Enzyme-based electrode

See also Enzymes Immobilized Enzymes Enzyme-Based Electrodes Enzyme-Based Assays. 

Enzyme electrodes are based on the coupling of a layer of an enzyme with an appropriate electrode. Such electrodes combine the specificity of the enzyme for its substrate with the analytical power of electrochemical devices. As a result of this coupling, enzyme electrodes have been shown to be extremely useful for monitoring a wide variety of substrates of analytical importance in clinical, environmental, and food samples. 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Particularly attractive for numerous bioanalytical applications are colloidal metal (e.g., gold) and semiconductor quantum dot nanoparticles. The conductivity and catalytic properties of such systems have been employed for developing electrochemical gas sensors, electrochemical sensors based on molecular- or polymer-functionalized nanoparticle sensing interfaces, and for the construction of different biosensors including enzyme-based electrodes, immunosensors, and DNA sensors. Advances in the application of molecular and biomolecular functionalized metal, semiconductor, and magnetic particles for electroanalytical and bio-electroanalytical applications have been reviewed by Katz et al. [142]. 

Vertically aligned CNT-modified electrodes are based on a more elaborated technique than other methods, and microscopic images are used to characterize the integrity of this type of electrode. The technique has been applied for the immobilization of enzymes and DNA, and the sensors based on this technique have shown a lower detection limit than those based on other methods. More research activities using this technique, particularly with low density CNT arrays, are expected in the near future because of its sensitivity and versatility. 

Potentiometric enzyme-based electrodes have found application in clinical, pharmaceutical, food and biochemical analyses to enable the selective determination of a wide range of important enzyme substrates, including amino acids, esters, amides, acylcholines, /Mactam antibiotics, sugars, enantioselective drugs and many others [74]. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Construction of enzyme-based amperometric biosensors usually involves coating the electrode surface with both the enzymes and the mediator. The method adopted and concentrations used are important factors in the performance of the device. For extended use, covalent immobilization is the most satisfactory method. 

Biological principles are also used in enzyme electrodes, where the sensor (usually an ion-selective electrode) is covered by a polymeric carrier containing an enzyme [32]. The determinand reacts in the enzyme layer yielding a product that causes a signal in the sensor. The bacterium electrode is based on a similar principle [84], as are electrodes using tissue in place of the enzyme layer [2]. 

The amygdaline electrode [47] that does not find extensive practical apph-cation is suitable as a model for investigation of the properties of enzyme electrodes in general [36], because of its specific response. The electrode is based on the reaction... 

Glucose sensors based on this electrochemistry are now commercially available. Furthermore, it seems likely that this concept will soon be expanded to other types of enzyme-based sensors. Hence, sensor development is proving to be one of the great success stories of the chemically modified electrode research area. 

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. 

The potentiometric biosensor is a combination of an ion-selective electrode (ISE) base sensor with a vegetable tissue (the source of enzyme), which provides a highly selective and sensitive method for the determination of a given substrate. Advantages of such potentiometric biosensors are simplicity of instrumentation (only a pH meter is needed),... 

F. Lucarelli, G. Marrazza and M. Mascini, Enzyme-based impedimetric detection of PCR products using oligonucleotide-modified screen-printed gold electrodes, Biosens. Bioelectron., 20 (2005) 2001-2009. 

Sensors of this type were constructed as multi-electrode arrays bearing chohnesterases of differing sensitivity to organo-phosphates, and the assay extended to milk [35]. Both spiked milk and milk from shops inhibited the activity of the electrodes. In two instances, estimates of the level of organo-phosphates in spiked milk made using sensors were very close to those made using GC-MS. This appears to have been a fortunate co-incidence as the response curves used for calibration were not linear. Nine out of ten milk samples from shops inhibited at least some members of the array. In only one case did the GC-MS assay find insecticides in the samples, but the insecticides were not organo-phosphates. The inhibition shown by the enzyme-based arrays was reversible by pyridine-2-aldoxime... 

A brief sample preparation procedure based on solid-phase extraction was developed [45]. Wool was extracted in acetonitrile for 30 min, the fluid then being filtered and passed through a C18 solid-phase cartridge. The acetonitrile was removed under vacuum and replaced with methanol for application of the putative inhibitors to enzyme-based electrodes. 

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-... 

There are three basic types of selective electrode those based on glass membranes, on inorganic salt solid membranes, and on ion exchange. Other more complex electrodes are sensitive to dissolved gases and enzymes. These various types are now described. 

In common with enzyme based biosensors (equations (7.3) and (7.4)) the reduced mediator can be re-oxidized when it diffuses to an electrode poised at a suitably oxidising potential, e.g.,... 

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