A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 ± 2.3% and 63.2 ± 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1–30 % v/v (10.0–238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034–0.087 g ethanol/L working with the on-line dilution integrated to the biosensor—FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.     

Bienzymatic analytical microreactors for glucose, lactate, ethanol, galactose and l-amino acid monitoring in cell culture media

A new alternative method for bioprocess monitoring based on bienzymatic analytical microreactors integrated in a flow injection analysis (FIA) system is described. Glucose-, alcohol-, lactate-, galactose- and l-amino acid oxidases (GO, AO, LacO, GalO and LAAO) and horseradish peroxidase (HRP) are immobilized on controlled pore glass (CPG) and used for the development of glucose, ethanol, lactate, galactose and amino acid sensors. The analytical methodology is based on HRP catalysed reaction of hydrogen peroxide produced by oxidases with phenol-4-sulfonic acid and 4-aminoantipyrine. The immobilized enzymes are characterized and used for preparation of the packed bed analytical microreactors. Shelf life and operational stability of the microeactors are determined. GO/HRP, AO/HRP and LAAO/HRP microreactors showed excellent shelf life, they could be stored and reused for more than 6 months with no or very little activity loss, while GalO/HRP and LacO/HRP could be stored for shorter periods of time (10-20 days). Operational stability of GO and LacO microreactors was very good: an equivalent to 16,900 FIA injections of 25 μl to a LacO microreactor resulted in loss of half of its activity, immobilized GO was so stable that it was impossible to evaluate enzyme halflife. Immobilized GalO and LAAO lose their operational activity much faster: approximately 1400 and 8000 FIA injections of the respective substrate solution in a FIA set-up resulted in 50% activity loss. The methods with all the described microreactors were successfully validated using off-line samples from S. cerevisiae, E. coli and mesenchymal stem cell cultures with HPLC as the reference method.     

Design of Block Copolymer Micellar Aggregates for Co‐Delivery of Enzyme and Anticancer Prodrug

Traditional enzyme–prodrug therapy (EPT) is a two‐step strategy, which has many serious deficiencies, so having a one‐step EPT treatment becomes a problem of immediate interest. This study aims to achieve an effective co‐delivery of horseradish peroxidase (HRP) as a kind of enzyme for prodrug activation and ethyl 3‐indoleacetate (EIA) as anticancer prodrug. A ternary block copolymer PEG‐PAsp(AED)‐CA consisting of poly(ethylene glycol) (PEG), reduction‐sensitive poly (N ‐(2,2′‐dithiobis(ethylamine)) aspartamide) PAsp(AED), and cholic acid (CA) was synthesized and assembled into spherical micelles which encapsulated EIA in its hydrophobic core and HRP in a reduction‐sensitive interlayer. TEM photographs show that the polymer micelle is around 40 nm, and the cell survival rate test shows that the EIA/HRP polymer micelle is highly lethal to human lung adenocarcinoma cells. Thus, co‐delivery of EIA and HRP demonstrates great potential in cancer therapy, offering a structurally simple and highly tunable platform for the synchronous delivery of enzymes and prodrugs in EPT.     

A novel non-invasive electrochemical biosensing device for in situ determination of the alcohol content in blood by monitoring ethanol in sweat

A non-invasive, passive and simple to use skin surface based sensing device for determining the blood's ethanol content (BAC) by monitoring transdermal alcohol concentration (TAC) is designed and developed. The proposed prototype is based on bienzyme amperometric composite biosensors that are sensitive to the variation of ethanol concentration. The prototype correlates, through previous calibration set-up, the amperometric signal generated from ethanol in sweat with its content in blood in a short period of time. The characteristics of this sensor device permit determination of the ethanol concentration in isolated and in continuous form, giving information of the BAC of a subject either in a given moment or its evolution during long periods of time (8 h). Moreover, as the measurements are performed in a biological fluid, the evaluated individual is not able to alter the result of the analysis. The maximum limit of ethanol in blood allowed by legislation is included within the linear range of the device (0.0005–0.6 g L⁻¹). Moreover, the device shows higher sensitivity than the breathalyzers marketed at the moment, allowing the monitoring of the ethanol content in blood to be obtained just 5 min after ingestion of the alcoholic drink. The comparison of the obtained results using the proposed device in the analysis of 40 volunteers with those provided by the gas chromatographic reference method for determination of BAC pointed out that there were no significant differences between both methods.     

