Water-soluble Mediator Research Articles

Novel Conductive Polymer Composite PEDOT:PSS/Bovine Serum Albumin for Microbial Bioelectrochemical Devices.

A novel conductive composite based on PEDOT:PSS, BSA, and Nafion for effective immobilization of acetic acid bacteria on graphite electrodes as part of biosensors and microbial fuel cells has been proposed. It is shown that individual components in the composite do not have a significant negative effect on the catalytic activity of microorganisms during prolonged contact. The values of heterogeneous electron transport constants in the presence of two types of water-soluble mediators were calculated. The use of the composite as part of a microbial biosensor resulted in an electrode operating for more than 140 days. Additional modification of carbon electrodes with nanomaterial allowed to increase the sensitivity to glucose from 1.48 to 2.81 μA × mM-1 × cm-2 without affecting the affinity of bacterial enzyme complexes to the substrate. Cells in the presented composite, as part of a microbial fuel cell based on electrodes from thermally expanded graphite, retained the ability to generate electricity for more than 120 days using glucose solution as well as vegetable extract solutions as carbon sources. The obtained data expand the understanding of the composition of possible matrices for the immobilization of Gluconobacter bacteria and may be useful in the development of biosensors and biofuel cells.

Development of Microdrip Enzyme Device Using Carbon-Coated Porous Silica Spheres

Carbon-coated porous silica spheres (C/PSSs) are particles with uniform particle and mesopore sizes, on which a thin layer of carbon is uniformly deposited on the surface by chemical vapor deposition. The continuous carbon deposit over the mesopore network allows the C/PSS to be electrically conductive and more chemically stable over its traditional counterparts, such as PSS, porous carbons, and carbon electrodes. As homogeneous particles, C/PSS can also be arranged uniformly on an electrode by screen printing to construct macroscopic networks that facilitate the diffusion of substances. Herein, C/PSS was synthesized and fabricated into a working electrode for a prototype glucose enzyme sensor to perform electrochemical measurements on a sample size of 20 μL. We selected the two enzymes, glucose dehydrogenase (GDH) and glucose oxidase (GOD), and water-soluble mediators, quinoline-5,8-dione (QD), to demonstrate the functionality of the sensor. The GDH sensor achieved a linear response up to 75 mM glucose concentration much higher than commercial sensor strips and the GOD sensor up to 25 mM. The results proved that the response current was mainly controlled by glucose diffusion. We succeeded in developing a sensor chip with C/PSS arranged on the surface with high efficiency by selecting a mediator that can smoothly transfer electrons to and from carbon, which enables various sensing applications. This work is the demonstration of the triple pore structure of the C/PSS electrode for enzyme electrode reactions.

The widest linear range of glucose test strips based on various mediators and membranes for whole blood analysis

We present a novel approach to increasing the upper limit of linearity in the calibration range of glucose test strips. Instead of using a diffusion membrane over an enzyme-containing layer, we propose membrane-forming mixtures for co-immobilization of enzymes and mediators in polymer matrices for a single-step electrode modification. Additionally, membrane components may be mixed into carbon paste and deposited on electrode supports in the course of screen-printing. We have shown that the transport of water-soluble mediators is controlled by the density of the immobilizing membrane as well as by electrostatic interactions between mediator and membrane-forming polymer. The resulting biosensor calibration range exceeds glucose concentrations in blood, due to prolongation of the linear range up to 50 mM. Furthermore, test strips, which are made via co-immobilization of mediator and enzyme in chitosan retain linear response in a wide range and 90% of the sensitivity, when tested in blood.

