Signal amplification using "spot-on-a-chip" technology for the identification of proteins via MALDI-TOF MS

The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 microm), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.     

Sample preparation for quantitation of tritium by accelerator mass spectrometry

The capability to prepare samples accurately and reproducibly for analysis of tritium (3H) content by accelerator mass spectrometry (AMS) greatly facilitates isotopic tracer studies in which attomole levels of 3H can be measured in milligram-sized samples. A method has been developed to convert the hydrogen of organic samples to a solid, titanium hydride, which can be analyzed by AMS. Using a two-step process, the sample is first oxidized to carbon dioxide and water. In the second step, the water is transferred within a heated manifold into a quartz tube, reduced to hydrogen gas using zinc, and reacted with titanium powder. The 3H/1H ratio of the titanium hydride is measured by AMS and normalized to standards whose ratios were determined by decay counting to calculate the amount of 3H in the original sample. Water, organic compounds, and biological samples with 3H activities measured by liquid scintillation counting were utilized to develop and validate the method. The 3H/1H ratios were quantified in samples that spanned 5 orders of magnitude, from 10(-10) to 10(-15), with a detection limit of 3.0 x 10(-15), which is equivalent to 0.02 dpm tritium/mg of material. Samples smaller than 2 mg were analyzed following addition of 2 mg of a tritium-free-hydrogen carrier. Preparation of organic standards containing both 14C and 3H in 2-mg organic samples demonstrated that this sample preparation methodology can also be applied to quantify both of these isotopes from a single sample.     

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.     

Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using laser fluorescence detection

Amino acids and peptides, from both standard solutions and biological samples, were successfully reacted with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, at low concentration, to form highly fluorescent isoindole derivatives. The formed mixtures are effectively separated by high-performance capillary electrophoresis and their constituents detected by their laser-induced fluorescence signals. The minimum detectable quantities in the low attomole (10(-18) mol) range are encountered.     

Non-thermal plasma-induced photocatalytic degradation of 4-chlorophenol in water

TiO(2) photocatalyst (P-25) (50mgL(-1)) was tentatively introduced into pulsed high-voltage discharge process for non-thermal plasma-induced photocatalytic degradation of the representative mode organic pollutant parachlorophenol (4-CP), including other compounds phenol and methyl red in water. The experimental results showed that rate constant of 4-CP degradation, energy efficiency for 4-CP removal and TOC removal with TiO(2) were obviously increased. Pulsed high-voltage discharge process with TiO(2) had a promoted effect for the degradation of these pollutants under a broad range of liquid conductivity. Furthermore, the apparent formation rates of chemically active species (e.g., ozone and hydrogen peroxide) were increased, the hydrogen peroxide formation rate from 1.10x10(-6) to 1.50x10(-6)Ms(-1), the ozone formation rate from 1.99x10(-8) to 2.35x10(-8)Ms(-1), respectively. In addition, this process had no influence on the photocatalytic properties of TiO(2). The introduction of TiO(2) photocatalyst into pulsed discharge plasma process in the utilizing of ultraviolet radiation and electric field in pulsed discharge plasma process enhanced the yields of chemically active species, which were available for highly efficient removal and mineralization of organic pollutants.     

A fully integrated monolithic microchip electrospray device for mass spectrometry

A novel microfabricated nozzle has been developed for the electrospray of liquids from microfluidic devices for analysis by mass spectrometry. The electrospray device was fabricated from a monolithic silicon substrate using deep reactive ion etching and other standard semiconductor techniques to etch nozzles from the planar surface of a silicon wafer. A channel extends through the wafer from the tip of the nozzle to a reservoir etched into the opposite planar surface of the wafer. Nozzle diameters as small as 15 microm have been fabricated using this method. The microfabricated electrospray device provides a reproducible, controllable, and robust means of producing nano-electrospray of a liquid sample. The electrospray device was interfaced to an atmospheric pressure ionization time-of-flight mass spectrometer using continuous infusion of test compounds at low nanoliter-per-minute flow rates. Nozzle-to-nozzle signal intensity reproducibility using 10 nozzles was demonstrated to be 12% with single-nozzle signal stability routinely less than 4% relative standard deviation (RSD). Solvent compositions have been electrosprayed ranging from 100% organic to 100% aqueous. The signal-to-noise ratio from the infusion of a 10 nM cytochrome c solution in 100% water at 100 nL/min was 450:1. Microchip electrospray nozzles were compared with pulled capillaries for overall sensitivity and signal stability for small and large molecules. The microchip electrospray nozzles showed a 1.5-3-times increase in sensitivity compared with that from a pulled capillary, and signal stability with the microchip was 2-4% RSD compared with 4-10% with a pulled capillary. Electrospray device lifetimes achieved thus far have exceeded 8 h of continuous operation and should be sufficient for typical microfluidic applications. The total volume of the electrospray device is less than 25 pL, making it suitable for combination with microfluidic separation devices.     

