Enzyme Layer Research Articles

Efficiency of a Bienzyme Sequential Reaction System Immobilized on Polyelectrolyte Multilayer-Coated Colloids

We assembled multilayer films of glucose oxidase (GOx) and horseradish peroxidase (HRP) coimmobilized together with polyelectrolyte layers on the surface of silica microparticles. The influence of different polyelectrolyte combinations on the immobilization and functionality of the enzymes was examined for several multilayer configurations. Precomplexation of the enzymes with a polyvinylpyridine-based polyamine allowed the stable adsorption of enzyme layers without affecting their catalytic activity. The efficiency of the sequential reaction between GOx and HRP on the surface of the colloids was quantitatively analyzed and rationalized in terms of the kinetic parameters of both enzymes and the reaction-diffusion kinetics of the system. In the optimized configuration, with GOx and HRP coimmobilized in the same layer, the overall rate of hydrogen peroxide conversion was around 2.5 times higher than for GOx and HRP in separate layers or for equivalent amounts of both enzymes free in solution.

Penicillin detection with nanocrystalline‐diamond field‐effect sensor

AbstractNanocrystalline‐diamond (NCD) films have been utilised for the detection of penicillin G for the first time. The developed penicillin‐sensitive biosensor consists of a field‐effect capacitive electrolyte‐diamond‐insulator‐semiconductor (EDIS) structure with an immobilised enzyme layer that covers the gate region of the sensor. Undoped NCD thin films of about 100 nm thickness were grown on a p‐Si‐SiO2 (50 nm thermally grown SiO2) structure by microwave plasma‐enhanced chemical vapour deposition. The enzyme penicillinase has been adsorptively immobilised directly onto the O‐terminated NCD surface. The EDIS biosensors have been characterised in buffer solutions with different content of penicillin G by means of capacitance–voltage and constant‐capacitance method. The developed penicillin biosensor possesses a low detection limit of 5 µM and a high sensitivity of 60–70 mV/decade in a wide linear range of 0.005–2.5 mM penicillin G concentration. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Modelling Amperometric Biosensors Based on Chemically Modified Electrodes.

The response of an amperometric biosensor based on a chemically modified electrode was modelled numerically. A mathematical model of the biosensor is based on a system of non-linear reaction-diffusion equations. The modelling biosensor comprises two compartments: an enzyme layer and an outer diffusion layer. In order to define the main governing parameters the corresponding dimensionless mathematical model was derived. The digital simulation was carried out using the finite difference technique. The adequacy of the model was evaluated using analytical solutions known for very specific cases of the model parameters. By changing model parameters the output results were numerically analyzed at transition and steady state conditions. The influence of the substrate and mediator concentrations as well as of the thicknesses of the enzyme and diffusion layers on the biosensor response was investigated. Calculations showed complex kinetics of the biosensor response, especially when the biosensor acts under a mixed limitation of the diffusion and the enzyme interaction with the substrate.

Hydrogen Peroxide Biosensor with a Supramolecular Layer-by-Layer Design

A new sensor design is reported for the construction of an amperometric enzyme biosensor toward H (2)O(2). It was based in the supramolecular immobilization of alternating layers of horseradish peroxidase (either modified with 1-adamantane or beta-cyclodextrin-branched carboxymethylcellulose residues) on Au electrodes coated with polythiolated beta-cyclodextrin polymer. The analytical response of the electrodes, using 1 mM hydroquinone as an electrochemical mediator, increases when the number of enzyme layers increases. The biosensor having three enzyme layers showed a sensitivity of 720 microA/M cm (2) and a detection limit of 2 microM and retained 96% of its initial activity after 30 days of storage. The host-guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.

Improved stability of redox enzyme layers on glassy carbon electrodes via covalent grafting

One of the challenges in the field of enzymatic biofuel cells is to significantly improve their current limited lifetime. In the present work, we report the covalent immobilization of enzyme layers on glassy carbon electrodes, functionalized via electrochemical reduction of in situ generated aryldiazonium salts bearing carboxylic acid groups. We present the performance and the stability over time of the modified electrodes. For glucose oxidase – modified electrodes, stable catalytic activity is observed for a minimum of 6 weeks.