Clinical use of plasmafiltration coupled with adsorption

Coupled plasma filtration adsorption is an extracorporeal therapy that combines plasma separation and adsorption of inflammatory mediators and toxins, followed by hemofiltration to control volume overload and removal of small and medium-sized water-soluble mediators. Although it is not new, there are still doubts about its clinical role. Evidence indicates that the coupled plasma filtration adsorption is highly efficient in removing inflammatory molecules in sepsis. So far it has not been shown to reduce mortality, but it is possible to achieve a better hemodynamic benefit over convective therapies. On the other hand, the absorptive component makes it possible to purify larger mediators associated with causes of non-septic acute kidney injury, such as myoglobin, toxins associated with liver failure and others, allowing that coupled plasma filtration adsorption indications to be expanded to various clinical settings with a high inflammatory component, particularly when it is required to manage several organ dysfunctions simultaneously.

Profiling of grazed cultures of the chlorophyte alga Dunaliella tertiolecta using an untargeted LC-MS approach.

Extracellular signals are reported to mediate chemical cross-talk among pelagic microbes, including microalgal prey and predators. Water-soluble mediator compounds play a crucial role in extracellular communication which is vital for prey recognition, attraction, capture, and predator deterrence. A range of exo-metabolites including oxylipins and vitamins are released by prey in response to grazing stress. The temporal dynamics of such exo-metabolites largely remains unknown, especially in large-scale cultivation of microalgae such as closed or open ponds. In open ponds, infestation of predators is almost inevitable but highly undesirable due to the imminent threat of culture collapse. The early production of exo-metabolites emitted by microalgal prey in response to predator attack could be leveraged as diagnostic markers of possible culture collapse. This study uses an untargeted approach for temporal profiling of Dunaliella tertiolecta-specific exo-metabolites under grazing pressure from Oxyrrhis marina. We report 24 putatively identified metabolites, belonging to various classes such as short peptides, lipids, indole-derivatives, and free amino acids, as potential markers of grazing-mediated stress. In addition, this study outlines a clear methodology for screening of exo-metabolites in marine algal samples, the analysis of which is frequently hindered by high salt concentrations. In future, a chemistry-based targeted detection of these metabolites could enable a quick and on-site screening of predators in microalgal cultures.

Disposable electrochemical glucose sensor based on water-soluble quinone-based mediators with flavin adenine dinucleotide-dependent glucose dehydrogenase

Glucose level measurement is essential for the point-of-care diagnosis, primarily for persons with diabetes. A disposable electrochemical glucose sensor is constructed using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and redox mediator for electron transfer from the enzyme to the electrode surface. Ideally, a suitable mediator should have high water solubility, high kinetic constant, high stability, and redox potential between −0.2 and 0.1 V vs. Ag|AgCl|sat. KCl. We designed and synthesized two new quinone-based water-soluble mediators: quinoline-5,8-dione (QD) and isoquinoline-5,8-dione (IQD). The formal potentials for both QD and IQD at pH 7.0 were −0.07 V vs. Ag|AgCl|sat. KCl. The logarithms of the electron exchange rate constants (k2/(M−1 s−1)) between QD/IQD and FAD-GDH were 7.7 ± 0.1 and 7.4 ± 0.1 for QD and IQD, respectively, which are the highest value among the water-soluble mediators for FAD-GDH reported to date. Disposable amperometric glucose sensors were fabricated by dropping FAD-GDH and QD or IQD onto a test strip. The sensor achieved a linear response up to glucose concentrations of 55.5 mM. The linear response was obtained even when the mediator loading was low (0.5 nmol/strip); loading was only 0.2 mol% of glucose. The results proved that the response current was primarily controlled by glucose diffusion. In addition, the sensor using QD exhibited high stability over 3 months at room temperature.

