Development of a Temperature-pulse Enhanced Electrochemical Glucose Biosensor and Characterization of its Stability via Scanning Electrochemical Microscopy

Glucose oxidase (GOx) is an enzyme frequently used in glucose biosensors. As increased temperatures can enhance the performance of electrochemical sensors, we investigated the impact of temperature pulses on GOx that was drop-coated on flattened Pt microwires. The wires were heated by an alternating current. The sensitivity towards glucose and the temperature stability of GOx was investigated by amperometry. An up to 22-fold increase of sensitivity was observed. Spatially resolved enzyme activity changes were investigated via scanning electrochemical microscopy. The application of short (<100 ms) heat pulses was associated with less thermal inactivation of the immobilized GOx than long-term heating.     

A 10,000-fold nuclear hyperpolarization of a membrane protein in the liquid phase via a solid-state mechanism

Several techniques rely on electron-nuclear interactions to boost the polarization of nuclear spins in the solid phase. Averaging out of anisotropic interactions as a result of molecular tumbling strongly reduces the applicability of such hyperpolarization approaches in liquids. Here we show for the first time that anisotropic electron-nuclear interactions in solution can survive sufficiently long to generate nuclear spin polarization by the solid-state photo-CIDNP mechanism. A 10,000-fold NMR signal increase in solution was observed for a giant biomolecular complex of a photosynthetic membrane protein with a tumbling correlation time in the submicrosecond regime, corresponding to a molecular weight close to 1 MDa.     

Determination of hydrazines by chip electrophoresis with contactless conductivity detection

In this report, a new approach for the fast determination of hydrazine compounds (hy) in complex matrices is presented. The experimental protocol is based on poly(methylmethacrylate) (PMMA) microchip separations with contactless conductivity detection using a compact portable device, which integrates all separation and detection components. Three hy (hydrozine (Hy), methylhydrazine (MH), and 1,1-dimethylhydrazine (UDMH)) were separated within < 30 s at a separation voltage of 3.8 kV using a L(-)-histidine/2-(N-morpholinoethanesulfonic acid) (His/MES) buffer (25:50 mM, pH 5.87). The contactless conductivity detection enables detection limits for Hy, MH, and UDMH of 11.9, 35.5, and 337.8 ng/mL, respectively, with linear concentration dependence up to 10 μg/mL. In complex matrices such as soil samples or river water, interferences were eliminated by implementing ultrasound-assisted headspace single-drop microextraction of hy under strongly alkaline conditions, using an aqueous drop of His/MES buffer as the extractant phase. The incorporation of this miniaturized sample preparation step led to improved limits of detection for Hy, MH, and UDMH of 6.5, 15.3, and 11.4 ng/mL, respectively. The overall protocol demonstrates a promising approach for interfacing chip electrophoresis with real-world applications.     

Solvation and crystal effects in bilirubin studied by NMR spectroscopy and density functional theory

The open-chain tetrapyrrole compound bilirubin was investigated in chloroform and dimethyl sulfoxide solutions by liquid-state NMR and as solid by (1)H, (13)C, and (15)N magic-angle spinning (MAS) solid-state NMR spectroscopy. Density functional theory (DFT) calculations were performed to interpret the data, using the B3LYP exchange-correlation functional to optimize geometries and to compute NMR chemical shieldings by the gauge-including atomic orbital method. The dependence of geometries and chemical shieldings on the size of the basis sets was investigated for the reference molecules tetramethylsilane, NH(3), and H(2)O, and for bilirubin as a monomer and in clusters consisting of up to six molecules. In order to assess the intrinsic errors of the B3LYP approximation in calculating NMR shieldings, complete basis set estimates were obtained for the nuclear shielding values of the reference molecules. The experimental liquid-state NMR data of bilirubin are well reproduced by a monomeric bilirubin molecule using the 6-311+G(2d,p) basis set for geometry optimization and for calculating chemical shieldings. To simulate the bilirubin crystal, a hexameric model was required. It was constructed from geometry-optimized monomers using information from the X-ray structure of bilirubin to fix the monomeric entities in space and refined by partial optimization. Combining experimental (1)H-(13)C and (1)H-(15)N NMR correlation spectroscopy and density functional theory, almost complete sets of (1)H, (13)C, and (15)N chemical shift assignments were obtained for both liquid and solid states. It is shown that monomeric bilirubin in chloroform solution is formed by 3-vinyl anti conformers, while bilirubin crystals are formed by 3-vinyl syn conformers. This conformational change leads to characteristic differences between the liquid- and solid-state NMR resonances.     

