Biosensor redox mediated

NADH. Immobilized redox mediators, such as the phenoxazine Meldola Blue or phenothiazine compoimds, have been particularly useful for this purpose (20) (see also Figure 4-12). Such mediation should be useful for many other dehydrogenase-based biosensors. High sensitivity and speed are indicated from the flow-injection response of Figure 3-21. The challenges of NADH detection and the development of dehydrogenase biosensors have been reviewed (21). Alcohol biosensing can also be accomplished in the presence of alcohol oxidase, based on measurements of the liberated peroxide product. 

The laccases, classed as polyphenol oxidases, catalyze the oxidation of diphenols, polyamines, as well as some inorganic ions, coupled to the four-electron reduction of oxygen to water see Fig. 12.4 for the proposed catalytic cycle. Due to this broad specificity, and the recognition that this specificity can be extended by the use of redox mediators [27], laccases show promise in a range of applications [28], from biosensors [29-32], biobleaching [27, 33-35] or biodegradation [36], to biocatalytic fuel cells [1-3, 18, 26, 37-42]. 

Electrochemical biosensors can be divided into mediated and non-mediated biosensors. Biosensors that do not require the participation of redox molecules with reversible electrochemistry are referred to as non-mediated biosensors, whereas the participation of redox mediators in signal transduction generates a category of mediated biosensors. 

Attempts to reduce interference and minimize the effect of variations in oxygen tension have resulted in the development of biosensors with improved linear ranges which operate at lower electrode potentials. They incorporate artificial electron acceptors, called mediators, to transfer electrons from the flavoenzyme (e.g. glucose oxidase) to the electrode and thus are not dependent on oxygen. Ferrocene (bis(i75-cyclopentadienyl)iron) and its derivatives are examples of redox mediators for flavoenzymes. The reaction now becomes... 

As far as the use of ferrocene molecules as amperometric sensors is concerned, they have found wide use as redox mediators in the so-called enzymatic electrodes, or biosensors. These are systems able to determine, in a simple and rapid way, the concentration of substances of clinical and physiological interest. The methodology exploits the fact that, in the presence of enzyme-catalysed reactions, the electrode currents are considerably amplified.61 Essentially it is an application of the mechanism of catalytic regeneration of the reagent following a reversible charge transfer , examined in detail in Chapter 2, Section 1.4.2.5 ... 

A further possibility is the use of a mediated amperometric biosensor incorporating activated sludge to provide rapid determination of toxicity [79]. The respiratory activity of this biosensor is determined by using the redox mediator p-benzoquinone. 

Figure 19.7—Amperometric detection of glucose. The reaction cycle is shown on the left. A sandwich-type biosensor involving glucose oxidase co-immobilised with an osmium-based redox mediator in a polyvinyl polymer is shown on the right.

Table 13.2 summarises the different approaches used to construct enzyme electrochemical biosensors for application to food analysis based on the different types of enzymes available. Generally, the main problems of many of the proposed amperometric devices have been poor selectivity due to high potential values required to monitor the enzyme reaction, and poor sensitivity. Typical interferences in food samples are reducing compounds, such as ascorbic acid, uric acid, bilirubin and acetaminophen. Electrocatalysts, redox mediators or a second enzyme coupled reaction have been used to overcome these problems (see Table 13.2), in order to achieve the required specifications in terms of selectivity and sensitivity. 

Perhaps the original hope for these polymers was that they would act simultaneously as immobilisation matrix and mediator, facilitating electron transfer between the enzyme and electrode and eliminating the need for either O2 or an additional redox mediator. This did not appear to be the case for polypyrrole, and in fact while a copolymer of pyrrole and a ferrocene modified pyrrole did achieve the mediation (43), the response suggested that far from enhancing the charge transport, the polypyrrole acted as an inert diffusion barrier. Since these early reports, other mediator doped polypyrroles have been reported (44t45) and curiosity about the actual role of polypyrrole or any other electrochemically deposited polymer, has lead to many studies more concerned with the kinetics of the enzyme linked reactions and the film transport properties, than with the achievement of a real biosensor. 

During the last decade, immobilization of oxidase type enzymes by physical entrapment in conducting or ionic polymers has gained in interest, particularly in the biosensor field. This was related to the possibility for direct electron tranfer between the redox enzyme and the electroconducting polymers such as polypyrrole (1,2), poly-N-methyl pyrrole (3), polyindole (4) and polyaniline (5) or by the possibility to incorporate by ion-exchange in polymer such as Nafion (6) soluble redox mediators that can act as electron shuttle between the enzyme and the electrode. 

More recently, a new series of water dispersed anionic polymers, the AQ 29D, 38D and 55D polymers were released by Eastman Kodak. Since that time, these polymers were used as electrode modifier (12, 13), as covering membrane (14) and as support for enzyme immobilization (15, 16). AQ polymers are high molecular weights (14,000 to 16,000 Da) sulfonated polyester type polymers (17, 18). Their possible structures have been recently presented (18). The AQ polymer serie shows many interesting characteristics useful for the fabrication of biosensors. They are water dispersed polymers and thus compatible with enzymatic activity. They have sulfonated pendant groups similar to Nafion and they can act as a membrane barrier for anionic interferring substances and they offer the possibility to immobilize redox mediators by ion exchange. 

Bennetto HP, Box J, Delaney GM, Mason JR, Roller SD, Stirling JL, Thurston CF (1987) Redox-mediated electrochemistry of whole microorganisms from fuel cells to biosensors. Oxford Scientific Publishers, Oxford, p 291... 

Redox Mediated Whole Cell Biosensors for Toxicity Assessment and Environmental Protection... 

Redox mediated whole cell biosensors for toxicity assessment... 

Fig. 7.5. Schematic representation of some of the redox mediator processes at a whole cell biosensor. Lipohilic mediators may be reduced at redox active sites in the plasma membrane or at sites within the cytoplasm or both processes may occur—depending on the cell type and the mediator. Lipophobic mediators can only be reduced at sites on the outside edge of the plasma membrane. The oxidized form of the mediator. O, may be present in excess, but much of the reduced form. R, may need to diffuse between packed cells (dotted arrows) or through the cytoplasm (squiggly arrows). The subscripts aq, cyt, elec, and surf represent mediator in the aqueous phase, within the cytoplasm, at the electrode surface, and at the plasma membrane-aqueous interface, respectively.

There are practical difficulties in producing redox mediated whole cell biosensors that are reproducible within and between batches [53]. Models such as the one here can provide insights into the influence of various physical factors on the sensor outputs. Perturbations such as those described by the function if/ may be used to describe both stimulation and inhibition of these biosensors which are of importance in rapid toxicity assessment. 

---