Photoelectric Current Enhancement via Millimeter‐Scale Bioelectrochemical Cell Using Iron Oxide Nanoparticles‐Modified Screen‐Printed Electrodes

Herein, photocurrent generating a millimeter‐scale bioelectrochemical cell (mBEC) is fabricated using a thylakoid membrane (TM) on a screen‐printed electrode (SPE). The mBEC is used to generate a direct and mediated photocurrent without using a selective membrane. The mediated photocurrent generation is tested with iron oxide nanoparticles (NPs) coated on the SPE surface and the water soluble mediators potassium hexacyanoferrate (FCN) and p‐Benzoquinone (BQ). The SPE/TM system produces a 4.35 μA cm−2 photocurrent without using a mediator obtained by direct electron transfer (DET). A significant enhancement reaches the current generation when an efficient amount of mediator is used in the system. The SPE/NP/BQ/TM system produces a 55 μA cm−2 photocurrent with an incorporation among TM, NP, and BQ. The performance of the mBECs is tested by taking the measurements from series and parallel connected cells. An enhanced photocurrent generation of 178 μA cm−2 is attained when the four cells are connected in series. Also, a higher light conversion rate is achieved by the parallel connected two cells. In addition, the maximum current density of mBEC obtained from the SPE/NP/BQ/TM cell at a pseudo‐steady state is 180 mA m−2 at a power density of 40 mWm−2.

Conjugation of native membranes via linear oligo-amines

We report the first observation of an efficient, native membrane conjugation mechanism via positively charged, linear oligo-amines. Clustering of membrane fragments relies on electrostatic interactions between the net negative charge of the membranes and the positively charged, water-soluble mediators. This conjugation principle is demonstrated with two different bacterial membranes in which are embedded either the intrinsic membrane protein (MP) bacteriorhodopsin (bR) or the more recently identified xanthorhodopsin (XR). As determined by their characteristic UV–vis absorption spectra and by circular dichroism, the MPs are not significantly perturbed by the oligo-amines carrying from +3 to +6 positive charges. Light microscopy and scanning electron microscope (SEM) imaging provide direct evidence for membrane conjugation. Process efficiency was found to be correlated with the net charge of the oligo-amine used. Membrane conjugation is accomplished within a wide range of pH values (7−2.5); is reversed by NaCl; and does not require the presence of a precipitant (e.g. PEG) nor Ca2+ ions. Some evidence for bilayer fusion is also observed, but only in the presence of the +6 oligo-amine analog.

Light-dependent photocurrent generation: Novel electrochemical communication between biofilm and electrode by ferrocene cored Poly(amidoamine) dendrimers

In this study, ferrocene (Fc) cored polyamidoamine (PAMAM) dendrimer generations were used as a mediator to produce photo-current by providing electrochemical communication between photosynthetic microorganisms and electrode. Photosynthetic microorganisms (PM) (algae and cyanobacteria mixture) were attached to the graphite electrode surface and photocurrent generation was demonstrated. A photocurrent density of 0.033 μA cm−2 was obtained by direct electron transfer of PM to the electrode without using any mediator. FcPAMAM structures with different redox potentials successfully interconnected the electrode and PM, resulting in a photocurrent density of 1.18 μA cm−2. Two different water-soluble mediators, p-benzoquinone and hexacyanoferrate (III), were used to accelerate photo-current through the reaction center and the photocurrent density was raised to 11.89 μA cm−2. Superoxide dismutase (SoD) and catalase (Cat) enzymes have been successfully used to remove reactive organic species (ROS) from the reaction center and further increase in photocurrent density was reached up to 20.04 μA cm−2.

How to engineer glucose oxidase for mediated electron transfer.

