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.     

Assay of α-glucosidase inhibitory activity using flow-biosensor system

A convenient and continuous method for the assay of α-glucosidase (AGH) inhibitory activity was developed using a continuous-flow/stopped-flow system combined with biosensors. The amount of glucose liberated from maltose by the action of AGH was quantified by an immobilized glucose oxidase (GOD) reactor with a Clark oxygen sensor in the downstream. The immobilized AGH reactor was set in the flow-line. When an inhibitor containing 10 mM maltose substrate was injected and as it reaches the center of the immobilized AGH reactor, the working solution (synthetic intestinal fluid) was stopped for a certain period. After the reaction of inhibitor and/or substrate with AGH, the working solution was propelled again, and the glucose liberated was passed through the immobilized GOD reactor. The inhibition ratios (%) were calculated as the percent inhibition, which were the glucose concentrations in the presence of maltose and inhibitor divided by those in the presence of maltose alone. The multi-channel stopped-flow (MCSF) system was also developed, in which a seven-port, six-positioned rotary valve was inserted and six immobilized AGH reactors were set in a parallel configuration. The IC₅₀ values of acarbose and 1-deoxynojirimycin, a medicinal inhibitor for diabetes, were 0.46±0.062 and 0.23±0.031 μM, respectively, and coincided well with those by our pseudo-in vivo method [Biol. Pharm. Bull. 232 (2000) 1084]. The time to assay the inhibitory activity of one unknown sample was estimated to be 11 min by the 6-channel modified-MCFS system. The proposed MCFS system offers a useful method to evaluate the inhibitory activity for AGH.     

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.     

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.     

Flow-through chemiluminescence sensor using immobilized histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007 and peroxidase for selective determination of histamine.

A flow sensor with immobilized oxidases is proposed for the determination of histamine in fish meat. Chemiluminometric measurement of histamine was based on the luminol reaction with hydrogen peroxide produced by immobilized histamine oxidase (EC 1.4.3.-.) and peroxidase (EC 1.11.1.7.) within a flow cell. Histamine oxidase was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The oxidase and peroxidase were coimmobilized covalently on tresylated hydrophilic vinyl polymer beads and packed into transparent PTFE; the tubing was used as the flow cell. One assay for histamine was done at intervals of 2 min without carryover. The calibration curve for histamine was linear from 0.1 microM to 50 microM. The response was reproducible within 1.25% of the relative standard deviation for 115-replicate injections of 50 microM histamine. The sensor system was applied to the determination of histamine in fish meat extracts.     

Computer-assisted optimization of an immobilized-enzyme flow-injection system for the determination of glucose

A flow-injection system utilizing the Trinder reaction for the determination of glucose is designed and optimized. Glucose is converted to gluconic acid by passing it through a single-bead-string reactor (SBSR) onto which glucose oxidase enzyme has been immobilized. As it flows through a second SBSR, the gluconic acid reacts with a reagent stream of horseradish peroxidase, 4-aminoantipyrine and 3,5-dichloro-2-hydroxyphenyl sulfonate. The absorbance of the quinoneimine dye produced is then monitored at 510 nm in a flow-through cell. Optimum operating conditions were sought by using both univariate and Composite Modified Simplex procedures. Seven variables were considered. The performance of the system was improved by a factor of 22.5 relative to the starting conditions. A calibration curve obtainedat the optimum conditions was linear for 0–3.3 mM glucose and usable for 0–5.5 mM glucose. The optimization procedures revealed some interesting aspects of the Trinder reaction. scatter diagrams generated from the simplex data showed definite trends for each of the seven variables. These are discussed.     

Use of a peroxidase reactor in flow injection analysis for the determination of chloramine and the inhibition kinetics

A method for the determination of chloramine using a flow injection peroxidase reactor based on the inhibition reaction of the enzyme is developed. The immobilisation of the horseradish peroxidase is performed on the commercial polymer carrier VA Epoxy Biosynth. The peroxidase activity is detected photometrically based on the dehydration of the dye 2,2′-azino-di-[3-ethylenebenzthiazoline-6-sulphonate] (ABTS). The calibration of the method gives a linear concentration range from 0.026 to 1.04 mmol l⁻¹ (SD_n=3=below 5%). The detection limit was calculated to 26 μmol l⁻¹. A mixture out of competitive and non-competitive inhibition was analysed based on the Lineweaver–Burk plots.     

A Mediator‐Free Bienzyme Amperometric Biosensor Based on Horseradish Peroxidase and Glucose Oxidase Immobilized on Carbon Nanotube Modified Electrode

Multiwalled carbon nanotube (CNT) modified glassy carbon electrode immobilized with horseradish peroxidase (HRP) in Nafion coating showed direct electron transfer between HRP enzyme and the CNT‐modified electrode. A mediator‐free bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase was constructed. The bienzyme biosensor exhibited a high sensitivity for glucose detection at zero applied potential.     

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.     

纳米增强型毛细管酶柱用于葡萄糖液滴生物传感器的研究

葡萄糖的检测在临床医学以及食品工业等领域中十分重要.以往的检测方法主要包括化学发光法_{[1]、吸光光度法[2]、电化学法[3]和荧光法[4]等.固定化酶柱的制作是发展葡萄糖传感器的关键技术之一.传统的固定化方法主要是将具有生物活性的酶通过物理吸附、共价键合和交联的方法固定于载体基质上或包埋于有机聚合物的基质中.近期研究[5,6]表明,采用溶胶凝胶(Sol-gel)法将蛋白质和酶等生物活性物质包埋于无机陶瓷或玻璃材料内,保持生物组分的活性,且SiO2作为基质材料具有较好的坚固性、抗磨性、化学惰性以及高的光稳定性和透过性,但目前该法多用于电化学型生物传感器[7,8].本文利用纳米颗粒的比表面积大和吸附能力强等特点,将酶吸附在SiO2纳米颗粒表面,用易成膜的聚乙烯醇缩丁醛(PVB)作辅助基质在毛细管上固定酶,并采用分立式酶柱,克服了以往混合型酶柱普遍存在的酶促效率不高和使用寿命较短的局限性.所制得的酶柱具有表面反应活性高、表面活性中心多和催化效率高等特点.结合自行设计的液滴光化学传感装置[9,10],建立了一种高效、快速、微量的葡萄糖实时检测方法.}     

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.     

A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase

This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested. Figure Preparation of apoGOx and restoration of enzyme activity using a soluton of FAD     

Enzymatic oxidation of tert-butylcatechol in the presence of sulfhydryl compounds: Application to the amperometric detection of penicillamine

The high sensitivity that can be attained using an enzymatic system and mediated by 4-tert-butylcatechol (4-TBC) has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of 4-TBC, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV. Thus, when penicillamine (PA) was added to the solution, these thiol-containing compounds participate in Michael type addition reactions with 4-TBC to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. The highest response for PA was obtained around pH 7. This method could be used to determine PA concentration in the range 0.02-80 μM (r = 0.998). The determination of PA was possible with a limit of detection of 7 nM, in the processing of as many as 50 samples per hour. The HRP-rotating biosensor was successfully applied to the determination of PA in pharmaceutical formulations.     

Oxidation of ferrocene and some substituted ferrocenes in the presence of horseradish peroxidase

The enzyme catalysed oxidation of ferrocene and some substituted ferrocenes to the corresponding ferricinium ions by hydrogen peroxide in the presence of native or immobilized horseradish peroxidase has been studied. Initial and maximum rates of oxidation have been determined. It was found that the oxidation was independent of the hydrogen peroxide concentration. The oxidation of ferrocene was effected also by horseradish peroxidase in a coupled system with glucose oxidase in the absence of any added hydrogen peroxide.     

