Determination of myo-inositol in a flow-injection system with co-immobilized enzyme reactors and amperometric detection

A selective and sensitive flow-injection system for the determination of myo-inositol (hexahydroxycyclohexane) is described. Inositol dehydrogenase, IDH, lactate dehydrogenase, LDH, and lactate oxidase, LOD, are co-immobilized on porous glass and used in a packed-bed enzyme reactor. myo-Inositol reacts to produce an equivalent amount of hydrogen peroxide, which oxidizes hexacyanoferrate(II) to hexacyanoferrate(III) in a second reactor containing immobilized peroxidase. The hexacyanoferrate(III) is then detected amperometrically at 0 mV vs. SCE in a flow-through detector. The system responds linearly to injected samples of myo-inositol (25 μl) in the concentration range 1–300 μM. The maximum throughput was 90 samples per hour. The IDH/LDH/LOD reactor was stable for at least 5 weeks.     

基于丙酮酸/还原型辅酶I/乳酸脱氢酶/氧化型辅酶I/乳酸正逆反应同时测定丙酮酸/乳酸的酶荧光毛细管分析

利用丙酮酸(PA)/还原型辅酶I(NADH)/乳酸脱氢酶(LDH)/氧化型辅酶I(NAD+)/乳酸(LA)荧光反应体系的正逆反应,建立了一种可直接用于临床检验、能同时测定血清中微量PA/LA的酶荧光毛细管分析法.本方法可在常规荧光光度计上,用普通玻璃毛细管同时实现了PA/LA的高灵敏分析,每次分析试剂和样品的用量仅9 ...     

Spectrophotometric cell comprising parallel rotating and stationary bioreactors: Application to the determination of glucose in serum samples

A spectrophotometric cell comprising parallel bioreactors facing each other and containing immobilized enzyme preparations is described. The lower reactor rotates to minimize diffusional constraints, and the upper reactor is fixed to provide an integrated design for the realization of coupled enzyme-catalyzed reactions. The operating characteristics of the cell are illustrated with the determination of glucose using glucose oxidase [EC 1.1.3.4] and horseradish peroxidase [EC 1.11.1.7] as immobilized enzymes (horseradish peroxidase on the rotating reactor and glucose oxidase on the stationary one). The H₂O₂ produced in the dissolved-oxygen oxidation of β- -glucose enters into oxidative coupling in a reaction with N,N-dimethylaniline and 4-aminophenazone which is catalyzed by horseradish peroxidase; the absorbance of the colored complex formed provides the basis for monitoring. The cell was incorporated into a continuous-flow/stopped-flow/continuous-flow operation, and the determination was based on the rate of response under stopped-flow conditions. The overall approach was applied to the determination of glucose in standards of human serum and samples of bovine blood serum.     

Novel Multiplexed Biosensor System for the Determination of Lactate and Pyruvate in Blood Serum

Multiplexing is one of the main current trends in biosensors, especially important for clinical diagnosis. However, simultaneous determination of several substances in one sample is often difficult due to different performance and working conditions of separate biosensors. This work was aimed at the development of a multiplexed biosensor system for the determination of lactate and pyruvate concentrations in liquid samples (i. e., blood serum). The system consisted of two amperometric biosensors based on lactate oxidase and pyruvate oxidase, which worked simultaneously in a single measuring cell. Conditions for the biosensor system work were selected. Linear range of lactate determination was 5–1000 μM, pyruvate – 10–5000 μM. Steady‐state response time was 30 s and 50 s for the lactate and pyruvate biosensors, respectively. After 2 weeks of storage biosensor responses decreased to 95 % (lactate biosensor) or 82 % (pyruvate biosensor) of the initial value. A scheme of analysis of the concentrations of lactate and pyruvate in human blood serum was proposed. The lactate and pyruvate concentrations as well as their ratio in human blood serum samples were determined and compared with the control method. The proposed biosensor system is suitable for the rapid detection of lactate, pyruvate and their ratio and can be used for clinical diagnosis, e. g., evaluation of the reasons of lactic acidosis and prognosis of patient's recovery.     

