Highly sensitive lactate biosensor by engineering chitosan/PVI-Os/CNT/LOD network nanocomposite

A novel chitosan/PVI-Os(polyvinylimidazole-Os)/CNT(carbon nanotube)/LOD (lactate oxidase) network nanocomposite was constructed on gold electrode for detection of lactate. The composite was nanoengineered by selected matched material components and optimized composition ratio to produce a superior lactate sensor. Positively charged chitosan and PVI-Os were used as the matrix and the mediator to immobilize the negatively charged LOD and to enhance the electron transfer, respectively. CNTs were introduced as the essential component in the composite for the network nanostructure. FESEM (field emission scan electron microscopy) and electrochemical characterization demonstrated that CNT behaved as a cross-linker to network PVI and chitosan due to its nanoscaled and negative charged nature. This significantly improved the conductivity, stability and electroactivity for detection of lactate. The standard deviation of the sensor without CNT in the composite was greatly reduced from 19.6 to 4.9% by addition of CNTs. With optimized conditions the sensitivity and detection limit of the lactate sensor was 19.7 μA mM⁻¹ cm⁻² and 5 μM, respectively. The sensitivity was remarkably improved in comparison to the newly reported values of 0.15–3.85 μA mM⁻¹ cm⁻². This novel nanoengineering approach for selecting matched components to form a network nanostructure could be extended to other enzyme biosensors, and to have broad potential applications in diagnostics, life science and food analysis.     

Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO)

An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order to lower the applied potential and minimise the matrix effects. Among them, 2,6-dichlorophenol indophenol (DPIP) and phenazine methosulfate (PMS) were chosen for further development. The working conditions (mediator concentration, applied potential and pH) were optimised for both DPIP and PMS. Detection limits for both types of biosensors were of 5 μM malic acid. Sensitivities obtained for the linear part of the calibration curve were 0.85 and 1.7 mA/M for the biosensors based on DPIP and PMS, respectively. Interferences due to non-specific oxidations were shown to be negligible when using PMS as mediator.     

Reagentless amperometric detection of l-lactate on an enzyme-modified conducting copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone)

We have constructed a reagentless lactate sensor using lactate oxidase (LOD) covalently attached to an electropolymerized copolymer film, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-thioacetic acid-1,4-naphthoquinone), poly(JUG-co-JUGA). Around 10(-12)Mcm(-2) of covalently bound enzymes are immobilized on these films. In aerated medium, the amperometric response versus lactate concentration shows a sensitivity of 350 +/- 50 microAM(-1)cm(-2) for an applied potential of +0.5V versus Ag|AgCl on a film-coated Pt electrode. In deaerated medium, the quinone group, conjugated with the polymer backbone, acts as an immobilized mediator. An amperometric response is observed on film-coated glassy carbon (GC) electrode at a potential as low as -0.1V versus Ag|AgCl, with a sensitivity of 110 +/- 40 microAM(-1)cm(-2).     

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E ₀) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E ₀, and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Needle-type lactate biosensor

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.     

Mediated electrochemistry of peroxidases-effects of variations in protein and mediator structures

The kinetics of a range of ferrocene derivatives with horseradish peroxidase (HRP), cytochrome c peroxidase (CCP) and 3 charge reversal mutants of cytochrome c peroxidase were measured using cyclic voltammetry. Substantial differences in rate constant (100 fold) were observed between HRP and CCP for the same mediator with smaller differences (4–5 fold) for different mediators with the same enzyme. The rate constant did not seem to be dependent on redox potential differences. Cluster analysis is proposed as a way of classifying mediator reactivity.     

