Electrochemical entrapment

Immobilisation of biomolecules on the surface of an effective matrix with maximum retention of their biological recognition properties is a crucial problem for the commercial development of a biosensor. Different methods of immobilisation have been used. One such method is electrochemical entrapment. Several conducting polymers can be deposited electrochemically and, in the process, a biological molecule can be entrapped. This process is also useful in the fabrication of microsensors in preparation of a multilayered structure with one or more enzymes/biomolecules layered within a multilayered copolymer for analysis of multiple analytes [133-135]. A number of reports have appeared on immobilisation of biomolecules using electrochemical entrapment [130, 131, 136-143]. 

Thin films of fullerenes, which were deposited on an electrode surface via, for example, drop coating, were largely heterogeneous, due to the entrapping of solvent molecules into their domains. Consequently, their electrochemical behaviour displayed different degrees of reversibility and stability depending on the time of electrolysis and the... 

Functionalized conducting monomers can be deposited on electrode surfaces aiming for covalent attachment or entrapment of sensor components. Electrically conductive polymers (qv), eg, polypyrrole, polyaniline [25233-30-17, and polythiophene/23 2JJ-J4-j5y, can be formed at the anode by electrochemical polymerization. For integration of bioselective compounds or redox polymers into conductive polymers, functionalization of conductive polymer films, whether before or after polymerization, is essential. In Figure 7, a schematic representation of an amperomethc biosensor where the enzyme is covalendy bound to a functionalized conductive polymer, eg, P-amino (polypyrrole) or poly[A/-(4-aminophenyl)-2,2 -dithienyl]pyrrole, is shown. Entrapment of ferrocene-modified GOD within polypyrrole is shown in Figure 7. 

Entrapment of biochemically reactive molecules into conductive polymer substrates is being used to develop electrochemical biosensors (212). This has proven especially useful for the incorporation of enzymes that retain their specific chemical reactivity. Electropolymerization of pyrrole in an aqueous solution containing glucose oxidase (GO) leads to a polypyrrole in which the GO enzyme is co-deposited with the polymer. These polymer-entrapped GO electrodes have been used as glucose sensors. A direct relationship is seen between the electrode response and the glucose concentration in the solution which was analyzed with a typical measurement taking between 20 to 40 s. 

Corrosion has been encountered infrequently to date and has been a surface type, as opposed to pitting corrosion that can result in perforations. Entrapped air in the beverage or in the cans headspace increases the corrosive action of the product according to Koehler et at (21). As with beer and other canned foods, aluminum ends provide electrochemical protection when combined with tinplate or tin-free-steel can bodies. The level of iron pickup is reduced while the amount of aluminum dissolved in soft drinks increases without detrimental effect. Aluminum containers with vinyl epoxy and vinyl organosol coatings are compatible with carbonated soft drinks. 

Similarly to the above-mentioned entrapment of proteins by biomimetic routes, the sol-gel procedure is a useful method for the encapsulation of enzymes and other biological material due to the mild conditions required for the preparation of the silica networks [54,55]. The confinement of the enzyme in the pores of the silica matrix preserves its catalytic activity, since it prevents irreversible structural deformations in the biomolecule. The silica matrix may exert a protective effect against enzyme denaturation even under harsh conditions, as recently reported by Frenkel-Mullerad and Avnir [56] for physically trapped phosphatase enzymes within silica matrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have been employed for this purpose, as extensively reviewed by Gill and Ballesteros [57], and the resulting materials have been applied in the construction of optical and electrochemical biosensor devices. Optimization of the sol-gel process is required to prevent denaturation of encapsulated enzymes. Alcohol released during the... 

Electron Transfer Type of Dehydrogenase Sensors To fabricate an enzyme sensor for fructose, we found that a molecular interface of polypyrrole was not sufficient to realize high sensitivity and stability. We thus incorporated mediators (ferricyanide and ferrocene) in the enzyme-interface for the effective and the most sensitive detection of fructose in two different ways (l) two step method first, a monolayer FDH was electrochemically adsorbed on the electrode surface by electrostatic interaction, then entrapment of mediator and electro-polymerization of pyrrole in thin membrane was simultaneously performed in a separate solution containing mediator and pyrrole, (2) one-step method co-immobilization of mediator and enzyme and polymerization of pyrrole was simultaneously done in a solution containing enzyme enzyme, mediator and pyrrole as illustrated in Fig.22. 

A platinum disk electrode was electrolytically platinized in a platinum chloride solution to increase the surface area and enhance the adsorption power. The platinized platinum electrode was then immersed in a solution containing 10 mg ml l ADH. 0.75 mM and 6.2 mM NAD. After sufficient adsorption of these molecules on the electrode surface, the electrode was transferred into a solution containing 0.1 M pyrrole and 1 M KC1. Electrochemical polymerization of pyrrole was conducted at +0.7 V vs. Ag/AgCl. The electrolysis was stopped at a total charge of 1 C cm 2. An enzyme-entrapped polypyrrole membrane was deposited on the electrode surface. 

DNA modification of a commercial carbon ink without any electrochemical preconditioning by dry-adsorption was also reported. The surface was modified by covering with dsDNA solution and leaving the electrode to dry overnight. A stable, thin (about 100 p,m) DNA layer was obtained [111, 112]. The dsDNA was also demonstrated to be stably adsorbed when entrapped in a cellulose-acetate-based film on the surface of a carbon-based ink [113]. 

Flowever, the focus of the major part of the chapters lies on the couphng chemistry used for DNA immobilization. Successful immobihzation techniques for DNA appear to either involve a multi-site attachment of DNA (preferentially by electrochemical and/or physical adsorption) or a single-point attachment of DNA (mainly by surface activation and covalent immobihzation or (strept)avidin-biotin linkage). Immobilization methods described here comprise physical or electrochemical adsorption, cross-linking or entrapment in polymeric films, (strept)avidin-biotin complexation, a surface activation via self-assembled monolayers using thiol linker chemistry or silanization procedures, and finally covalent coupling strategies. 

Wang, S.F., Chen, T., Zhang, Z.L., Pang, D.W., and Wong, K.Y., Effects of hydrophobic room-temperature ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate on direct electrochemistry and biocatalysis of heme proteins entrapped in agarose hydrogel films, Electrochem. Commun., 9, 1709-1714, 2007. 

Many different types of techniques for protein immobilization have been developed using, in most cases, enzyme sensors. Early studies of enzyme biosensors often employed thick polymer membranes (thickness 0.01-1 mm) in which enzymes are physically entrapped or chemically anchored. The electrode surface was covered with the enzyme-immobilized polymer membranes to prepare electrochemical enzyme sensors. Although these biosensors functioned appropriately to... 

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