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l-glutamate is an umami component found in various foods, and l-glutamate oxidase (LGOX, EC 1.4.3.11) is a FAD-dependent oxidoreductase that catalyzes the oxidative deamination of l-glutamate. A recently reported single-chain LGOX mutant from Streptomyces sp. X-119-6 is a potential material for l-glutamate sensors because this mutant is easier to prepare than the wild type. Herein, we report the development of a simple analytical kit using this LGOX mutant for the on-site quantification of l-glutamate. The kit named PACKTEST was developed using a colorimetric method to estimate the l-glutamate concentration in the range of 1–50 mg/L through the visual comparison of the resulting sample color with a standard color sequence. In addition, the PACKTEST kit was combined with a portable photometer for the spectrophotometric quantification of l-glutamate in the range of 0.5–12.0 mg/L. It was used to quantify l-glutamate in commercially available soy sauces, and the results were in good agreement (r = 0.97) with the values obtained by post-column derivatized ion-exchange chromatography. Furthermore, we loaded and dried all the reagents, including enzymes, on a 96- well plate to prepare an enzyme-based sensing device, which showed potential for the high-throughput quantification of l-glutamate. [ABSTRACT FROM AUTHOR]

Introduction: Glutamate oxidase (GluOx; EC 1.4.3.11), as an oxidoreductase enzyme, catalyzes the oxidation of glutamate to α-ketoglutarate, ammonia, and hydrogen peroxide (H2O2). In this reaction, the amount of H2O2 is proportional to the concentration of glutamate and its concentration can be measured by using an electrochemical biosensor. The same as other enzyme-based biosensors, glutamate oxidase is one of the key elements in the construction of glutamate biosensors. Such biosensors are fully capable of identifying biological analytes, such as glutamine, ammonia, and creatinine. In addition, glutamate oxidase-based biosensors have many applications in quantitative and qualitative measurements in analytical chemistry, determining the quality of food products, as well as early detection of heart and liver disorders in clinical biochemistry. Considering the importance of the glutamate neurotransmitter in various brain functions, this study investigated its measurement in the brain of male Wistar rats by using a glutamate oxidase-based biosensor. Material & Methods: In order to measure glutamate, initially, a glutamate oxidase-based biosensor was made by simultaneously immobilizing the enzyme and chitosan on the platinum electrode surface. Then, the animals were sacrificed, and their brains were removed and placed in a phosphate buffer. Afterward, the contents of the brain were centrifuged to create a completely uniform mixture. Finally, the concentration of glutamate in the prepared brain samples was measured using the fabricated biosensor by the cyclic voltammetry technique. Findings: The results of cyclic voltammetry experiments showed that the cathodic peak current of the fabricated biosensor was 0.812 µA. Moreover, the calibration curve indicated that the biosensor response was linear up to 1 mM and glutamate concentration in brain samples was also equal to 63.5 µM. Discussion & Conclusion: The cyclic voltammetry experiments showed that the concentration of H2O2, which is produced during the catalytic activity of glutamate oxidase, is completely proportional to the concentration of glutamate oxidase. Furthermore, this study showed that, despite the very low concentration of glutamate neurotransmitters, the glutamate oxidase-based electrochemical biosensor can precisely identify it qualitatively and quantitatively in biological samples, such as brain samples.

The aim of this work was to create the new biosensor of alanine transaminase (ALT) activity detection in water samples. A platinum disk electrode was used to transduce chemical reactions into amperometry measured signal. Nanosize polyphenylendiamine (PPD) was applied to improve the selectivity of the transducer. In the work, the most effective isomer PD was selected and the stability of the additional membrane based on it was checked. The bioselective element was formed by the immobilization of glutamate oxidase on the surface of the transducer covered by a semipermeable P-meta-PD membrane. There was reported the method of ALT activity measurement. There was also reported the optimal conditions to create this biosensor (including enzyme immobilization parameters and substrates concentrations). The analytical characteristics were investigated including the sensitivity, the linear range, the minimum limit of detection, time of measurement, duration of analysis, stability, etc. Also, there was explored the biosensor selectivity to different interferents, e.g., electroactive molecules and amino acids. For the purpose to test the fundamental possibility of this biosensor to measure the ALT activity in real samples, the operation of the biosensor was tested in a solution with an ionic composition close to serum. [ABSTRACT FROM AUTHOR]

Herein, a simple method has been used in the fabrication of a microneedle electrode (MNE). To do this, firstly, a commercial self-dissolving microneedle patch has been used to make a hard-polydimethylsiloxane-based micro-pore mold (MPM). Then, the pores of the MPM were filled with the conductive platinum (Pt) paste and cured in an oven. Afterward, the MNE made of platinum (Pt-MNE) was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). To prove the electrochemical applicability of the Pt-MNE, the glutamate oxidase enzyme was immobilized on the surface of the electrode, to detect glutamate, using the cyclic voltammetry (CV) and chronoamperometry (CA) methods. The obtained results demonstrated that the fabricated biosensor could detect a glutamate concentration in the range of 10–150 µM. The limits of detection (LODs) (three standard deviations of the blank/slope) were also calculated to be 0.25 µM and 0.41 µM, using CV and CA, respectively. Furthermore, the Michaelis–Menten constant (KMapp) of the biosensor was calculated to be 296.48 µM using a CA method. The proposed biosensor was finally applied, to detect the glutamate concentration in human serum samples. The presented method for the fabrication of the mold signifies a step further toward the fabrication of a microneedle electrode. [ABSTRACT FROM AUTHOR]

