On-line microdialysis assay of l-lactate and pyruvate in vitro and in vivo by a flow-injection system with a dual enzyme electrode

A flow-injection biosensor system with an on-line microdialysis sampling is proposed for the simultaneous assay of l-lactate and pyruvate in serum and rat brain. The dialysate collected in the sample loop by perfusing Ringer’s solution through the microdialysis probe is automatically injected into the flow-injection line with a dual enzyme electrode arranged in parallel for the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film to two sensing parts, which respond selectively to l-lactate and pyruvate, respectively, without any cross-reactivity. Both the sensing parts respond linearly to the concentrations of both analytes between 0.01 and 5 mM, without any interference from oxidizable species and low-molecular weight proteins present in the dialysate. The proposed flow-injection analysis (FIA) method can be successfully applied to the simultaneous in vitro and in vivo assays of both analytes in serum and rat brain, respectively. The system can be automatically processed at an analytical speed of 19 dialysates h⁻¹ over a period of 5 h.     

Amperometric assay of glucose and lactic acid by flow injection analysis

A computer-controlled flow-injection system is described for the assay of D-glucose and L-lactic acid in undiluted plasma. Glucose or lactate is quantified by coupling an immobilized glucose oxidase or lactate oxidase membrane with an amperometric sensor; the hydrogen peroxide generated is directly related to the concentration of glucose or lactate. The linear range is 0–40 mM and 0–10 mM for glucose and lactic acid, respectively. The sample frequency is 60 h^?1 with a standard deviation of less than 1.5%. Correlation with the results for blood plasma obtained by routine clinical analyzers was good for both glucose and lactic acid.     

Optically specific detection of D- and L-lactic acids by a flow-injection dual biosensor system with on-line microdialysis sampling.

A flow-injection dual biosensor system with microdialysis sampling is proposed for the simultaneous determination of D-lactic and L-lactic acids. The dialysate from the microdialysis tube is delivered to a sample loop of the six-way autoinjector and then automatically injected into the flow-injection line with a dual enzyme electrode arranged in perpendicular to the flow direction. The dual enzyme electrode is constructed by hybridizing a poly(1,2-diaminobenzene) film into two sensing parts which respond selectively to D-lactic and L-lactic acids, respectively, without any cross-reactivity. The proposed flow-injection analysis method can be successfully applied to the simultaneous determination of D,L-lactic acids in alcoholic beverages.     

Simultaneous determination of sulfite and phosphate in wine by means of immobilized enzyme reactions and amperometric detection in a flow-injection system

A flow-injection system is proposed for the simultaneous determination of sulfite and phosphate in wine. A sulfite oxidase immobilized reactor and purine nucleoside phosphorylase-xanthine oxidase co-immobilized reactor are incorporated at fixed positions (parallel configuration) in the flow line, which is based on the splitting of the flow after sample injection and subsequent confluence. A poly(1,2-diaminobenzene)-coated platinum electrode is used as an amperometric detector to detect selectively hydrogen peroxide generated enzymatically in the enzyme reactors, without any interference from oxidizable species and proteins present in wine. Because each channel has a different residence time, two peaks are obtained. The first peak corresponds to sulfite and the second peak to phosphate. The peak current is linearly related to the concentrations of sulfite between 1 × 10⁻⁵ and 2 × 10⁻³M and phosphate between 2 × 10⁻⁵ and 5 × 10⁻³M. The simultaneous determination of sulfite and phosphate in wine can be performed at a rate of 30 samples/hr with satisfactory precision (less than 1.2% RSD) and no pretreatment except for the sample dilution.     

Simultaneous assay of glucose,lactate, <Emphasis Type="SmallCaps">L</Emphasis>-glutamate and hypoxanthine levels in a rat striatum using enzyme electrodes based on neutral red-doped silica nanoparticles

An electrochemical method suitable for the simultaneous measurement of cerebral glucose, lactate, L-glutamate and hypoxanthine concentrations from in vivo microdialysis sampling has been successfully performed for the first time using a neutral red-doped silica (NRDS) nanoparticle-derived enzyme sensor system. These uniform NRDS nanoparticles (about 50±3 nm) were prepared by a water-in-oil (W/O) microemulsion method, and characterized by a TEM technique. The neutral red-doped interior maintained its high electron-activity, while the exterior nano-silica surface prevented the mediator from leaching out into the aqueous solution, and showed high biocompability. These nanoparticles were then mixing with the glucose oxidase (GOD), lactate oxidase (LOD), L-glutamate oxidase (L-GLOD) or xanthine oxidase (XOD), and immobilized on four glassy carbon electrodes, respectively. A thin Nafion film was coated on the enzyme layer to prevent interference from molecules such as ascorbic acid and uric acid in the dialysate. The high sensitivity of the NRDS modified enzyme electrode system enables the simultaneous monitoring of trace levels of glucose, L-glutamate, lactate and hypoxanthine in diluted dialysate samples from a rat striatum.     

