Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 μA mmol⁻¹ l cm^-2 and a limit of detection of 0.20 mmol l⁻¹ being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H₂O₂ produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.     

C NMR relaxation studies of 1:2 LiCl-ethylaluminum dichloride solutions

A new room-temperature molten salt, 1:2 LiCl-ethylaluminum dichloride (LiCl-EtAlCl₂, f.p. about 178 K), is examined using ¹³C relaxation methods at 7.05 T (−25 to + 80 °C). The methylene carbon undergoes scalar relaxation of the ‘second kind’ as it is coupled to a faster relaxing (quadrupolar) nucleus. LiCl-EtAlCl₂ undergoes a significant liquid-state phase change between 5 and 15 °C as evidenced by observed changes in the relaxation properties of the methylene and methyl carbons and J(¹³C−²⁷Al). The J(¹³C−²⁷Al) coupling constants are 75 (− 10 to + 5 °C) and 11 Hz (15–65 °C), indicating a change in structure between 5 and 15 °C. Chemical shift anisotropies of 56 and 48 ppm are obtained for the methylene and methyl carbons in the EtAlCl₂ dimer part of the 1:2 LiCl-EtAlCl₂ solution.     

Activity and storage stability of immobilized glucose oxidase onto magnesium silicate

Glucose oxidase (GOD) was covalently immobilized onto florisil (magnesium silicate) carrier via glutaraldehyde. Immobilization conditions were optimized: the amount of initial GOD per grams of carrier as 5 mg, pH as 5.5, immobilization time as 120 min and temperature as 10 °C. Under the optimized reaction conditions activities of free and immobilized GOD were measured. Free and immobilized GOD samples were characterized with their kinetic parameters, and thermal and storage stabilities. K_M and V_max values were 68.2 mM and 435 U mg GOD⁻¹ for free and 259 mM and 217 U mg GOD⁻¹ for immobilized enzymes, respectively. Operational stability of the immobilized enzyme was also determined by using a stirred batch type column reactor. Immobilized GOD was retained 40% of its initial activity after 50 reuses. Storage stabilities of the immobilized GOD samples stored in the mediums with different relative humidity in the range of 0–100% were investigated during 2 months. The highest storage stability was determined for the samples stored in the medium of 60% relative humidity. Increased relative humidity from 0% to 60% caused increased storage stability of immobilized GODs, however, further increase in relative humidity from 80% to 100% caused a significant decrease in storage stability of samples.     

Structure of Co2(CO)6(dppm) and Co2(CO)5(CHCO2Et)(dppm) (dppm = Ph2PCH2PPh2) and exchange reaction with CO: An experimental and computational study

Crystal structures of Co₂(CO)₆(dppm) (1) and Co₂(CO)₅(CHCO₂Et)(dppm) (2) (dppm = Ph₂PCH₂PPh₂) show asymmetry with respect to the orientation of the phenyl groups in 1 and owing to the bridging ethoxycarbonylcarbene ligand in 2. The effect of this asymmetry was recognized in the solid-state ³¹P NMR spectra of 1 and 2 and in the solid-state and solution ¹³C NMR spectra of 2 as well, but not in the solid-state and solution ¹³C NMR spectra of 1. In CH₂Cl₂ solution under an atmosphere of ¹³CO, the CO ligands of both complexes exchange with ¹³CO. The overall rate of ¹³CO exchange at 10 °C was found to be k_obs = 0.107 × 10⁻³ s⁻¹ for 1 and k_obs = 0.243 × 10⁻³ s⁻¹ for 2. Two-layered ONIOM(B3LYP/6-31G(d):LSDA/LANL2MB) studies revealed fluxional behavior of 1 with rather small barriers of activation of the rearrangements. Four possible isomers have been computed for 2, close to each other energetically.     

Effect of chloride ions on the S-state distribution and deactivation kinetics in preparations of the cyanobacterium Anacystis nidulans

The roles of Ca²⁺ and Cl⁻ on the photosynthetic O₂ yield under flash illumination have been examined in EDTA-washed preparations of the cyanobacterium Anacystis nidulans. Especially the effect of Cl⁻ deficiency on the O₂ yield and on the S-state distribution was analyzed. As the results show, omission of both Ca²⁺ and Cl⁻ (Mn²⁺ present) almost totally inhibited O₂ evolution. When Ca²⁺ was replaced by Na⁺, a substantial reduction of the O₂ yield was observed, but only a minor change in the S-state distribution occurred. However, when Cl⁻ was displaced by NO⁻₃, which is equivalent to Cl⁻ deficiency of the water-splitting complex, a substantial reduction of the O₂ yield and in addition a significant change in the S-state distribution was observed. The comparison of deactivation kinetics in NO⁻₃ containing samples with those in control samples indicated that Cl⁻ deficiency allowed accumulation of oxidizing equivalents up to the S₃ state but modified the final step of O₂ evolution. Moreover, those centers which advanced to the S₃ state in the absence of Cl⁻ deactivated in a special way which involved a faster deactivation of S₂ and an increased formation of S₋₁.     