Introduction: Glutamate oxidase (GluOx; EC 1.4.3.11), as an oxidoreductase enzyme, catalyzes the oxidation of glutamate to α-ketoglutarate, ammonia, and hydrogen peroxide (H2O2). In this reaction, the amount of H2O2 is proportional to the concentration of glutamate and its concentration can be measured by using an electrochemical biosensor. The same as other enzyme-based biosensors, glutamate oxidase is one of the key elements in the construction of glutamate biosensors. Such biosensors are fully capable of identifying biological analytes, such as glutamine, ammonia, and creatinine. In addition, glutamate oxidase-based biosensors have many applications in quantitative and qualitative measurements in analytical chemistry, determining the quality of food products, as well as early detection of heart and liver disorders in clinical biochemistry. Considering the importance of the glutamate neurotransmitter in various brain functions, this study investigated its measurement in the brain of male Wistar rats by using a glutamate oxidase-based biosensor. Material & Methods: In order to measure glutamate, initially, a glutamate oxidase-based biosensor was made by simultaneously immobilizing the enzyme and chitosan on the platinum electrode surface. Then, the animals were sacrificed, and their brains were removed and placed in a phosphate buffer. Afterward, the contents of the brain were centrifuged to create a completely uniform mixture. Finally, the concentration of glutamate in the prepared brain samples was measured using the fabricated biosensor by the cyclic voltammetry technique. Findings: The results of cyclic voltammetry experiments showed that the cathodic peak current of the fabricated biosensor was 0.812 µA. Moreover, the calibration curve indicated that the biosensor response was linear up to 1 mM and glutamate concentration in brain samples was also equal to 63.5 µM. Discussion & Conclusion: The cyclic voltammetry experiments showed that the concentration of H2O2, which is produced during the catalytic activity of glutamate oxidase, is completely proportional to the concentration of glutamate oxidase. Furthermore, this study showed that, despite the very low concentration of glutamate neurotransmitters, the glutamate oxidase-based electrochemical biosensor can precisely identify it qualitatively and quantitatively in biological samples, such as brain samples. [ABSTRACT FROM AUTHOR]

A biosensor based on glutamate oxidase (GluOx) was developed to measure glutamate concentration. The main function of this type of biosensor is related to the structure and catalytic activity of GluOx. Since radiofrequency, as the widest spectrum of electromagnetic fields, can affect the catalytic activity and structure of GluOx, in this study, the effect of these fields on the analytical parameters of the fabricated biosensor was investigated. To build the biosensor a sol-gel solution of chitosan and native GluOx were prepared and then immobilized on the surface of the platinum electrode. Similarly, to investigate the effect of radiofrequency fields on the analytical parameters of the biosensor, instead of the native GluOx, irradiated GluOx was used to build the biosensor. To evaluate the biosensor responses, cyclic voltammetry experiments were performed and voltammograms were considered as biosensor responses. To determine the analytical parameters including detection limit, linear range, and saturation region of the responses, calibration curves were drawn for each of the biosensors. Also the long-term stability and selectivity of the fabricated biosensor were evaluated. Thereafter, the optimum pH and temperature for each of these two biosensors were examined. The results showed that radiofrequency waves harmed the detection and response of biosensors in the saturation region, while they had little effect on the linear region. Such results could be due to the effect of radiofrequency waves on the structure and function of glutamate oxidase. In general, the results indicate that when a glutamate oxidase-based biosensor is used to measure glutamate in radiofrequency fields, corrective coefficients for this type of biosensor should be considered to accurately measure glutamate concentration.

In this work, we report a simple electrochemical sensor that detects proteinuria upon immersion into a sample. We immobilized peptidases in conjunction with glutamate oxidase (GluOx) on a Prussian blue-modified glassy carbon (PB-GC) electrode. In the sensor, albumin, which is a major protein in urine, was degraded by peptidases to produce glutamate that was detected by GluOx on PB-GC. Initially, we validated the strategy for the detection of proteinuria using glutamate sensing. We quantified the amount of glutamate produced by degrading human serum albumin with HCl and carboxypeptidase A (CPA). The results indicated that an increase in the enzyme degradation rate was necessary for daily proteinuria sensing that needs to be completed within 1 h. Therefore, we investigated the use of endopeptidase in conjunction with CPA. The combination of proteinase K and CPA liberated glutamate from albumin at 17.5 times higher comparing to solely CPA. The subsequent glutamate assay using peptidase-modified GluOx-PB-GC electrode did not exhibit changes in the electrochemical signal with increases in the glutamate concentration, because peptidase degraded the GluOx on the modified electrode. To prevent this, GluOx was encapsulated in ZIF-8. Glutamate was successfully detected using peptidase and GluOx encapsulated in the ZIF-8-modified PB-GC (peptidase-ZIF-8/GluOx-PB-GC) electrode. Finally, an albumin assay was performed using the peptidase-ZIF-8/GluOx-PB-GC electrode, wherein albumin was detected with a sensitivity of 0.1 mg/mL within 30 min. Our simple and easy-to-use method for albumin detection provides a viable sensor for daily urinalysis.

