Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems

Biosensors containing cytochrome P450cam in a didodecyldimethylammonium bromide vesicular system were prepared by cross-linking onto a glassy carbon electrode (GCE) with glutaraldehyde in the presence of bovine serum albumin. Cyclic voltammetric responses of the sensor in air-free buffer solution showed that the sensor exhibited reversible electrochemistry due to direct electron exchange between the haem Fe(3+/2+) redox system and the GCE surface. In air-saturated solution containing camphor, the biosensor gave an irreversible electrocatalytic current which is compatible with the monooxygenation of the substrate. Steady state amperometric experiments with camphor, adamantanone and fenchone were performed with a biosensor prepared by cross-linking P450cam with glutaraldehyde onto a Pt disc electrode. The sensor was characterised by fast amperometric responses, attaining steady-state in about 20 s in a cobalt sepulchrate mediated electrochemical system. The kinetic parameters of the biosensor were analysed using the electrochemical Michaelis Menten equation. The estimated apparent Michaelis-Menten constant, Km, values for the biosensors were in the range of 1.41-3.9 mM.     

Utility of wiring nitrate reductase by alkylpyrroleviologen-based redox polymers for electrochemical biosensor and bioreactor applications

The purpose of this work was to see if the alkylpyrroleviologen redox polymer technology previously developed for a reagentless nitrate biosensor based on nitrate reductase (NaR) from Escherichia coli (Cosnier, S.; Innocent, C.; Jouanneau, Y. Anal. Chem. 1994, 66, 3198-3201) could be applied to the isozyme from Aspergillus niger. In particular, the enzyme viability after immobilization was of great interest, as Cosnier et al. reported a residual activity of only 0.33% of the amount initially applied. The present work showed that A. niger NaR lost 99.2% of soluble activity on vacuum-drying in the presence of 2.5 nM N-methyl-N'-(12-[pyrrol-1-yl]dodecyl)-4,4'-bipyridinium ditetrafluoroborate monomer (C12V2+) and that most of this loss was due to monomer inhibition (91%). The loss due to dehydration was only 8%. In the biosensor configuration, the enzyme gave a residual activity of 0.18% of the amount originally applied and a specific response of 1.7 mA M-1 cm-2, but all activity was lost after 4 d storage at 4 degrees C in phosphate buffer. It was concluded that for practical biosensors and bioreactors, modification of the redox polymer format was needed, for example by covalent immobilization, to effect higher loading of viable NaR and improved enzyme stability.     

Anoxic LTP is mediated by the redox modulatory site of the NMDA receptor

1. The effects of redox reagents, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and tris(carboxyethyl)phosphine (TCEP), on anoxia-induced long-term potentiation (LTP) were investigated in CA1 hippocampal neurons using extracellular recording techniques. Experiments were performed in the presence of 0.1 mM MgCl2 and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to pharmacologically isolate N-methyl-D-aspartate (NMDA) receptor-mediated responses. 2. DTNB (200 microM), a thiol oxidizing reagent, reduces by 52 +/- 9% (mean +/- SE) (n = 9/9) NMDA-receptor field potentials evoked by electrical stimulation of Schaffer collaterals and this effect could not be reversed by extensive washing. Nearly the same reduction of the initial response was obtained with different concentrations of DTNB (100 and 500 microM), but the time required to reach the maximal inhibition was concentration-dependent. 3. In keeping with an earlier study oxygen and glucose deprivation for 2-3 min induced a long-term potentiation (LTP) of the NMDA receptor response (+65 +/- 16%, n = 4/6). This potentiation was reversed by DTNB (100-500 microM) (-47 +/- 18%; n = 4/4) and the initial LTP could not be restored upon extensive washing of the drug. 4. TCEP (200 microM), a reagent which reduces S-S bond, amplified the electrically evoked NMDA-receptor EPSP (+27 +/- 12%; n = 3). In addition, TCEP (200 microM), nearly completely reversed the effect of DTNB (200 microM) on anoxia-induced LTP (+56 +/- 19%; n = 3/3). Preliminary results also indicate that TCEP occlude anoxic-LTP (n = 3/4). 5. Following DTNB (200 microM) treatment, oxygen and glucose deprivation did not generate anoxic LTP and extensive washing did not restore a potentiated NMDA field potential. 6. These observations strongly suggest that the redox site of the NMDA receptor is involved in the induction and the maintenance of the anoxic LTP of the NMDA receptor-mediated response in CA1.     