Glucose oxidase (GOx) is of high industrial interest for glucose sensing because of its high β-d-glucose specificity. The efficient and specific electrochemical communication between the redox center and electrodes is crucial to ensure accurate glucose determination. The efficiency of the electron transfer rates (ETR) with GOx, together with quinone diamine based mediators, is low and differs even among mediator derivatives. To design optimized enzyme-mediator couples and to describe a mediator binding model, a joint experimental and computational study was performed based on an oxygen-independent GOx variant V7 and two quinone diimine based electron mediators (QDM-1 and QDM-2), which differ in polarity and size, and ferrocenemethanol (FM). A site saturation library at position 414 was screened with all three mediators and yielded four beneficial substitutions Tyr, Met, Leu, and Val. The variants showed increased mediator activity for the more polar QDM-2 with a simultaneously decreased activity for the less polar and smaller QDM-1 and for FM. The variant GOx V7-I414Y exhibited the biggest change for the quinone diimine derivatives compared with V7 (QDM-1: 55.9 U/mg V7, 33.2 U/mg V7-I414Y; QDM-2: 2.7 U/mg V7, 12.9 U/mg V7-I414Y). TheoreticalETR calculated based on the Marcus theory were in good agreement with the experimental results. Molecular docking studies revealed a preferable binding of the two QD mediators directly in the active site, 3.5 Å away from the N5 atom of the flavin adenine dinucleotide (FAD) and in direct vicinity to position 414. In summary, position 414 in the active site was identified to modulate the electron shuttling from the FAD of the GOx to small water-soluble mediators dependent on the polarity and size of residue 414 and on the polarity and size of the mediator. The presented mediator binding model offers a promising possibility for the design of optimized enzyme-mediator couples.

Horseradish peroxidase and toluidine blue covalently immobilized leak-free sol-gel composite biosensor for hydrogen peroxide

The enzyme horseradish peroxidase and the water-soluble mediator toluidine blue were covalently immobilized to 3-aminopropyl trimethoxy silane precursor through glutaraldehyde crosslinker. A rigid ceramic composite electrode was fabricated from this modified silane along with graphite powder, which resulted in an amperometric biosensor for H2O2. The electrochemical behaviour of the modified biosensor was monitored using cyclic voltammetry in the potential range of 0.2V to −0.4V vs SCE. The biosensor exhibited a stable voltammogram with cathodic peak at −0.234V and anodic peak at −0.172V, with a formal potential of −0.203V. Various factors influencing the performance of the biosensor such as buffer solution, pH, temperature and potential were examined for optimizing the working conditions. The modified biosensor exhibited a good catalytic behaviour for the reduction of H2O2 at a lower potential of −0.25V without any barrier from possible interferents. The analytical working range was found to be 0.429μM to 0.455mM of H2O2 with a detection limit of 0.171μM. The fabricated biosensor is robust for long-term usage in addition to the high sensitivity, rapid response and having an advantage of surface renewability by simple mechanical polishing.