Capillary electrophoresis of amino sugars with laser-induced fluorescence detection

3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde has been utilized as a precolumn derivatization agent for various amino sugars. Constituents of various biological mixtures can be converted to highly fluorescent isoindole derivatives, separated by high-performance capillary electrophoresis and determined at attomole (10(-18) mol) levels by a laser-induced fluorescence detector. This method has been applied to the analysis of monosaccharides and acid-hydrolyzed polysaccharides. Carbohydrate moieties derived from a glycoprotein were also tagged and determined.     

Realization of vertical silicon nanowire networks with an ultra high density using a top-down approach

In this paper, we demonstrate the top-down fabrication of vertical silicon nanowires networks with an ultra high density (4 x 10(10) cm(-2)), a yield of 100%, and a precise control of both diameter and location. Firstly, dense and well-defined networks of nanopillars have been patterned by e-beam lithography using a negative tone e-beam resist Hydrogen SylsesQuioxane (HSQ). A very high contrast has been obtained using a high acceleration voltage (100 kV), very small beam size at a current of 100 pA and a concentrated developer, 25% Tetramethylammonium Hydroxide. The patterns were transferred by reactive ion etching. Using chlorine based plasma chemistry and low pressure, etching anisotropy was guaranteed while avoiding the so-called 'grass effect'. This approach enabled the production of vertical silicon nanowires networks with a 20 nm diameter and a pitch of 30 nm. Lastly, the self-limited oxidation phenomenon in 1D structure has been used to perfectly control the shrinking of NWs and to obtain a Si surface free of defects induced by reactive ion etching. The silicon nanowires networks have been tapered by wet oxidation (850 degrees C) down to a diameter of 10 nm with a high aspect ratio 11.     

Determination of total organic carbon in water by thermal combustion-ion chromatography

A sensitive method for determining total organic carbon at microgram per liter levels in industrial, environmental, and drinking waters by thermal combustion ion chromatography was developed using tube furnace and readily accessable HPLC equipment. To achieve complete oxidation, persulfate (0.25%) was added to oxidize nonvolatile organic compounds in solution and cupric oxide heated at 900 °C to convert volatile organic compounds to CO(2), which was scrubbed in a 20 mL solution of 50 mM KOH with 10 drops of butanol added. The carbonate anion obtained was determined by nonsuppressed ion chromatography using 0.6 mM potassium hydrogen phthalate (KHP) as the eluent. Both surfactants and volatile and nonvolatile organic compounds commonly found in environmental waters give highly repeatable recoveries close to 100%. The detection limit (S/N = 2) and linear range for a 1 L water sample are 2 μg of C L(-)(1) and 10-2500 μg of C L(-)(1), respectively, and they can be adjusted using samples ranging from 100 mL to 2 L. Good repeatablity (RSD less than 10%) and close to 100% recoveries were obtained for KHP added to real samples such as deionized, mineral, tap, and river water and seawater. Compared with the ASTM D2579 method, the method developed is 3 orders of magnitude more sensitive, more accurate, and reliable in determining samples with low total organic carbon values and more flexible in adjusting the linear range and sensitivity using variable sample sizes.     

Electrochemistry in nanovials fabricated by combining screen printing and laser micromachining

The coupling of screen-printing and laser micromachining technology has been used to create a nanovial with "built-in" working and reference electrodes. The volume of the nanovial was calculated to be 7.2 nL using dimensions determined by SEM. The electrochemical nanovial was characterized using the ferri/ferrocyanide redox couple. Cyclic voltammetry and chronoamperometry experiments were performed with electrochemical nanovials utilizing 5% (v/v) glycerin in the solutions and a humidified headspace to control evaporation of the small-volume samples. Chronoamperometry experiments gave results consistent with a diffusion-limited process and revealed a working electrode surface area of 2.6 x 10(4) micron 2. The ultrasmall-volume cells represent a simple, reliable, low-cost approach for the fabrication of complete electrochemical nanovials.     

