A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

Alcohol dehydrogenases (ADHs; E.C. 1.1.1.1) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)⁺‐dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium‐chain family (medium‐chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme‐binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged ADH was overexpressed and purified by Ni²⁺‐nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12 % sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone.     

Solid‐state nuclear magnetic resonance investigation of neurosteroid compounds and magnesium interactions

The neurosteroid trans‐dehydroandrosterone (DHEA) and its analogs with slightly different modifications in the side chain attached to C17, that is, (3S)‐acetoxypregn‐5‐en‐20‐one ( 1 ) and (3S,20R)‐acetoxypregn‐5‐en‐20‐ol ( 2 ), have been synthesized to investigate DHEA–cation interactions. In this study, we applied solid‐state ¹H/¹³C cross‐polarization/magic‐angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy to a series of DHEA analog/Mg²⁺ mixtures at different Mg²⁺ concentrations. The high‐resolution ¹³C NMR spectra of 1 /Mg²⁺ mixtures exhibit two distinct ¹³C spectral patterns, one attributable to 1 free from Mg²⁺, and the other attributable to 1 with bound Mg²⁺. For 2 , the ¹³C NMR spectra exhibit three distinct spectral patterns; besides that of the free form, the other two can be assigned to Mg²⁺‐bound forms. Based on the analysis of the chemical shift deviations (CSDs), we conclude that both 1 and 2 might be subject to a cation–π interaction via the C5–C6 double bond, in contrast to that observed previously for DHEA. As demonstrated, DHEA possesses two Mg²⁺ binding sites, that is, C17–O and C5–C6 double bond, in which the binding affinity of the former is at least three times stronger than that of the latter. The solid‐state ¹³C NMR investigation allows better understanding of the underlying cation binding effects of neurosteroid molecules in vitro.     

A new fabrication process for a microchip electrophoresis device integrated with a three-electrode electrochemical detector

We report here a novel and simple process for the fabrication of a poly(methyl methacrylate) (PMMA)-based microchip electrophoresis device, integrated with a screen-printed three-electrode electrochemical detector that does not require a replicate mold. In this approach, a photoresist layer constitutes both an adhesion layer and side walls of 50 mum wide and 50 mum tall microfluidic channels on a screen-printed three-electrode PMMA substrate. Openings were drilled for buffer reservoirs on an additional piece of PMMA, then the final device was bonded in a PMMA/photoresist/PMMA sandwich configuration. This process is inexpensive, less time-consuming, and simpler compared with traditional fabrication methods. The combination of this PMMA-based microchip fabrication together with screen-printed electrode technology holds great promise for the mass production of a single-use micrototal analytical system. Successful determination of uric acid and L-ascorbic acid with the presented system validates its utility. In combination with a suitable electrochemical detector, this device holds much promise for the determination of other analytes in various biological samples for medical and clinical diagnosis.     

Vertically well aligned P-doped ZnO nanowires synthesized on ZnO-Ga/glass templates

Vertically well aligned P-doped ZnO nanowires were prepared on ZnO-Ga/glass templates at 550 degrees C by reactive evaporation without metal catalysts and the nanowires were found to be single crystalline with the würtzite structure, oriented in the c-axis direction; the P-doping shortened the physical lengths of the ZnO nanowires without changing their diameter, and furthermore, the introduction of P atoms resulted in a much weaker and broader ZnO band edge emission.     

Sample-stacking techniques in non-aqueous capillary electrophoresis

In sample-stacking techniques, the detection limit cannot be improved by simply increasing the length of the sample solution, because the individual electrophoretic parameters must be optimized. In an attempt to increase the amount of sample injected, as well as to focus them onto a small zone, two novel methods are proposed. One of these employs an "ultra-high conductivity zone", which was inserted between the sample zone and background solution to build an unequal conductivity gradient. The other employs a "low temperature bath". A portion of the capillary (near the junction between the sample solution and the background solution) was immersed in a low temperature bath, which served as a "pseudo-high-conductivity zone" due to the fact that conductivity would increases when the temperature is decreased. As a result, a large volume of sample injection can be achieved. Using 3,4-methylenedioxymethamphetamine as a model compound, the detection limit was determined to be 1.6 x 10(-6) M (S/N = 3) by means of normal non-aqueous capillary electrophoresis (NACE). This could be improved to 3.0 x 10(-8) M, 4.8 x 10(-9) M and 5.0 x 10(-9) M, respectively, when the normal stacking, ultra-high conductivity zone NACE-stacking and the low-temperature zone NACE-stacking methods were applied.     

Non-quadratic-intensity dependence of two-photon absorption induced fluorescence of organic chromophores in solution

We have provided a model to interpret the non-quadratic-intensity dependence behavior commonly observed in the two-photon fluorescence (TPF) experiment excited with high laser intensity. The model also provides one with a different technique to measure the two-photon absorption cross section of an organic chromophore in solution. In contrast to the commonly used low intensity technique that depends on the quadratic-intensity law, the present technique is based on the non-quadratic-intensity dependence of two-photon fluorescence. Auxiliary data such as two-photon quantum efficiency and fluorescence collection efficiency, essential in the low intensity method, are not required in the present technique. TPF measurements of Rhodamine B in methanol are carried out to demonstrate the validity of the present method. The method is used to determine the two-photon absorption cross section of a new chromophore attached with tricyano-derivatized furan as the electron acceptor. The two-photon absorption cross section measured using this method is also compared with that using a conventional transmission technique.     