A Holocene environmental record from the southern Yangtze River delta, eastern China

The Holocene sedimentary record of core ZX-1, recovered west of mid-Holocene Chenier ridges on the monsoon-controlled Southern Yangtze delta, eastern China, consists of lagoon, salt marsh, upper tidal flat, and limnic facies, reflecting low-energy depositional environments. Holocene deposits mainly originated from the surrounding Tai Lake drainage basin. The temporal variation of most geochemical element percentages corresponds with the climatic phases inferred from the pollen record, i.e., the relatively low values of SiO₂, Na₂O, CaO and high values of Al₂O₃, K₂O, MgO, Fe₂O₃, FeO, FeO + Fe₂O₃ and TiO₂ generally concur with the warm and humid climate, and vice versa. Three geochemical indices − Al₂O₃/Na₂O, K₂O/Na₂O, and CaO/K₂O − are found to be sensitive to past precipitation in the monsoon-controlled southern Yangtze River delta. Based on fine sediment analysis of core ZX-1, the samples deposited under a dry climate with a mean annual precipitation (MAP) < 500–900 mm tend to have Al₂O₃/Na₂O values less than 12.2, and the samples deposited under a moist climate with an MAP 1000–1800 mm mostly have Al₂O₃/Na₂O values higher than 12.2. Similarly, the K₂O/Na₂O boundary value is 2.0. However, for CaO/K₂O, the dry climate sediments likely show values higher than 0.9, and those wet climate ones generally have values less than 0.9. This geochemical response suggests the potential application of these indices in the interpretation of palaeoclimate variation of monsoon-controlled eastern China.

Holocene climatic variation history is reconstructed for the southern Yangtze delta. From 8000 to 7000 yr BP, regional climate demonstrated frequent fluctuations, with warm and wet periods (8000–7700; 7500–7200 yr BP) alternating with cool and less humid periods (7700–7500; 7200–7000 yr BP). From 7000 to 6000 yr BP, the climate was relatively warm and humid. Since then, it had turned cool and dry, climaxing in an intense cold event around 4000 yr BP. After the cold event, it became warm and humid until around 2500 yr BP.     

CYP450 biosensors based on gold chips for antiepileptic drugs determination

Three-electrode configuration chips containing a Pt, Au and a screen-printed Ag/AgCl as counter, working and reference electrode, respectively, have been developed. Selective determination of Phenobarbital (PB) has been carried out by Cytochrome P450 2B4 (CYP450) immobilization into a polypyrrole matrix onto the gold working electrode. Chronoamperometric experiments show a PB diffusion coefficient of 2.42 × 10⁻⁶ cm² s⁻¹, a reproducibility and repeatability in terms of residual standard deviation (RSD) of 13% and 5.51%, respectively, and a limit of detection (LOD) of 0.289 μmol dm⁻³ ( = β = 0.05) for the developed CYP450-biosensor chip. Its performance has been showed by the determination of PB in pharmaceutical drugs. HPLC has been used as reference technique.     

Body temperature in arboreal and nonarboreal anuran amphibians: Differential effects of a small change in water vapor density

1. 1.|Body temperatures (T_b) and contaneous evaporative water loss rates (CWL) were measured in tree frogs (Hyla cinerea) and toads (Bufo valliceps) exposed to cyclical ramp changes in water vapor density (WVD) between 7.5 and 9.8 gm⁻³ (1 cycle h⁻¹ at an air temperature of 27.0°C.

2. 2.|CWL was 3.3 times greater in toads than in tree frogs.

3. 3.|T_b in toads cycled directly with WVd; WVD accounted for 98% of the variation in toad T_b.

4. 4.|T_b in tree frogs was independent of WVD, probably due to changes in skin resistance to water loss.

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Template synthesis of highly ordered Prussian blue array and its application to the glucose biosensing

In this paper, we propose a strategy to form nanoelectrode arrays by electrochemical deposition of the Prussian blue (PB) through highly ordered porous anodic alumina (PAA) membrane. The structure and morphology of the nanoarrays were characterized by scanning electron microscopy (SEM). As the highly ordered PB arrays can behave as an ensemble of closely spaced but isolated nanoelectrodes, the nanostructured PB arrays are successfully applied to improve the analytical performances of glucose by electrocatalytic reduction enzymatically liberated H₂O₂. The resulting PB based nanoelectrode arrays show a wide linear calibration range over three orders of magnitude of glucose concentrations (5.0 × 10⁻⁶ to 8.0 × 10⁻³ M) and a low detection limit of 1 μM. Moreover, the biosensor exhibits other good characteristics, such as short response time, high selectivity, excellent operation stability. In addition, effects of the glucose oxidase (GOx) loading, applied potential and pH on the biosensor performance were also discussed.     

