Glutamate Oxidase Research Articles

Disposable glutamate biosensor based on platinum nanoparticles, carbon quantum dots and poly-L-aspartic acid

This study reports the design and development of a disposable amperometric biosensor for the determination of L-glutamate. Glutamate oxidase (GlOx) was immobilized onto a screen-printed carbon electrode (SPE) modified with poly-L-Aspartic acid (PAsp), carbon quantum dots (CQD), and platinum nanoparticles (PtNP) for the construction of the biosensor. The surface composition of the modified SPE was optimized using the one variable at a time method. The morphological properties of the biosensor were characterized by scanning electron microscopy and energy-dispersive X-ray spectroscopy. The electrochemical behavior of the modified electrodes was studied by cyclic voltammetry. Under the optimized experimental conditions the linear working range, detection limit and sensitivity of the GlOx/PtNP/CQD/PAsp/SPE were found to be 1.0 − 140 µM, 0.3 µM and 0.002 µA µM−1, respectively. The GlOx/PtNP/CQD/PAsp/SPE biosensor also exhibited good measurement repeatability. The as-developed biosensor was applied for the determination of L-glutamate in spiked serum samples and the average analytical recovery of added glutamate was 98.9 ± 3.9%.

Development, optimization, and comparison of two versions of ALT-sensitive biosensor based on glutamate oxidase and pyruvate oxidase

Development, optimization, and comparison of two versions of ALT-sensitive biosensor based on glutamate oxidase and pyruvate oxidase

Measurement of Neuropeptides and Amino Acids with Carbon-Fiber Multielectrode Array Enzyme-Modified Biosensors

Carbon fiber microelectrodes (CFMEs) have been used to detect neurotransmitters and other biomolecules using fast-scan cyclic voltammetry (FSCV) for the past few decades. These assays typically measure small molecule neurotransmitters such as dopamine and serotonin. The carbon fiber is relatively small, biocompatible, and makes minimally invasive measurements at high spatial and temporal resolution. Carbon Fiber Multielectrode arrays have been utilized to measure multiple neurotransmitters in several brain regions simultaneously with multi-waveform application on each electrode. We have extended this work to measure larger molecule neuropeptides such as Neuropeptide Y and Oxytocin, a pleiotropic peptide hormone, is physiologically important for adaptation, development, reproduction, and social behavior. This neuropeptide functions as a stress-coping molecule, an anti-inflammatory agent, and serves as an antioxidant with protective effects especially during adversity or trauma. Here, we measure tyrosine using the Modified Sawhorse Waveform (MSW), enabling enhanced electrode sensitivity for the amino acid and peptide, decreased surface fouling, and codetection with other catecholamines. As both oxytocin and Neuropeptide Y contain tyrosine, the MSW was also used to detect these neuropeptides. Additionally, we demonstrate that applying the MSW on CFMEs allows for real time measurements of exogenously applied neuropeptides on rat brain slices. These results may serve as novel assays for neuropeptide detection in a fast, sub-second timescale with possible implications for in vivo measurements and further understanding of the physiological role of neuropeptides such as Neuropeptide Y and oxytocin.Moreover, we have also developed enzyme modified microelectrodes for the measurement of glutamate (L-glutamic acid), which is an important excitatory amino acid and biomarker for epilepsy along with the inhibitory GABA. Since glutamate is not redox active at carbon electrodes, we modified CFMEs with glutamate oxidase enzyme to metabolize glutamate to hydrogen peroxide, which was then oxidized at carbon electrodes to produce readout cyclic voltammograms (CVs). The enzyme coating was optimized by varying the concentration of enzyme, chitosan binder, solvent, and deposition time. The coating was further analyzed electrochemically and imaged with scanning electron microscopy (SEM) for thickness and uniformity of surface coverages. Energy-Dispersive Spectroscopy (EDS/EDX) was utilized for chemical surface functionalization analysis. Glutamate oxidation was found to be adsorption controlled to CFMEs and characterized at various scan rates, concentrations, and stability times as well with an approximate 100 nM limit of detection. Glutamate was co-detected in complex mixtures with several monoamines such as dopamine, serotonin, norepinephrine, and others. Glutamate will furthermore be measured in several food samples and ex vivo in rat coronal brain slices and in vivo in anesthetized and freely behaving animals.