Herein, a simple method has been used in the fabrication of a microneedle electrode (MNE). To do this, firstly, a commercial self-dissolving microneedle patch has been used to make a hard-polydimethylsiloxane-based micro-pore mold (MPM). Then, the pores of the MPM were filled with the conductive platinum (Pt) paste and cured in an oven. Afterward, the MNE made of platinum (Pt-MNE) was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). To prove the electrochemical applicability of the Pt-MNE, the glutamate oxidase enzyme was immobilized on the surface of the electrode, to detect glutamate, using the cyclic voltammetry (CV) and chronoamperometry (CA) methods. The obtained results demonstrated that the fabricated biosensor could detect a glutamate concentration in the range of 10–150 µM. The limits of detection (LODs) (three standard deviations of the blank/slope) were also calculated to be 0.25 µM and 0.41 µM, using CV and CA, respectively. Furthermore, the Michaelis–Menten constant (KMapp) of the biosensor was calculated to be 296.48 µM using a CA method. The proposed biosensor was finally applied, to detect the glutamate concentration in human serum samples. The presented method for the fabrication of the mold signifies a step further toward the fabrication of a microneedle electrode.

The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.

Aim. Alanine aminotransferase (ALT) is an enzyme mostly found in heart and liver cells. Therefore, its blood content is often used as a non-selective biomarker of damage to these organs and helps diagnose such diseases as cirrhosis, hepatitis, myocardial infarction, stroke, etc. Since these diseases usually result in a patient’s critical condition requiring immediate medical aid, the availability of a fast, accurate, portable and easy-to-use method for ALT blood level detecting is a necessity. Currently, clinical research of ALT level is conducted using such methods as spectrometry and immunoassay, which can’t meet all the needs of doctors. In contrast, biosensors have the potential for miniaturization, automation, and cost reduction of analysis. Therefore, it was decided to develop biosensors for determining the level of ALT in biological solutions. Methods. The measurement system was designed using a platinum disk electrode as an electrochemical transducer, connected to a PalmSense potentiostat using a three-electrode scheme (working electrode, additional electrode, reference Ag/ AgCl electrode). In order to ensure additional selectivity of the converter, a poly(phenylenediamine) membrane was formed. The bioselective membrane on the electrode surface was formed by covalent cross-linking between enzyme and additional protein using glutaraldehyde. The level of enzyme activity in the solution was measured by determining the increase in current in the system per unit of time as a result of the oxidation of the ALT reaction product (glutamate or pyruvate, respectively). Results. In the work, two versions of a biosensor based on two different bioreactive systems with glutamate oxidase and pyruvate oxidase are proposed, and laboratory prototypes of these biosensors for measuring ALT are made. The optimal conditions for bioselective membrane creation and functioning conditions for both biosensors were selected, in particular the method of immobilization of each enzyme and concentration of coenzymes pyruvate oxidase and ALT. The main analytical characteristics of both versions of the developed biosensors were studied and compared. Conclusions. It was shown that the biosensor based on glutamate oxidase is easier to use and more stable due to the smaller number of coenzymes. However, the biosensor based on pyruvate oxidase is preferred when creating more advanced biosensor system for the simultaneous determination of ALT and AST (another biomarker of heart and liver diseases, which is often determined simultaneously with ALT for more accurate diagnosis and has one common stage of the enzymatic reaction), as it allows to separate the signal from these two enzymes. [ABSTRACT FROM AUTHOR]

In this work, an amperometric biosensor for ALT detection, which will be part of a biosensor system for simultaneous measurements of AST and ALT and their ratios, has been developed. A platinum disk electrode was used as the electrochemical transducer. To form a bioselective element, a mixture based on glutamate oxidase was applied to the surface of transducer. A universal method for measuring ALT activity using the developed biosensor was proposed and described. During the study, the optimal concentrations of ALT substrates (α-ketoglutarate – 50 μM, alanine – 4 mM) and coenzyme (50 μM of pyridoxal phosphate) were selected. The analytical characteristics of the developed biosensor were also investigated, namely, sensitivity, linear range, minimum limit of determination, selectivity, stability, etc. It was shown the fundamental possibility of using this biosensor to measure the content of ALT in real samples. [ABSTRACT FROM AUTHOR]

