High-throughput enzyme assay on a multichannel microchip using optically gated sample introduction

To meet the requirements for high-throughput screening for drug discovery research, it is very important to develop techniques with the ability of performing multiple enzyme assays simultaneously. Using optically gated sample introduction on a multichannel microchip, multiple enzyme assays have been demonstrated in four parallel channels. The hydrolysis of fluorescein mono-beta-D-galactopyranoside by beta-galactosidase and the inhibition of this reaction by the competitive inhibitor phenylethyl beta-D-thiogalactoside were initially studied to determine the effect of system movement using the voice coil actuator on the enzyme assay reaction. The results from these two studies are consistent with the results from the assay using a single-channel microchip, and they demonstrate that the system using optically gated sample introduction on multichannel microchip can be used to perform multiple enzyme assays. Three unique enzyme assays were also performed in different channels, which show this technique could be competitive for high-throughput screening in drug discovery with other traditional techniques.     

Proteolytic enzyme-immobilization techniques for MS-based protein analysis

Protein digestion utilizing proteases (e.g., trypsin, Lys C and other proteolytic enzymes) is one of the key sample-preparation steps in contemporary proteomics, followed by liquid chromatography coupled to mass spectrometry (MS). Tryptic digestion is traditionally performed in aqueous solutions, usually applying the enzyme and the sample in a 50:1 protein-to-protease ratio. Long digestion times (up to 24 h), auto-digestion sub-products and poor enzyme-to-substrate ratio are common issues with liquid-phase protein-digestion processes. The use of enzymes immobilized onto solid supports can minimize these problems by increasing enzyme-to-substrate ratios, significantly speeding up digestion times and reducing autolysis. The other main goal of protease immobilization is to obtain rugged, efficient enzyme reactors.In this article, we review the most important proteolytic enzyme-immobilization techniques with the main emphasis on fabrication of trypsin microreactors and nanoreactors and their utilization in bottom-up proteomics. We also discuss data reportedly obtained using the various immobilization protocols with respect to enzyme activity and MS-sequence coverage.     

Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity

The development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1-0.5; rocket immunoelectrophoresis, 0.5-0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico-chemical differences between different molecular forms of the lipase, e.g. forms with different pI.     

Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.     

Methods for the determination of adenosine triphosphate and other adenine nucleotides

The following methods for the determination of adenosine triphosphate reported in the past 25 years are considered: bioluminescence methods with the use of the firefly luciferase enzyme (with sensitivity to 10^?14 M); chromatographic methods (ion-exchange, thin-layer, and high performance liquid chromatography) for the determination of adenine nucleotides in mixtures with other nucleotides, nucleosides, and nitrogen bases; and fluorescence, spectrophotometric, and electrochemical techniques (including those with the use of sensors), which are promising but not commonly used for the determination of adenine nucleotides. The advantages and disadvantages of these methods are demonstrated.     

Purification and determination of glutamine synthetase by high-performance immunoaffinity chromatography.

High-performance immunoaffinity chromatography (HPIAC) with anti-glutamine synthetase polyclonal antibodies bound to epoxy-activated silica was used to purify and determine this enzyme from the cyanobacterium Synechocystis. A single-step HPIAC procedure with cell-free extracts yielded electroporetically homogeneous glutamine synthetase. In the determination of glutamine synthetase by HPIAC a linear response in the range 10-60 micrograms of enzyme was observed. Recoveries of 70% of the loaded enzymatic activity and 100% of protein were obtained. The determination of glutamine synthetase protein by HPIAC was compared with that obtained by rocket immunoelectrophoresis. The chromatographic method is proposed as a possible alternative to other immunochemical quantitative techniques, particularly when non-limiting amounts of samples are available.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Immobilized enzyme studies in a microscale bioreactor

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of > 90% were observed.     

Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves

A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions.     

Microdrop analysis of a bead-based immunoassay

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.     