Clinical implications of genetic polymorphisms and drug interactions mediated by cytochrome P-450 enzymes

Hepatic oxidation is a major drug metabolising process and is carried out by the cytochrome P-450 monooxygenase system. This system consists of a variety of isoenzymes among which the cytochromes 1A2, 2C8, 2C9/10, 2C19, 2D6, 2E1 and 3A4 are involved in the oxidative metabolism of drugs. Interindividually, large differences in capacities are found. These differences are partly due to genetic constitution (genetic polymorphism, which has been proved to exist for CYP2D6 and CYP2C19) and partly due to environmental factors, among which the administration of interfering drugs can play a major role.     

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the alpha-helix structure of the protein and induces slight changes at the active-site environment: however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (delta G0) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode: on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology: the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.     

Polishable and renewable DNA hybridization biosensors

Routine applications of DNA hybridization biosensors are often restricted by the need for regenerating the single-stranded (ss) probe for subsequent reuse. This note reports on a viable alternative to prolonged thermal or chemical regeneration schemes through the mechanical polishing of oligonucleotide-bulk-modified carbon composite electrodes. The surface of these biocomposite hybridization biosensors can be renewed rapidly and reproducibly by a simple extrusion/polishing protocol. The immobilized probe retains its hybridization activity on confinement in the interior of the carbon paste matrix, with the use of fresh surfaces erasing memory effects and restoring the original target response, to allow numerous hybridization/measurement cycles. We expect that such reusable nucleic acid modified composite electrodes can be designed for a wide variety of biosensing applications.     

Biosynthesis of inositol phosphoceramides and remodeling of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae are mediated by different enzymes

Metabolic labeling of cells with [3H]dihydrosphingosine ([3H]DHS) allows us to follow the incorporation of this tracer into ceramides (Cer), inositol phosphoceramides (IPCs), and mannosylated IPCs and at the same time to assess the remodeling of glycosylphosphatidylinositol proteins during which preexisting anchor lipid moieties are replaced by [3H]Cer-containing anchors. The results indicate that the remodelases in the endoplasmic reticulum and Golgi use as their substrate Cers that are not generated by the breakdown of IPCs but are newly synthesized. Aureobasidin A, an inhibitor of the IPC synthase Aur1p completely blocks IPC biosynthesis at 0.5 micrograms/ml but does not block remodeling of glycosylphosphatidylinositol anchors even at concentrations up to 10 micrograms/ml. In addition, a synthetic Cer analogue, N-hexanoyl-[3H]DHS, is used as a substrate by Aur1p but not by the remodelases. Thus, remodeling is not mediated by Aur1p although remodeling presumably proceeds by an analogous reaction. Studies with secretion mutants deficient in COPII or COPI coat proteins show that all COPII mutants are unable to introduce [3H]Cer by the Golgi remodelase at the restrictive temperature. This suggests that Cer has to be transported by a COPII-dependent way from the endoplasmic reticulum to Golgi for Golgi remodeling to occur. Golgi remodeling is also not operating in the erd2 mutant and is significantly reduced in COPI mutants, suggesting a dependence of Golgi remodeling on retrotransport.     

Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase

Cytosolic and lysosomal acid phosphatases have the ability to hydrolyze orthophosphoric monoesters below pH 5-6. However, it is thought they may have different intracellular roles. To clarify their properties, substrate specificity, inhibitor sensitivity and the modulation of enzyme by redox conditions were determined using bovine liver enzymes. DEAE-cellulose chromatography following (NH4)2SO4 fractionation revealed three forms of cytosolic acid phosphatases as in the KCl gradient (0-500 mM). After Sephadex G-75 gel filtration, the enzymes appeared as single bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their activities for D-erythrose 4-phosphate co-purified with p-nitrophenylphosphatase activities in all steps. In contrast the lysosomal enzyme was purified by Octyl-Sepharose column chromatography after n-butanol treatment, (NH4)2SO4 fractionation, Bio gel P-200 gel filtration and DE-52 chromatography. The relative molecular masses (M(r)) determined by SDS-PAGE indicated that M(r) of the cytosolic enzymes (16,000) was less that of lysosomal enzyme (160,000). The cytosolic enzymes were active against sugar phosphates and were inhibited by 1 mM Cu2+. In addition, the cytosolic enzymes were inactivated by 5 mM oxidized glutathione and protected by 10 mM reduced glutathione (in the presence or absence of thioltransferase), suggesting that sensitive cysteinyl residue(s) existed. The lysosomal enzyme was active against various substrates and was strongly inhibited by 1 mM Cu2+ and 2 mM fluoride. The results presented here suggest that cytosolic enzymes have different properties from those of lysosomal enzyme with respect to substrates, inhibitors and regulation of activity.     

