achieved and characterized under different powers applied to the SAW device, focusing more on the jetting behaviors induced by the ZnO SAW.

Modelling of self-limiting laser ablation of rough surfaces: application to the polishing of diamond films

Abstract We present a theoretical model for the interaction of excimer laser radiation with rough polycrystalline CVD diamond films, showing the influence of angle of incidence, irradiation intensity and number of laser pulses. A new regime of laser treatment — self-limiting laser ablation — was found, which allows faceted films to be smoothed without wasteful ablation of the bulk. Multipulse XeCl laser (308 nm) irradiation of our diamond films, grown on silicon wafers, confirms that such polishing is accomplished.