Whole-cell systems offer many benefits for biochemical production, such as relatively easy enzyme control and higher tolerance toward harsh environments, than purified enzymes. These systems can be applied to many bioconversion reactions, but they sometimes require cofactor regeneration units to support reactions at high substrate concentrations. Here, we examined l-glutamate oxidase (GOX) from Streptomyces sp. X119-6, which produces α-ketoglutarate (α-KG) from l-glutamate, and catalase (KatE) from Escherichia coli, which removes hydrogen peroxide generated by GOX. After optimizing the expression vector, pH, strains, culture conditions, and isopropyl β-d-1-thiogalactopyranoside concentration, we compared their efficiency to that of a previously reported GOX from Streptomyces mobaraensis. Our results indicated that GOX from Streptomyces sp. X119-6 and KatE increased α-KG production by 2.76-fold. This GOX required high levels of α-KG as an amino donor to convert 5-aminovaleric acid to glutaric acid. Performing the reaction at pH 8 enabled us to avoid the exogenous addition of catalase, but severe substrate inhibition was observed, resulting in the production of 287 mM glutaric acid. This α-KG regeneration system has potential for improving production in various aminotransferase systems. [ABSTRACT FROM AUTHOR]

Today, the concentration of glutamate in the patient′s blood is an important indicator in medical diagnostics; therefore, it is necessary to have a simple, accurate, and fast tool to obtain the data. Here, a recently developed amperometric glutamate‐sensitive biosensor was optimized to improve its characteristics. The platinum disk electrode was used as a transducer. As a bioselective element we used the enzyme glutamate oxidase, covalently crosslinked with bovine serum albumin by glutaraldehyde. Circumstances of enzyme immobilization on the transducer's surface were optimized (enzyme and glutaraldehyde concentrations and immobilization duration). To test the ability of this biosensor to work in biological environments containing complex biological substances, the influence of the working solution was investigated (concentration of the working buffer, its temperature, presence of the protein in the analyzed sample). The linear range of biosensor was from 5 to 600 μM of glutamate and the sensitivity was 150–200 nA/mM. Measurements of glutamate concentrations in the blood plasma were performed by biosensor and glutamate dehydrogenase assay. The linear correlation between the methods was found in a range of 50.4–182.5 μM (R2=0.99). Thus, it has been shown that the developed biosensor makes it possible to measure the concentration of glutamate in blood plasma. [ABSTRACT FROM AUTHOR]

The aim of the work were the optimization of an amperometric glutamate-sensitive biosensor and its utilization for the determination of the glutamate concentrations in food samples. Amperometric method of measurements was used. The biosensor was based on immobilized glutamate oxidase and platinum disc electrode. The biosensor was connected to the working cell with auxiliary (platinum wire) and reference (Ag/AgCl) electrodes. The biosensor exhibited high sensitivity to glutamate, duration of one analysis was about 5 min. An influence of the ionic strength, pH, and buffer capacity on the biosensor operation was investigated. The sensitivity of biosensor to various possible interfering substances, including amino acids, was studied; high selectivity to glutamate was shown. The reproducibility of analysis of food samples and an impact of sample dilution was evaluated. Glutamate concentrations in different sauces and seasonings were measured by the developed biosensor; the results correlated well with those obtained by the spectrophotometric method (R2 = 0,988). Thus, the amperometric biosensor for glutamate determination was successfully optimized and used for measurement of glutamate concentrations in sauces and seasonings.

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We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 − 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products. Schematic representation of the enzyme quantification system for GABA. Serial reaction catalyzed by the three enzymes enables colorimetric measurement of GABA content in biological samples. [ABSTRACT FROM AUTHOR]

Glutamate (GLU) is a primary excitatory neurotransmitter, and its dysregulation is associated with several neurodegenerative disorders. A major challenge in GLU estimation is the existence of other biomolecules in the brain that could directly get oxidized at the electrode. Hence, highly selective electroenzymatic biosensors that enable rapid estimation of GLU are needed. Initially, a copolymer, poly(2-dimethylaminoethyl methacrylate- styrene) was synthesized through reversible addition-fragmentation chain transfer polymerization to noncovalently functionalize reduced graphene oxide (rGO), named DS–rGO. Glutamate oxidase macromolecule immobilized DS–rGO formed enzyme nanosheets, which was drop-coated over Prussian blue electrodeposited disposable electrodes to fabricate the GLU biosensor. The interconnectivity between the enzyme nanosheets and the Prussian blue endows the biosensor with enhanced conductivity and electrochemical activity. The biosensor exhibited a linearity: 3.25–250 μM; sensitivity: 3.96 μA mM−1 cm−2, and a limit of detection: 0.96 μM for GLU in the Neurobasal Medium. The biosensor was applied to an in vitro primary rat cortical model to discriminate GLU levels in Neurobasal Medium, before and after KCl mediated depolarization, which provides new insights for elucidating neuronal functioning in the brain. Amperometric biosensor for detecting GLU in the in vitro model of primary rat cortical neurons before and after KCl mediated depolarization. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract In this work, we developed a new amperometric biosensor for glutamate detection using a typical method of glutamate oxidase (GlOx) immobilization via adsorption on silicalite particles. The disc platinum electrode (d = 0.4 mm) was used as the amperometric sensor. The procedure of biosensor preparation was optimized. The main parameters of modifying amperometric transducers with a silicalite layer were determined along with the procedure of GlOx adsorption on this layer. The biosensors based on GlOx adsorbed on silicalite demonstrated high sensitivity to glutamate. The linear range of detection was from 2.5 to 450 μM, and the limit of glutamate detection was 1 μM. It was shown that the proposed biosensors were characterized by good response reproducibility during hours of continuous work and operational stability for several days. The developed biosensors could be applied for determination of glutamate in real samples.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

