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.     

Exchangeable immobilized enzyme reactor for enzyme inhibition tests in flow-injection analysis using a magnetic device. Determination of pesticides in drinking water

A flow-injection system for rapid automated enzyme inhibition testing is described. Exchange of inactivated enzyme immobilized on magnetic particles was performed with magnetic devices which could be electrically switched off to release all bound material. The flow resistance of the reactor was excellent. Inhibition of immobilized acetylcholinesterase was used to determine pesticides in drinking water. Concentrations of 0.5 μg l^?1 of the pesticides carbofuran and malaoxon were detected. A complete cycle of analysis, including calibration, took 20 min.     

A sensitive continuous-flow bioluminescent system for determining ethanol in serum and saliva

A continuous-flow system, based on NAD(P)H:FMN oxidoreductase and bacterial luciferase co-immobilized on a nylon coil placed in front of a photomultiplier, and alcohol dehydrogenase separately immobilized on a second nylon coil, is described for the assay of ethanol in serum and saliva. The flow is air-segmented and 5–50-μl samples are required. The sample throughput is 25–30 h^?1 with no carryover. The detection limit is 1 μmol l^?1 ethanol (50 pmol injected) and the response is linear between 50 and 2500 pmol of ethanol. The relative standard deviation is 3–10% for both within-day and between-day assays, and serum and saliva can be analyzed directly. The immobilized enzymes are satisfactorily stable and up to 900 samples can be analyzed with one enzyme reactor.     

Spectrophotometric flow-injection determination of urea in body fluids by using an immobilized urease reactor

A flow system involving a packed-bed enzyme reactor (volume 180 μl) with urease immobilized covalently on poly(glycidyl methacrylate)-coated porous glass is used for determining urea in blood serum and urine. Enzymatically produced ammonia is converted to an indophenolate dye (by oxidative coupling with hypochlorite and sodium salicylate), which is detected spectrophotometrically at 700 nm. The calibration graph is rectilinear for 25–500 μM urea when injecting samples (75 μl) diluted 1:50 for serum or 1:1000 for urine at a frequency of 60 h^?1; the relative standard deviation is 1.1% for ten injections of 300 μM urea. The immobilized urease is stabilized by the addition of disodium EDTA, sodium azide and 2-mercaptoethanol to a 0.2 M phosphate buffer (pH 6.9) used as the carrier stream, which serves also as a preservative for longterm storage of the urease reactor packing at 4°C.     

Flow injection analysis for glucose and urea with enzyme reactors and on-line dialysis

A flow injection system for glucose and urea determination is described. The glucose determination uses immobilized glucose oxidase in a reactor designed to give 100% substrate conversion. The hydrogen peroxide formed is converted to a coloured complex with 4-aminophenazone and N,N-dimethylaniline. The coupling is catalysed by a reactor containing immobilized peroxidase. The coloured complex is measured in a flow-through spectrophotometric cell. Urea is converted to ammonia in a reactor with immobilized urease and detected with an ammonia gas membrane electrode. Proteins and other interfering species from serum samples are removed in an on-line dialyzer. Calibration curves are linear for glucose in the range 1.6 × 10^-4–1.6 × 10^-2 M and for urea in the range 10^-4–10^-1 M. The samples are 25 μl for glucose determination and 100 μl for urea determination. Linear ranges can be changed by varying the sample sizes. The effects of the dialyser, enzyme reactors and detectors on dispersion are evaluated.     

Continuous-Flow Determination of Phenol With Chemically Immobilized Polyphenol Oxidase (Tyrosinase)

Abstract

The use of covalently bound mushroom polyphenol oxidase (tyrosinase, EC 1.10.3.1) for the determination of μg/mL and ng/mL concentrations of phenol in water samples with use of continuous-flow sample/reagent processing is described. Immobilization on controlled-pore glass, CPG, was accomplished via diazo coupling. Detection was effected with hexacyanoferrate(II) as a redox mediator and was either spectrophotometric or amperometric. the immobilized enzyme preparation was part of an open tubular reactor (CPG thermally embedded on Tygon tubing). the redox mediator was used either in solution or as part of a thin-layer cell and immobilized on poly(4-vinylpyridine) incorporated in a carbon paste electrode. Different spectrophotometric and amperometric strategies are compared and the method is applied to the determination of phenol in water samples and quality control standards.     

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β-d-glucose. The detection limit was 0.25 μM β-d-glucose for 50-μl samples. The electrode was kept at ?50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.     