The superoxide production mediated by the redox cycling of xenobiotics in rat brain microsomes is dependent on their reduction potential

Responding effectively to trauma survivors who engage in self-injury can be challenging, even for experienced therapists. This paper outlines therapeutic goals and appropriate clinical responses, including remaining present at and open to communication about disclosures of self-injury, helping clients to intervene in their own process of self-injury, and working with clients to resolve underlying issues. Alternatives to self-injury are discussed and cautions are offered about common therapeutic responses likely to be particularly unhelpful.     

Clinical assessment applications of ambulatory biosensors.

Ambulatory biosensor assessment includes a diverse set of rapidly developing and increasingly technologically sophisticated strategies to acquire minimally disruptive measures of physiological and motor variables of persons in their natural environments. Numerous studies have measured cardiovascular variables, physical activity, and biochemicals such as cortisol in psychopathology and treatment research. The physiological concomitants of many behavior and medical disorders and the benefits of a multimethod assessment strategy provide strong rationales for clinical applications of ambulatory biosensor measurement. A number of psychometric dimensions of evaluation are important in clinical applications of biosensor measurement, including accuracy and validity, reliability and consistency, clinical utility, incremental validity and utility, sensitivity to change, generalizability, cost benefits, and the conditional nature of dimensions of biomeasure evaluation. The authors review ambulatory biosensor methods and make recommendations for use of the technology. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Characterization and electrochemical performance of CeO2 and Eu-doped CeO2 films as a manganese redox flow battery component

Hexagonal CeO_2 and Eu-doped CeO_2 nanoparticles were obtained using a facile microwave-hydro thermal method under mild conditions and their application towards manganese redox flow battery component were studied. The structural properties were studied by X-ray diffraction and indicate that samples present a fluorite structure. Raman spectroscopy shows Eu3+ ions substitute Ce~(4+) and generate oxygen vacancies. Electrochemical properties of pure and Eu-doped CeO_2 films deposited at graphite substrates investigated by cyclic voltammetry and galvanostatic charge—discharge indicate that dopant concentration affects the electrochemical properties of CeO_2. The increase in the reversibility redox of electrochemical systems observed is attributed to coexistence of Ce~(4+)/Ce~(3+) redox couple confirmed by XPS.Charge—discharge tests display coulombic and voltage efficiency values of above 80% and 90%, respectively. The obtained specific capacity for Ce_(0.99)Eu_(0.01)O_2(372.49 mAh/g) and pure oxide(334.84 mAh/g)indicates that both samples are promising for application in Mn-batteries.     

双氧水脱硫工艺设计与应用

从双氧水的工艺流程及特点进行阐述,详细介绍了双氧水脱硫设计思路、应用、成本、安全、环保等。根据硫酸尾气、有色冶炼炉窑环集烟气、再生铜冶炼炉窑烟气的特点与条件,明确双氧水脱硫工艺不同烟气条件的设计方案,并结合具体的烟气条件,进行合理的工艺改进,设计方案优化,更好地适应不同烟气工况,从而降低生产成本,消除安全环保隐患。     

纳米金在生物传感领域的应用研究进展

近年来,由于纳米金具有容易制备以及良好的生物相容性和相对较大的比表面积等特点,在生物传感领域的应用研究得到了飞速的发展.文中综述了纳米金在构建DNA、免疫、酶、糖等各类生物传感器方面的应用研究进展.     

MSI brain scan machines, the grandfather of biosensors

We all know about MRIs. You hurt your knee and for $800 the doctor can have a close look inside, from the outside, and the resulting images can often prevent costly and painful knee surgery. A similar technological breakthrough has been achieved for use in diagnosing brain disorders such as epilepsy. Instead of electrode insertion into one's brain at a cost of $50,000, Biomagnetic Technologies' (BTi) totally passive Magnetic Source Imaging (MSI) machines can detect incredibly subtle differences, and provide the critical information for 10% of that cost.     