Determination of H2O and CO2 concentrations in fluid inclusions in minerals using laser decrepitation and capacitance manometer analysis

Water and carbon dioxide concentrations within individual and selected groups of fluid inclusions in quartz were analyzed by using laser decrepitation and quantitative capacitance manometer determination. The useful limit of detection (calculated as ten times the typical background level) is about 5 x 10(-10) mol of H2O and 5 x 10(-11) mol of CO2; this H2O content translates into an aqueous fluid inclusion approximately 25 micrometers in diameter. CO2/H2O determinations for 38 samples (100 separate measurements) have a range of H2O amounts of 5.119 x 10(-9) to 1.261 x 10(-7) mol; CO2 amounts of 7.216 x 10(-10) to 1.488 x 10(-8) mol, and CO2/H2O mole ratios of 0.011 to 1.241. Replicate mole ratio determinations of CO2/H2O for three identical (?) clusters of inclusions in quartz have average mole ratios of 0.0305 +/- 0.0041 1 sigma. Our method offers much promise for analysis of individual fluid inclusions, is sensitive, is selective when the laser energy is not so great as to melt the mineral (laser pits approximately 50 micrometers in diameter), and permits rapid analysis (approximately 1 h per sample analysis).     

Development of advanced-type multi-functional electronic personal dosemeter

An advanced-type small, light, multi-functional electronic personal dosemeter has been developed using silicon semiconductor radiation detectors for dose management of workers at nuclear power plants and accelerator facilities. This dosemeter is 62 x 82 x 27 mm(3) in size and approximately 130 g in weight, which is capable of measuring personal gamma ray and neutron dose equivalents, Hp(10), simultaneously. The neutron dose equivalent can be obtained using two types of silicon semiconductors: a slow-neutron sensor (<1 MeV) and a fast-neutron sensor (>1 MeV). The slow neutron sensor is a 10 x 10 mm(2) p-type silicon on which a natural boron layer is deposited around an aluminium electrode. The fast neutron sensor is also a 10 x 10 mm(2) p-type silicon crystal on which an amorphous silicon hydride is deposited. The neutron energy response corresponding to the fluence-to-dose-equivalent conversion coefficient given by ICRP Publication 74 has been evaluated using a monoenergetic neutron source from 250 keV to 15 MeV at the Fast Neutron Laboratory of Tohoku University. As the result, the Hp(10) response to neutrons in the energy range of 250 keV and 4.4 MeV within +/-50% difference has been obtained.     

Photocatalytic dehalogenation coupled on-line to a reversed micellar-mediated chemiluminescence detection system: application to the determination of iodinated aromatic compounds

The effects of illumination time, temperature, catalyst concentration, and pH on the on-line photocatalytic dehalogenation of iodinated aromatic compounds in a near-UV-illuminated titanium dioxide (anatase type) aqueous suspension were monitored via the iodine-luminol chemiluminescence (CL) reaction in a reversed micellar medium, and a new, automated, rapid, and efficient method was developed. A water-cooled, 400-W high-pressure Hg lamp was used as an internal light source. The flow procedure involved the following: (1) photocatalytic dehalogenation/degradation of the iodinated compound by the near-UV-illuminated titanium dioxide and the production of iodide species, (2) oxidation of iodide into iodine, (3) extraction of iodine into cyclohexane, (4) membrane separation of the iodine-containing organic phase from the aqueous phase, and (5) the detection of iodine using the luminol CL reaction in the reversed micellar solution of cetyltrimethylammonium chloride in 6:5 (v/v) chloroform-cyclohexane/water buffered with sodium carbonate. Results for the dehalogenation of the iodinated compounds, o-iodobenzoic acid and L-thyroxine (3,5,3',5'-tetraiodothyronine) sodium, were compared with a standard inorganic iodide solution. After establishing the optimum chemical and instrumental conditions, detection limits of 0.8 x 10(-9) and 0.2 x 10(-9) M and linear calibration graphs were obtained with dynamic ranges from 0.79 x 10(-7) to 7.9 x 10(-7) M and from 0.20 x 10(-7) to 2.0 x 10(-7) M for o-iodobenzoic acid and L-thyroxine, respectively. A precision of approximately 4% relative standard deviation (n = 6) was provided at an o-iodobenzoic acid concentration of 0.79 x 10(-7) M. The method developed was applied to the on-line determinations of iodinated aromatic compounds such as L-thyroxine sodium and iopamidol ((S)-N,N'-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hydroxy-1-oxopropyl)amino]-2,4,6-triiodoisophthaldiamide) in pharmaceuticals.     

Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.     