Alternative Hydride Sources for Ene‐Reductases: Current Trends

Asymmetric hydrogenations are important reactions in industrial synthesis, as up to two stereogenic centres can be generated. Given the current trend towards more “green” or sustainable chemistry, biocatalytic approaches are becoming more important in the production of fine chemicals, pharmaceuticals, and agrochemical products. There are at least four known classes of enzymes that have been explored for their biocatalytic applicability, collectively known as ene-reductases (ERs). Each enzyme requires NAD(P)H as the hydride donor, whereas the mechanism, substrate scope and product stereoand/or enantiospecificity differs between biocatalyst classes. The most widely investigated class is the old yellow enzyme (OYE; EC 1.6.99.1) family and many recent reviews have summarised the (potential) applications of these enzymes for industrial syntheses, for example, Refs. [1–3] . These flavin mononucleotide (FMN)-containing enzymes catalyse the C=C reduction of a wide variety of a,b-unsaturated aldehydes, ketones, nitroalkenes, maleimides, dicarboxylic acids and their esters, and nitriles. A second less well-characterised enzyme class catalysing biocatalytic reductions are enoate reductases (EC 1.3.1.31). These oxygen-sensitive enzymes contain both FAD and [4Fe-4S], and are members of the NADH:flavin oxidoreductase/NADH oxidase family. They have been shown to catalyse the reduction of a variety of a,b-unsaturated monoacids and esters. 5] A more recent class of enzymes investigated are the medium-chain dehydrogenase/reductase (MDR) family of oxidoreductases (1.3.1.-). For example, the flavin-independent double-bond reductase from Nicotiana tabacum (NtDBR) catalyses the reduction of a wide variety of a,b-unsaturated aldehydes, ketones and nitroalkenes. There are a few examples in the literature of potential applications of a fourth class of enzymes, namely the flavin-independent short-chain dehydrogenase/reductases (SDR) from plants. Examples include two menthol dehydrogenases, ( )-menthol dehydrogenase (EC 1.1.1.207) and (+)-neomenthol dehydrogenase (EC 1.1.1.208), which catalyse the reduction of specific menthone isomers to menthol. Unfortunately, the high cost of NAD(P)H nicotinamide coenzymes makes them uneconomical for industrial-scale syntheses. Therefore, alternative methodologies have been adopted to supply the necessary hydride equivalents for alkene reduction. One such method is to include a coenzyme recycling system, in which catalytic levels of NAD(P) are constantly regenerated to NAD(P)H. Several systems have been developed and routinely employed for ER biocatalytic reactions, such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH/glucose-6-phosphate), formate dehydrogenase and phosphite dehydrogenase, and reviewed elsewhere. In each case, only catalytic levels of NAD(P) are needed but stoichiometric levels of a co-substrate are required to drive the recycling enzyme. Faber and coworkers have performed comparative biotransformations by using multiple cofactor recycling systems, substrates and ERs, to determine the ideal hydride source. Product yield and/or enantioselectivity can vary considerably, depending on which coenzyme recycling system has been employed. Additionally, the inclusion of a second enzyme system into large-scale (bio)synthesis may present problems, for example in maintenance of the activity and stability of two enzymes and the high cost of some co-substrates. Electrochemical regeneration of nicotinamide coenzymes has also been investigated, for example in the direct cathodic reduction of NAD(P) . Unfortunately, this simple regeneration method is hampered by low selectivity and the formation of undesired NAD(P) dimeric side products. Several techniques have been developed to bypass the need for nicotinamide coenzymes by using alternative hydride donors to reduce the flavin cofactor of some ERs. Reetz and coworkers described a method whereby the FMN cofactor of the OYE homologue YqjM from Bacillus subtilis was photoreduced, employing free flavin and a sacrificial electron donor. The YqjM bound oxidised FMN was reduced by the free photoreduced FMN, thereby generating the active enzyme. This NAD(P)H-free system was successful in converting ketoisophorone to (R)-levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co-workers described the photoenzymatic flavin reduction of YqjM and NEMR (N-ethylmalemide reductase from E. coli) by using alternative sacrificial electron donors formate and phosphite. These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen. Early attempts at NAD(P)H-independent alkene reduction focussed on the use of artificial mediators, such as N,N-dimethyl4-4-bipyridinium [methyl viologen (MV)] , as a means of reducing the flavin in clostridial enoate reductases. A variety of reduced mediator recycling systems were employed, including direct electron transfer from a cathode and enzymatic-based recycling systems. Successful enzyme-coupled mediator recycling was achieved used the following systems: 1) hydroge[a] Dr. H. S. Toogood, Dr. T. Knaus, Prof. N. S. Scrutton Manchester Institute of Biotechnology, Faculty of Life Sciences University of Manchester 131 Princess Street, Manchester M1 7DN (UK) Fax: (+44)01613068918 E-mail : nigel.scrutton@manchester.ac.uk

Bioelectrocatalytic dioxygen reduction at hybrid silicate–polyallylamine film with encapsulated laccase

Abstract The tin-doped indium oxide (ITO) electrode served as a suitable support for the mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and extracellular laccase from Cerrena unicolor encapsulated in sol–gel processed thin hydrophilic silicate/polyallylamine film. For some experiments the electrode was premodified with tin-doped indium oxide nanoparticles (ca. 20 nm in diameter). After initial decrease of the current the electrode shows stable voltammetry of the immobilised mediator. This indicates that silicate/polyallylamine film to prevent water soluble mediator ions expulsion into the aqueous solution. The modified electrode exhibits electrocatalytic response towards dioxygen reduction. Both the magnitude of voltammetric current corresponding to the redox process of mediator and the catalytic current is dependent on the order of deposited components. The magnitude of the catalytic current with immobilised mediator is larger than when mediator is present only in the buffer solution. The efficiency of the electrocatalytic process is also more than twice increased (reaching 0.25 mA cm −2 ) by electrode premodification with ITO nanoparticles.