Flow injection analysis for glucose by the combined use of an immobilized glucose oxidase reactor and a peroxidase electrode

The amperometric peroxidase electrode measures hexacyanoferrate(III), produced by hydrogen peroxide, which is generated by injecting a 2μl sample into a reactor of immobilized glucose oxidase covalently bound to silica gel. The peak current is linearly related to the glucose concentration in the range 0.05–10 g l^?1; sample throughput is about 100 h^?1. Ascorbic acid (? 0.5 mM) does not interfere.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

Characterization of immobilized enzymes by flow-injection techniques with variable forward flow

The use of variable forward flow with small packed enzyme reactors is shown to be valuable for improving the efficiency of enzymatic conversion. Designs with stopped flow, oscillating flow and variable (fast/slow/fast) flow are compared for the spectrophotometric determination of glucose with glucose oxidase and horseradish peroxidase immobilized on controlled-pore glass in the same reactor. Variable forward flow increased the sensitivity considerably without excessive time consumption. The technique is also useful for characterizing the activity of immobilized enzyme reactors, e.g., peroxidase reactors for hydrogen peroxide determinations.     

Immobilized enzymes in clinical and biochemical analysis : Applications to the Simultaneous Determination of Acetylcholine and Choline and to Determination of Lipids

Uses of immobilized enzyme mini-columns in flow-injection systems are described Simultaneous determination of ? × 10^?5 M choline and acetylcholine is achieved by using acetylcholinesterase and choline oxidase columns. A home-made amperometric detector is used to detec the hydrogen peroxide produced enzymatically. An ion-exchange column is used on-line to remove interferences at the amperometric detector during analysis of blood and brain samples. Immobilization of the lipid enzymes phospholipase-C and -D is described. These enzymes are used for the determination of phospholipids. Total phospholipids (1– mM) are determined with a combination of phospholipase-D, lipase and glycerol-3-phosphate oxidase. All the methods described are simple and reproducible and the immobilized enzymes show good stability.     

Amperometric determination of glucose in undiluted food samples

Glucose oxidase is immobilized onto a cellulose acetate membrane by glutaraldehyde linkage, and the membrane is used to cover the platinum electrode of a hydrogen peroxide sensor. A silanized polycarbonate membrane then covers the enzyme layer, and extends the linear calibration range to higher concentrations. The sensor, when incorporated into a flow-injection system, allows the determination of glucose at levels up to 1 M in soft drinks at a rate of 60 samples h^?1 without sample dilution.     

Development of biosensors based on hexacyanoferrates

Ferric and copper hexacyanoferrates (PB and CuHCF, respectively) were electrodeposited on glassy carbon electrodes providing a suitable catalytic surface for the amperometric detection of hydrogen peroxide. Additionally glucose oxidase was immobilized on top of these electrodes to form glucose biosensors. The biosensors were made by casting glucose oxidase-Nafion layers onto the surface of the modified electrodes. The operational stability of the films and the biosensors were evaluated by injecting a standard solution (5 muM H(2)O(2) for PB, 5 mM H(2)O(2) for CuHCF and 2.5 mM glucose for both) over 5-10 h in a flow-injection system with the electrodes polarized at -50 (PB) and -200 mV (CuHCF) versus Ag/AgCl, respectively. The glucose biosensors demonstrated suitability for glucose determination: 0.0-2.5 mM (R(2)=0.9977) for PB and 0.0-10 mM (R(2)=0.9927) for CuHCF, respectively. The visualization of the redox catalyst modifiers (PB and CuHCF films) was presented by scanning electron micrographs.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.     

Micro-flow in vivo analysis of L-glutamate with an on-line enzyme amplifier based on substrate recycling.

A micro-flow enzyme system with a microdialysis probe is proposed for the amperometric detection of trace amounts of neurotransmitter L-glutamate released from rat brain cells. The L-glutamate oxidase (EC 1.4.3.11)/glutamate dehydrogenase (EC 1.4.1.4) coimmobilized reactor was used to enhance the sensitivity of L-glutamate as an on-line amplifier based on substrate recycling. A poly(1,2-diaminobenzene) film-coated platinum electrode was also used to selectively detect only the hydrogen peroxide generated into a upstream enzyme reactor, without interference from oxidizable species, such as L-ascorbate, the adsorption of low molecular-weight proteins in a dialysate, and NADPH added to the carrier solution to initiate substrate recycling. By the present in vivo system, L-glutamate was selectively assayed with about a 600-fold increase in sensitivity compared with the unamplified responses. The detection limit was 0.08 mumol dm-3. This method was applied to an in vivo assay of L-glutamate in the extracellular space of rat brain; also, monitoring of the L-glutamate level changed after a continuous stimulation of KCl to demonstrate the reliability of the system.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Comparative study of first-, second- and third-generation amperometric glucose enzyme electrodes in continuous-flow analysis of undiluted whole blood