Quantitative amino acid analysis of individual snail neurons by open tubular liquid chromatography

A method is described for the determination of amino acids in individual cells. The amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. It was possible to quantitatively determine 17 amino acids in three different neurons of the land snail Helix aspersa. Quantitation was accomplished through the use of two internal standards and a calibration curve. Alanine was found to be the most abundant amino acid by about a factor of 2 over glutamine in all three types of neurons. This method has the advantages of sensitivity in the attomole range (5 X 10(-9) M) and selectivity for a specific class of compounds and is at least as reliable as other methods used for single-cell analysis.     

A fiber-optic fluorescence sensor for lithium ion in acetonitrile

A novel fiber-optic fluorescence sensor based on a controlled-release reagent for the determination of lithium ion in organic solvents is proposed. The fluorogenic indicator 2-(2-hydroxyphenyl)benzoxazole is contained in a mini-polyethylene tube as the reagent reservoir and is brought into contact with the analyte solution by diffusion across a poly(vinyl chloride) (PVC) membrane to form a strongly fluorescent complex at the membrane/solution interface. The fluorescence signals produced are measured via two joined optical fibers positioned closely to the backside of the PVC membrane for light illumination and collection. The sensor is useful for measuring Li+ at concentrations in acetonitrile ranging from 1.0 x 10(-6) to 1.0 x 10(-2) M with a detection limit of 3.0 x 10(-7) M. The steady-state response can be reached within seconds, and the signal changes are fully reversible. The sensor shows minimal interference effects from other alkali metal and alkaline earth metal cations and has good stability and durability when stored in acetonitrile solutions.     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.     

Magnetophoretic velocimetry of manganese(II) in a single microdroplet in a flow system under a high gradient magnetic field generated with a superconducting magnet

An experimental system for magnetophoretic velocimetry, which could determine the volume magnetic susceptibility of a single particle dispersed in a liquid phase from a magnetophoretic velocity, has been developed. A micrometer-sized high-gradient magnetic field could be generated in a capillary by a pair of iron pole pieces in a superconducting magnet (10 T). The magnetophoretic behavior of a single particle in a capillary flow system was investigated under the inhomogeneous magnetic field. From the magnetophoretic velocity of a polystyrene latex particle dispersed in a MnCl2 aqueous solution, the product of the magnetic flux density and the gradient, B(dB/dx), was determined as a function of the position along the capillary. The maximum value of B(dB/dx) was 4.7 x 10(4) T2 m(-1), which was approximately 100 times higher than that obtained by two Nd-Fe-B permanent magnets (0.4 T). Organic droplets extracting manganese(II) with 2-thenoyltrifluoroacetone and tri-n-octylphosphine oxide from MnCl2 solution were used as test samples. The difference of the volume magnetic susceptibility between the droplet and the medium could be determined from the magnetophoretic velocity. This method allowed us to continuously measure a volume magnetic susceptibility of 10-6 level for a picoliter droplet and to determine manganese(II) in the single droplet at the attomole level.     

Thermal stability of metal-silicide nanocrystal nonvolatile memory with barrier engineered tunnel layers

WSi2 nanocrystal nonvolatile memory devices were fabricated with a silicon oxide-nitride-oxide (SiO2: 2 nm/Si3N4:2 nm/SiO2:3 nm) tunnel layer. WSi2 nanocrystals of 2.5 nm diameters and a density of 3.6 x 10(12) cm(-2) were formed using radio frequency magnetron sputtering and annealing processes. The WSi2 nanocrystal nonvolatile memory device exhibited strong thermal stability during writing/erasing operations at temperatures up to 125 degrees C. When the writing/erasing voltages were applied at +10 V/-10 V for 500 ms, the memory window of the initial approximately 2.6 V decreased by approximately 1.1 V at 25 degrees C and 0.4 V at 125 degrees C after 10(4) sec, respectively. These results show that WSi2 nanocrystals with barrier-engineered tunnel layers are possible for application in nonvolatile memory devices.     

Development of the fast neutron standard using a Be({alpha},n) reaction at the National Metrology Institute of Japan

This paper describes the 8-MeV neutron field where the neutrons are generated in the (9)Be(alpha,n)(12)C reaction by bombardment of a beryllium target with a 2.4-MeV (4)He(+) beam from a Van de Graaff accelerator. The neutron field is being prepared for a new national standard on neutron fluence in Japan. Absolute measurement of the neutron fluence was taken using a proton recoil neutron detector, consisting of a silicon surface barrier detector with a polyethylene radiator. Neutron spectra were measured using a newly developed recoil proton spectrometer and a liquid organic scintillation detector. The gamma rays existing in the field were also characterised using a liquid organic scintillation detector. The ambient dose equivalents of the gamma rays were estimated to be <100 microSv at the neutron fluence of 10(7) neutrons cm(-2).