Performance of an emulsion enzyme membrane reactor combined with premix membrane emulsification for lipase-catalyzed resolution of enantiomers

Enzyme membrane reactors combined with the large interfacial area of a fine emulsion, called emulsion enzyme membrane reactors (E-EMRs), were investigated for the optical resolution of ( R, S)-naproxen methyl esters to produce ( S)-naproxen. Preparation of a fine emulsion by continuous premix membrane emulsification (ME) was studied and compared to direct ME. By continuous ME, an emulsion with 1.32 μm of average droplet size and 46.5% (v/v) of organic phase content was obtained using a Shirasu porous glass (SPG) membrane of 5 μm pore size under 0.12 MPa pressure. The performance of the E-EMR, including mass transfer, reaction rate, enzyme activity, productivity, enantiomeric excess and enantioselectivity, were studied with different loads of lipase on the membrane or with different substrate concentrations in the emulsion. The immobilized enzyme layer caused the main mass transfer resistance in E-EMRs. An increase of the amount of lipase in the membrane did not result in a production increase but an increase in substrate concentration led to an increase in reaction rate. Up to 100% of enantiomeric excess and more than 100 of enantioselectivity was obtained in E-EMR.

XPS and AFM Characterization of the Enzyme Glucose Oxidase Immobilized on SiO2 Surfaces

A process to immobilize the enzyme glucose oxidase on SiO2 surfaces for the realization of integrated microbiosensors was developed. The sample characterization was performed by monitoring, step by step, oxide activation, silanization, linker molecule (glutaraldehyde) deposition, and enzyme immobilization by means of XPS, AFM, and contact angle measurements. The control of the environment during the procedure, to prevent silane polymerization, and the use of oxide activation to obtain a uniform enzyme layer are issues of crucial importance. The correct protocol application gives a uniform layer of the linker molecule and the maximum sample surface coverage. This result is fundamental for maximizing the enzyme bonding sites on the sample surface and achieving the maximum surface coverage. Thin SiO2 layers thermally grown on a Si substrate were used. The XPS Si 2p signal of the substrate was monitored during immobilization. Such a signal is not completely shielded by the thin oxide layer and it is fully suppressed after the completion of the whole protocol. A power spectral density analysis on the AFM measurements showed the crucial role of both the oxide activation and the intermediate steps (silanization and linker molecule deposition) to obtain uniform immobilized enzyme coverage. Finally, enzymatic activity measurements confirmed the suitability of the optimized protocol.

Needle Enzyme Electrode for Lactate MeasurementIn Vivo

Electrochemical lactate needle enzyme electrodes were fabricated based on lactate oxidase with a conventional hydrogen peroxide detection regimen with a linear range up to 7 mM, response time ~ 3 min, and sensitivity ~ 1 nA/mM. A negatively charged inner (sulphonated polyether ether sulphone-polyether sulphone) membrane was applied for ensuring selectivity by limiting oxidazible anion diffusion to the Pt working electrode; polyurethane outer membrane layers were dip coated over the enzyme layer to limit substrate diffusion to the enzyme layer to achieve: (1) stir independence and (2) a low oxygen requirement. Lactate was monitored subcutaneously in rats during controlled haemorrhage and hypovolaemic shock. Tissue lactate showed agreement with blood lactate before haemorrhage and for limited haemorrhage (up to 2 ml blood withdrawal from 16 ml total blood volume) but with blood loss above 3 ml the tissue lactate rise was less pronounced than in blood. Loss of intercompartmental equilibrium due to diffusion limitation is suggested as a factor in causing this difference. An experimental in vitro model was developed which employed the needle lactate electrode within a cylindrical collagen gel to monitor inward diffusion of lactate as a basis for determining lactate diffusion coefficient. The high precision measurement gave a diffusion coefficient consistent with report values 3.54 times 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-6</sup> cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> /s. The simplified experimental approach could allow lactate transport studies across tissue analogues.

An approach to in situ detection of hydrogen peroxide: application of a commercial needle-type electrode

The reactive oxygen species are thought to play major roles in developing different physiological disorders. A commercial, needle-type amperometric glucose enzyme sensor manufactured for human patients was investigated. This sensor measures glucose by detecting hydrogen peroxide evolved in the enzymatic reaction of glucose. In the experiments, the immobilized enzyme layer of the sensor was inactivated. The applicability of this ‘inhibited’ glucose sensor for detecting hydrogen peroxide was tested. The simple battery powered, single purpose electronic unit was replaced by an advanced electrochemical workstation. The sensitivity, selectivity and lower limit of detection of the hydrogen peroxide measurements were investigated. Voltammetric measurements were carried out in intensively stirred buffered aqueous media, in plasma samples as well as in subcutan areas of anesthetized Wistar rats. Preliminary measurements carried out with the amperometric and periodically interrupted amperometric technique predicted that the human clinical sensor, after our enzyme inhibition step, can be used for checking the elevation of the hydrogen peroxide level in different subcutan areas of human subjects.