A novel approach for the separation of neutral analytes by means of electrochemically assisted injection coupled to capillary electrophoresis-mass spectrometry

A novel concept for capillary electrophoresis-mass spectrometry (CE-MS) studies of neutral analytes is presented. The experimental approach is based on the implementation of electrochemically assisted injection (EAI) which enables the generation of charged species from neutral analytes. In this way electrophoretic separations can be performed without the use of surfactants leading to an excellent compatibility with ESI-MS. Various ferrocene species have been used as model compounds to demonstrate the potential of the EAI-CE-MS technique.     

Determination of enkephalin peptides by nonaqueous capillary electrophoresis with electrochemical detection

Nonaqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the analysis of enkephalin peptides. The effect of different buffer compositions on the electrophoretic behavior of methionine enkephalin, leucine enkephalin, and [D-Ala2]-leucine enkephalin was studied. Separation of the protonated and the deprotonated peptides was obtained using ACN/methanol-based electrolyte systems. The electrochemical behavior of the enkephalins was studied by the capillary batch injection analysis technique. NACE-ED yielded well-defined signals in the oxidation mode only for the negatively charged analytes. The optimized BGE for the counterelectroosmotic separation consisted of 10 mM ammonium acetate in ACN/methanol (3:1 v/v). Using a platinum microdisk electrode set to an actual potential of +0.65 V detection limits in the submicromolar range were observed which are about one order of magnitude lower compared to UV detection. Problems concerning EOF instability and electrode fouling caused by water and other neutral sample impurities transported by the EOF can be avoided in the EOF-inverted mode using poly(ethylene glycol)-coated capillaries and an actual working electrode potential of +1.0 V. For the quantification of the enkephalins [D-Ala2]leucine enkephalin was used as internal standard. The practical utility for the determination of enkephalins in spiked plasma samples after SPE was demonstrated.     

15N MAS NMR studies of cph1 phytochrome: Chromophore dynamics and intramolecular signal transduction

Solid-state nuclear magnetic resonance (NMR) is applied for the first time to the photoreceptor phytochrome. The two stable states, Pr and Pfr, of the 59-kDa N-terminal module of the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 containing a uniformly 15N-labeled phycocyanobilin cofactor are explored by 15N cross-polarization (CP) magic-angle spinning (MAS) NMR. As recently shown by 15N solution-state NMR using chemical shifts [Strauss, H. M.; Hughes, J.; Schmieder, P. Biochemistry 2005, 44, 8244], all four nitrogens are protonated in both states. CP/MAS NMR provides two additional independent lines of evidence for the protonation of the nitrogens. Apparent loss of mobility during photoactivation, indicated by the decrease of line width, demonstrates strong tension of the entire chromophore in the Pfr state, which is in clear contrast to a more relaxed Pr state. The outer rings (A and D) of the chromophore are significantly affected by the phototransformation, as indicated by both change of chemical shift and line width. On the other hand, on the inner rings (B and C) only minor changes of chemical shifts are detected, providing evidence for a conserved environment during phototransformation. In a mechanical model, the phototransformation is understood in terms of rotations between the A-B and C-D methine bridges, allowing for intramolecular signal transduction to the protein surface by a unit composed of the central rings B and C and its tightly linked protein surroundings during the highly energetic Pfr state.