Measurement of unbound cocaine in blood, brain and bile of anesthetized rats using microdialysis coupled with liquid chromatography and verified by tandem mass spectrometry

To investigate the disposition of unbound cocaine in the rat blood, brain and bile, we demonstrate an in vivo multiple sampling microdialysis system coupled with liquid chromatography for cocaine assay and verified by tandem mass spectrometry. Three microdialysis probes were concurrently inserted into the jugular vein, bile duct and brain striatum of each anesthetized rat. After a period of 2 h post-surgical stabilization, cocaine (10 mg kg(-1)) was administered through the femoral vein. Separation of unbound cocaine from various biological fluids was applied to a reversed-phase C(18) column (250 x 4.6 mm I.D., 5 microm). The mobile phase consisted of acetonitrile--10 mm potassium dihydrogen phosphate buffer (25:75, v/v, pH 4.0) and 0.8% diethylamine at a flow rate of 1 mL min(-1). The UV detector wavelength was set at 235 nm. The results indicate that cocaine penetrates the blood--brain barrier with a rapid distribution. However, unbound cocaine in the bile dialysate was not detectable in the UV detection. We therefore use LC--tandem mass spectrometry to detect the bile fluid after cocaine administration (3 mg kg(-1), i.v.). The results indicate that cocaine goes through hepatobiliary excretion.     

Isothermal equilibrium adsorption of concanavalin A on dextran-modified poly(methyl methacrylate) latex particles

Isothermal equilibrium adsorption experiments were carried out to study the adsorption of concanavalin A (Con A) on dextran-modified poly(methyl methacrylate) (PMMA) latex particles. Three PMMA particles with various levels of dextran modification were selected for study: 0% (designated as D0), 1.24% (D20), and 2.45% (D75) based on total polymer weight. The Langmuir model is applicable to both D0 and D20 systems, although the data for the D20 system are somewhat scattered. On the other hand, the amount of Con A adsorbed per gram polymer particles (q*) versus the Con A concentration in water (c*) curve for the D75 system cannot be described by the Langmuir model. The deviation is attributed to the formation of a crosslinked network structure, caused by specific binding of the dimeric Con A molecules onto two neighboring particles with grafted dextran. The ratio of the initial number of Con A molecules to the initial number of active binding sites on the dextran-modified particle surface plays an important role in determining the structure of flocs formed. The maximum amount of Con A adsorbed on the particle surface (q _max) is of the order of 10⁻¹ μmol per gram particles and q _max in decreasing order is D75 > D20 > D0. The dissociation constant of the Con A-D20 (or Con A-D75) pair is of the order of 10⁻¹ μmol dm⁻³ which is 1 order of magnitude smaller than that of the Con A-D0 pair. Thus, the electrostatic interaction between Con A and D0 is much weaker than the affinity interaction between Con A and D20 (or D75). An empirical model is proposed to qualitatively explain the q* versus c* data. Received: 18 June 1998 Accepted in revised form: 24 December 1998     

Heterostructured composite of NiFe-LDH nanosheets with Ti4O7 for oxygen evolution reaction

Developing oxygen evolution reaction (OER) electrocatalyst based on earth-abundant materials holds great promise for ascertaining water-splitting to surmount its deprived kinetics. In this regard, NiFe-LDH (layered double hydroxide) receives considerable attention owing to their layered structure. However, they still suffer from poor electronic conductivity and structural stability. We combined NiFe-LDH nanosheets with Magnéli phase Ti₄O₇ into a heterostructured composite. A series of analyses reveal that decorating Ti₄O₇ facilitates charge transfer to enhance the conductivity of NiFe-LDH-Ti₄O₇. During electrochemical measurement, Ni²⁺ is transformed to metastable Ni³⁺ (Ni (OH)→ NiOOH) before the OER onset potential. Thus, the presence of Ni³⁺ as the main active sites could improve the chemisorption of OH^? to facilitate OER. As a result, the NiFe-LDH-Ti₄O₇ catalyst delivers as low as onset potential (1.43 V). Combining the holey structure (NiFe-LDH and Ti₄O₇) and the defect engineering generated on NiFe-LDH-Ti₄O₇ as a synergistic effect improves the OER performance. The inclusion of Ti₄O₇ in the composite leads to more vacancy sites, as evidenced by the extended X-ray absorption fine structure (EXAFS) analysis. The obtained defective structure with a low coordination environment would improve the electronic conductivity and facilitate the adsorption process of H₂O onto metal cations, thereby increasing the intrinsic catalytic activity of NiOOH. The strong coupling of NiFe-LDH and Ti₄O₇ also increases the stability, and the heterostructured composite helps maintain the structural robustness of the LDH.     

Effect of selected dopants on conductivity and moisture stability of Li3PS4 sulfide solid electrolyte: a first-principles study

The first principle computational screening was performed to investigate the effect of selected dopants for Li₃PS₄ sulfide solid electrolyte on its ionic conductivity and stability toward moisture. The results suggest that substitution P⁵⁺ using isovalent cations whose electronegativity (EN) value is closer to the value of S has more significant effects on the ionic conductivity, whereby W⁵⁺ and Sb⁵⁺ can improve most. Similarly, aliovalent cation substitutions with compensating changes in the lithium-ion concentration, particularly those with a lower oxidation state and higher EN, such as Cu²⁺, effectively enhance the lithium-ion conductivity in this structure. For cation dopants, it is found that ionic conductivity improvement of Li₃PS₄ is the synergetic effect of EN and oxidation number of the dopant as well as the material's lattice parameter change. Oxides of the considered cation dopants can also improve the ionic conductivity of the material but have much lower lithium-ion conductivity than the cases of cation dopants. However, the metal oxide dopants, particularly those derived from soft Lewis' acid cations, show a marginal improvement in moisture stability of the Li₃PS₄ electrolyte. The effect of halides and metal halide dopants on the lithium-ion conductivity and moisture stability of Li₃PS₄ electrolyte are also studied. It is found that metal halides are more effective than any other dopants in improving the ionic conductivity of Li₃PS₄.