Improving Sensitivity and Longevity of In Vivo Glutamate Sensors with Electrodeposited NanoPt.

In vivo glutamate sensing has provided valuable insight into the physiology and pathology of the brain. Electrochemical glutamate biosensors, constructed by cross-linking glutamate oxidase onto an electrode and oxidizing H2O2 as a proxy for glutamate, are the gold standard for in vivo glutamate measurements for many applications. While glutamate sensors have been employed ubiquitously for acute measurements, there are almost no reports of long-term, chronic glutamate sensing in vivo, despite demonstrations of glutamate sensors lasting for weeks in vitro. To address this, we utilized a platinum electrode with nanometer-scale roughness (nanoPt) to improve the glutamate sensors' sensitivity and longevity. NanoPt improved the GLU sensitivity by 67.4% and the sensors were stable in vitro for 3 weeks. In vivo, nanoPt glutamate sensors had a measurable signal above a control electrode on the same array for 7 days. We demonstrate the utility of the nanoPt sensors by studying the effect of traumatic brain injury on glutamate in the rat striatum with a flexible electrode array and report measurements of glutamate taken during the injury itself. We also show the flexibility of the nanoPt platform to be applied to other oxidase enzyme-based biosensors by measuring γ-aminobutyric acid in the porcine spinal cord. NanoPt is a simple, effective way to build high sensitivity, robust biosensors harnessing enzymes to detect neurotransmitters in vivo.

Enzyme-modified Pt nanoelectrodes for glutamate detection.

We present here a glutamate oxidase (GluOx)-modified platinum (Pt) nanoelectrode with a planar geometry for glutamate detection. The Pt nanoelectrode was characterized using electrochemistry and scanning electron microscopy (SEM). The radius of the Pt nanoelectrode measured using SEM is ∼210 nm. GluOx-modified Pt nanoelectrodes were generated by dip coating GluOx on the Pt nanoelectrode in a solution of 0.9% (wt%) bovine serum albumin (BSA), 0.126% (wt%) glutaraldehyde, and 100 U mL-1 GluOx. An increase in current was observed at +0.7 V vs. Ag/AgCl/1 M KCl with adding increasing concentrations of glutamate. Two-sample t-test results showed that there is a significant difference for current at +0.7 V between the blank and the added lowest glutamate concentration, as well as between adjacent glutamate concentrations, confirming that the increase in current is related to the increased glutamate concentration. The experimental current-concentration curve of glutamate detection fitted well to the theoretical Michaelis-Menten curve. At the low concentration range (50 μM to 200 μM), a linear relationship between the current and glutamate concentration was observed. The Michaelis-Menten constants of Imax and Km were calculated to be 1.093 pA and 0.227 mM, respectively. Biosensor efficiency (the ratio of glutamate sensitivity to H2O2 sensitivity) is calculated to be 57.9%. Enzact (Imax/H2O2 sensitivity, an indicator of the amount of enzyme loaded on the electrode) of the GluOx-modified Pt nanoelectrode is 0.243 mM. We further compared the sensitivity of a GluOx-modified Pt nanoelectrode with a GluOx-modified carbon fiber microelectrode (7 μm diameter and a sensing length of ∼350 μm). Glutamate detection on the GluOx-modified carbon fiber microelectrode fitted well to a Michaelis-Menten like response. Based on the fitting, the GluOx-modified carbon fiber microelectrode exhibited an Imax of 0.689 nA and a Km of 301.2 μM towards glutamate detection. The best linear range of glutamate detection on the GluOx-modified carbon fiber microelectrode is from 50 μM to 150 μM glutamate. The GluOx-modified carbon fiber microelectrode exhibited a higher potential requirement for glutamate detection compared to the GluOx-modified Pt nanoelectrode.