Modelling an amperometric biosensor acting in a flowing liquid

AbstractThis paper presents a mathematical model of the amperometric biosensor based on an electrode covered with an enzyme membrane. The model involves three regions: the enzyme layer where enzymatic reaction as well as mass transport by diffusion takes place, a diffusion‐limiting region where only diffusion takes place, and a convective region, where the analyte concentration is maintained constant. Using computer simulation the influence of the biosensor geometry as well as the flow intensity on the biosensor response was investigated. This paper also deals with the conditions when the mass transport in the exterior region may be neglected to simulate the biosensor response assuming that the buffer solution is in intense flow and in powerful motion. The digital simulation was carried out using the finite difference technique. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Cellulose Fiber−Enzyme Composites Fabricated through Layer-by-Layer Nanoassembly

Cellulose microfibers were coated with enzymes, laccase and urease, through layer-by-layer assembly by alternate adsorption with oppositely charged polycations. The formation of organized polyelectrolyte and enzyme multilayer films of 15-20 nm thickness was demonstrated by quartz crystal microbalance, zeta-potential analysis, and confocal laser scanning microscopy. These biocomposites retained enzymatic catalytic activity, which was proportional to the number of coated enzyme layers. For laccase-fiber composites, around 50% of its initial activity was retained after 2 weeks of storage at 4 degrees C. The synthesis of calcium carbonate microparticles on urease-fiber composites confirmed urease functionality and demonstrated its possible applications. This strategy could be employed to fabricate fiber-based composites with novel biological functions.

Viscoelastic change following adsorption and subsequent molecular reorganisation of a nitroreductase enzyme on a gold surface: A QCM study

A quartz crystal shear mode resonator operating at 5 MHz has been used to study viscoelastic changes which result from adsorption from solution of a layer of the enzyme nitroreductase on a gold electrode. The enzyme is anchored to the gold via thiol end-groups of cysteine residues the number of which, per molecule, has been varied by genetic modification. Using an appropriate model for the acoustic shear wave load on the resonator, the viscoelasticity of the adsorbed enzyme layer have been monitored as the layer develops. A negative viscosity is deduced for all the enzymes and is attributed to amplification of shear waves in the enzyme layer by energy transfer from a compression mode also produced by the crystal. In addition to gradual changes in the viscoelastic parameters as adsorption proceeds, spontaneous changes in viscoelasticity are observed, which indicate a rapid reorganization of the enzyme layer. These various changes, reflecting alterations in the mechanical response of the adsorbed enzyme to shear forces acting at 5 MHz, suggest that the quartz crystal shear wave resonator has considerable potential for studying structural motions in adsorbed proteins at frequencies not readily accessible by other techniques.

Characterisation of poly(neutral red) modified carbon film electrodes; application as a redox mediator for biosensors

The polymer redox mediator, poly(neutral red) (PNR), has been synthesised and characterised electrochemically to investigate the best electropolymerisation and mediation conditions for application in enzyme biosensors and to clarify the mechanism of action. Neutral red was electropolymerised by potential cycling on carbon film electrode substrates by allowing the monomer to be oxidised during the full 20 cycles of polymerisation or reducing the positive limit of the potential window after the first 2 cycles to impede monomer oxidation with a view to obtaining longer polymer chains and a lesser degree of branching. Comparison was made with glassy carbon substrates. The PNR films on carbon film electrodes were characterised using cyclic voltammetry and electrochemical impedance spectroscopy, as well as in glucose biosensors prepared with PNR. Glucose oxidase enzyme was immobilised by encapsulation in silica sol-gel and compared with that obtained by cross-linking with glutaraldehyde. The biosensors were evaluated by chronoamperometry in 0.1 M phosphate buffer saline solution, pH 7.0, and showed evidence of electron transfer between the enzyme cofactor flavin adenine dinucleotide and PNR dissolved in the enzyme layer competing with PNR-mediated electrochemical degradation of H2O2 formed during the enzymatic process.

Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832unitsmg−1) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na+–K+ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110s) was characterized using the 0.2M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1×10−10 to 1×10−6M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5×10−11M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.