Optimization of flow-injection systems for determination of substrates by means of enzyme amplification reactions and chemiluminescence detection

A flow-injection system is described that incorporates a small column reactor containing two co-immobilized, synergistically operating oxidoreductases, allowing determination of minute amounts of substrates by means of enzyme amplification and subsequent chemiluminescence detection of the hydrogen peroxide generated in the repeated redox cycling. With lactate oxidase and lactate dehydrogenase, and taking advantage of the fact that the enzymatic degradation step and the ensuing detection step can be individually optimized, the FIA-system has been optimized by factorial experiments to yield an amplification factor of over 140 for each of the two substrates lactate and pyruvate. With a linear calibration range of 0-6muM, the limits of detection for the two species were 48 and 103nM, respectively, and the sampling rate was 50-60/hr. The optimized system has also been employed for assay of glucose by utilizing a column reactor with immobilized glucose oxidase and glucose dehydrogenase, but yielded amplification factors of only 3-4. The large discrepancy in the performance of the two enzyme systems is discussed.     

Bioluminescent flow sensor for the determination of L-(+)-lactate

The amount of L-lactate in biological fluids (serum, plasma and cerebrospinal fluid) was determined by monitoring the reduced form of nicotinamide adenine dinucleotide (NADH) produced by immobilised lactate dehydrogenase (LDH), with bacterial bioluminescent enzymes immobilised on a separate nylon coil. The LDH catalysed the reaction of L-lactate with NAD; this reaction took place in a nylon coil that preceded the coil for the bioluminescent detection. The co-immobilisation of alanine aminotransferase (ALT) with LDH improved the lactate transformation by 117-183%. The response was linear from 0.1 to 50 micron mol l(-1) at 25 degrees C for the LDH - ALT reactor. The intra- and inter-assay coefficients of variation were less than 5% and the recoveries ranged from 93 to 106%. The results agreed well with those obtained with a spectrophotometric method and with the normal reference values.     

Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.     

流动注入式乳酸生物传感器

研制了一种测定L－乳酸的生物传感器,将乳酸氧化酶（LOD）通过共价键固定在尼龙钢上制备乳酸氧化酶膜,将制得的酶膜固定在氧电极上构成乳酸生物传感器;将透析膜放在氧化酶膜上产生对L－乳酸扩散高度限制来改变该生物传感器的响应;酶膜机械强度高,在氧电极上反复装卸而不损坏,所构成的乳酸生物传感器的校正曲线的乳酸定量上限达5mmol/L,响应时间小于30s;初步血样测试的结果显示该乳酸生物传感器用于临床血乳酸的测定具有可行性。     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Galactose determination in an automated flow-injection system containing enzyme reactors and an on-line dialyzer

d-Galactose is oxidized in a reactor containing immobilized galactose oxidase to give hydrogen peroxide, which is determined spectrophotometrically after an oxidative colorforming reaction in a peroxidase reactor. On-line treatment in a dialyzer, a metal-chelate column and a catalase reactor makes the method essentially free from interferences for serum samples, for example. The effect of catalase impurities in the galactose oxidase preparation is illustrated. The linear range for the determination of d-galactose was from 10 μM to 14 mM, and the recovery from spiked serum samples, at low galactose levels, was close to 100%. The sample frequency was 45 h^?1 and the sample volume was 160 μl. The sampler, the pump and the valves in the flow systm were controlled by a personal computer which also collected and analyzed the data.     