On-line microfluidic sensor integrated with an enzyme-modified pre-cell for the monitoring of paracetamol in pharmaceutical samples

An on-line microfluidic sensing device with an enzyme-modified pre-cell coupled to an amperometric detector for the monitoring of paracetamol in pharmaceutical formulations is described. Horseradish peroxidase (HRP) [EC 1.11.1.7], immobilized on a 3 μl pre-cell, in presence of hydrogen peroxide catalyses the oxidation of paracetamol to N-acetyl-p-benzoquinoneimine. The electrochemical reduction back to hydroquinone is detected on glassy carbon electrode surface at −0.10 V. The recovery of paracetamol from 10 samples ranged from 99.00 to 101.10%. This method could be used to determine paracetamol concentration in the range 0.35-100 μM (r = 0.997) with a limit of detection of 3.0 × 10⁻⁷ M and a relative standard deviation was less than 4.1% (n = 8). The method was successfully applied for the processing of as many as 20 samples per hour of paracetamol in pharmaceutical formulations.     

Attachment of horseradish peroxidase (HRP) onto the poly(styrene/acrolein) latexes and onto their derivatives with amino groups on the surface; activity of immobilized enzyme

The polystyrene (P(S)), poly(styrene/acrolein) (P(SA)), and polyacrolein (P(A)) latexes, with varied fraction of polyacrolein in the surface layer (f _A=0, 0.50, 0.63, 0.84, 1.00), were used for the attachment of horseradish peroxidase. Surfaces of latexes were modified by reaction with ethylenediamine. In this step the aldehyde groups from polyacrolein were blocked and the primary amino groups were introduced. The carbohydrate portion of HRP was oxidized in the reaction leading to formation of aldehyde groups. The adsorption and covalent immobilization of HRP onto the P(S), P(SA), and P(A) latexes and of the oxidized HRP (HRP-OX) onto the modified latex particles, with amino groups on the surface (P(SA)-M and P(A)-M), were investigated. The activities of parent and oxidized HRP were compared with activities of the corresponding enzymes in solution. It has been found that whereas HRP is not suitable for the covalent immobilization on P(SA) latex and loses its activity after adsorption onto P(S) latex, HRP-OX can be adsorbed onto P(S) latex and is readily immobilized covalently onto the ethylenediamine modified P(SA) and P(A) latexes, retaining much of its former enzymatic reactivity.This work was supported by the KBN Grant 2 0624 91 01     

Preparation,characterization, and potential application of an immobilized glucose oxidase

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.     

集成化蛋白质组定量分析平台的构建及应用

为提高蛋白质组定量分析的准确度、通量和自动化程度,构建了由微升级混合离子交换色谱、亲水型固定化酶反应器（hIMER）和纳升级反相色谱-电喷雾串级质谱（nanoRPLC-ESI-MS/MS）组成的集成化蛋白质定量分析平台。该平台实现了二甲基化标记蛋白质样品在线分离、酶解、肽段分离鉴定和定量分析。采用质量比为1：1的轻、重标记的蛋白质样品考察该平台的定量性能,发现蛋白质水平二甲基化标记效率为90%;蛋白质经hIMER在线酶解10 min产生的漏切及酶解产物在hIMER柱上的非特异性吸附对定量准确度的影响较小,所有定量到的重/轻标记的蛋白质质量比的平均值为1.01。最后将该平台应用于小鼠腹水型肝癌淋巴道高、低转移细胞系差异蛋白质的分析,发现了12种蛋白质在高转移细胞系中低表达,15种蛋白质在高转移细胞系中高表达。以上结果证明了该平台可以实现高准确度和高通量的蛋白质组定量分析。     

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4 min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5 μg mL⁻¹. The limit of detection (LOD) (S/N = 3) was 0.005 μg mL⁻¹. Finally, the automatic CL system could also be used for the detection of HRP in HRP–protein conjugates, which showed its practical application in immunoassay.     

Bacterial acyltransferases as an alternative for lipase-catalyzed acylation for the production of oleochemicals and fuels

Bacterial acyltransferases are a new class of enzymes, and the first member was identified as WS/DGAT in Acinetobacter baylyi ADP1. Their unspecificity have been used in several biotechnological applications for lipid modification, a field that has been dominated by the use of lipases. Examples are the biosynthesis of jojoba-like wax esters and fatty-acid ethyl esters. In addition, these enzymes are also capable of synthesizing acylthioesters. Acyloxoesters and acylthioesters can thus be produced in vivo by whole-cell fermentations rather than in vitro in an enzyme reactor. In this Minireview, we focus on the biotechnological utilization of acyltransferases for the production of modified lipids from renewable resources.     

Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system

Streptomyces SP.N 14, isolated from soil samples, produced extracellular L-glutamate oxidase (GOD) in liquid culture. After a two-step ammonium sulfate purification and dextran G-150 chromatography, the specific activity was reached at 28.2 U/mg. The partial purified enzyme and horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass (CPG) by means of glutaraldehyde. About 200–300 U/g of immobilized GOD and 300–400 U/g of immobilized HRP were obtained. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) for glutamate in broth. A good linear range was observed for this immobilized enzyme system at 0.1–2.0 mM, and the precision was 2.8% (n = 25). More than 80 samples were measured within an hour. One enzyme column with about 4 U of immobilized GOD and 5 U of immobilized HRP, applied for 50 assays/d, has been used for more than 50 d. The concentration of L-glutamate remaining lower than 2.0 mM, the determination of glutamate in this system was not affected by pH and temperature within the range of 6.0–7.0 and 25–35‡C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation, and good correlation was achieved between results obtained with the system and with the Warburg’s method.     