Holographic glucose sensors

A novel holographic sensor system capable of detecting dynamic changes in glucose concentration has been developed. The hologram is recorded within a bio-compatible hydrogel matrix containing phenylboronic acid derivatives. On binding glucose, the colour of the hologram red-shifts to longer wavelengths as the hydrogel expands and this colour change is used to quantify glucose concentration. However, phenylboronic acids are non-selective and bind a wide variety of cis-diols. In blood, glucose is the only sugar found free at high concentration, whilst other sugars are typically found as part of glycoproteins and macromolecular structures. Although glycoproteins have been shown to have no effect on the sensor, phenylboronic acids can bind lactate much more readily than glucose. We have designed two polymer hydrogel systems to increase the selectivity of the sensor for glucose over lactate. The first involved the use of high concentrations of 3-acrylamidophenylboronic acid (3-APB) whilst the second system utilised 2-acrylamido-5-fluorophenylboronic acid (5-F-2-MAPB). Both systems displayed an increased selectivity to glucose over lactate at physiological pH and ionic strength and could be deployed as selective holographic sensors for glucose detection in physiological fluids.     

Improved postischemic function following acute exercise is not mediated by nitric oxide synthase in the rat heart

The mediators of acute exercise-induced preconditioning against ischemia-reperfusion injury are not understood. This study assesses the role of nitric oxide synthase (NOS), a reported mediator of other forms of preconditioning. Male Fischer 344 rats were divided into five groups (n = 6-7): sedentary (Sed); exercised 2 days on a treadmill at 20 m/min, 6 degrees grade, for 60 min (Run); sedentary, perfused with 100 microM N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME) to inhibit NOS (Sed/L-N); exercised, perfused with l-NAME (Run/L-N); and exercised in a 4 degrees C environment, perfused with l-NAME (CRun/L-N). Twenty-four hours following exercise, isolated, perfused working hearts were subjected to 22.5 min of global ischemia plus 30 min of normoxic reperfusion. Left ventricle contents of several putative preconditioning mediators were determined. Postischemic recovery of cardiac output times systolic pressure was better in Run than Sed (78.4 vs. 50.2% of preischemia, P < 0.05). Inhibition of NOS did not abrogate the improved recovery in the exercise groups or alter recovery in Sed. All exercise groups also displayed improved myocardial efficiency (cardiac output times systolic pressure/oxygen consumption) postischemia and less lactate dehydrogenase release (P < 0.05). l-NAME appeared to lower lactate dehydrogenase release independent of exercise. The only change in antioxidant enzyme activity was a decrease in manganese superoxide dismutase in CRun/L-N (P < 0.05). Heat shock protein 72 expression increased only in Run and Run/L-N and endothelial NOS only in CRun/L-N (P < 0.05). Acute exercise-induced preconditioning of the Fischer 344 rat heart is not mediated by NOS and does not require increases in heat shock protein 72 or antioxidant enzymes.     

Enzyme electrodes based on sono-gel containing ferrocenyl compounds

An amperometric-mediated glucose sensor has been developed by employing a silica sono-gel carbon composite electrode (SCC). The chosen mediators, ferrocene (Fc) and 1,2-diferrocenylethane (1), have been immobilized in the sono-gel composite matrix. The complex 1 has been employed for the first time as an electron transfer mediator for signal transduction from the active centre of the enzyme to the electrode conductive surface. After the optimisation of the construction procedure the best operative conditions for the analytical performance of the biosensor have been investigated in terms of pH, temperature and applied potential. Cyclic voltammetric and amperometric measurements have been used to study the response of both the glucose sensors, which exhibit a fast response and good reproducibility. The sensitivity to glucose is quite similar (6.7+/-0.1 microA/mM versus 5.3+/-0.1 microA/mM) when either Fc or 1 are used as mediators as are the detection limit ca. 1.0 mM (S/N=3) and the range of linear response (up to 13.0 mM). However, the dynamic range for glucose determination results wider when using 1 (up to 25.0 mM). The apparent Michaelis-Menten constants, calculated from the reciprocal plot under steady state conditions, are 27.7 and 31.6 mM for SCC-Fc/GOx and SCC-1/GOx electrodes, respectively, in agreement with a slightly higher electrocatalytic efficiency for the mediator 1.     