Immobilization of lactate as an electroactive indicator on pencil graphite electrode for the development of a new electrochemical biosensor for the detection of lactate dehydrogenase

A biosensor was developed for the detection of lactate dehydrogenase (LDH) enzyme using a lactate modified pencil graphite electrode (PGE). The sensor relies on the immobilization of the lactate on PGE, and LDH detection is based on the decrease of lactate peak current following oxidation to pyruvate in the presence of LDH. Square wave voltammetric technique was used for the assay of signals in the range of ?0.6 to 0.8 V and a frequency of 25 Hz for the determination of LDH. The dependence of the response was investigated in terms of reaction time, washing time and LDH and NAD⁺ amounts. Also, the electrochemical behavior of LDH treatment on the lactate modified PGE was studied. The electrode showed good selectivity, repeatability and an operational stability of about 90% of its original response for two weeks. Moreover, the sensor displayed a linear response range from 0.36?C2.13 U ??l^?1 for LDH with a detection limit of 0.16 U ??l^?1. The response time of the LDH-treated lactate modified PGE was found to be 2 s. The relative standard deviation (RSD) obtained was 3.5% (for LDH 0.71 U ??l^?1 and n = 3).     

Amperometric determination of lactate dehydrogenase based on a carbon fiber microcylinder electrode modified covalently with Toluidine Blue O by acylation

A method has been developed for the modification of a carbon fiber microcylinder electrode with acylation. The stability and surface coverage of the Toluidine Blue O-modified microelectrode were studied by cyclic voltammetry. The modified electrode showed significant activity for the electrocatalytic oxidation of NADH in pH 6.8-7.8 solution. The catalytic current increased linearly with increasing concentration of NADH from 4.0 x 10(-5) to 1.5 x 10(-3) M. A simple amperometric determination based on electrochemical detection of NADH produced from the enzymatic reaction of lactate with NAD(+) under the catalysic effect of lactate dehydrogenase (LDH) is reported. The experimental factors which had primary influence on the analytical performance were studied. The sensor had a linear response over a range of LDH concentrations from 5.0 U l(-1) to 200 U l(-1) at -0.2 V vs. SCE under optimum conditions. A satisfactory result was obtained for the determination of LDH in clinical blood samples.     

微量乳酸脱氢酶毛细管电泳在线反应电化学检测方法的研究

以乳酸脱氢酶催化乳酸与NAD⁺反应生成丙酮酸与NADH和安培法检测NADH为基础,利用电泳中介微分析(EMMA)技术,研究了超微量乳酸脱氢酶的毛细管电泳在线反应的电化学检测方法,并从理论上对EMMA电泳图中的平台宽度和高度作了初步探讨.结果表明,在EMMA的恒高压和零高压两种模式下,使用直径为150μm和束状碳纤维电极,在+0.8V检测电位下,对LDH活性检测灵敏度分别为1.1nU和0.6nU;所导出的平台高度、宽度与实验条件的关系式对提高毛细管电泳分离效率和检测灵敏度有一定指导意义.     

Chronoamperometric stripping analysis following biocatalytic preconcentration

Electrodes containing glucose oxidase or xanthine oxidase adsorbed on modified glassy carbon electrodes or on conductive complexes, accumulate charge in the presence of substrates, the discharge of which gives the chronoamperometric stripping current. This current is 15 times higher than the stationary current after preconcentration for 8 min. A ten-fold increase in sensitivity of the determination of glucose or hypoxanthine is observed. The stripping current of electrodes based on cytochrome b₂ adsorbed on a graphite electrode is 25 times higher than the stationary current. The electrode is useful for lactate determinations for more than 7 days.     

Prussian Blue-Based Thin-Layer Flow-Injection Multibiosensor for Simultaneous Determination of Glucose and Lactate

A planar multibiosensor for the simultaneous determination of glucose and lactate is developed by combining the Prussian Blue-based electrocatalyst and the protocol for immobilization of glucose oxidase and lactate oxidase enzymes from solutions with a high content of an organic solvent. High sensitivity coefficients (>80 mA M^–1 cm^–2 for lactate and >20 mA M ^–1 cm^–2 for glucose) are demonstrated by the multibiosensors operating in the flow-injection mode in a thin-layer measuring cell. The linear range of the analyzed concentration is 1–500 μM for lactate and 5–1000 μM for glucose. A multibiosensor can be used repeatedly (the exhibited operational stability is not less than 100 measurements without the need for recalibration), which allows using it for the analysis of diluted blood samples and blood serum. The electrocatalytic system with a multibiosensor demonstrates performance characteristics that are superior to the commercial analyzers.     