Surface renewable sol–gel composite electrode derived from 3-aminopropyl trimethoxy silane with covalently immobilized thionin

Sol–gel technique has been used for the covalent immobilization of the water-soluble mediator, thionin to construct a bulk modified, leak free composite electrode. This renewable composite electrode provides stable immobilization matrix for thionin via glutaraldehyde crosslinking. In the electrode composition the sol–gel precursor 3-aminopropyltrimethoxy silane serves as the host for immobilization of thionin, thereby preventing its leakage. An additional precursor methyl trimethoxy silane endows hydrophobicity and limits the wetting section of the modified electrode. Cyclic voltammetric characterization of the modified electrode in the potential range of 0.2 to −0.6 V exhibited stable redox peaks with a formal potential of −0.273 V, corresponding to immobilized thionin. This chemically modified electrode exhibits good electrocatalytic activity for the reduction of H 2O 2 at a lower potential of −0.35 V. The reduction current of the modified electrode increases linearly in the range of 3.44 × 10 −6 M to 3.07 × 10 −3 M H 2O 2 with a detection limit of 1.38 × 10 −6 M. The stable and quick response (5 s) during chronoamperometry shows the potential application of the modified electrode for flow system analysis. The low potential operation (−0.35 V) favoured selective determination of H 2O 2. The composite electrode exhibits distinct advantages of polishing in the event of surface fouling as well as simple preparation, good chemical and mechanical stability, economical and remarkable long-term stability (more than 1 year). The applicability of the present sensor for H 2O 2 determination proposes a method for the detection of other biologically significant analytes.

Amperometric hydrogen peroxide sensor based on a sol-gel-derived ceramic carbon composite electrode with toluidine blue covalently immobilized using 3-aminopropyltrimethoxysilane

A carbon composite amperometric hydrogen peroxide sensor has been developed using a sol-gel technique. Toluidine blue (TB), which acts as the redox mediator, was covalently immobilized via glutaraldehyde crosslinking with an organically modified silane, namely 3-aminopropyltrimethoxysilane (APTMOS). Methyltrimethoxysilane (MTMOS) was used as the additional monomer; this controls the hydrophobicity of the electrode surface, thus limiting the wettability. The immobilization of TB within the sol-gel matrix was confirmed with FTIR studies. The sol-gel mixture containing TB immobilized in APTMOS and MTMOS was mixed with graphite powder in order to prepare the carbon composite electrode. The electrode was characterized using voltammetric techniques and its electrocatalytic activity for the reduction of hydrogen peroxide was also studied. The carbon composite electrode has the advantage of sensing H(2)O(2) at a lower potential and with a higher sensitivity, and interferences due to ascorbic acid, uric acid and acetaminophen were greatly minimized. The linear range for the determination of H(2)O(2) extends from 5.37 x 10(-6) to 6.15 x 10(-3) M, with a correlation coefficient of 0.9981. The detection limit was found to be 2.15 x 10(-6) M. The covalent immobilization of TB effectively prevents the leakage of the water-soluble mediator during measurements. The modified electrode, aside from electrocatalyzing the reduction of H(2)O(2), exhibits distinct advantages in terms of surface renewal in the event of surface fouling, as well as simple preparation, good chemical and mechanical stability, and good reproducibility.

