A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting.

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of porcine trypsin via ε-amino groups. The function was assessed using the artificial substrate Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonuclease A and a human plasma sample. A 10-membrane flow-through reactor was used for fragmentation and MS analysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and by coupling on-line to the instrument. The peptide pattern allowed correct identification of the single target protein in both cases, and of >70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained 76% of the initial activity after 14days of storage and repeated use at room temperature. SIGNIFICANCE: This manuscript describes the design of a stable enzyme reactor that allows efficient and fast digestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in an online-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

Phase Formation Behavior in Ultrathin Iron Oxide.

Nanostructured iron oxides, and especially hematite, are interesting for a wide range of applications ranging from gas sensors to renewable solar hydrogen production. A promising method for deposition of low-dimensional films is atomic layer deposition (ALD). Although a potent technique, ALD of ultrathin films is critically sensitive to the substrate and temperature conditions where initial formation of islands and crystallites influences the properties of the films. In this work, deposition at the border of the ALD window forming a hybrid ALD/pulsed CVD (pCVD) deposition is utilized to obtain a deposition less sensitive to the substrate. A thorough analysis of iron oxide phases formation on two different substrates, Si(100) and SiO2, was performed. Films between 3 and 50 nm were deposited and analyzed with diffraction techniques, high-resolution Raman spectroscopy, and optical spectroscopy. Below 10 nm nominal film thickness, island formation and phase dependent particle crystallization impose constraints for deposition of phase pure iron oxides on non-lattice-matching substrates. Films between 10 and 20 nm thickness on SiO2 could effectively be recrystallized into hematite whereas for the corresponding films on Si(100), no recrystallization occurred. For films thicker than 20 nm, phase pure hematite can be formed directly with ALD/pCVD with very low influence of the substrate on either Si or SiO2. For more lattice matched substrates such as SnO2:F, Raman spectroscopy indicated formation of the hematite phase already for films with 3 nm nominal thickness and clearly for 6 nm films. Analysis of the optical properties corroborated the analysis and showed a quantum confined blue-shift of the absorption edge for the thinnest films.

Glutathione transferases immobilized on nanoporous alumina: flow system kinetics, screening, and stability.

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

Dye-sensitized solar cells employing a SnO2-TiO2 core-shell structure made by atomic layer deposition.

This paper describes the synthesis and characterization of core-shell structures, based on SnO2 and TiO2, for use in dye-sensitized solar cells (DSC). Atomic layer deposition is employed to control and vary the thickness of the TiO2 shell. Increasing the TiO2 shell thickness to 2 nm improved the device performance of liquid electrolyte-based DSC from 0.7% to 3.5%. The increase in efficiency originates from a higher open-circuit potential and a higher short-circuit current, as well as from an improvement in the electron lifetime. SnO2-TiO2 core-shell DSC devices retain their photovoltage in darkness for longer than 500 seconds, demonstrating that the electrons are contained in the core material. Finally core-shell structures were used for solid-state DSC applications using the hole transporting material 2,2',7,7',-tetrakis(N, N-di-p-methoxyphenyl-amine)-9,9',-spirofluorene. Similar improvements in device performance were obtained for solid-state DSC devices.

Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina.

Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H2O2/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.

Self-supported three-dimensional nanoelectrodes for microbattery applications.

A nanostructured three-dimensional (3D) microbattery has been produced and cycled in a Li-ion battery. It consists of a current collector of aluminum nanorods, a uniform layer of 17 nm TiO(2) covering the nanorods made using ALD, an electrolyte and metallic lithium counter electrode. The battery is electrochemically cycled more than 50 times. The increase in total capacity is 10 times when using a 3D architecture compared to a 2D system for the same footprint area.

Hyperbaric laser chemical vapor deposition of carbon fibers from the 1-alkenes, 1-alkynes, and benzene.

The growth of long carbon fibers was investigated using hyperbaric-pressure laser chemical vapor deposition (HP-LCVD). Precursors included the unbranched alkenes with linear structure 1-C(x)H(2x) (where x = 2,4,5,6,7,8), the unbranched alkynes, i.e., 1-C(x)H(2)(x-2) (where x = 3,4,5,6,8), and benzene. Rate constants, reaction orders, and apparent activation energies were derived for each precursor over a range of experimental conditions. Axial growth rates from the alkenes were 1-2 orders of magnitude greater than for the alkynes, while growth rates for benzene exceeded 10 mm s(-1). Generalized expressions for the growth rate vs molecular weight were determined. For the alkenes, the growth rate was directly proportional to the square root of the precursor molecular weight, while the alkynes exhibited an inverse relationship. Two regions of differing reaction order were identified for the alkynes; at pressures less than 2.0-2.5 bar, the average reaction order was 3.07, while above 2.0-2.5 bar, reaction orders diverged. Expressions were derived for the fraction of carbon atoms deposited per alkyne molecule transported; the deposition efficiency decreased with molecular weight for the alkynes, due in part to the Soret effect. In contrast, the reaction order for the alkenes was 1.65, and for benzene was 2.25. A phase change in the deposit was observed for both the alkenes and alkynes, with the exceptions of pentene and pentyne. Complete axial rate equations for the alkenes and alkynes were derived, as well as volumetric growth equations for the alkynes. It was shown that the volumetric rate increases nonlinearly with laser power at sufficiently high pressures.

Fabrication of high-aspect-ratio Prussian blue nanotubes using a porous alumina template.

Prussian blue nanotubes were fabricated by using a sequential deposition technique inside the 60-nm well-ordered pores of anodic alumina. By varying the deposition parameters and the dimensions of the template, we could tailor the length and the outer as well as the inner diameter of the tubes. The nanotubes were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD).