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.     

Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes

In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system.     

新型乳酸固定化酶荧光毛细生物传感器

对长45 mm、内径0.9 mm的医用毛细管进行γ-氨丙基三乙氧基硅烷氨基化和戊二醛醛基化后,再将乳酸脱氢酶(LDH)的氨基与戊二醛的醛基结合,使其固定在毛细管内壁,构成一种新型固定化酶乳酸荧光毛细生物传感器(IE-LFCBS),实现了对乳酸的微量、快速测定.IE-LFCBS吸入辅酶Ⅰ与乳酸的混合液,在固定化酶催化下使乳酸与辅酶Ⅰ反应,生成荧光物质还原型辅酶Ⅰ;激发波长353 nm、发射波长466 nm.适用于IE-LFCBS的优化条件为:辅酶Ⅰ浓度4 mmol/L、用于固定化的LDH浓度60 kU/L、反应时间15 min、反应温度38 ℃、测定范围为1.0～5.0 mmol/L、回收率95%～98%,IE-LFCBS的相对标准偏差为RSD<1.5%(n=11),检出限为0.45 mmol/L.IE-LFCBS的试液用量极少(18 μL),并能重复使用,可望用于发酵食品、药品、血液标本等各类样品中乳酸的快速检测.     

Determination of acetaldehyde by flow injection analysis with soluble or immobilized aldehyde dehydrogenase

Acetaldehyde (0.18–7.7 × 10^?4 M) in water is determined by using a double injection technique with the soluble enzyme or with a mini-column of aldehyde dehydrogenase immobilized on cyanogen-activated Sepharose 4B. The NADH produced is monitored spectrophotometrically. The sample throughput is ca. 40 h^?1, and the immobilized enzyme is stable for at least a month. Ethanol up to 5% (v/v) does not interfere.     

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.     

Flow-injection determination of branched-chain L-amino acids with immobilized leucine dehydrogenase

L-Leucine, L-isoleucine and L-valine are determined by passage through a column of the enzyme immobilized on polystyrene beads, at pH 11.0. The NADH produced is monitored fluorimetrically. The detection limit is 0.1 nmol of L-leucine.     

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

Amperometric probe for 3-hydroxybutyrate with immobilized 3-hydroxybutyrate dehydrogenase

The enzyme 3-hydroxybutyrate dehydrogenase is immobilized on a graphite electrode suitable for determination of NADH. Of the several procedures tested for the immobilization, direct adsorption on the electrode surface was most satisfactory, with useful lifetimes of up to 3 days. The best calibration graphs for the NADH, and for 3-hydroxybutyrate were obtained at pH 7.5 (phosphate buffer). 3-Hydroxybutyrate was determined in the range 5–100 μmol l^?1 at pH 7.5 with good precision. Interferences are discussed.     

Enzymatic methods for the determination of ethanol based on linear and cyclif flow-injection systems

Linear and cyclic systems are described for the determination of ethanol (ca. 0.17–30×10^?3 M). In the linear system, the solution passes either through a minicolumn of yeast alcohol dehydrogenase (YADH) immobilized on controlled-pore glass or through minicolumns of the immobilized YADH and of yeast aldehyde dehydrogenase immobilized on cyanogen bromide-activated Sepharose-4B. The NADH formed is monitored either spectrophotometrically or spectrofluorimetrically. In the cyclic system, the solution passes through the same enzyme columns, and the NADH produced is monitored similarly before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on cyanogen bromie-activated Sepharose-4B in the presence of α-ketoglutarate and ammonium ions also present in the flow system. the sample throughout for both systems is ca. 40 h^?1 and 50 h^?1 for spectrophotometric and spectrofluorimetric detection, respectively. An on-line double-injection technique is described as an alternative to the cyclic system for limiting the consumption of NAD⁺.     

Determination of l-phenylalanine in serum by flow-injection analysis using immobilized phenylalanine dehydrogenase and fluorimetric detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of l-phenylalanine. Phenylalanine dehydrogenase from Rhodoccus sp. M4 was immobilized on tresylated poly (vinyl alcohol) beads (13 mum) and packed into a stainless-steel column (5 cm x 4 mm i.d.). Serum sample was deproteinized with tungstic acid and filtered through an ultrafiltration membrane. The sample solution (30 mul) was injected into the carrier stream (water). The NADH formed was detected at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 mum l-phenylalanine; the detection limit was 0.3 mum. The sample throughout was 25 h(-1) without carryover. The half-life period of the immobilized enzyme was 23 days.     

Flow-injection determination of 3-hydroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and fluorescence detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm x 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 mul) is injected into the carrier stream [4mM NAD(+) in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7-500muM 3-hydroxybutyrate; the detection limit is 0.5muM.     

A regenerable immobilized NADH model. II. Reduction of α,α,α-trifluoroacetophenone by immobilized 1,4-dihydronicotinamide in aprotic solvent

A regenerable dihydronicotinamide, immobilized on a macroreticular polystyrene resin was used for the reduction of trifluoroacetophenone (TFA) in acetonitrile. Practical reutilization of the polymeric reagent (91% per cycle) was obtained when magnesium perchlorate was added to the reaction of the immobilized dihydropyridine with TFA. A possible explanation of the role of Mg ion in the reaction of benzyldihydronicotinamide and its polymeric analog is presented. This explanation also accounts for the results of other NADH model reactions, described in the literature, and for the role of the Zn ion in the enzyme liver alcohol dehydrogenase (LADH).     

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.     

Augmentation of enzyme electrode sensitivity using biocatalytic preconcentration

A method for increasing the sensitivity of enzyme sensors based on biocatalytic accumulation of an intermediate product was investigated using a biospecific electrode consisting of an immobilized glucose dehydrogenase-lactate dehydrogenase-lactate monooxygenase membrane and an electrochemical oxygen probe. Addition of the analyte (glucose) and an excess of NAD⁺ to the background solution permits NADH to be biocatalytically preconcentrated in the enzyme membrane. When this reaction has approached equilibrium, the sensor signal is generated by injection of an excess of pyruvate, thus starting oxygen consumption catalysed by the sequential lactate dehydrogenase-lactate monooxygenase reaction. Glucose can be determined at concentrations between 10 and 100 μM. Compared with operation of the sensor without NADH preconcentration, the increase in the sensitivity to glucose is 18-fold in the current-time mode and 40-fold in the derivative current-time mode. The measuring regime permits interferences from the sample solution to be avoided.     

Enzymatic production and analysis of d-xylulose with a recirculating flow system and post-column liquid chromatographic detection using a co-immobilized enzyme reaction and a chemically modified electrode

A pure d-xylulose and standard was produced by isomerization of d-xylose in a recirculating flow system incorporating an enzyme reactor containing immobilized xylose isomerase. The d-xylulose formed was purified chromatographically. A selective chromatographic detection system was used in the post-column mode. It consisted of a co-immobilized enzyme reactor (CIMER) with xylose isomerase, mutarotase and glucose dehydrogenase on-line with a chemically modified electrode for selective elctrochemical oxidation of NADH. The pure standard was compared with commercially available d-xylulose, which was confirmed to contain impurities of d-glucose and d-xylose.     

Cooperative Catalysis of an Alcohol Dehydrogenase and Rhodium‐Modified Periodic Mesoporous Organosilica

The combined use of a metal‐complex catalyst and an enzyme is attractive, but typically results in mutual inactivation. A rhodium (Rh) complex immobilized in a bipyridine‐based periodic mesoporous organosilica (BPy‐PMO) shows high catalytic activity during transfer hydrogenation, even in the presence of bovine serum albumin (BSA), while a homogeneous Rh complex exhibits reduced activity due to direct interaction with BSA. The use of a smaller protein or an amino acid revealed a clear size‐sieving effect of the BPy‐PMO that protected the Rh catalyst from direct interactions. A combination of Rh‐immobilized BPy‐PMO and an enzyme (horse liver alcohol dehydrogenase; HLADH) promoted sequential reactions involving the transfer hydrogenation of NAD⁺ to give NADH followed by the asymmetric hydrogenation of 4‐phenyl‐2‐butanone with high enantioselectivity. The use of BPy‐PMO as a support for metal complexes could be applied to other systems consisting of a metal‐complex catalyst and an enzyme.     

中空纤维膜固定化甲酸脱氢酶催化CO_2合成甲酸

以紫外光表面接枝改性的聚乙烯(PE)中空纤维膜为载体,采用共价结合的方式固定化甲酸脱氢酶(FDH),考察了CO2通入方式、溶液pH值、缓冲液种类和还原型烟酰胺腺嘌呤二核苷酸(NADH)的浓度对酶催化CO2合成甲酸反应的影响.结果表明,与加压法相比,CO2鼓泡法更有利于甲酸的生成;磷酸盐缓冲液优于Tris-HCl和盐酸三乙醇胺缓冲液;体系pH值对反应的影响较大,固定化FDH的最佳pH值仍为6.0,但pH耐受性增强;随着辅酶NADH浓度的增加,反应初速度加快,收率下降;游离酶和固定化酶的最大酶活分别为0.246和0.138mmol/(L.h);固定化FDH在4℃贮存两周后活性仅下降4%,而游离酶活性下降50%.FDH催化膜重复利用10次后,活性没有明显降低.     

Amperometric biosensor for ethanol based on immobilization of alcohol dehydrogenase on a nickel hexacyanoferrate modified microband gold electrode

Alcohol dehydrogenase (ADH) has been immobilized on a nickel hexacyanoferrate modified microband gold electrode surface by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new amperometric sensor for the assay of ethanol. The resulting enzyme electrode exhibits excellent electrocatalysis for the oxidation of reduced nicotinamide-adenine dinucleotide (NADH). The amperometric determination is based on the electrochemical detection of NADH which is generated in the enzymatic reaction of ethanol with NAD(+) under catalysis of ADH. The influence of various experimental conditions was examined for the determination of the optimum analytical performance. The sensor responds rapidly to ethanol with a detection limit of (5.0 +/- 0.3) x 10(-7) mol 1(-1). The response current increases linearly with ethanol concentration up to 5 mmol 1(-1). The sensor remains relatively stable for about 1 week.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

Determination of Glycerol and Serum Triglycerides with Collagen-Immobilized Glycerol Dehydrogenase and Amperometric Detection

Abstract

An amperometric procedure is described for the determination of glycerol and triglycerides in aqueous samples and in serum using glycerol dehydrogenase immobilized on a collagen membrane. Glycerol is determined by measurement of the steady-state oxidation currents generated at a platinum electrode by NADH produced in the enzyme-catalyzed reaction. The triglycerides were first hydrolyzed by the enzyme lipase in solution and the resulting glycerol determined similarly. Olive oil, determined to contain 78 % triolein, was used as the source of triglycerides in this study. For both glycerol and triglycerides the calibration plots are linear in the range from 0 to 12 μM, with detection limits of 0.2 and 0.7 μM, respectively. The immobilized glycerol dehydrogenase retained high operational activity for a period longer than 30 days.