Enhanced performance of enzymes confined in biocatalytic hydrogen-bonded organic frameworks for sensing of glutamate in the central nervous system

Glutamate (Glu) is a key excitatory neurotransmitter associated with various neurological disorders in the central nervous system, so its measurement is vital to both basic research and biomedical application. In this work, we propose the first example of using biocatalytic hydrogen-bonded organic frameworks (HOFs) as the hosting matrix to encapsulate glutamate oxidase (GLOD) via a de novo approach, fabricating a cascaded-enzyme nanoreactor for Glu biosensing. In this design, the ferriporphyrin ligands can assemble to form Fe-HOFs with high catalase-like activity, while offering a scaffold for the in-situ immobilization of GLOD. Moreover, the formed GLOD@Fe-HOFs are favorable for the efficient diffusion of Glu into the active sites of GLOD via the porous channels, accelerating the cascade reaction with neighboring Fe-HOFs. Consequently, the constructed nanoreactor can offer superior activity and operational stability in the catalytic cascade for Glu biosensing. More importantly, rapid and selective detection can be achieved in the cerebrospinal fluid (CSF) collected from mice in a low sample consumption. Therefore, the successful fabrication of enzyme@HOFs may offer promise to develop high-performance biosensor for further biomedical applications.

Glutamate oxidase sheets-Prussian blue grafted amperometric biosensor for the real time monitoring of glutamate release from primary cortical neurons

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.

Comparison of molecular properties and docking exploration of ternary organic compounds of 2, 4-Di-substituent-aniline calculated by DFT method

Investigating the molecular structure, vibration methods, nonlinear optical, electronic and thermo dynamical properties of 2, 4-dichloroaniline (2, 4DCA), 2, 4-difluoroaniline (2, 4DFA) and 2, 4-diiodoaniline (2, 4DIA) gives comparable hypothetical properties to decide a desired material. Density Functional Theories (DFTs) have the basic guidelines to affect the Schrödinger equation for several body systems. Comparison is drawn among theoretical properties like stability and charge delocalization of the molecules which are studied by Natural Bond Orbital (NBO) analysis, Nonlinear Optical (NLO) behavior in terms of first-order hyperpolarizability, Molecular Electrostatic Potential (MEP) and dipole moment of title compounds. The chance of the title atoms being bio-active hypothetically incontestable by the electrophilicity index prompts additional property studies for the chosen compounds. The title molecules are found to suit well with L – glutamate oxidase inhibitor utilizing molecular docking techniques. The outcomes of the chosen organic alkane series are obtained utilizing higher basis set (6-311++G (d, 2p)) to realize results.

A Facile Method for the Fabrication of the Microneedle Electrode and Its Application in the Enzymatic Determination of Glutamate.

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.

A chemo-biocatalyst based on glutamate oxidase-integrated biomimetic trimanganese tetraoxide as cascade composite nano-catalyst for synthesis of α‑Ketoglutaric acid

The combination of chemo- and biocatalysts to perform one-pot synthetic route has presented great challenges for decades. Herein, glutamate oxidase (GLOX) and trimanganese tetraoxide (Mn3O4) nanocrystals were combined for the first time by one-step biomineralization to construct a mimic multi-enzyme system (GLOX@Mn3O4) for chemoenzymatic synthesis of α‑ketoglutaric acid (α‑KG). Mn3O4 not only served as a support for the enzyme immobilization, but also contributed its catalytic activity to co-operate with natural enzymes for the cascade reactions. The as-synthesized chemo-enzyme catalysts with directly contacted catalytic sites of the enzyme and inorganic catalyst maximizes the substrate channeling effffects for in situ rapid decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of sodium glutamate, thus relieving the inhibition of H2O2 accumulation for GLOX. Benefiting from the excellent stability and reusability of GLOX@Mn3O4, a nearly 100% conversion (99.7%) of l-glutamate to α-KG was achieved, over 4.7 times higher than that of the free GLOX system (21.2%). This work provides a feasibility for constructing a high-performance chemo-enzyme catalyst for cascade catalysis, especially for those reactions with toxic intermediates.