Interpreting the Mechanisms of Continuous Renal Replacement Therapy in Sepsis: The Peak Concentration Hypothesis

Severe sepsis and septic shock are the primary causes of multiple organ dysfunction syndrome (MODS), which is the most frequent cause of death in intensive care unit patients. Many water-soluble mediators with pro- and anti-inflammatory action such as TNF, IL-6, IL-8, and IL-10 play a strategic role in septic syndrome. In intensive care medicine, blocking any one mediator has not led to a measurable outcome improvement in patients with sepsis. CRRT is a continuously acting therapy, which removes in a nonselective way pro- and anti-inflammatory mediators; "the peak concentration hypothesis" is the concept of cutting peaks of soluble mediators through continuous hemofiltration. Furthermore, there is evidence of increased efficacy of high-volume hemofiltration compared to conventional CVVH, and other blood purification techniques that utilize large-pore membranes or sorbent plasmafiltration are conceptually interesting.

Design of novel electron transfer mediators based on indophenol derivatives for lactate sensor

Novel indophenol derivatives which have limited solubility in water solution functioned as excellent electron-transfer mediators for lactate oxidase and a lactate sensor, showing good sensor performance, including a high sensitivity and good durability. These less water-soluble mediators were prepared by the O-alkylation or acylation of indophenols in order to prevent the mediator being leached from the sensor. Consequently the characteristic substituent effects of indophenol derivatives were found to be follows, (1) modification of the phenolic OH group improved sensor durability; (2) the presence of chloride groups at the 2,6-positions of the phenol ring improved the sensitivity of the sensor; (3) the introduction of substituents at the quinoide moiety led to a deterioration in durability. A mediator based lactate sensor using these derivatives was found to be nearly independent of oxygen concentration, showed a low level of interference effect, and a quite long durability.

A flow-injection (FI) mediated biosensor for on-line monitoring of lactate in mammalian cell culture

A flow-injection (FI) biosensor system has been developed for on-line monitoring of lactate formation during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and, after cell separation by a steam sterilizable ceramic microfilter, the cell-free filtrate was fed to the FI system. Lactate oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with a water-soluble mediator 1,1′-dimethylferricinium (DMFe +)-cyclodextrin inclusion complex to recycle the reduced lactate oxidase to its original active state. 1.1′-Dimethylferrocene (DMFe) was then reoxidized to DMFe + at the surface of the platinum electrode poised at +0.15 V vs. silver/silver chloride. The FI biosensor was linear up to 3 mM lactate with a detection limit of 0.05 mM, and possessed ±1.5% reproducibility over 154 repeated analyses during a 77 h continuous operation. Due to a significant accumulation of lactate during fed-batch cultivation of 293S mammalian cells, a dialysis membrane with 1000 molecular weight cut-off was placed over the lactate oxidase membrane to extend the linear detection range up to 40 mM. Excellent reproducibility (±1.3%) was observed for 96 repeated hourly analyses with culture medium containing 15 mM lactate. When applied to (5–10)-day fed-batch cultivation of 293S mammalian cells, the results obtained from the biosensor system compared well with HPLC and spectrophotometric assay data.

DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus.

Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatus coded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the -10 and -35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) M. Binding of CrtJ to the bchC promoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizing versus reducing conditions.

Glucose sensor based on carbon paste electrode incorporating poly(ethylene glycol) -modified glucose oxidase and various mediators

Amperometric bioelectrodes constituted of carbon paste electrode incorporating poly(ethylene glycol)-modified glucose oxidase and mediators are studied. Influences of the nature of the mediators (anthraquinone, methylene blue, 2,6-dichloro-phenolindophenol, tetracyanoquinodimethane, 1,1'-dimethylferrocene) on the characteristics (potential window, apparent Michaelis constant, response time, stability) of the bioelectrodes are investigated. It is possible to show that the behaviour of the mediator is influenced by the pasting liquid contained in the carbon paste. Moreover, the water solubility of the mediator is the cause of a decrease of the apparent Michaelis constant in the case of methylene blue. A comparison between the stability of a low water soluble mediator (tetracyanoquinodimethane) and a more water soluble mediator (methylene blue) shows that the leakage of mediator from the carbon paste is not the cause of the lack of stability of such bioelectrodes. Among the various mediators tested, tetracyanoquinodimethane seems the most appropriate for the construction of a bioelectrode.