Transition metal half-sandwich complexes as redox mediators to glucose oxidase

Chromium and manganese half-sandwich complexes are evaluated as mediators to glucose oxidase (GOx) since they are of similar size to ferrocene derivatives (sandwich complexes) and contain a single pi-ligand for interaction with the enzyme co-factor. A series of seven amino derivatives of [(eta-C(6)H(6))Cr(CO)(3)] were investigated of which only [[eta-C(6)Me(4)(NH(2))(2)]Cr(CO)(3)] (7), with the lowest oxidation potential of +40 mV (versus SCE), was found to display reversible electrochemistry. Small catalytic currents were recorded in the presence of GOx and glucose when complex (7) was incorporated in a screen-printed carbon electrode. Manganese cyclopentadienyl (Cp) half-sandwich complexes were found to be more effective GOx mediators and comparable in efficacy to ferrocene derivatives. A mediator rate constant k(M) of 2.1 x 10(5)M(-1)s(-1) was determined for the water-soluble complex [(eta-MeC(5)H(4))Mn(NO)(CN)(2)]Na (11) compared to a range of 3 x 10(4) to 8 x 10(6)M(-1)s(-1) previously determined for ferrocenes under the same experimental conditions. beta-Cyclodextrin (beta-cd) was found to be helpful in solubilising hydrophobic complexes such as [(eta-MeC(5)H(4))Mn(NO)(S(2)CNMe(2))] (15) and the neutral oxidised form of [MeCpMn(NO)[(SCCN)(2)]]NEt(4) (14), either directly as an inclusion adduct or in situ during cyclic voltammetry. Screen-printed amperometric electrodes, containing a mediator and GOx immobilised in an organic conducting carbon layer, were useful in assessing the mediation ability of complex (15) where aqueous insolubility precluded any kinetic studies with GOx in solution. This work was briefly extended to other oxidoreductase enzymes apart from GOx. Thus, rotating ring-disk voltammetry demonstrated that the beta-cd complex of compound (15) is also a useful mediator to Horseradish peroxidase (HRP) since it displays an identical catalytic current to the ferrocene ethanolamine derivative (1) used in the MediSense ExacTech and Precision QID blood glucose biosensor electrodes.     

Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp

The natural phenolic compounds syringaldehyde and vanillin were compared to the synthetic mediators 1-hydroxybenzotriazole, violuric acid and promazine in terms of boosting efficiency in a laccase-assisted biobleaching of eucalyptus kraft pulp. Violuric acid and 1-hydroxybenzotriazole revealed to be the most effective mediators of the bioprocess. Nevertheless, laccase-syringaldehyde system also improved the final pulp properties (28% delignification and 63.5% ISO brightness) compared to the process without mediator (23% and 61.5% respectively), in addition to insignificant denaturation effect over laccase. The efficiency of the biobleaching process was further related to changes in non-conventionally used optical and chromatic parameters of pulp, such as (L( *)), chroma (C( *)) and dye removal index (DRI) showing good correlation. Adverse coupling reactions of the natural phenolic mediators on pulp lignin were predicted by electrochemical studies, demonstrating the complexity of the laccase-mediator reaction on pulp.     

Immobilized redox mediators for electrochemical NAD(P)<Superscript>+</Superscript> regeneration

The applicability of dissolved redox mediators for NAD(P)⁺ regeneration has been demonstrated several times. Nevertheless, the use of mediators in solutions for sensor applications is not a very convenient strategy since the analysis is not reagentless and long stabilization times occur. The most important drawbacks of dissolved mediators in biocatalytic applications are interferences during product purification, limited reusability of the mediators, and their cost-intensive elimination from wastewater. Therefore, the use of immobilized mediators has both economic and ecological advantages. This work critically reviews the current state-of-art of immobilized redox mediators for electrochemical NAD(P)⁺ regeneration. Various surface modification techniques, such as adsorption polymerization and covalent linkage, as well as the corresponding NAD(P)⁺ regeneration rates and the operational stability of the immobilized mediator films, will be discussed. By comparison with other existing regeneration systems, the technical potential and future perspectives of biocatalytic redox reactions based on electrochemically fed immobilized mediators will be assessed.     

