Ferrocenes as mediators

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. 

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. 

Since the first report on the ferrocene mediated oxidation of glucose by GOx [69], extensive solution-phase studies have been undertaken in an attempt to elucidate the factors controlling the mediator-enzyme interaction. Although the use of solution-phase mediators is not compatible with a membraneless biocatalytic fuel cell, such studies can help elucidate the relationship between enzyme structure, mediator size, structure and mobility, and mediation thermodynamics and kinetics. For example, comprehensive studies on ferrocene and its derivatives [70] and polypy-ridyl complexes of ruthenium and osmium [71, 72] as mediators of GOx have been undertaken. Ferrocenes have come to the fore as mediators to GOx, surpassing many others, because of factors such as their mediation efficiency, stability in the reduced form, pH independent redox potentials, ease of synthesis, and substitutional versatility. Ferrocenes are also of sufficiently small size to diffuse easily to the active site of GOx. However, solution phase mediation can only be used if the future biocatalytic fuel cell... 

A first application using ferroceneboronic acid as mediator [45] was described for the transformation of p-hydroxy toluene to p-hydroxy benzaldehyde which is catalyzed by the enzyme p-cresolmethyl hydroxylase (PCMH) from Pseudomonas putida. This enzyme is a flavocytochrome containing two FAD and two cytochrome c prosthetic groups. To develop a continuous process using ultrafiltration membranes to retain the enzyme and the mediator, water soluble polymer-bound ferrocenes [50] such as compounds 3-7 have been applied as redox catalysts for the application in batch electrolyses (Fig. 12) or in combination with an electrochemical enzyme membrane reactor (Fig. 13) [46, 50] with excellent results. 

Attempts have been performed to analyse (in terms of rate constants) the steric-to-electrochemical factors which can influence the interaction of ferrocenium derivatives with reduced GOD, or the ability of ferrocenes to act as mediators in the enzymatic oxidation of glucose as a function of cyclopentadienyl substituents.63... 

Fig. 37.6. Experimental example of an unintended feedback imaging, (a) Schematic showing (1) the intended GC imaging of HRP activity (2) hindered diffusion above a densely packed monolayer using the ferrocinium derivative (Fc+) as mediator. Fc+ is present in low concentration possibly due to partial chemical oxidation by 02 or H202 (3) the regeneration of Fc+ at the gold electrode surface under a less-organized monolayer, (b) Experimental line scan in the presence of ferrocene methanol (and traces of Fc+) and H202. (c) Same as (b) but in absence of H202.

Di Gloria et al. [54] proposed the use of electrode-enzyme coupling for the amplified readout of a redox label, using a lidocaine-ferrocene conjugate in combination with GOx. The use of ferrocene as a mediator for glucose determination within GOx enzyme electrodes provided the... 

Scheme 3 Some water-soluble polymer-bound ferrocene derivatives used as mediators for the anaerobic reactivation of FAD enzymes.

According to the reaction scheme given in Fig. 26, the anaerobic oxidation of l-glycerol 3-phosphate was performed under the conditions of an indirect electrochemical process using a water-soluble ferrocene derivative as mediator in the presence of o-fruc-tose-1,6-diphosphate aldolase from rabbit muscle for the in situ generation of the carbohydrate product. With a mediator concentration fo 1.5 mM using about 80 U of immobilized enzyme, after 25 h a 75% turnover of the substrate L-glycerol phosphate... 

An interesting investigation concerns the use of PPI dendrimers functionalized with both ferrocene and cobaltocenium moieities for glucose biosensor [105, 127], Such dendrimers can exhibit a double function while the ferrocene units act as mediators in enzymatic processes under anaerobic conditions, the cobaltocenium moieties take part in the electrocatalysis in the presence of oxygen. Another major advantage cited of these electrodes is that a large amount of enzyme can be immobilized due to electrostatic interactions between the positive... 

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