L -glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L -glutamate deamination. LGOX from Streptomyces sp. X-119–6 is used widely for L -glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119–6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications. [Display omitted] • Novel single-chain L -glutamate oxidase was developed by inserting flexible loops. • This L -glutamate oxidase showed similar activity to mature form of wild-type from Streptomyces sp. X-119–6. • There was little conformational difference between the single-chain enzyme and the mature Streptomyces sp. X-119–6. • Efficient enzyme production of L -glutamate oxidase will be expected by not using protease treatment to mature the enzyme. [ABSTRACT FROM AUTHOR]

A simple method that relies only on an electrochemical workstation has been investigated to fabricate a highly sensitive glutamate microbiosensor for potential neuroscience applications. In this study, in order to develop the highly sensitive glutamate electrode, a 100 µm platinum wire was modified by the electrochemical deposition of gold nanoparticles, Prussian blue nanocubes, and reduced graphene oxide sheets, which increased the electroactive surface area; and the chitosan layer, which provided a suitable environment to bond the glutamate oxidase. The optimization of the fabrication procedure and analytical conditions is described. The modified electrode was characterized using field emission scanning electron microscopy, impedance spectroscopy, and cyclic voltammetry. The results exhibited its excellent sensitivity for glutamate detection (LOD = 41.33 nM), adequate linearity (50 nM–40 µM), ascendant reproducibility (RSD = 4.44%), and prolonged stability (more than 30 repetitive potential sweeps, two-week lifespan). Because of the important role of glutamate in neurotransmission and brain function, this small-dimension, high-sensitivity glutamate electrode is a promising tool in neuroscience research.

This paper describes an electrochemiluminescent (ECL) based method for chiral recognition and detection of both glutamate (Glu) enantiomers. The luminophore luminol (Lum) was used as both the reductant and stabilizer of Ag nanoparticles (AgNP-Lum) which were combined with carbon quantum dots (C-dots) and placed on a glassy carbon electrode (GCE) along with the enzyme glutamate oxidase (GluOx). The use of these materials is found to result in strong amplification of ECL. The nanomaterials used were characterized by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR). The stepwise fabrication of the electrode was verified by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under optimized conditions and by applying a typical potential of 0.6 V, the ECL increases linearly in the 5.0 μM to 5.0 mM Glu concentration range, with a 1.6 μM lower detection limit and satisfactory selectivity. A Glu logic gate idea has been designed that is based on this enzymatic biosensor. [ABSTRACT FROM AUTHOR]

[Display omitted] • SPR strategy allows to control and describe molecular loading on sensor surface. • Linker type influences on the loading of bioreceptors on the sensor surface. • PAMAM generation has effect on the enzyme activity and amperometric sensor response. • The highest sensitivity and the lowest LOD was obtained for interlayer PAMAM D4. In this paper, we discuss dendrimer usage in enzyme-based electrochemical biosensors, particularly with respect to biomolecule loading on the sensing surface. A novel approach to design bioactive layers with immobilized enzymes for electrochemical biosensors using the surface plasmon resonance (SPR) method in combination with electrochemical impedance spectroscopy was presented. The gold surface was modified with linear linkers (various mercaptoalkanoic acids and aminoalkanethiols) and poly(amidoamine) dendrimers from the first- to fifth-generation. The best functionalization procedure was established by detailed SPR studies and transferred onto gold electrodes to electrochemically examine the model enzymatic reaction catalysed by glutamate oxidase. In the case of the chronoamperometric method, the determined sensitivity was 3.36 ± 0.08 μA∙mM−1, and the low limit of detection (LOD) was 1.52 μM. Comparing the sensitivity and LOD obtained for CV measurements, the values of these parameters were 2.5 times higher and 4 times lower, respectively, for the fourth-generation dendrimer-based biosensor and the biosensor with a linear linker. The positive impact of the dendrimer interlayer on the long-term enzyme activity was also confirmed. The research results indicate the possibility of a significant increase in the sensor response using the dendrimer itself without enriching it with electrochemical components. [ABSTRACT FROM AUTHOR]

Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method 1 have faster response time (

[Display omitted] • A minitype electrochemical sensor for detecting l -glutamate is constructed using GluOx/PMPD/Pt modified GRE. • In vivo monitoring of l -Glu level can be realized in cucumber. • Accurate detection of l -Glu concentration in cucumber juice in vitro. • There are good performances in terms of sensitivity, linear range and anti-interference capability. l -glutamate (l -Glu) is the basic unit for the synthesis of protein, metabolic substances and chlorophyll, and plays an important role in the carbon and nitrogen metabolism in plants. The in vivo monitoring of l -Glu level is of great significance to study the growth and development of plants. Although a variety of methods have been reported for the in vitro determination of l -Glu, there are very limited research focused on the determination of l -Glu in plants. Here we firstly report a simple method to construct a minitype glutamate sensor for in vivo monitoring of l -glutamate in cucumber. The minitype graphite rod electrode (GRE) allows its application for in vivo monitoring, and modification of Pt nanoparticles (PtNPs) and poly (m -phenylenediamine) film (PMPD) significantly improves the sensitivity and anti-interference ability, and immobilization of the glutamate oxidase (GluOx) could effectively catalyze the l -Glu into a detectable H 2 O 2. The resulting l -Glu sensor exhibits a good performance in terms of high sensitivity, good selectivity and excellent stability with a linear range of 2–550 μM and limit of detection (LOD) as low as 0.536 μM. More importantly, the as-developed sensor was successfully applied for the determination of l -Glu in cucumber juice and the in vivo monitoring of l -Glu level in cucumber fruit. We envision that the constructed sensor will show great potentials for studying the growth mechanisms of plants. [ABSTRACT FROM AUTHOR]