Lactate Biosensor Based on Hydrotalcite‐Like Compounds: Performances and Application to Serum Samples

A lactate biosensor based on lactate oxidase supported onto a hydrotalcite, electrochemically deposited on a platinum surface, was developed for the first time. For the best electrode configuration, a linear response up to 0.8 mM, with a limit of detection of 14 μM and a sensitivity of 91 mA M^?1 cm^?2, was obtained. The influence of some interferents due to the oxidation of hydrogen peroxide (at +0.35 V vs. SCE) was also studied. By controlling carefully the experimental conditions, the determination of lactate in a commercial serum sample in the presence of interferents was successfully accomplished.     

Potentiometric determination of glucose by enzymatic oxidation in a flow system

A potentiometric determination is described for glucose based on oxidation by 1,4-benzoquinone with immobilized glucose oxidase as catalyst in an enzyme reactor. The electrode is preceded by an analytical dialysis unit to remove proteins. The ratio of quinone to hydroquinone was measured with a flow-through gold electrode. Another gold electrode preceded the enzyme reactor to correct for serum components (e.g. ascorbic acid) which can also reduce quinone. The operating range is 0.04–10 × 10^-3 M β-D-glucose. The dialysis proceeds with a linear dependence on glucose concentration, and the dialysis ratio can be adjusted by changing the buffer flow rate.     

A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate

A novel replaceable dual-enzyme capillary microreactor was developed and evaluated using magnetic fields to immobilize the alcohol dehydrogenase (ADH)- and lactate dehydrogenase (LDH)-coated magnetic beads at desired positions in the capillary. The dual-enzyme assay was achieved by measuring the two consumption peaks of the coenzyme β-nicotinamide adenine dinucleotide (NADH), which were related to the ADH reaction and LDH reaction. The dual-enzyme capillary microreactor was constructed using magnetic beads without any modification of the inner surface of the capillary, and showed great stability and reproducibility. The electrophoretic resolution for different analytes can be easily controlled by altering the relative distance of different enzyme-coated magnetic beads. The apparent K(m) values for acetaldehyde with ADH-catalyzed reaction and for pyruvate with LDH-catalyzed reaction were determined. The detection limits for acetaldehyde and pyruvate determination are 0.01 and 0.016 mM (S/N = 3), respectively. The proposed method was successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. The results indicated that combing magnetic beads with CE is of great value to perform replaceable and controllable multienzyme capillary microreactor for investigation of a series of enzyme reactions and determination of multisubstrates.     

Efficient mixing and reactions within microfluidic channels using microbead-supported catalysts

A strategy for efficiently mixing solutions and carrying out multistep catalytic reactions in microfluidic systems is described. The approach involves immobilizing catalysts on microbeads, placing the beads into well-defined microreactor zones, and then passing reactants through one or more of the reactor zones to yield products. The catalyst-modified beads effectively mix reactants and increase the effective surface area of the channel interior, both of which improve reaction velocities compared to open channels. This approach is demonstrated using two sequential reactions catalyzed by glucose oxidase and horseradish peroxidase. In addition to providing a general route to chemical synthesis within microfluidic systems, this design strategy may also be applicable to modeling reaction pathways within cells and to bio/chemical sensing applications.     

Determination of lactate or oxalate using injected lactate oxidase and peroxidase by capillary electrophoresis with UV detection

Two reactions, catalyzed by lactate oxidase (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2'-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the run buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD(+) which is separated and detected in less than 2 min at 266 nm with a sample throughput of 7 min (including wash steps between samples). Simplex trade mark software is used to optimize the enzyme concentrations and reaction temperature. Consumption of the more expensive LO enzyme is only 1.4 x 10(-3) U per assay assuming 27 nL per injection. Linearity is established within the range from 0.0025 to 1 mM with R(2) of 0.9982. Recoveries of lactate from five spiked serum samples averaged 101%. Application of this method for the determination of oxalate as an inhibitor of LO is demonstrated.