Phosphorescence of adenosine and poly(riboadenylic acid). Evidence for dual emissions and n pi states

Phosphorescence from the 9-adenylyl group in the fom of microcrystalline powders of adenosine films of poly(riboadenylic acid) (poly(rA)) in hyaluronic acid has been studied at 77 K. For adenosine, clearly resolved vibronic structure consists of two progressions, A and B, with _A ‡ 1363 cm⁻¹ and _B ‡ 1575 cm ⁻¹, correlated with in-plane C₅-N₇ and in-plane C₄-C₅ stretch, respectively. The relative strength of the progressions varies with excitation wavelength and this, together with the absence of a common origin, indicates the existence of two independent emitting states with 0-0' levels separated by either 300 or 1000 cm⁻¹. Two different excitation spectra are observed lying below the normal (ππ*) adsorption and one is assigned as a previously undetected ¹(nπ*) transition. For poly(rA) films the emission band envelope is identical with that of adenosine but the vibronic structure is lost. Only one excitation peak is observed at 32.9×10³ cm⁻¹, identical with one of the adenosine spectra. The second adenosine excitation spectrum probably represents an intermolecular charge transfer transition. Comparison is made with the predictions of six semi-empirical MO calculations.     

INFLUENCE OF RELATIVE HUMIDITY ON SURVIVAL OF THE CITRUS LEAF-MINER, PHYLLOCNZSTZS CITRELLA STAINTON (LEPIDOPTERA: PHYLLOCNISTIDAE)

Abstract The relationshLps between relative humidity (RH) and survival rates of eggs, all larval stages and pupae of the citrus leaf-miner, Phyllooiistis citrella Stainton, were determined by laboratory experiments. The survival of the citrus leaf-miner was observed at seven levels of relative humidity from 35% RH to 95% RH at intervals of 10% RH, with 12 L: 12 D photoperiod and temperatiure (29±0.5) C. The relative humidity was controlled by saturated solutions of MgCl₂ 6H₂O, K₂CO₃ 2H₂O, C₆H₁₂O₆, NaNO₂, NaCl, KCl, and Pb(NO₃)₂. The results showed that lower relative humidity is unfavorable for incubation of the eggs, survival of the larvae and eclosion of the pupae. The survival rates increased generally with rising of relative humidity within the range of 35% - 85% RH, and the maximum survival rates occurred at 85% RH for different life stages. The variations in hatching rates of the eggs, survival rates of the larvae and emergence rates of the pupae were great, but unimodal at different relative humidity. The effect of relative humidity on survival rates of the citrus leaf-miner could be simulated by regression analysis, using a polynomial function of three orders, and the results of fitting the model to the observed data are presented and discussed.     

Diffusion of KCl in an amylose film

A method has been developed measuring the diffusion coefficient of KCl in amylose films. The films were soaked in potassium chloride solutions, then immersed in pure water and conductivity measured as a function of time. Different concentrations of the soaking solution were used and the measurements were made at several temperatures. The diffusion coefficient of KCl was found to be independent of the soaking solution KCl concentration, but found to increase with increasing temperature. The diffusion coefficient values were about one quarter of those found in water and varied from 4.8×10⁻¹⁰ to 11×10⁻¹⁰ m² s⁻¹. The activation energy of diffusion was close to that found in water. Two values for the activation energy were obtained, 20.1 and 14 kJ mol⁻¹, indicating a change in the film structure at 45 °C. Amylose films swelled equally in KCl-solutions and water. The thickness of amylose films doubled and the increase in mass was 100–200% corresponding the decrease of amylose content from about 87 to 37%, when the conditions changed from normal humidity conditions to water.     