Alanine aminotransferase detection using TIT assisted four tapered fiber structure-based LSPR sensor: From healthcare to marine life

Alanine aminotransferase (ALT), a type of inactive enzyme largely present in fish liver cells, is essential for the tricarboxylic acid (TCA) cycle. Monitoring ALT activity in the blood/hepatocellular layer has been demonstrated to be a sensitive sign of liver dysfunction and an essential method for determining the health status of fish. This study details the development of a multi-layer material (hybrids of graphene oxide and multi-walled carbon nanotubes (GO/MWCNTs), gold nanoparticles (AuNPs), and glutamate oxidase (GluOx) enzyme) immobilized localized surface plasmon resonance based unique fiber structure biosensor for the quantitative determination of ALT biomolecules at concentrations ranging from 0 to 1000 U/L. For this kind of detection, a novel taper-in-taper with four tapered (TIT4T) structure based on single-mode fiber has been developed. In addition to AuNPs, GO/MWCNTs were immobilized in the probe's sensing region to increase its LSPR efficiency and sensitivity. Synthesis of AuNPs was carried out utilizing the Turkevich method. The selectivity of the sensor is ensured by the effective immobilization of GluOx on the surface treatment. The linearity of sensor is in the range of 0–1000 U/L, whereas the sensitivity, limit of detection, and detection time are individually found at 7.5 p.m./(U/L), 4.84 U/L and 20 min, respectively. After evaluating the prospective applications of the sensors, the sensors' reusability, reproducibility, stability, pH test, and selectivity have all been found to be satisfactory. Proposed fiber optic biosensors have high sensitivity, robustness, reliability, fast detection, no electromagnetic interference, low cost, real-time monitoring, and biocompatible.

Electrochemical study of the effect of radiofrequency on glutamate oxidase activity using a glutamate oxidase-based biosensor

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.

PtNPs/rGO-GluOx/mPD Directionally Electroplated Dual-Mode Microelectrode Arrays for Detecting the Synergistic Relationship between the Cortex and Hippocampus of Epileptic Rats.

Precise and directional couplings of functional nanomaterials with implantable microelectrode arrays (IMEAs) are critical for the manufacture of sensitive enzyme-based electrochemical neural sensors. However, there is a gap between the microscale of IMEA and conventional bioconjugation techniques for enzyme immobilization, which leads to a series of challenges such as limited sensitivity, signal crosstalk, and high detection voltage. Here, we developed a novel method using carboxylated graphene oxide (cGO) to directionally couple the glutamate oxidase (GluOx) biomolecules onto the neural microelectrode to monitor glutamate concentration and electrophysiology in the cortex and hippocampus of epileptic rats under RuBi-GABA modulation. The resulting glutamate IMEA exhibited good performance involving less signal crosstalk between microelectrodes, lower reaction potential (0.1 V), and higher linear sensitivity (141.00 ± 5.66 nA μM-1 mm-2). The excellent linearity ranged from 0.3 to 68 μM (R = 0.992), and the limit of detection was 0.3 μM. For epileptic rats, the proposed IMEA sensitively obtained synergetic variations in the action potential (Spike), local field potentials (LFPs), and glutamate of the cortex and hippocampus during seizure and RuBi-GABA inhibition. We found that the increase in glutamate preceded the burst of electrophysiological signals. At the same time, both changes in the hippocampus preceded the cortex. This reminded us that glutamate changes in the hippocampus could serve as important indicators for early warning of epilepsy. Our findings provided a new technical strategy for directionally stabilizing enzymes onto the IMEA with versatile implications for various biomolecules' modification and facilitated the development of detecting tools for understanding the neural mechanism.

Development of an Enzymatic Biosensor Using Glutamate Oxidase on Organic-Inorganic-Structured, Electrospun Nanofiber-Modified Electrodes for Monosodium Glutamate Detection.