Electrochemical Measurements of Glucose Using a Micro Flow‐Through Immobilized Enzyme Reactor

A new fast, cheap and efficient epoxy‐amine immobilized enzyme reactor (IMER) is demonstrated. Polyethyleneimine (PEI) was used as the curing agent of an epoxy resin (bisphenol‐A diglycidyl ether (BADGE)) in order to introduce a high number of reactive –NH₂ groups at the substrate. A ratio mixture of 3:2 PEI:BADGE (w/w) was shown to be the most suitable for curing, taking into account the number of immobilization sites and the resistance of the material towards channeling. Electrochemical measurements made by a three electrode system, adapted at the IMER, showed that the K_m value for immobilized glucose oxidase (GOx) was half the value commonly reported for GOx immobilized at PEI derived substrates. The IMER response decreased by around 41 % after one week of intense usage. Even so, the remaining activity was sufficient for quantifications of approximately 0.1 mmol L⁻¹, merely requiring a new calibration of the reactor before its utilization. The developed system was applied to D‐glucose quantification of beverage samples, requiring an injection volume of only 10 μL and a flow rate of 150 μL min⁻¹ (almost 60 samples per hour). Low detection and quantification limits (1.94 and 5.89 μmol L⁻¹, respectively) and a wide linear range (from 8 μmol L⁻¹ to 2 mmol L⁻¹) were also found, which are useful characteristics for quality control analysis.     

Bound coefactor/dual enzyme electrode system for l-alanine

A dual enzyme-bound coenzyme electrode system for quantifying l-alanine is described. Commercially available dextran-bound NAD was incorporated into an l-alanine dehydrogenase (E.C. 1.4.1.1)/l-lactate dehydrogenase (E.C. 1.1.1.27) enzyme system and held at the surface of a potentiometric ammonia gas sensor. Using this system, l-alanine calibration curves with a slope of 45 mV/decade and 10^?5 M detection limit were obtained with a sensor lifetime of at least 10 days. This system is potentially useful for the clinical determination of l-alanine in serum.     

An enzymatic flow injection method for the determination of oxygen

An enzymatic flow injection method for the determination of dissolved oxygen is described. Oxygen from the sample is reduced quantitatively to hydrogen peroxide in a packed-bed reactor containing immobilized glucose oxidase β-d-Glucose is used as a cosubstrate. The effluent is mixed with a stream containing chromogens and fed into a second reactor containing immobilized peroxidase. The coloured product formed is monitored spectrophotometrically. The response is linear from the detection limit (2–5 μM O₂ to air-saturated samples (0.3 mM O₂) when the peak areas are plotted versus the O₂ content in the samples (50 μl). The maximum speed for 1% carry-over is 60 samples per hour and the results are available 25 s after the start of sampling. Broadening of the peak is caused by adsorption of the coloured product in the peroxidase reactor.     

Spectrophotometric determination of ethanol in blood using a flow-injection system with an immobilized enzyme (alcohol dehydrogenase) reactor coupled to an on-line dialyser

The determination of ethanol in whole blood without pretreatment using spectrotometric detection with an immobilized enzyme reactor coupled to an on-line dialyser is described. Optimum working conditions are given. The effects of the injection volume and the flow-rate on the peak height using the dialyser were investigated. A good linear calibration graph over the ethanol concentration range 3–40 μg ml^?1 was observed; these concentrations correspond to 0.3?4.0 g of ethanol per 1000 g of whole blood prior to dilution. For comparison, whole blood samples were analysed by gas chromatography with direct injection. The effect of temperature on the peak height was also studied in a system without the dialyser.     

A post-column enzyme reactor for detection of oxidized cholesterols in h.p.l.c. separations

An enzyme reactor is used as a post-column detector after the h.p.l.e. separation of a mixture of cholesterol and some auto-oxidation products. The enzyme reactor contains cholesterol oxidase immobilized on controlled-pore glass. A method for purification of the enzyme is described. The properties and the solvent dependence of the reactor are discussed. A post-column system for dilution of the eluent with aqueous buffer is described.     

A sensitive and accurate automatic coulometric detector system for enzymatically produced carbon dioxide

A system for the determination of carbon dioxide liberated in an enzyme reactor is described. The properties of the system are illustrated by the determination of urea. Enzymatically generated carbon dioxide is expelled by boiling, dried and determined coulometrically. The lowest concentration of urea which can be determined with a relative standard deviation of 5% is 1 μM. Compared with other methods based on immobilized enzymes for the determination of compounds as carbon dioxide, this sensitivity constitutes an improvement of about two orders of magnitude. Possible application to sea water is discussed.     