Immobilization of laccase on gold, silver and indium tin oxide by zirconium–phosphonate–carboxylate (ZPC) coordination chemistry

In this paper we present a simple method allowing for stable laccase immobilization on various conducting surfaces that retains the activity of the enzyme. The strategy for laccase immobilization presented in this paper relies on Zr 4+ ion coordination chemistry that involves –COO − terminal groups present on the protein. Using a host of techniques, including surface plasmon resonance (SPR), quartz crystal microbalance (QCM) gravimetry, atomic force microscopy (AFM), surface enhanced Raman scattering (SERS), resonance Raman scattering (RR) and electrochemical techniques, we show that laccase bound to a surface coordinatively through zirconium phosphonate/carboxylate (ZPC) functionalities forms a stable enzymatic layer with the enzyme retaining its activity to a significant extent.

Immobilization Techniques to Avoid Enzyme Loss from Oxidase-Based Biosensors: A One-Year Study

Continuous amperometric sensors that measure glucose or lactate require a stable sensitivity, and glutaraldehyde crosslinking has been used widely to avoid enzyme loss. Nonetheless, little data is published on the effectiveness of enzyme immobilization with glutaraldehyde. A combination of electrochemical testing and spectrophotometric assays was used to study the relationship between enzyme shedding and the fabrication procedure. In addition, we studied the relationship between the glutaraldehyde concentration and sensor performance over a period of one year. The enzyme immobilization process by glutaraldehyde crosslinking to glucose oxidase appears to require at least 24-hours at room temperature to reach completion. In addition, excess free glucose oxidase can be removed by soaking sensors in purified water for 20 minutes. Even with the addition of these steps, however, it appears that there is some free glucose oxidase entrapped within the enzyme layer which contributes to a decline in sensitivity over time. Although it reduces the ultimate sensitivity (probably via a change in the enzyme's natural conformation), glutaraldehyde concentration in the enzyme layer can be increased in order to minimize this instability. After exposure of oxidase enzymes to glutaraldehyde, effective crosslinking requires a rinse step and a 24-hour incubation step. In order to minimize the loss of sensor sensitivity over time, the glutaraldehyde concentration can be increased.

Enzyme Electrodes for Glucose Oxidation Prepared by Electropolymerization of Pyrrole

In this work, a method of fabricating enzyme electrodes with the potential application for biofuel cells and biosensors was investigated. Enzyme electrodes were prepared by entrapping native glucose oxidase (GOx) and ferrocene wired GOx (FeFcGOx), as the dopants, in conducting polypyrrole matrices by electropolymerization. Furthermore, the developed polymer enzyme layer was characterized by physical and electrochemical analysis, and glucose oxidation activity on both electrodes was studied. Both electrodes showed direct electrical communication between the enzyme and electrode surface. Low glucose oxidation current was observed from the electrode containing GOx. Higher glucose oxidation current was obtained with FeFcGOx, and the current increased with the escalating glucose concentration suggesting that efficient electron transfer between the enzyme and the electrode surface were achieved. A maximum sensitivity of 20mM for glucose concentration was obtained for the polymer electrode prepared from FeFcGOx.

Enzyme-based Field-Effect Transistor for Adenosine Triphosphate (ATP) Sensing

Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage.

Prototype Amperometric Biosensor for Sialic Acid Determination

This paper describes the first report on the development, characterization, and applications of a prototype amperometric biosensor for free sialic acid (SA). The sensor was constructed by the coimmobilization of two enzymes, i.e., N-acetylneuraminic acid aldolase and pyruvate oxidase, on a polyester microporous membrane, which was then mounted on top of a platinum disk electrode. The SA biosensor operation was based on the sequential action of the two enzymes to ultimately produce hydrogen peroxide, which was then detected by anodic amperometry at the platinum electrode. The surface of the platinum electrode was coated with an electropolymeric layer to enhance the biosensor selectivity in the presence of interfering oxidizable species. Optimization of the enzyme layer composition resulted in a fast and steady current response in phosphate buffer pH 7.2 at 37 degrees C. The limit of detection was 10 microM, and the response was linear to 3.5 mM (r = 0.9987). The prepared SA biosensors retained approximately 85% of their initial sensitivity after 8 days and showed excellent response reproducibility (CV = 2.3%). Utilization of a third enzyme, sialidase, expanded the scope of the present SA biosensor to determine bound sialic acid as well. The merits of the described biosensor allowed its successful application in determining SA in biological and pharmaceutical samples. The obtained results indicated that the presented SA biosensor should be a useful bioanalytical tool in several biological and clinical applications such as screening of SA as a nonspecific tumor marker as well as monitoring of tumor therapy.