Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 μM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 μM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 μM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 μM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 μM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 μM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate.     

Amperometric glucose sensor based on catalytic reduction of dissolved oxygen at soluble carbon nanofiber

This work shows excellent catalytic activity of soluble carbon nanofiber (CNF), which was obtained with a simple nitric acid treatment, toward the electroreduction of dissolved oxygen at a low operating potential. Thus the CNF was applied in the construction of amperometric biosensors for oxidase substrates using glucose oxidase as a model. The good dispersion of CNF led to convenient preparation and acceptable repeatability of the proposed sensors. UV-vis spectra, Fourier transform infrared spectra, X-ray photoelectron spectra and titration curves demonstrated that the good dispersion resulted from the large numbers of surface oxygen-rich groups produced in the treatment process. The membrane of CNF showed good stability and provided fast response to dissolved oxygen with a linear range from 0.1 to 78 microM and detection limit of 0.07 microM. The proposed glucose biosensor could monitor glucose ranging from 10 to 350 microM with detection limit of 2.5 microM and sensitivity of 36.3 nA cm(-2) microM(-1). The coefficients of variation for intra-assay were 4.7 and 3.2% at glucose concentrations of 20 and 210 microM, respectively. The use of a low operating potential (-0.3 V) and Nafion membrane produced good selectivity toward the glucose detection. CNF-based biosensors would provide wide range of bioelectrochemical applications in different fields.     

Glutamate optical biosensor based on the immobilization of glutamate dehydrogenase in titanium dioxide sol-gel matrix

A simple and novel titania sol-gel derived optical biosensor coupled with carboxy seminaphthorhodamine-1-dextran (SNARF-1-dextran) as the fluorescent dye was fabricated for the determination of glutamate in water and biological samples. The NADH-dependent glutamate dehydrogenase (GLDH) was trapped in titania sol-gel derived matrix prepared by vapor deposition method. In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the surface morphology of the spots. SEM and AFM images showed that the deposition of titania precursor at 27 degrees C for 6.5h was found to be suitable to form transparent titania sol-gel matrix to encapsulate GLDH and fluorescent probe. AFM images showed that the roughness of TiO(2) surface increased from 2.16 nm in the absence of GLDH and SNARF to 37.8 nm after the immobilization. The developed titania biosensor has good analytical performance with water samples. A dynamic range between 0.04 and 10mM with the detection limit of 5.5 microM were observed. The responses to glutamate in biological samples also showed good performances, and the dynamic range and detection limit were 0.02-10mM and 6.7 microM, respectively. High precision with relative standard deviations of 4.2 and 10.7% in water and biological samples, respectively, were also demonstrated. In addition, the biosensor showed a relatively high storage stability over more than 1 month. Results obtained in this study clearly demonstrate that this simple vapor deposition method can be successfully used to form transparent titania sol-gel film for the fabrication of glutamate biosensors that are suitable for optical detection of glutamate in water and biological samples.     

A novel glucose biosensor based on the nanoscaled cobalt phthalocyanine-glucose oxidase biocomposite

We report on the utilization of a novel nanoscaled cobalt phthalocyanine (NanoCoPc)-glucose oxidase (GOD) biocomposite colloid to create a highly responsive glucose biosensor. The biocomposite colloid is constructed under enzyme-friendly conditions by adsorbing GOD molecules on CoPc nanoparticles via electrostatic interactions. The glucose biosensor can be easily achieved by casting the biocomposite colloid on a pyrolytic graphite electrode (PGE) without any auxiliary matter. It has been found that GOD can be firmly immobilized on PGE surface and maintain its bioactivity after conjugating with NanoCoPc. NanoCoPc displays intrinsic electrocatalytic ability to the oxidation of the product of enzymatic reaction H2O2 and shows a higher catalytic activity than that of bulk CoPc. Under optimal conditions, the biosensor shows a wide linear response to glucose in the range of 0.02-18 mM, with a fast response (5s), high sensitivity (7.71 microA cm(-2) mM(-1)), as well as good thermostability and long-term life. The detection limit was 5 microM at 3 sigma. The general interferences coexisted in blood except ascorbic acid and L-cysteine do not affect glucose determination, and further coating Nafion film on the surface of the biosensor can effectively eliminate the interference from ascorbic acid and L-cysteine. The biosensor with Nafion film has been used for the determination of glucose in serum with an acceptable accuracy.     

