Enzyme based films

Second, sensors are often intended for a single use, or for usage over periods of one week or less, and enzymes are capable of excellent performance over these time scales, provided that they are maintained in a nfild environment at moderate temperature and with minimal physical stress. Stabilization of enzymes on conducting surfaces over longer periods of time presents a considerable challenge, since enzymes may be subject to denaturation or inactivation. In addition, the need to feed reactants to the biofuel cell means that convection and therefore viscous shear are often present in working fuel cells. Application of shear to a soft material such as a protein-based film can lead to accelerated degradation due to shear stress [Binyamin and Heller, 1999]. However, enzymes on surfaces have been demonstrated to be stable for several months (see below). 

Chemiluminescent labeling systems have been developed, based on the incorporation of fluorescein-11-dUTP into a DNA probe. An anti-fluorescein antibody covalently bound to the enzyme HRP is then bound to the incorporated fluorescein label. HRP catalyses the breakdown of luminol and the chemiluminescent signal is detected by autoradiography with X-ray film or by fluorescence scanning instrumentation. Chemiluminescence is more sensitive than enzyme-based color detection systems. Furthermore the labeled gene probes are stable and give results quickly (160). 

Chapters 1 to 5 deal with ionophore-based potentiometric sensors or ion-selective electrodes (ISEs). Chapters 6 to 11 cover voltammetric sensors and biosensors and their various applications. The third section (Chapter 12) is dedicated to gas analysis. Chapters 13 to 17 deal with enzyme based sensors. Chapters 18 to 22 are dedicated to immuno-sensors and genosensors. Chapters 23 to 29 cover thick and thin film based sensors and the final section (Chapters 30 to 38) is focused on novel trends in electrochemical sensor technologies based on electronic tongues, micro and nanotechnologies, nanomaterials, etc. 

This book on Electrochemical (Bio)Sensor Analysis, edited by S. Alegret and A. Merko< i, is an additional step to advance the field of rapid analysis. It presents advanced sensor developments as well as practical applications of electrochemical (bio)sensors in various fields in a single source. The book contains 38 chapters grouped into seven sections (a) Potentiometric sensors, (b) Yoltammetric (bio)sensors, (c) Gas sensors, (d) Enzyme based sensors, (e) Affinity biosensors, (f) Thick and thin film biosensors, and (g) Novel trends. This interdisciplinary book has contributions from well-known specialists in the field and will be a useful resource for professionals with an interest in the application of electrochemical (bio)sensors. 

Part I contains general reviews on the theoretical and practical aspects related to the application of (bio) sensors in various fields. Its 38 chapters are grouped in seven sections 1) Potentiometric sensors, 2) Voltammetric sensors, 3) Gas sensors, 4) Enzyme based sensors, 5) Affinity biosensors, 6) Thick and thin film (bio) sensors and 7) Novel trends. 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

In an attempt to improve the selectivity of local dopamine measurements in the complex extracellular matrix of brain fluid, an implantable enzyme-based dopamine microbiosensor has been constructed based on the immobilization of tyrosinase in a thin-film chitosan coating of carbon-fiber disc microelectrodes [357]. o-Dopaquinone, which is the product of the tyrosinase reaction with dopamine, was monitored via its reduction at the modified microelectrode surface. The application of these cathodic tyrosinase dopamine microbiosensors was reported for the continuous real-time in vivo visualization of electrically stimulated dopamine release in the brain of anesthetized laboratory rats. Remarkably, due to the cathodic potential the sensor response was not significantly disturbed by the presence of typical interferences such as ascorbic and uric acid, serotonin, norepinephrine, and epinephrine. 

Electropolymerization of polymers directly onto the surface of an electrode has been used for a number of enzyme-based biosensors. By polymerizing from a solution containing the monomer, as well as the other components of the sensor, enzymes for example, a multifunctional polymer film can be fabricated. As the polymer film grows on the electrode, the enzyme and other components are entrapped in the film [9]. GOD and other enzymes have been incorporated into sensors using electropolymerization. Advantages of electropolymerization are that the film thickness can be easily controlled by the amount of polymerization charge passed, and that the polymer film is deposited only on the sensing electrode. 

In a biosensor application, Wolfson and his research group [10] employed microfabrication techniques to produce miniature electrodes for potential implantable, non-enzyme-based glucose sensors. Thin-film techniques, such as DC magnetron-sputtered processing, were used to deposit platinum film or platinum film enhanced with titanium film on quartz substrates. The geometric shapes of the quartz substrate include cylindrical rods and rectangular plates in a... 

Matsue et al. [27] were the first to explore an enzyme-based OECT biosensor. They used Diaphorase as the entrapped enzyme in a polypyrrole transducing layer for the detection of NADH via a redox mediator (the sodium salt of anthraquinone-2-sulfonic acid). The net result was the conversion of polypyrrole from its conducting state to its insulating state in the presence of NADH. The device showed a response time of 15--20 min in the presence of NADH. Later Nishizawa et al. [26] exploited the pH sensitivity of the polypyrrole film for the design and fabrication of OECT sensors for pH and for pencillin. The Penicillinase enzyme was entrapped in a membrane which was coated with a polypyrrole film, in which a decrease in pH was observed in the presence of penicillin due to the hydrolysis of penicillin by Penicillinase. 

Enzymes-based biosensors are well reported in the literature for chemical toxicity screening. The sensor devices produced using enzymes are usually simple and easy to fabricate, inexpensive, and sensitive to low levels of toxicants. Immobilization of enzymes on the electrode surface can include adsorption, covalent attachment, or film deposition using a range of procedures [68-70]. The sensor system relies primarily on two enzyme mechanisms catalytic transformation of a pollutant and detection of pollutants that inhibit or mediate the enzyme s activity. In catalytic enzyme biosensor, the enzyme specific for the substrate of interest (toxin in this case)... 

Pritchard et al. [131] describe the development of a single thiocholine enzyme-based biosensor. This biosensor is a sonochemically fabricated enzyme microelectrode array in order to impart stir-independent (convection) responses that are characteristic of microelectrodes. Microelectrode arrays with up to 2 x 10 microelectrode elements may be fabricated via the sonochemical ablation of noncondnc-tive polymer films [132,133], which coat and thereby insnlate nnderlying condnctive snrfaces [134]. Paraoxon is determined down to concentrations of 1 x 10 M via the nse of sonochemically fabricated acetylcholine/polyaniline microelectrode array-based sensors. These sensors were fabricated via the electropolymerization of thin... 

An approach which is now becoming of more importance in the development of enzyme-based biosensors is that of immobilisation of the active protein in a polymer matrix. We have now carried out a preliminary investigation into the incorporation of anti-HSA into an electrochemically-generated conducting polypyrrole film. Conducting polymers such as polypyrrole (I)... 

Kumagai, Y., and Doi, Y., 1992, Enzymic degradation of poly(3-hydroxybutyrate>based films poly(3-hydroxybutyrate)/poly(ethylene oxide) blend. Polym. Degrad. Stab. 35 87-93. 

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARE-1 into polyelectrolyte multilayer capsules. The co-precipitation of calcium caibonate, urease, and dextran followed up by multilayer film coating and Ca- extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10 to 10 M. Sensitivity to urea in concentration range of 10 to 10 M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by confocal fluorescent microscopy. 

Sun, Z. and H. Tachikawa (1992). Enzyme-based bilayer conducting polymer electrodes consisting of polymetallophthalocyanines and polypyrrole-glucose oxidase thin films. Ana/. Chem. 64, 1112-1117. 

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