Determination of total 3α-hydroxy bile acids in serum by a bioluminescent flow-injection system using a hollow-fibre reactor

A continuous-flow bioluminescence method for measuring total 3α-hydroxy bile acids in serum is described. A bacterial luciferase and NADH:FMN oxidoreductase are covalently co-immobilized on a CNBr-activated Sepharose 4B. A permeable membrane (hollow fibre) reactor is used for the introduction of NAD⁺ and bioluminescent reagent. The reagent permeates into the flow-stream due to the existing difference in ionic strength. The continuous-flow light-emitting system, in which the column filled with the immobilized bioluminescent enzyme is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. The technique was tested by comparing results with those obtained by fluorimetry. More than 20 samples an hour can be analyzed. Normal values for total bile acids content serum ranged from 1.0 to 7.5 μM, in agreement with those obtained by the other method. Excellent reproducibility, precision, and sensitivity are achieved.     

Determination of fructosyl amino acids and fructosyl peptides in protease-digested blood sample by a flow-injection system with an enzyme reactor.

A flow-injection system with an enzyme reactor was proposed for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. A fructosyl-amino acid oxidase (FAOX-TE) and two fructosyl-peptide oxidases (FPOX-CE and FPOX-CET) were covalently immobilized onto an inert support. They were used as the enzyme reactor in a FIA system with a hydrogen peroxide electrode. In particular, the FPOX-CET reactor possessed high selectivity for the detection of fructosyl valine (FV) and fructosyl valyl histidine (FVH) and an excellent operational stability. The proposed FIA system with the FPOX-CET reactor responded linearly to the concentration of FV over the dynamic range of 7.8 x 10(-6) to 5.8 x 10(-4) M. The present method could be successfully applied to the assay of FV and FVH in the protease-digested blood samples.     

Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure. The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaCl solution. Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e., the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product from the unreacted substrate. The measured peak area of the product then represented the enzyme activity. For enzyme inhibitor screening, the mixture solution of the substrate and the inhibitor was injected and assayed the reduction of the enzyme activity. The immobilized enzyme could withstand 100 consecutive assays by only losing 10% activity. The reproducibility in terms of time-to-time, day-to-day, and batch-to-batch was measured with RSD% less than 4.7%. Furthermore, the screening system was validated by a known inhibitor. Finally, screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.     

The use of an immobilized enzyme nylon tube reactor incorporating a four enzyme system for creatinine analysis

A single immobilized enzyme nylon tube reactor was produced incorporating a four enzyme system for the analysis of creatinine. The enzyme activity ratios in the coupling solution used to prepare the reactor were found to be of extreme importance in governing the activity of the latter. The reactor was incorporated into a continuous flow analysis system used to assay creatinine in urine samples and the results were correlated with a manual technique employing the same enzyme system in solution. The precision, correlation, high specificity, simplicity, and speed of the analysis were concluded to be factors in favor of the method's suitability for urine creatinine determinations.     

l-Glutamate enzyme electrode involving amplification by substrate recycling

An enzyme electrode with an amplified response for l-glutamate is constructed by co-immobilizing l-glutamate oxidase and glutamate-pyruvate transaminase on a platinum disk electrode. With l-alanine (1 mM) in the solution, the sensitivity of the electrode to l-glutamate is greatly increased by the substrate recycling reaction between the two enzymes; the detection limit is 0.2 nM in the presence of l-alanine compared with 0.2 μM in its absence. The amplification factor for 1 μM l-glutamate is ca. 53.     

基于中心辐射树枝状介孔二氧化硅构筑CPO固定化酶反应器及应用

首先制备粒径均匀的具有开放的三维中心辐射树枝状结构的介孔二氧化硅(DSP)粒子, 再通过静电相互作用在孔道内负载氯过氧化物酶(CPO)构筑了CPO@DSP固定化酶反应器. 通过改变硅源正硅酸乙酯(TEOS)和模板剂十六烷基三甲基氯化铵(CTAC)的浓度调控孔径大小, 研究了孔径对固定化酶反应器催化活性的影响; 同时基于酶促反应动力学分析探讨了孔道内酶催化反应的限域效应, 并进一步在CPO@DSP表面包覆海藻酸钠(SA)水凝胶薄膜以抑制酶反应器在使用过程中酶分子的泄露, 所得SA-CPO@DSP固定化酶反应器的重复使用性显著提高, 循环使用10次后, 仍能保持90%以上的催化活性. 将SA-CPO@DSP酶反应器用于环境水体中残留抗生素左氧氟沙星的降解, 对100 μg/mL的底物在25 min内降解率可达88%以上; 将该反应器用于苯酚的视觉比色检测, 裸眼可检测到5 μmol/L的苯酚, 表明SA-CPO@DSP酶反应器在环境保护方面具有良好的应用前景.     

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.