解析参数对活性焦再生过程及再生效果的影响

 活性焦的热解析参数对再生活性焦的脱硫脱硝性能和机械强度至关重要。为了明确解析参数对活性焦再生过程和再生效果的影响规律,通过热解析试验探究活性焦硫残余比例、CO₂和CO生成量及再生活性焦脱硫脱硝性能随解析温度和解析时间的变化规律,继而明确适宜的活性焦热解析参数。结果表明,活性焦升温解析过程中,脱硫产物在317 ℃左右迅速分解,随后分解速率下降;在进入恒温解析阶段后脱硫产物分解速率先快速下降,而后进入缓慢解析状态。硫残余比例随恒温解析温度的升高而下降,在530 ℃下解析3 h可使脱硫产物完全解析;解析温度高于430 ℃后,活性焦表面的酚基、醌基、内酯基等含氧官能团分解量明显增加,并随恒温解析温度的升高而持续增加,分解所生成的CO和CO₂也随之大幅增加,这将使活性焦的孔隙结构进一步发展,继而不利于活性焦机械强度的保持;解析温度低于530 ℃时,硫残余比例随解析温度的升高而持续降低,使再生活性焦的脱硫脱硝性能持续提高;解析温度高于530 ℃后,含氧官能团分解量随解析温度的升高而持续增加,这将有利于提高活性焦表面SO₂氧化反应速率,继而使再生活性焦的脱硫性能持续升高,但酚基、内酯基等酸性含氧官能团的分解使再生活性焦对NH₃的吸附性能降低,进而使其脱硝性能降低。在兼顾再生活性焦脱硫脱硝性能、机械强度和生产效率等多方面因素时,430 ℃恒温解析3 h是相对较优的解析参数。在此解析条件下,再生活性焦的硫残余比例仅为1.8%,含氧官能团尚未发生大量分解,脱硫脱硝性能相对较为优良。     

The use of neuronal networks on multielectrode arrays as biosensors

Mammalian spinal neuronal networks growing on arrays of photoetched electrodes in culture provide a highly stable system for the long-term monitoring of multichannel, spontaneous or evoked electrophysiological activity. In the absence of the homeostatic control mechanisms of the central nervous system, these networks show remarkable sensitivities to minute chemical changes and mimic some of the properties of sensory tissue. These sensitivities could be enhanced by receptor up-regulation and altered by the expression of unique receptors. The fault-tolerant spontaneous network activity is used as a dynamic platform on which large changes in activity signify detection of chemical substances. We present strategies for the expression of novel supersensitivities to foreign molecules via genetic engineering that involves the grafting of ligand binding cDNA onto truncated native receptor DNA and the subsequent expression of such chimeric receptors.     

Rates of Trace Mineral-Catalyzed Decomposition of Hydrogen Peroxide

Rapid hydrogen peroxide (H2O2) decomposition is a significant limitation of catalyzed H2O2 propagations (i.e., modified Fenton’s reagent) for the remediation of contaminated soil and groundwater by in situ chemical oxidation. Rates of H2O2 decomposition mediated by seven trace minerals and four iron and manganese oxides were evaluated in batch reactors containing slurries of H2O2 and each of 11 minerals. At pH 3, the dominant catalysts in the decomposition of H2O2 on a per surface area basis were the manganese and iron oxides pyrolusite, goethite, and hematite, while decomposition rates in the presence of the manganese oxyhydroxide manganite and the trace mineral siderite were one to two orders of magnitude lower. At pH 7, the dominant catalysts were hematite and pyrolusite, and decomposition rates were one to two orders of magnitude lower in the presence of goethite, manganite, and siderite. The trace minerals anatase, bauxite, cuprite, ilmenite, magnesite, and willemite provided the least activity for decomposing H2O2 at both pH regimes. The results of this study document that the trace minerals anatase, bauxite, cuprite, ilmenite, magnesite, siderite, and willemite do not provide a significant pathway for H2O2 decomposition in the subsurface, and efforts to stabilize H2O2 for ISCO should focus on reactions occurring on the surfaces of iron and manganese oxides.     

过氧化物促进作用的研究和探讨

探讨了H2O2和CaO2在去离子水、矿浆和氰化浸出液中的稳定性和去毒作用．通过对H2O和CaO2放氧速度的测定，得出；H2O2在弱碱性条件下的稳定性较好；在强碱性条件下，CaO2的稳定性较好；在矿浆和氰化浸出液中，H2O2能迅速分解而放氧，而CaO2却以相对较为缓慢的速度放氧．通过测定溶液和矿浆中的游离CN－，得出：在溶液中H2O2和CaO2的去毒作用随pH值的增大而减小；在矿浆中只要控制过氧化物的适宜用量，不但不会引起去毒作用，反而会节省大量氰化物．以此为依据，对全泥氰化和金精矿氰化的两个典型矿石进行了试验，表明利用H2O2或CaO2助浸均可提高浸出速度，缩短浸出时间．通过试验和理论分析证明，浸出速度得以提高的原因是溶解氧和过氧化物本身作为氧化剂都参与了浸金反应．本文最后对过氧化物促浸法进行了评价，并提出了应用前景．