Application of Carbon Nanotubes in the Extraction and Electrochemical Detection of Organophosphate Pesticides: A Review

Recent trends and challenges in developing carbon nanotubes (CNT) based sensors and biosensors for the detection of organophosphate (OP) pesticides and other organic pollutants in water are reviewed. CNT have superior electrical, mechanical, chemical, and structural properties over conventional materials such as graphite. At the same time CNT based sensors and biosensors are more efficient compared to the existing traditional techniques such as high-performance liquid chromatography or gas chromatography, because they can provide rapid, sensitive, simple, and low-cost on-field detection. The measurement protocols can be based on enzymatic and non-enzymatic detection. The enzyme acetylcholinesterase (AChE) is used with CNT for fabricating ultrasensitive biosensors for OP detection involving different immobilization schemes such as adsorption, crosslinking, and layer-by-layer self-assembly. This protocol relies on measuring the degree of enzyme inhibition as means of OP quantification. The other enzyme used along with CNT for OP detection is organophosphate hydrolase (OPH) which hydrolyzes the OP into detectable species that can be measured by amperometric or potentiometric methods. Different forms of CNT electrode materials can be used for fabricating such electrodes such as pure CNT and composite CNT. Due to their large surface area and hydrophobicity, CNT have also been used for the extraction and non-enzymatic electrochemical detection of OP with very high efficiency. The application of CNT and their novel properties for the adsorption and electrochemical detection of OP compounds is discussed in detail.     

A transition path sampling study of the reaction catalyzed by the enzyme chorismate mutase

The study of the chemical steps in enzyme-catalyzed reactions represents a challenge for molecular simulation techniques. One concern is how to calculate paths for the reaction. Common techniques include the definition of a reaction coordinate in terms of a small set of (normally) geometrical variables or the determination of minimum energy paths on the potential energy surface of the reacting system. Both have disadvantages, the former because it presupposes knowledge of which variables are likely to be important for reaction and the latter because it provides a static picture and dynamical effects are ignored. In this paper, we employ the transition path sampling method developed by Chandler and co-workers, which overcomes some of these limitations. The reaction that we have chosen is the chorismate-mutase-catalyzed conversion of chorismate into prephenate, which has become something of a test case for simulation studies of enzyme mechanisms. We generated an ensemble of approximately 1000 independent transition paths for the reaction in the enzyme and another approximately 500 for the corresponding reaction in solution. A large variety of analyses of these paths was performed, but we have concentrated on characterizing the transition state ensemble, particularly the flexibility of its structures with respect to other ligands of the enzyme and the time evolution of various geometrical and energetic properties as the reaction proceeds. We have also devised an approximate technique for locating transition state structures along the paths.     

Theory and practice of enzyme bioaffinity electrodes. Direct electrochemical product detection

The use of enzyme labeling techniques to convert biorecognition events into high sensitivity electrochemical signals may follow two different strategies. One, in which the current is the electrocatalytic response of a redox couple serving as cosubstrate to a redox enzyme label and another that consists in the detection of an electrochemically active product of the enzyme label. The theoretical relationships that link, in the latter case, the electrochemical current response to the amount of recognized labeled target analyte are established for steady-state diffusion-convection chronoamperometric regimes. Two governing parameters thus emerge. One measures the Michaelis-Menten competition in the enzyme kinetics. The other characterizes the competition between the enzymatic kinetics and the diffusion of the substrate. The electrochemical response is finally related to the labeled target analyte concentration in solution through the recognition isotherm. The direct electrochemical product detection thus provides a route to the characteristics of the recognition isotherm, which serves as a calibration curve in analytical applications. The establishment of further theoretical relationships allows one to surmise the increase in sensitivity that may be obtained by using cyclic voltammetry instead of steady-state chronoamperometry in standard electrochemical cells or by accumulation of the enzyme-product in cells of small volume/surface ratios. The theoretical predictions are tested with the example of the avidin-biotin recognition process in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichlorophenyl phosphate as substrate, generating 4-amino-2,6-dichlorophenol as electrochemically active product. The advantages of the dichloro-substitution are discussed. The theoretical analysis is a requisite for a rational and realistic discussion of the analytical performances of the steady-state chronoamperometric and cyclic voltammetric approaches. These are shown to compare favorably with the best heterogeneous bioaffinity assays so far reported.     

Potentiometric determination of proteins with an ammonia sensor

A flow system is described for the cleavage of proteins with immobilized protease enzyme to L-amino acids which are then converted to ammonia with glass-immobilized L-amino acid oxidase. An ammonia gas electrode is used as detector. Immobilization techniques are discussed, as are optimum conditions for L-phenylalanine. Bovine serum albumin was determined in the range 0.1–100 μg ml^-1. Human blood sera required dilution for analysis.     

Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (n_HRP/n_DA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with n_HRP/n_DA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.      

