Enzyme-mediated sensors

There are a number of reports of mismatch detection strategies, in which enzymatic reactions are exploited to amplify the electrochemical signal. 

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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. 

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. 

Schuhmann, W., Wohlschlager, H., Lammert, R., Schmidt, H.-L, Loftier, U., Wiemhofer, H.-D., and Gopel, W. (1990) Leaching of dimethylferrocene, a redox mediator in amperometric enzyme electrodes. Sensors and Actuators B, BI, 571-574. 

Electrochemical biosensors have been divided into two basic types enzyme-based sensor and electrochemical probe-based sensor. Alkaline phosphatase (ALP) and horse radish peroxidase (HRP) have been often employed for enzyme-based biosensors using p-nitrophenyl phosphate (PNP), a-naphtyl phosphate, 3-3, 5,5 -tetramethylbenzidine (TMB) and 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrates of electrochemically active species, and ferrocene (Fc) and methylene blue as the electrochemical mediators. In general, enzymatic amplification of electrochemical signals enables highly sensitive detection of analytes. On the other hand, a direct detection of analytes by using electrochemical probes allows a more rapid time-response onto the detector surface and needs no enzymatic reaction. Based on the reason, a direct detection of analytes by using electrochemical probes has been... 

As described above, the introduction of ferrocenes as mediators for enzyme electrodes was subsequently developed as the versatile chemistry of these molecules was exploited both in redox mediation sensors and also through the use of ferrocene derivatives as labels in affinity sensors based upon electrochemical detection. 

On the other hand, a half-sandwich complex of manganese with j -CyD [( /-MeC5H4)Mn(NO)(S2CNMe2)] (Fig. 16.4.3) was found to be an efficient mediator in enzyme-based sensors including those useful for blood glucose sensing such as those involving GOx and horseradish oxidase [44]. 

The wealth of natural examples provides immense inspiration for the molecular design of novel peptide-based materials that can be potentially applied as devices, sensors, and biomaterials for medical applications. In addition to hierarchical self-assembly, nature uses other mechanisms, for example, enzyme-mediated covalent cross-linking, to build up structural proteins and higher-ordered structures. In the following sections we will focus on manmade peptide-based materials that belong to the three classes listed below. They will be split with respect to the underlying design concept into materials formed by ... 

Amperometric biosensors are generally composed of conventional metal electrodes and mediators that can shuttle electrons from the electrode to the active centre commonly buried deep within the polypeptide structure of the enzyme. Synthetic metals, including poly aniline, have also attracted considerable attention as electrochemical transducers for a variety of enzyme-based sensor devices. Significantly, these electrodes facilitate electron transfer to an enzyme without an added mediator species. This approach has simplified the construction of biosensors and also obviates sluggish kinetics, which are deleterious to precise detection (Figure 12.27)... 

Oxygen present in the analyte solution presents a potential problem for enzyme/mediator-based sensors of this type. This is because O2 can also oxidize... 

Electrocatalytic enzyme mediation has been demonstrated using quinones, viol-ogens, 2,2-azinobis(3-ethylbenzothiazohne-6-sulfonate) (ABTS), and complexes of iron, ruthenium, cobalt, osmium, and many other compounds [22-24]. Much early work concerned the GOx anode, intended for a glucose sensor. In 1974, Schlapfer et al. tested 11 different mediators for a GOx electrode with a semipermeable membrane [25]. Ten years later, Cass et al. reported membrane-bound electrodes that operated in whole blood [26]. In 1986, Bourdillon et al. presented an analysis arguing that immobilized enzyme electrodes have higher efficiency than those with free enzyme in solution [27]. These examples demonstrate several possible enzyme/ mediator configurations. Both enzyme and mediator can exist as firee species in the liquid electrolyte, or one or both can be immobilized on the electrode surface. As an alternative to immobilization, enzyme and mediator can also be confined near the electrode by a semipermeable membrane. 

Saito T, Watanabe M (1998) Characterization of poly(vinylferrocene-co-2-hydroxyethyl methacrylate) for use as elecnon mediator in enzymic glucose sensor. React Funct Polym 37 263-269... 

Yabuki S, Mizutani F, Katsura T (1994) Choline-sensing electrode based on polyethylene glycol-modified enzyme and mediator. Sensor Actuat B Chtan 20 159-162... 

Figure 11.39 summarizes the reactions taking place in this amperometric sensor. FAD is the oxidized form of flavin adenine nucleotide (the active site of the enzyme glucose oxidase), and FAD1T2 is the active site s reduced form. Note that O2 serves as a mediator, carrying electrons to the electrode. Other mediators, such as Fe(CN)6 , can be used in place of O2. 

By changing the enzyme and mediator, the amperometric sensor in Figure 11.39 is easily extended to the analysis of other substrates. Other bioselective materials may be incorporated into amperometric sensors. For example, a CO2 sensor has been developed using an amperometric O2 sensor with a two-layer membrane, one of which contains an immobilized preparation of autotrophic bacteria. As CO2 diffuses through the membranes, it is converted to O2 by the bacteria, increasing the concentration of O2 at the Pt cathode. 

The advancement in our understanding of mediated enzyme electrochemistry since the pioneering work of Hill and colleagues can be easily appreciated when it is realised that a commercial blood glucose sensor, the size of a pen, became commercially available only about a decade later. 

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