Fabrication of Bienzymatic Glucose Biosensor Based on Novel Gold Nanoparticles‐Bacteria Cellulose Nanofibers Nanocomposite

An exploration of gold nanoparticles–bacterial cellulose nanofibers (Au‐BC) nanocomposite as a platform for amperometric determination of glucose is presented. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) were immobilized in Au‐BC nanocomposite modified glassy carbon electrode at the same time. A sensitive and fast amperometric response to glucose was observed in the presence of electron mediator (HQ). Both of GOx and HRP kept their biocatalytic activities very well in Au‐BC nanocomposite. The detection limit for glucose in optimized conditions was as low as 2.3 µM with a linear range from 10 µM to 400 µM. The biosensor was successfully applied to the determination of glucose in human blood samples.     

Enzyme sequence and competition electrodes based on immobilized glucose oxidase,peroxidase and catalase

The toxicologically important peroxidase substrates bilirubin and aminopyrine can be determined by combination of immobilized glucose oxidase, horseradish peroxidase and catalase, forming so-called enzyme sequence and enzyme competition electrodes. Bilirubin and aminopyrine are determined in the concentration range 5–50 μM.     

Immobilization of Glycosylated Enzymes on Carbon Electrodes,and its Application in Biosensors

A novel approach was used to immobilize glycosylated enzymes on a glassy carbon electrode (GCE) based on the interaction of boronic acid and carbohydrate moiety within the glycoproteins. 4-Aminomethylphenylboronic acid (4-AMBA) was covalently grafted on a glassy carbon electrode (GCE) by amine cation radical formation in the electrooxidation process of the amino-containing compound. The boronic acid group immobilized in this way could recognize glycoproteins such as glucose oxidase, horseradish peroxidase, dehydrogenase and others. X-ray photoelectron spectroscopy measurement proved the presence of a 4-AMBA monolayer on the GCE. The adsorptions of three kinds of enzymes were investigated by cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The activity of the immobilized horseradish peroxidase was also studied.     

Study of the enhanced chemiluminescence from the luminol-horseradish peroxidase-hydrogen peroxide-p-coumaric acid system at very short times: stopped flow selective determination of p-coumaric acid in beers

p-Coumaric acid has a greater enhancing effect on the chemiluminescence of the luminol-H₂O₂-horseradish peroxidase system, at low concentration, than other phenolic acids studied. We have used this effect to study the variations of the chemiluminescent signal with luminol, hydrogen peroxide, p-coumaric acid, horseradish peroxidase concentrations and pH, using the stopped-flow technique, by monitoring the initial reaction rate. The interference effects of other phenolic acids on the enhanced chemiluminescence with p-coumaric acid (25 nM) were negligible at similar concentrations of phenolic acid. We monitored the chemiluminescence intensity at 10 s for the determination of p-coumaric acid in beers. The detection limit was ca. 0.7 nM and the linear range was 0–12.5 nM. The precision of the method, expressed as a relative standard deviation, was 2.5%.     

Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

Enzymatic rotating biosensor for ciprofloxacin determination

The high sensitivity that can be attained using an enzymatic system and mediated by catechol has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP [EC 1.11.1.7], immobilized on a rotating disk, in the presence of hydrogen peroxide, catalyzed the oxidation of catechol, whose back electrochemical reduction was detected on a glassy carbon electrode surface at −200 mV. Thus, when ciprofloxacin (CF) was added to the solution, this piperazine-containing compound participate in Michael addition reactions with catechol to form the corresponding piperazine-quinone derivatives, decreasing the peak current obtained, in proportion with the increase of its concentration. The highest response for CF was obtained around pH 7. This method could be used to determine CF concentration in the range of 0.02-65 μM (r = 0.999). The determination of CF concentration was possible with a detection limit of 0.4 nM, in the processing of as many as 25 samples per hour. Application of this analysis to different pharmaceutical samples containing CF supports the utility of the HRP-rotating biosensor.