Adult rabbits with experimental glaucoma were used in this study. We studied glutamate and glutamine levels and glutamine synthetase, glutamate oxidase and glutaminase activity in the eye tissue during glaucoma process. Results suggest increase of both glutamate level and glutaminase activity, as well as decrease of both glutamine level and glutamine synthetase and glutamate oxidase activity compared to normal ranges over all period of observation. This influence was significally prevented using neuroprotective drugs.

A biosensor based on glutamate oxidase (GluOx) was developed to measure glutamate concentration. The main function of this type of biosensor is related to the structure and catalytic activity of GluOx. Since radiofrequency, as the widest spectrum of electromagnetic fields, can affect the catalytic activity and structure of GluOx, in this study, the effect of these fields on the analytical parameters of the fabricated biosensor was investigated. To build the biosensor a sol-gel solution of chitosan and native GluOx were prepared and then immobilized on the surface of the platinum electrode. Similarly, to investigate the effect of radiofrequency fields on the analytical parameters of the biosensor, instead of the native GluOx, irradiated GluOx was used to build the biosensor. To evaluate the biosensor responses, cyclic voltammetry experiments were performed and voltammograms were considered as biosensor responses. To determine the analytical parameters including detection limit, linear range, and saturation region of the responses, calibration curves were drawn for each of the biosensors. Also the long-term stability and selectivity of the fabricated biosensor were evaluated. Thereafter, the optimum pH and temperature for each of these two biosensors were examined. The results showed that radiofrequency waves harmed the detection and response of biosensors in the saturation region, while they had little effect on the linear region. Such results could be due to the effect of radiofrequency waves on the structure and function of glutamate oxidase. In general, the results indicate that when a glutamate oxidase-based biosensor is used to measure glutamate in radiofrequency fields, corrective coefficients for this type of biosensor should be considered to accurately measure glutamate concentration., Competing Interests: The authors declare no conflict of interest.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

In this study, a miniaturized biosensor based on permselective polymer layers (overoxidized polypyrrole (Ppy) and Nafion®) modified and enzyme (glutamate oxidase (GlutOx)) immobilized micro-platinum wire electrode for the detection of alanine aminotransferase (ALT) was fabricated. The proposed ALT biosensor was measured electrochemically by constant potential amperometry at +0.7 V vs. Ag/AgCl. The ALT biosensor provides fast response time (~5 s) and superior selectivity towards ALT against both negatively and positively charged species (e.g., ascorbic acid (AA) and dopamine (DA), respectively). The detection range of the ALT biosensor is found to be 10-900 U/L which covers the range of normal ALT levels presented in the serum and the detection limit and sensitivity are found to be 8.48 U/L and 0.059 nA/(U/L.mm2) (N = 10), respectively. We also found that one-day storage of the ALT biosensor at -20°C right after the sensor being fabricated can enhance the sensor sensitivity (1.74 times higher than that of the sensor stored at 4°C). The ALT biosensor is stable after eight weeks of storage at -20°C. The sensor was tested in spiked ALT samples (ALT activities: 20, 200, 400, and 900 U/L) and reasonable recoveries (70%~107%) were obtained. [ABSTRACT FROM AUTHOR]

The development of biosensors for the determination of glutamate has been of great research interest for the past 25 years due to its importance in biomedical and food studies. This review focusses on the various strategies used to fabricate glutamate biosensors as well as their performance characteristics. A brief comparison of the enzyme immobilisation method employed and the performance characteristics of a range of glutamate biosensors are described in tabular form and then described in detail throughout the review: some selected examples have been included to demonstrate the various applications of these biosensors to real samples. [ABSTRACT FROM AUTHOR]

In this paper, a novel amperometric glutamate biosensor with glutamate oxidase (GlOx) immobilized directly on NH 2 functionalized, platinum doped tetrahedral amorphous carbon (ta-C) film, has been successfully developed. First, we demonstrate that direct GlOx immobilization is more effective on amino-groups than on carboxyl- or hydroxyl-groups. Second, we show that anodizing and plasma treatments increase the amount of nitrogen and the proportion of protonated amino groups relative to amino groups on the aminosilane coating, which subsequently results in an increased amount of active GlOx on the surface. This effect, however, is found to be unstable due to unstable electrostatic interactions between GlOx and NH 3 + . We demonstrate the detection of glutamate in the concentration range of 10 µM−1 mM using the NH 2 functionalized Pt doped ta-C surface. The biosensor showed high sensitivity (2.9 nA μM −1 cm −2 ), low detection limit (10 μM) and good storage stability. The electrode response to glutamate was linear in the concentrations ranging from 10 µM to 500 µM. In conclusion, the study shows that GlOx immobilization is most effective on aminosilane treated ta-C surface without any pre-treatments and the fabricated sensor structure is able to detect glutamate in the micromolar range. [ABSTRACT FROM AUTHOR]