An amperometric inhibitor biosensor for the determination of reduced glutathione (GSH) without any derivatization in some plants

In this study, an amperometric biosensor based on cucumber tissue homogenate was developed for the determination of glutathione. Cucumber (Cucumis sativus L.) tissue homogenate was used as the biological material. The cucumber tissue homogenate was cross-linked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of ascorbate oxidase in the biological material by glutathione. Determinations were carried out by standard curves which were obtained by the measurement of the decrease in the consumed oxygen level related to glutathione concentration. Optimization and characterization studies of the biosensor were carried out and a linearity in the gamma-L-glutamyl-L-cysteinyl-glycine (GSH) concentration range 0.1-2 microM was obtained when 600 microM ascorbic acid was used as a substrate. The repeatability experiments (n = 7) revealed that for 1.5 microM GSH, the average value (x), standard deviation (S.D.) and variation coefficient (C.V.) were 1.517 microM, 4.72 x 10(-5) 3.11%, respectively. The biosensor useful lifetime was at least 2 months. The results of some plant samples analyzed with the presented biosensor agreed well with the spectrophotometric method (Ellman's reagent) used as a reference.     

Nanocrystalline diamond modified gold electrode for glucose biosensing

Boron-doped diamond has drawn much attention in electrochemical sensors. However there are few reports on non-doped diamond because of its weak conductivity. Here, we reported a glucose biosensor based on electrochemical pretreatment of non-doped nanocrystalline diamond (N-NCD) modified gold electrode for the selective detection of glucose. N-NCD was coated on gold electrode and glucose oxidase (GOx) was immobilized onto the surfaces of N-NCD by forming amide linkages between enzyme amine residues and carboxylic acid groups on N-NCD. The anodic pretreatment of N-NCD modified electrode not only promoted the electron transfer rate in the N-NCD thin film, but also resulted in a dramatic improvement in the reduction of the dissolved oxygen. This performance could be used to detect glucose at negative potential through monitoring the current change of oxygen reduction. The biosensor effectively performs a selective electrochemical analysis of glucose in the presence of common interferents, such as ascorbic acid (AA), acetaminophen (AP) and uric acid (UA). A wide linear calibration range from 10 microM to 15 mM and a low detection limit of 5 microM were achieved for the detection of glucose.     

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.     

Covalent conjugation of avidin with dye-doped silica nanopaticles and preparation of high density avidin nanoparticles as photostable bioprobes

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Na?on film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062-14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM(-1)cm(-2)). The Michaelis-Menten constant (K(m)) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.     

Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel

A novel amperometric biosensor, based on electrodeposition of platinum nanoparticles onto multi-walled carbon nanotube (MWNTs) and immobilizing enzyme with chitosan-SiO(2) sol-gel, is presented in this article. MWNTs were cast on the glass carbon (GC) substrate directly. An extra Nafion coating was used to eliminate common interferents such as acetaminophen and ascorbic acids. The morphologies and electrochemical performance of the modified electrodes have been investigated by scanning electron microscopy (SEM) and amperometric methods, respectively. The synergistic action of Pt and MWNTs and the biocompatibility of chitosan-SiO(2) sol-gel made the biosensor have excellent electrocatalytic activity and high stability. The resulting biosensor exhibits good response performance to glucose with a wide linear range from 1 microM to 23 mM and a low detection limit 1 microM. The biosensor also shows a short response time (within 5s), and a high sensitivity (58.9 microAm M(-1)cm(-2)). In addition, effects of pH value, applied potential, rotating rate, electrode construction and electroactive interferents on the amperometric response of the sensor were investigated and discussed in detail.     

Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversal

Na(+)-dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. L-threo-beta-hydroxyaspartate (L-TBHA), DL-threo-beta-benzyloxyaspartate (DL-TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC) (+/-)-cis-4-methy-trans-pyrrolidine-2,4-dicarboxylic acid (cis-4-methy-trans-2,4-PDC) and L-antiendo-3,4-methanopyrrolidine-2,4-dicarboxylic acid (L-antiendo-3,4-MPDC) inhibited L-[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 microM (DL-TBOA and L-TBHA) - 43 microM (cis-4-methy-trans-2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While L-TBHA, L-trans-2,4-PDC, cis-4-methy-trans-2,4-PDC and L-antiendo-3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, DL-TBOA was a substrate only in astrocytes. This effect of DL-TBOA was concentration-dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal DL-TBOA uptake velocity (< or = 10 microM), DL-TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial DL-TBOA uptake (> 10 microM). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions.     

Monitoring of alpha-ketoglutarate in a fermentation process using expanded bed enzyme reactors.

A bienzyme flow injection system is presented for the monitoring of alpha-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, alpha-ketoglutarate was converted to L-glutamate by GDH in the presence of ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to alpha-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of alpha-ketoglutarate in the coupled packed bed reactors was 1 microM (defined as 3 S/N), the linear range 0-100 microM, and the sensitivity 0.80 nA/microM (R(2) 0.99). In the coupled expanded bed reactors, the detection limit of alpha-ketoglutarate was 7 microM (defined as 3 S/N), the linear range and the sensitivity being 0-500 microM and 0.11 nA/microM (R(2) 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

A sensitive NADH and glucose biosensor tuned by visible light based on thionine bridged carbon nanotubes and gold nanoparticles multilayer

A NADH and glucose biosensor based on thionine cross-linked multiwalled carbon nanotubes (MWNTs) and Au nanoparticles (Au NPs) multilayer functionalized indium-doped tin oxide (ITO) electrode were presented in this paper. The effect of light irradiation on the enhancement of bioelectrocatalytic processes of the biocatalytic systems by the photovoltaic effect was investigated. This bioelectrode exhibited excellent catalytic activity of the oxidation towards dihydronicotinamide adenine dinucleotide (NADH). Most interesting, the performance of this NADH sensor could be tuned by the visible light. When the biosensor was performed in the dark, the anodic current increased linearly with NADH concentration over the range from 0.5 to 237 microM with detection limit 0.1 microM and sensitivity 17 nA microM(-1). The sensitivity became 115 nA microM(-1) with detection limit 0.05 microM with the light irradiation. Compared with the reaction in dark, the sensitivity increased around 7 folds while the detection limit decreased 2 folds. The glucose biosensor also exhibited the same behavior. The linear range was from 10 microM to 2.56 mM with the sensitivity of 7.8 microAmM(-1) and detection limit 5.0 microM in the dark. After the light irradiation, the linear range was from 1 microM to 3.25 mM with the sensitivity of 18.5 microA mM(-1) and detection limit 0.7 microM. It indicated a potential to provide an operational access to develop new kinds of photocontrolled dehydrogenase enzyme-based bioelectronics.     

Development of amperometric biosensor for glucose based on a novel attractive enzyme immobilization matrix: calcium carbonate nanoparticles

Calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 microM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mAcm-2M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.     

Enzymatic degradation protects neurons from glutamate excitotoxicity

Several enzymes with the capacity to degrade glutamate have been suggested as possible neuroprotectants. We initially evaluated the kinetic properties of glutamate pyruvate transaminase (GPT; also known as alanine aminotransferase), glutamine synthetase, and glutamate dehydrogenase under physiologic conditions to degrade neurotoxic concentrations of glutamate. Although all three enzymes initially degraded glutamate rapidly, only GPT was able to reduce toxic (500 microM) levels of glutamate into the physiologic (<20 microM) range. Primary cultures of fetal murine cortical neurons were subjected to paradigms of either exogenous or endogenous glutamate toxicity to evaluate the neuroprotective value of GPT. Neuronal survival after exposure to added glutamate ranging from 100 to 500 microM was improved significantly in the presence of GPT (> or =1 U/ml). Cultures were also exposed to the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC), which produces neuronal injury by elevating extracellular glutamate. GPT significantly reduced the toxicity of PDC. This reduction was associated with a reduction in the PDC-dependent rise in the medium concentration of glutamate. These results suggest that enzymatic degradation of glutamate by GPT can be an alternative to glutamate receptor blockade as a strategy to protect neurons from excitotoxic injury.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.