Genetic studies of free-ranging macaques of Cayo Santiago. I. Description of the population and some nonpolymorphic red cell enzymes.

Phenotypes of eight red cell enzymes at nine genetic loci were determined in the semi-free-ranging population of rhesus macaques; Macaca mulatta, that inhabit Cayo Santiago. The following enzymes were examined electrophoretically: adenosine deaminase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, indophenol oxidase, lactate dehydrogenase, malate dehydrogenase, phosphoglucomutase-1, phosphoglumutase-2, and purine nucleoside phosphorylase. Hemolysates from at least 372 animals were analyzed, and no variants of the enzymes were observed with the exception of malate dehydrogenase. Three animals displaying a variant form of malate dehydrogenase were found.     

Novel system for real-time ex vivo lactate monitoring in human whole blood

The objective of the study was to evaluate the performance of an amperometric enzyme based lactate sensor and to investigate the possibility of replacing a double lumen catheter based blood withdrawal system with a heparin coated single lumen system. The inner lumen of a double lumen catheter which was placed in a peripheral vein was perfused with heparin solution. The outer lumen was used to collect heparinized blood samples at a defined flow rate. The single lumen system was attached to a heparinized catheter which was also placed in a peripheral vein. The undiluted blood samples were collected at a specified flow rate. A sensor flow chamber incorporating an amperometric thin-film lactate microbiosensor was placed in the sampling line for real-time lactate monitoring. Plasma lactate concentrations were measured during frequently performed hyperlactatemia bicycle ergometer experiments in six healthy volunteers (age 25.8±2.8 years, BMI 22.7±1 kg/m²). Additionally, plasma lactate was measured in real-time using the lactate sensors. The first three experiments were performed with a double lumen based catheter system whereas the following three experiments were performed with a heparin coated catheter system. The correlation coefficients of sensor readings and laboratory analyzer results in all six experiments were between 0.93 and 0.99, respectively (P<0.001). The miniaturized lactate sensors showed a linear range up to 25 mmol/l lactate concentration and 95% response times <30 s in undiluted serum. During the experiments maximum lactate concentrations of 14 mmol/l were achieved. Improvements of system performance using heparin coated catheter systems could be shown. The overall SD of the sensor readings compared to laboratory results using three double lumen catheter based systems was 0.91 mmol/l whereas the SD using three heparin coated systems was 0.65 mmol/l. In summary, real-time monitoring of lactate in human whole blood is feasible with such a device and can be improved by using heparin coated catheter systems.     

Interactions of glucose oxidase with various metal polypyridine complexes as mediators of glucose oxidation

The interaction between glucose oxidase (GOx) and a typical metal complex, which is chemically stable in both oxidized and reduced forms, has been investigated by a voltammetric method. The evaluation of an electron-transfer mediator useful for glucose oxidation is discussed from thermodynamic and kinetic points of view, i.e. the redox potentials of various metal complexes and the second-order rate constants for the electron transfer between GOx in reduced form and the metal complexes in oxidized form. No mediation of glucose oxidation by [Co(bpy)(3)](2+) (bpy=2,2'-bipyridine) or [Cu(bpy)(2)](2+) occurred, in spite of their appropriate redox potentials. This was attributed mainly to the lower electron-self-exchange rates of the mediator and the reaction with GOx. All three types of osmium(II) complexes, [Os(PP) (n)](2+) ( n=2 or 3; PP=polypyridine), [OsL(2)(PP)(2)](2+) (L=imidazole and its derivatives), and [OsClL(bpy)(2)](+), acted as excellent electron-transfer mediators for the glucose oxidation. Mixed ligand complexes, [OsL(2)(PP)(2)](2+) and [OsClL(bpy)(2)](+), have been concluded to be more efficient electron-transfer mediators. The electron-transfer rates between the mediator and GOx have been found to be accelerated by intermolecular electrostatic interactions or hydrogen bonds.     