L-Glutamate oxidase (LGOX; EC 1.4.3.11) is the important enzyme that catalyzes the deamination of L-glutamic acid to 2-oxo glutarate and ammonium ions. In this study, the microbial production of L-glutamate oxidase enzyme from Hypocrea jecorina pure culture was carried out and the optimum conditions and calculation of some kinetic parameters of the produced enzyme were also determined by using Nesslerization reaction. The enzyme shows maximum activity at pH 8.5 and 40°C in TRIS buffer. The kinetic parameters of the enzyme, KM and Vmax values, were determined as 2.58 mM and 83.33 U/mg protein, respectively. The inactivation rate constants and half life (t1/2) were determined from the slope of thermal stability plots of the enzyme at 60, 70 and 80°C temperatures. The activation energy (Ea) was found to be 1.40 kcal mol-1. [ABSTRACT FROM AUTHOR]

Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method 1 have faster response time (<2 s) and lower detection limit (2.5 ± 1.1 μM) compared to that prepared by Method 2 (response time: <5 sec and detection limit: 6.5 ± 1.7 μM); glutamate sensors prepared by Method 2 have a larger linear detection range (20-352 μM) and higher sensitivity (86.8 ± 8.8 nA.μM-1.cm-2, N = 12) compared to those prepared by Method 1 (linear detection range: 20-217 μM and sensitivity: 34.9 ± 4.8 nA.μM-1.cm-2, N = 8). The applicability of the glutamate sensors in vivo was also demonstrated. The glutamate sensors were implanted into the rat brain to monitor the stress-induced extracellular glutamate release in the hypothalamus of the awake, freely moving rat. [ABSTRACT FROM AUTHOR]

Highlights: [•] Constructed a PPyNPs/PANI/AuE hybrid film. [•] Fabricated an improved amperometric glutamate biosensor based on this film. [•] Biosensor had a detection limit of 0.1nM and linear range 0.02–400μM. [•] Employed for glutamate determination in different food stuffs. [•] It also showed improved storage stability and reusability. [ABSTRACT FROM AUTHOR]

Abstract: A method is described for the construction of a novel amperometric glutamate biosensor based on covalent immobilization of glutamate oxidase (GluOx) onto, carboxylated multi walled carbon nanotubes (cMWCNT), gold nanoparticles (AuNPs) and chitosan (CHIT) composite film electrodeposited on the surface of a Au electrode. The GluOx/cMWCNT/AuNP/CHIT modified Au electrode was characterized by scanning electron microscopy (SEM), fourier transform infra-red (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The biosensor measured current due to electrons generated at 0.135V against Ag/AgCl from H2O2, which is produced from glutamate by immobilized GluOx. The biosensor showed optimum response within 2s at pH 7.5 and 35°C. A linear relationship was obtained between a wide glutamate concentration range (5–500μM) and current (μA) under optimum conditions. The biosensor showed high sensitivity (155nA/μM/cm2), low detection limit (1.6μM) and good storage stability. The biosensor was unaffected by a number of serum substances at their physiological concentrations. The biosensor was evaluated and employed for determination of glutamate in sera from apparently healthy subjects and persons suffering from epilepsy. [Copyright &y& Elsevier]

Abstract: The potentiometric glutamate biosensor based on ammonium-selective poly(vinylchloride) (PVC) membrane electrode was constructed by chemically immobilizing glutamate oxidase. Ammonium ions produced after an enzymatic reaction were determined potentiometrically. We determined the optimum working conditions of the biosensor such as buffer concentration, buffer pH, lifetime, response time, linear working range, kinetic constants (Km and Vmax) of glutamate oxidase enzyme used for biosensor construction values, and other response characteristics. Additionally, glutamate assay in some real samples such as chicken bullion, healthy human serum, and commercial multipower amino acid mixture were also successfully carried out. The results showed good agreement with previously reported values. [ABSTRACT FROM AUTHOR]

Abstract: Novel electrochemical platform based on Pt nanoparticle modified ordered three-dimensional gold nanowire arrays (PtNP/NAEs) for the amperometric sensing of H2O2 and glutamate is developed. Pt nanoparticle (PtNP) is fabricated by electrodeposition onto the 3D nanowires and characterised using scanning electron microscopy (SEM) and cyclic voltammetry. The deposited nanoparticles have an average size of 20nm. The PtNP/NAE shows a linear response of up to 20mM for H2O2 detection with a sensitivity of 194.60μAmM−1 cm−2 at 20°C. It can detect 1μM (S/N=3) of H2O2 at normal condition without using any enzyme or mediator. Analytical performance of this electrode is tested by immobilising glutamate oxidase (GlutOx) through cross-linking in the matrix of bovine serum albumin (BSA), Nafion and glutaraldehyde. At physiological pH, the biosensor showed the sensitivity of 10.76μAmM−1 cm−2, with a linear range of up to 0.8mM. [ABSTRACT FROM AUTHOR]