First-, second- and third-generation amperometric glucose enzyme electrodes were compared under flow-injection and steady-state conditions for the monitoring of undiluted whole blood. First-generation electrodes, based on the detection of hydrogen peroxide at a platinum electrode, are generally unsuitable because of the eventual poisoning of the electrode and because of their susceptibility to oxygen variation. Second-generation electrodes in which a mediator is used for the reoxidation of glucose oxidase are more suitable for the analysis of whole blood under both steady-state and flow-injection conditions. However, the choice of mediator is important. The best results with regard to linear range and stability were obtained with tetrathiafulvalene, whereas dimethylferrocene required considerable pretreatment before use. A third-generation electrode based on tetrathiafulvalene-tetracyanoquinodimethane where direct oxidation of glucose oxidase occurs also proved useful but showed lower stability and a smaller dynamic range compared with the second-generation devices. Flow-injection and steady-state studies were carried out using wall-jet cell geometry.     

Fluorometric determination of ethanol in liquor samples by flow-injection analysis using an immobilized enzyme-reactor column with packing prepared by coupling alcohol oxidase and peroxidase onto chitosan beads

A flow-injection system was developed for the determination of ethanol with an immobilized enzyme-reactor column. This system, which consisted of hand-made reactor columns packed with alcohol oxidase and horseradish peroxidase immobilized onto chitosan beads, and a fluorometric detector, was applied to the determination of ethanol in liquor samples. Under the recommended conditions, the ethanol, which was present in the pretreated samples, was converted to hydrogen peroxide when it was passed through the immobilized alcohol oxidase (AOD) column with 0.1 mol/dm3 phosphate buffer (pH 7.0). A sample can be analyzed with this system in <10 min. The calibration curve for ethanol was linear from 2.0 to 0.1 mg/dm3. The determination limit, which was defined by the difference between the sample peak and blank peak, was estimated to be 50 microg/dm3 for ethanol. Interferences from some substances present in actual liquor samples decreased the analytical response and activity of the immobilized AOD-reactor column, but they were removed by dilution and pretreatment with an octyldecylsilane cartridge.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Determination of fructose using immobilized glucose isomerase in an on-line analyzer

The determination of fructose using a continuous analyzer based on analyte conversion in enzyme reactors followed by amperometric oxygen measurement is described. Two experimental setups were compared, allowing determinations in the ranges 0–180 and 0–25 mM fructose. In the former, fructose was continuously dialyzed versus a buffer stream conducting fructose to an enzyme reactor. This reactor contained two immobilized enzyme preparations, one with immobiized glucose isomerase (E.C. 5.3.1.5) that isomerized fructose to glucose and another that subsequently oxidized the former glucose by immobilized glucose oxidase (E.C. 1.1.3.4) with the consumption of dissolved oxygen. In the latter set-up, fructose was first isomerized in a glucose isomerase reactor, then glucose was continuously dialyzed and oxidized by glucose oxidase as above. This set-up was run in continuous operation for 1000 measurement cycles with a total decrease in response less than 15%.     

Highly sensitive detection of l-glutamate by on-line amperometric micro-flow analysis based on enzymatic substrate recycling

A highly selective and sensitive on-line monitoring system is proposed for amperometric assay of trace amounts of l-glutamate. The system includes a microdialysis probe, immobilized enzyme reactor, and poly(1,2-diaminobenzene)-coated platinum electrode. The enzyme reactor prepared by the co-immobilization of l-glutamate oxidase and glutamate dehydrogenase are here employed to enhance the sensitivity of l-glutamate as an on-line amplifier based on the substrate recycling. The l-glutamate in the dialysate from the probe are recycled enzymatically during passage through the reactor in the presence of sufficient amounts of NADH and oxygen to produce a large amount of hydrogen peroxide, which is detected if selectively at a downstream poly(1,2-diaminobenzene)-coated platinum electrode without interference from oxidizable species such as l-ascorbate in the sample and NADH added to the carrier buffer. The cycle is also initiated with 2-oxoglutarate, and so saccharopine dehydrogenase reactor is positioned in series before the amplifier reactor to remove 2-oxoglutarate in the dialysate. By the present method, l-glutamate is selectively assayed with a 160-fold increase in sensitivity compared with the unamplified responses. The detection limit is 0.5x10(-7) M of l-glutamate.