华北低丘山区栓皮栎人工林冠层CO2浓度和δ13C变化及其影响因素

采用离轴积分腔输出光谱技术测定华北低丘山区栓皮栎人工林冠层上缘(11 m)和下部(6 m)大气CO₂浓度和δ¹³C值,在小时尺度上分析冠层CO₂浓度和δ¹³C变化及其影响因素.结果表明： 冠层CO₂浓度呈先高后低再升高的日变化趋势,而δ¹³C值没有明显一致的日变化规律.白天大气不稳定状态出现的频率为70.2%,在光合作用和林内湍流的共同作用下,栓皮栎冠层下部CO₂浓度高于冠层上缘约1.70 μmol·mol^-1,而δ¹³C值低于冠层上缘约0.81‰.晚上大气稳定状态出现的频率为76.2%,湍流弱,冠层叶片呼出的CO₂不易流动,导致冠层下部CO₂浓度高于上缘约1.24 μmol·mol^-1,δ¹³C值低于冠层上缘约0.58‰.白天和晚上冠层上下缘的CO₂浓度差值与δ¹³C差值均呈显著的相关关系.逐步回归分析表明,白天太阳辐射和相对湿度是影响冠层CO₂浓度和δ¹³C值差异的主要环境因子,晚上温度显著影响冠层下部与上缘δ¹³C值的变化,这些环境因子通过增强或减弱光合和呼吸作用来影响冠层大气中CO₂浓度和δ¹³C值的变化.     

Synthesis and characterization of trisodium salt of bis(thiosalicylato)aurate(I): Na3[Au(TSA)2]·5H2O

Sodium salt of a water-soluble, anionic, and monomeric 1:2 complex of Au(I) with a dianion of thiosalicylic acid TSA²⁻(Hin2TSA) = o-HS(C₆H₄)COOH) was first prepared and isolated as colorless needle crystals through a stoichiometric reaction of NaAuCl₄:H₂TSA:NaOH = 1:4:8 molar ratio in aqueous/EtOH solution. In this reaction, TSA²⁻ ligand has played a role of a reducing agent for the starting Au(III) ion and also of donor ligands coordinating to the reduced Au(I). This compound was characterized by complete elemental analyses, TG/DTA, FT-IR, 2D-NMR (¹H-¹H COSY, ¹H-¹³C HMBC, and ¹H-¹³C HMQC) spectroscopy, and the molmass measurement based on the cryoscopic method. It was shown that this complex was a monomeric species of Au(I) with a formula of Na₃[Au(TSA)₂]·5H₂O in the solid state, but not a polymeric species even in aqueous solution. A full assignment of seven carbon and four proton resonances in the coordinated TSA²⁻ ligand was achieved by the 2D ¹H-¹³C HMBC NMR technique.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.     

Effect of iron concentration on hydrogen fermentation

The effect of the iron concentration in the external environment on hydrogen production was studied using sucrose solution and the mixed microorganisms from a soybean-meal silo. The iron concentration ranged from 0 to 4000 mgFeCl₂ l⁻¹. The temperature was maintained at 37°C. The maximum specific hydrogen production rate was found to be 24.0 mlg⁻¹ VSSh⁻¹ at 4000 mgFeCl₂ l⁻¹. The specific production rate of butyrate increased with increasing iron concentration from 0 to 20 mgFeCl₂ l⁻¹, and decreased with increasing iron concentration from 20 to 4000 mgFeCl₂ l⁻¹. The maximum specific production rates of ethanol (682 mgg⁻¹ VSSh⁻¹) and butanol (47.0 mgg⁻¹ VSSh⁻¹) were obtained at iron concentrations of 5 and 3 mgFeCl₂ l⁻¹, respectively. The maximum hydrogen production yield of 131.9 mlg⁻¹ sucrose was obtained at the iron concentration of 800 mgFeCl₂ l⁻¹. The maximum yields of acetate (389.3 mgg⁻¹ sucrose), propionate (37.8 mgg⁻¹ sucrose), and butyrate (196.5 mg g⁻¹ sucros) were obtained at iron concentrations of 3, 200 and 200 mgFeCl₂ l⁻¹, respectively. The sucrose degradation efficiencies were close to 1.0 when iron concentrations were between 200 and 800 mgFeCl₂ l⁻¹. The maximum biomass production yield was 0.283 gVSSg⁻¹ sucrose at an iron concentration of 3000 mgFeCl₂ l⁻¹.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

大连市黑松树木水分利用效率的环境响应

以生长于大连城区的黑松为研究对象,建立了1951—2010年间的树木径向生长、树轮稳定碳同位素比率(δ¹³C)和水分利用效率的时间序列,研究了三者的变化特点及其与主要气候因子的关系.结果表明: 1980年以来,黑松树木径向生长有减缓趋势,δ¹³C值降低,但是水分利用效率显著增加(P<0.05).年轮宽度、稳定同位素比率和水分利用效率的变化均受气候因素的影响,并随季节波动:夏季温度与树木径向生长呈负相关,而冬季则呈正相关;6月降水和相对湿度的波动与年轮宽度变化基本呈正相关;3—9月各月温度与δ¹³C和水分利用效率呈弱正相关,其他月份基本呈弱负相关;全年降水和相对湿度分别与δ¹³C和水分利用效率基本呈负相关.快速暖干化的城市气候环境促进了树木水分利用效率的提高.