细胞固定化方法制备微藻光电极的研究

通过将微藻细胞固定在平面多孔碳纸上,制备微藻光电极,并在三电极体系电解液中加入电子介体进行测试,可产生与光照同步的光电流响应。考察了不同固定化方法、不同微藻及不同电子介体的光电流响应,结果表明硅溶胶-凝胶法制备的光电极光电流响应最佳,且对于亚心形四爿藻、金藻、莱茵衣藻、蛋白核小球藻、聚球藻等 5 种微藻都适用,表明该制备方法对不同微藻具有较好的通用性。电子介体的研究表明苯醌及其衍生物由于氧还电位较高,具有较好的阳极光电流响应特性,而甲基紫精氧还电位较低,具有较好的阴极光电流响应。     

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.     

The synthesis of deuterium-substituted, spin-labeled analogues of AMP and NAD+ and their use in ESR studies of lactate dehydrogenase

Two spin-labeled analogues of AMP and NAD+ were synthesized, in which a perdeuterated nitroxide radical (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, TEMPAMINE) was attached to C-6 or C-8 position of the adenine ring. The ESR spectra of these derivatives exhibit a 4-fold increase in sensitivity and a concomitant decrease in line-width as compared to the corresponding protonated analogues. The improved resolution of composite spectra consisting of freely tumbling and immobilized components is demonstrated in ternary complexes of the spin-labeled NAD+ derivatives with lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and oxalate.     

Oxamate Improves Glycemic Control and Insulin Sensitivity via Inhibition of Tissue Lactate Production in db/db Mice

Oxamate (OXA) is a pyruvate analogue that directly inhibits the lactate dehydrogenase (LDH)-catalyzed conversion process of pyruvate into lactate. Earlier and recent studies have shown elevated blood lactate levels among insulin-resistant and type 2 diabetes subjects and that blood lactate levels independently predicted the development of incident diabetes. To explore the potential of OXA in the treatment of diabetes, db/db mice were treated with OXA in vivo. Treatment of OXA (350–750 mg/kg of body weight) for 12 weeks was shown to decrease body weight gain and blood glucose and HbA1c levels and improve insulin secretion, the morphology of pancreatic islets, and insulin sensitivity in db/db mice. Meanwhile, OXA reduced the lactate production of adipose tissue and skeletal muscle and serum lactate levels and decreased serum levels of TG, FFA, CRP, IL-6, and TNF-α in db/db mice. The PCR array showed that OXA downregulated the expression of Tnf, Il6, leptin, Cxcr3, Map2k1, and Ikbkb, and upregulated the expression of Irs2, Nfkbia, and Pde3b in the skeletal muscle of db/db mice. Interestingly, LDH-A expression increased in the islet cells of db/db mice, and both treatment of OXA and pioglitazone decreased LDH-A expression, which might be related to the improvement of insulin secretion. Taken together, increased lactate production of adipose tissue and skeletal muscle may be at least partially responsible for insulin resistance and diabetes in db/db mice. OXA improved glycemic control and insulin sensitivity in db/db mice primarily via inhibition of tissue lactate production. Oxamic acid derivatives may be a potential drug for the treatment of type 2 diabetes.     

A long-term flexible minimally-invasive implantable glucose biosensor based on an epoxy-enhanced polyurethane membrane

This paper describes the preparation method as well as the in vitro and in vivo evaluation of a novel flexible glucose biosensor designed for long-term subcutaneous implantation. An epoxy-enhanced polyurethane membrane, which includes ca. 30–40% epoxy resin adhesive and 50–70% polyurethane, has been developed and used for the first time as the outer protective membrane of the sensor. This new membrane was developed to increase the in vivo durability and lifetime of implantable biosensors. This epoxy-polyurethane membrane was shown to be porous and is of excellent durability. A sensor with such a membrane shows excellent long-term stability and can last for 4–8 months in solutions at room temperature. To verify the in vivo performance of the sensor, nine sensors were implanted in three rats and tested regularly. Eight sensors kept functioning well in the rats for 10–56 days. The ninth sensor was damaged during implantation. All original sensitivity data as well as four response curves obtained at days 7, 17, 52 and 56, respectively are presented.