Herein, dendrimer-modified montmorillonite (Mt)-decorated poly-Ɛ-caprolactone (PCL) and chitosan (CHIT)-based nanofibers were prepared. Mt was modified with a poly(amidoamine) generation 1 (PAMAMG1) dendrimer, and the obtained PAMAMG1-Mt was incorporated into the PCL-CHIT nanofiber's structure. The PCL-CHIT/PAMAMG1-Mt nanofibers were conjugated with glutamate oxidase (GluOx) to design a bio-based detection system for monosodium glutamate (MSG). PAMAMG1-Mt was added to the PCL-CHIT backbone to provide a multipoint binding side to immobilize GluOx via covalent bonds. After the characterization of PCL-CHIT/PAMAMG1-Mt/GluOx, it was calibrated for MSG. The linear ranges were determined from 0.025 to 0.25 mM MSG using PCL-CHIT/Mt/GluOx and from 0.0025 to 0.175 mM MSG using PCL-CHIT/PAMAMG1-Mt/GluOx (with a detection limit of 7.019 µM for PCL-CHIT/Mt/GluOx and 1.045 µM for PCL-CHIT/PAMAMG1-Mt/GluOx). Finally, PCL-CHIT/PAMAMG1-Mt/GluOx was applied to analyze MSG content in tomato soup without interfering with the sample matrix, giving a recovery percentage of 103.125%. Hence, the nanofiber modification with dendrimer-intercalated Mt and GluOx conjugation onto the formed nanocomposite structures was performed, and the PCL-CHIT/PAMAMG1-Mt/GluOx system was successfully developed for MSG detection.

Effect of dendrimer-based interlayers for enzyme immobilization on a model electrochemical sensing system for glutamate

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.

A dual enzyme-phosphate hybrid nanoflower for glutamate detection

The enzyme hybrid nanoflower has gained interests in biosensors due to their simple synthesis and high efficiency. In this study, glutamate oxidase (GLOX) and horseradish peroxidase (HRP) hybrid nanoflowers (GLOX&HRP-HNFs) were successfully prepared for the detection of glutamic acid (Glu). The effects of the synthesis conditions on the activity of GLOX & HRP-HNFs were investigated. Results revealed that the maximum activity of GLOX&HRP-HNFs was under 4 mM phosphate radical, 2.5 mM MnSO4, 0.04 mg/mL GLOX, and 0.16 mg/mL HRP. After immobilization, no significant differences were observed in optimum pH and temperature values of the GLOX and HRP. The GLOX&HRP-HNFs exhibited higher storage stability and resistance to organic solvents than free GLOX and HRP. Additionally, the GLOX&HRP-HNFs maintained 69% of its primary activity after 6 cycles. More important, the GLOX&HRP-HNFs exhibited a good linear range from 1 to 100 μM (R2 = 0.9979) and a low limit of detection (LOD) of 0.59 μM for glutamate. These results suggest that the GLOX&HRP-HNFs is a promising candidate for applications in biosensing for the detection of glutamate.

Construction of minitype glutamate sensor for in vivo monitoring of l-glutamate in plant

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 H2O2. 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.

Electrochemical Analysis Reveals Chlorpyrifos-Induced Dysregulation of Net Astrocytic Glutamate Uptake