Fibre optic fluorometric enzyme sensors for hydrogen peroxide and lactate,based on horseradish peroxidase and lactate oxidase

An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H₂O₂. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum.     

Al-MOF基多级孔Al2O3固定化酶反应器的构筑及构效关系研究

针对生物酶在固相载体负载后存在的催化活性与稳定性之间“此消彼长”的问题, 本工作采用“自牺牲模板”策略以铝基金属有机骨架材料(Al-MOF)为前驱体设计制备多级孔Al₂O₃ (MHAl₂O₃)材料, 再以“聚多巴胺(PDA)”仿生膜对材料表面进行功能化修饰后用以固载辣根过氧化物酶(HRP). 通过调节前驱体的煅烧温度来实现载体孔径大小的调控, 探讨了载体的孔道限域效应对固定化酶反应器催化活性的影响, 所得固定化酶反应器的热稳定性和重复使用性显著提高. 为了解析固定化酶反应器的构效关系, 采用酶动力学和热动力学参数研究了固定化酶反应器催化过程中酶与底物的相互作用, 结果表明固载后酶分子对底物的亲和性和专一性得到提升. 将固定化酶反应器用于模拟废水中苯胺黑药的催化降解时, 表现出非常高效的催化效率.     

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.     

Development of an Amperometric Enzymatic Biosensor Based on Gold Modified Magnetic Nanoporous Microparticles

Gold modified nanoporous silica based magnetic microparticles have been prepared as support for the immobilization of the enzyme horseradish peroxidase (HRP). The enzyme modified gold microparticles were retained onto the surface of a solid carbon paste electrode with the help of a permanent magnet. The analytical performances of the resulting biosensor were characterized by studying hydroquinone (HQ) and hydrogen peroxide. The former was monitored by the direct electroreduction of the biocatalytically generated quinone. Several experimental parameters influencing the biosensor response were investigated. A linear response to HQ was obtained in the concentration range comprised between 5×10^?7 and 4.5×10^?6 M with a detection limit of 4×10^?7 M. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range comprised between 5×10^?7?1.3×10^?4 M with a detection limit of 4×10^?7 M. The inhibition of the biosensor response in the presence of thiols e.g. cysteine, captopril, glutathione and Nacystelyn (NAL) has been pointed out.     

An improved ELISA for the determination of southern bean mosaic virus with linear sweep voltammetry detection based on new system of PAP-H_2O_2-HRP

An improved enzyme-linked immunosorbent assay (ELISA) for the determination of southern bean mosaic virus (SBMV) with linear sweep voltammetry based on a new system of p-aminophenol (PAP)-H2O2-horseradish peroxidase (HRP) has firstly been developed. The enzymatic product 3-[(4-hydrox-yphenyl) amino]-4-(2-amino-5-hydroxyphenyl)-6-[ (4-hydrox-yphenyl)imino]-2,4-cyclohexadiene-l-one, produced from the oxidation of PAP with H2O2 catalyzed by HRP, yielded a sensitive linear sweep voltammetric response at - 0.45 V ( vs. SCE) in Britton-Robinson (BR) buffer solution. Based on the voltammetric peak, HRP can be measured with a detection limit of 0.4 mU/L and a linear range of 1.0-1.0 × 102 mU/ L. The detection limit for the SBMV is 8.0 ng/mL and the highest dilution ratio for the detection of infected leaf sap is 1: 1.5×105.     

Multicommutated Flow Analysis System for Determination of Horseradish Peroxidase and Its Inhibitors

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47–200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.     

Development and application of a system for analysis of mixed cultures of microorganisms

Development and application of a system for real-time quantitative assessment of individual cell activities in a mixed culture system was investigated. This was based on a concept that the activities of individual cells in a mixed culture can be assessed if the cells are physically separated (in separate compartments) in a vessel while the culture conditions, including the broth components, are maintained the same in all the compartments during the cultivation. On this basis, three different apparatus (M-1, M-2, and M-3) were constructed using various types of membranes. In terms of mass transfer characteristics and membrane fouling, the M-3 apparatus was the most effective system for analysis of mixed cultures at high cell densities. With the M-3 apparatus, the interrelationships between two alcohol-producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) under anaerobic and aerobic conditions were studied. Under anaerobic condition, except for possible competition for nutrients, there were no significant effects of the activities of one microorganism on the other. However, under aerobic condition, amensalism was observed because acetaldehyde that was produced by Z. mobilis inhibited the growth of S. cerevisiae.     

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.     

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