Abstract: A highly sensitive and selective glutamate microbiosensor based on polypyrrole (PPy), multiwalled carbon nanotubes (MWCNT) and glutamate oxidase (GluOx) deposited on the transducer platinum electrode (Pt) is described. The sensor consists of a permselective membrane of polypyrrole for the rejection of interferences, followed by a layer of multiwalled carbon nanotubes and glutamate oxidase deposited by asymmetrical alternating current electrophoretic deposition (AC-EPD). The biosensor has a high sensitivity (3.84nA/(μMmm2)), low response to interferences such as ascorbic acid, uric acid and acetaminophen, a fast response time (7s), low detection limit (∼0.3μM), a linear range of 140μM and a satisfactory stability. In order to improve the linear range and the stability, a thin layer of polyurethane (PU) was applied to the Pt/PPy/MWCNT/GluOx sensor. The resulting sensor with the PU outer membrane showed an increase in the linear range up to ∼500μM glutamate and has a better stability at the expense of a decrease in sensitivity (2.5nA/(μMmm2)) and an increase in the response time (15s). [Copyright &y& Elsevier]

Abstract: Glucose oxidase and glutamate oxidase lines, with typical width of 100 µm, were patterned on gold surfaces using a micro-dispensing system, by shooting 100 pl droplets of the corresponding enzyme solutions. The probe of a scanning electrochemical microscope (SECM) was then carefully positioned in the close proximity of the enzyme microstructure and poised to +600 mV vs. Ag/AgCl, KCl 0.1 M. The H2O2, generated by the enzyme lines at different concentrations of glucose and glutamate in the surrounding solution, was sequentially monitored. Reproducible calibration curves for glucose and glutamate were obtained in one single experiment, proving that the combination of enzyme microstructures with SECM can provide a new way of achieving multianalyte detection. [Copyright &y& Elsevier]

Abstract: Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been developed for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release. On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip. In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-l-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4°C. A glutamate detection limit of 10−7 M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters. [Copyright &y& Elsevier]

Background: Microdialysis is a technique to monitor extracellular changes in living tissue. Substances present in the extracellular space, such as neurotransmitters and metabolites transported between cells and capillaries in the extracellular fluid (ECF), are major object. Results: Since its introduction to the research of the nervous system, microdialysis has become a popular method for the measurements of brain chemistry and greatly affected in the fields of neuropharmacology, neuroanatomy and neurophysiology. Most of published papers using microdialysis have focused on the area of neuroscience, recently more biomedical application. Conclusion: In this review, we focused on cerebral microdialysis as a monitoring tool for physiologic and pathophysiologic changes in chemical processes in the brain. Then we presented the principle and various applications of cerebral microdialysis. [ABSTRACT FROM AUTHOR]

Abstract: Biosensors were fabricated at neutral pH by sequentially depositing the polycation polyethyleneimine (PEI), the stereoselective enzyme l-glutamate oxidase (GluOx) and the permselective barrier poly-ortho-phenylenediamine (PPD) onto 125-μm diameter Pt wire electrodes (Pt/PEI/GluOx/PPD). These devices were calibrated amperometrically at 0.7V versus SCE to determine the Michaelis–Menten parameters for enzyme substrate, l-glutamate (Glu) and co-substrate, dioxygen. The presence of PEI produced a 10-fold enhancement in the detection limit for Glu (∼20nM) compared with the corresponding PEI-free configurations (Pt/GluOx/PPD), without undermining their fast response time (∼2s). Most remarkable was the finding that, although some designs of PEI-containing biosensors showed a 10-fold increase in linear region sensitivity to Glu, their oxygen dependence remained low. [Copyright &y& Elsevier]

Abstract: A sensitive biosensor has been developed for the neurotoxin β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) contained in the seeds of grass pea (Lathyrus sativus) and for l-glutamate based on glutamate oxidase (GlOx) and a Prussian blue (PB) modified glassy carbon (GC) electrode. The configuration of the system is so as to detect the hydrogen peroxide released during the enzymatic cycle at a low applied potential, −50mV versus Ag|AgCl, in the flow injection mode. For this purpose GlOx was coupled to PB electrodeposited onto a glassy carbon electrode and stabilised by treatment with tetrabutylammonium toluene-4-sulfonate (TTS) during one of the steps in the electrodeposition. GlOx was cross-linked with glutaraldehyde (GA), bovine serum albumin (BSA) and Tween-20 on the surface of the PB modified GC electrodes. Addition of 0.01% and 0.001% polyethyleneimine (PEI) to the immobilisation mixture resulted in an enhancement of the response signal with about 35% and 62% for glutamate and β-ODAP, respectively, when using 0.01% PEI and with 164% and 200% for glutamate and β-ODAP, respectively, when using 0.001% PEI. The linear response range for β-ODAP was extended from 0.05–0.5mM to 0.01–1mM, when 0.001% PEI was used. However, a higher concentration of PEI, 0.1%, caused a decrease in the sensitivity of the biosensor. [Copyright &y& Elsevier]