Chlorpyrifos (CPF), an organophosphate pesticide widely used on food crops and in households, has been a critical public health concern due to its neurotoxic effects. Investigations of CPF-induced neurotoxicity have focused on dysregulation of acetylcholinergic signaling but recent work suggests that alterations in glutamatergic neurotransmission and astrocytic glutamate metabolism may occur. L-glutamate is a major excitatory neurotransmitter that is mostly removed from the synaptic cleft by astrocytic glutamate transporters. Here we assessed the effects of CPF on net glutamate uptake by human-induced pluripotent stem cell derived astrocytes using electrochemical analysis. Our microclinical analyzer (µCA) paired with screen-printed electrodes (SPE) offers an automated sensing system that can be used to monitor cellular metabolism and signaling. In this work, a platinum based SPE combined with the µCA was used to electrochemically quantify glutamate uptake in human astrocytes. Immobilized glutamate oxidase on electrode surfaces provided stable and sensitive glutamate detection. The temperature modification of these electrodes (37°C) led to a nearly four-fold improvement in sensitivity (0.84±0.04 nA/µM), with low limits of detection (2.8±0.1 µM), and quantitation (9.3±0.5 µM). The linear range of the sensor (1-667 µM) is on the same order as typical extracellular glutamate concentrations. These sensors were used to assess the net glutamate uptake efficiency by using escalating concentrations of glutamate (10-200 µM). Glutamate uptake was most efficient when astrocytes were exposed to 50 µM glutamate for 30min (50% net uptake). We are investigating acute and chronic effects of net glutamate uptake using sublethal doses of CPF (0-100 µM). Identifying these cellular mechanisms affected by CPF is critical for the understanding of the impact of organophosphate toxicity.

An optimized method for estimating glutaminase activity in biological samples

Spectrophotometric methodologies have been used to assess glutaminase activity, for which coloured complexes have been developed that measure spectrophotometry across the visible spectrum using different reagents. The present paper describes a precise, simple and reliable procedure for quantifying glutaminase activity, which is a key enzyme in glutamine hydrolysis and also involved in glutamine metabolism regulation.The procedure presented here measures glutaminase activity by incubating glutaminase enzyme at 37 °C for 20 min with a glutamine substrate dissolved in a buffer (pH 8.6). The enzymatic reaction contains suitable activity of glutamate oxidase, which acts to convert glutamate to hydrogen peroxide and 2-oxoglutarate. To terminate the enzymatic activity, a working solution containing pyridine-2,6-dicarboxylic (PDA) acid and ammonium vanadate (AV) was added following incubation.Oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV), a stable orange-coloured chelate complex measuring 435 nm spectrophotometrically, was produced by the interaction between the generated hydrogen peroxide and the supplied reagent. Using the response surface methodology (RSM) as an indicator of the assay's accuracy, we employed the Box-Behnken design (BBD) to improve the method's design (the OPDV-Glutaminase assay). Improvement factors were the volume of working reagent solution (PDA/AV), volume of glutamate oxidase solution (GO), and incubation time. In matched samples, this novel method was verified against a Bland-Altman plot assessment of glutaminase activity using the indophenol methodology.A correlation value of 0.99 between the two methods' comparisons showed that the novel protocol was equally applicable to the reference method.

Enzyme Nanosheet-Based Electrochemical Aspartate Biosensor for Fish Point-of-Care Applications.

Bacterial infections in marine fishes are linked to mass mortality issues; hence, rapid detection of an infection can contribute to achieving a faster diagnosis using point-of-care testing. There has been substantial interest in identifying diagnostic biomarkers that can be detected in major organs to predict bacterial infections. Aspartate was identified as an important biomarker for bacterial infection diagnosis in olive flounder (Paralichthys olivaceus) fish. To determine aspartate levels, an amperometric biosensor was designed based on bi-enzymes, namely, glutamate oxidase (GluOx) and aspartate transaminase (AST), which were physisorbed on copolymer reduced graphene oxide (P-rGO), referred to as enzyme nanosheets (GluOx-ASTENs). The GluOx-ASTENs were drop casted onto a Prussian blue electrodeposited screen-printed carbon electrode (PB/SPCE). The proposed biosensor was optimized by operating variables including the enzyme loading amount, coreactant (α-ketoglutarate) concentration, and pH. Under optimal conditions, the biosensor displayed the maximum current responses within 10 s at the low applied potential of −0.10 V vs. the internal Ag/AgCl reference. The biosensor exhibited a linear response from 1.0 to 2.0 mM of aspartate concentrations with a sensitivity of 0.8 µA mM−1 cm−2 and a lower detection limit of approximately 500 µM. Moreover, the biosensor possessed high reproducibility, good selectivity, and efficient storage stability.