Enzyme Inhibition and Chromatographic Techniques: Comparative Studies and Application to Pesticide Residue Analysis

Abstract

The development of enzyme inhibition techniques in relation to pesticide analysis is discussed, along with discussion of (a) principles of thin-layer chromatograph-enzyme inhibition (TLC-EI) technique, (b) general procedures of the technique, (c) the use of enzymes in combination with TLC and colorimetry, and (d) merits and limitations of the techniques. TLC-EI techniques and gas-liquid chromatography are compared based on sensitivity of detection, selectivity, and applicability to pesticide analysis.

The TLC-EI technique is being used and developed further in the Research Laboratories, Health Protection Branch, Ottawa for determination and confirmation of some organophosphorus and carbamate pesticides. Recently, it has been developed to detect methomyl (Lannate^(R)) residues in rapeseeds, oils, and meals.     

Enzyme Based Electrochemical Biosensor for Ethanolamine

Enzyme based electrochemical detection method developed for chemical toxicant ethanolamine (EA). Monoamine oxidase A (MAO‐A) enzyme was used for the oxidation of EA. A direct electron transfer from the electrode to EA without any mediator with the help of MAO‐A enzyme was attained and this confirms the application of this methodology for the development of third generation biosensor for EA sensing well below the IDLH (30 ppm) value of EA. Moreover, heterogeneous rate constant (0.021s^?1) and the number of electron involved (5.2) were deduced for EA in PBS buffer. The calibration plot showed linearity 2.02×10^?4 M to 10.10×10^?4 M of EA in PBS buffer with detection limit 4.1 ppm. The modified electrodes are characterized by Raman and Electrochemical impedance spectroscopy (EIS) techniques. The outcome of this work indicates about the utility of this methodology for the sensing of EA in the environment if it is present as well as to degrade EA into other compounds without using any indicator or mediator.     

Spread monolayers of proteins

The study of spread monolayers of proteins is of interest for understanding the fundamental behavior of proteins as well as the many phenomena resulting from their ubiquitous presence at interfaces in nature. Spread monolayers of proteins is a branch of the developing field of membrane mimetic chemistry. In recent times, it has been somewhat neglected in comparison to other branches (such as bilayers, liposomes and vesicles), despite the unique advantage that the arrangement and packing of molecules in monolayers may be measured and controlled. Methods for spreading proteins and techniques used for their manipulation are outlined. As well as the more traditional methods (such as surface pressure, potential and viscosity), more recent innovations, including removal of monolayers on slides for study by radiotracer techniques, electron diffraction and infrared (IR) spectroscopy, are discussed. Direct optical methods for the study of monolayers in situ are also available (e.g., multiple reflectance spectroscopy, ellipsometry). The use of measurements in the low pressure region to measure molecular weights is discussed. At higher pressures, configurational changes, surface coagulation and desorption are all observed. Experimental and theoretical work on the desorption of proteins from the air/water interface is reviewed. The introduction of multicompartment film balances has proved valuable for the study of reactions occurring in monolayers. This instrumentation has been applied to the study of enzyme reactions at the surface, of direct relevance to reactions where the enzyme is immobilized in the cell membrane. Some applications of monolayer studies are briefly illustrated with reference to biological membranes, foams and emulsions and biomedical problems.     

Redox enzyme linked electrochemical sensors: Theory meets practice

Information from theoretical models of redox enzyme linked biosensors highlights the importance of membrane thickness and enzyme loading on signal response in regard to both sensitivity and reproducibility. The conclusions are substantiated by examination of practical examples in the literature and, with a view to the importance of the character of the enzyme layer and overlayers, immobilization techniques are assessed which are in current use.     

A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum

Different techniques have been used (physical adsorption, physically entrapped sandwich and microencapsulation) for the immobilization of urease enzyme in tetramethylorthosilicate (TMOS) derived sol-gel matrix on the sensing surface of glass-pH-electrode. No significant leaching of enzyme occurs from the microencapsulated and physically entrapped enzyme sandwich films. Potentiometric techniques have been used for the estimation of urea concentration in each instance. Various parameters of biosensor performance have been studied which indicates that microencapsulation technique is a better method of enzyme immobilization in sol-gel films derived from TMOS. The advantage of microencapsulated biosensor over others include higher sensitivity (dpH/dp(C) = 2.4), lower detection limit of 52 μg mL⁻¹, larger linear range (0.01-30 mM) of urea determination and reasonably long-term stability of about 25 days with 80% response signal.