Immobilization redox mediator

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. 

Controlled potential electrolysis of the substrates, at —1.4V vs. SCE, at a carbon felt electrode on which only 3.6x10" mol Bjj were immobilized (approx. 1 X 10 ° mol cm" ) resulted in the production of 76pmol of valeronitrile 16, corresponding to a turnover number of 2100 This example shows that the combination of inner sphere redox mediators and high surface electrodes is promising. 

In redox mediation, to have an effective electron exchange, the thermodynamic redox potentials of the enzyme and the mediator have to be accurately matched. For biocatalytic electrodes, efficient mediators must have redox potentials downhill from the redox potential of the enzyme a 50 mV difference is proposed to be optimal [1, 18]. The tuning of these potentials is a compromise between the need to have a high cell voltage and a high catalytic current. Furthermore, an obvious requirement is that the mediator must be stable in the reduced and oxidized states. Finally, for operation of a membraneless miniaturized biocatalytic fuel cell, the mediators for both the anode and the cathode must be immobilized to prevent power dissipation by solution redox reactions between them. 

A remaining crucial technological milestone to pass for an implanted device remains the stability of the biocatalytic fuel cell, which should be expressed in months or years rather than days or weeks. Recent reports on the use of BOD biocatalytic electrodes in serum have, for example, highlighted instabilities associated with the presence of 02, urate or metal ions [99, 100], and enzyme deactivation in its oxidized state [101]. Strategies to be considered include the use of new biocatalysts with improved thermal properties, or stability towards interferences and inhibitors, the use of nanostructured electrode surfaces and chemical coupling of films to such surfaces, to improve film stability, and the design of redox mediator libraries tailored towards both mediation and immobilization. 

Li L, Wang J, Zhou J et al (2008) Enhancement of nitroaromatic compounds anaerobic biotransformation using a novel immobilized redox mediator prepared by electropolymerization. Bioresour Technol 99 6908-6916... 

MesoScale Discovery (MSD) succeeded in introducing product with a similar technology approach based upon ruthenium redox-mediated electrochemical detection (Figure 2.14). MSD is a joint venture of its parent company, MesoScale, and IGEN, a company that pioneered much of fhe work on electrochemical detechon based on the ruthenium redox system. MSD s Multi-Spot plates contain antibodies immobilized on multiple working electrode pads within each well, allowing each spot within the well to serve as an individual assay. Multiplexed cytokine immxmoassays can be performed in 96-well (4,7, or 10 spots per well) patterns with detection limits of 1 to 10 pg/mL and a linear dynamic range up to 3,000 pg/mL. Both 24-and 384-well electrode systems are available. 

LiP catalyzes the oxidation of a low-molecular-weight redox mediator, veratryl alcohol, which in mrn mediates one-electron oxidation of lignin to generate aryl cation radicals [100]. The radicals facilitate a wide variety of reactions such as carbon-carbon cleavage, hydroxylation, demethylation, and so on. Dezotti et al. [101] reported enzymatic removal of color from extraction stage effluents using lignin and horseradish peroxidases immobilized on an activated silica gel support. 

One of the most fruitful trends in the comprehension and control of electrochemical reaction kinetics and electrocatalysis has been the development of modified electrodes to achieve redox mediators of solution processes. This strategy is based on the electrochemical activation (through the application of an electrical perturbation to the electrode) of different sites at a modified surface. As a result of this activation, the oxidation or the reduction of other species located in the solution adjacent to the electrode surface (which does not occur or occurs very slowly in the absence of the immobilized catalyst) can take place4 [40, 69, 70]. 

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. 

The approaches that have been proposed to immobilize artificial mediators include the adsorption of the redox mediator (9), the immobilization in carbon paste (75), the covalent linkage on electroinactive (75) or conducting polymer backbone (10), the covalent attachement to the enzyme structures (3) and... 

Since mainly the E° of the mediator dictates at what potential the heterogenous electron transfer occurs, the oxidation of NADH can now take place at a much lower potential. The different mediator structures used to produce CMEs for NADH oxidation at a decreased overpotential are summarized in Table I. As is seen in the table, not only chemically modified electrodes based on only immobilized redox mediators have been used for this purpose, but also electrodes based on the combination of redox mediators and NADH oxidizing enzymes (diaphorase and NADH dehydrogenase) as well as electrodes made of the conducting radical salts of tetrathiafulvalinium-7,7,8,8-tetracyanoquinodimethan (TTF-TCNQ) and W-methyl-phenazin-5-ium-7,7,8,8-tetracyanoquinodimethan (NMP-TCNQ). 

Mediated Ehzyme Electrodes. Further improvements in the performance of immobilized enzyme sensors stem from the use of redox mediators which shuttle electrons from the redox centre of the enzyme to the surface of the indicator electrode according to the following reaction sequences depicted for glucose oxidase ... 

Using platinum electrodes (167, 238) requires +0.6 V versus SCE to oxidize H2O2. However, this potential precludes selective measurements of uric acid because it is also oxidized at the electrode surface (167). Thus, to improve the selectivity, bienzyme amperometric devices using a redox mediator (hexa-cyanoferrate) have been constructed (239). The enzymes uricase and peroxid ise are immobilized together and the hexacyanoferrate(III) is measured at 0.0 V versus Ag/AgCl. Alternatively, a carbon dioxide selective electrode is used for the detection of the enzymatically liberated CO2 (240, 241). 

First the immobilized GOD-anti-HCG is jdlowed to react with the antigen (equation 45). In a second step the antibody electrode is utilized to capture the GOD-anti-HCG complex (equation 46). After electrode capture, the catalytic activity of the bound enzyme label is measured by cyclic voltammetry in the presence of a redox mediator. The electrode is regenerated by soaking in urea for 5 min to break the Ab/Ag bond. Sensitivity of the assay is 9 mlU HCG/mL in serum. 

Wittstock, G., Emons, H., and Heineman, W. R. Electron transfer through an immunoglobulin layer via an immobilized redox mediator. Electroanalysis 1996, 8, 143-146. 

Fig. 10.2. Sensing principles for the determination of the biochemical oxygen demsind (BOD), (a) Immobilized cells on the surface of a Clark electrode and (b) batch assay with cells in suspension and with a redox mediator.

When constructing biosensors, which are to be used continuously in vivo or in situ, maintaining sensor efficiency while increasing sensor lifetime are major issues to be addressed. Researchers have attempted various methods to prevent enzyme inactivation and maintain a high density of redox mediators at the sensor surface. Use of hydrogels, sol-gel systems, PEI and carbon paste matrices to stabilize enzymes and redox polymers was mentioned in previous sections. Another alternative is to use conductive polymers such as polypyrrole [123-127], polythiophene [78,79] or polyaniline [128] to immobilize enzymes and mediators through either covalent bonding or entrapment in the polymer matrix. Application to various enzyme biosensors has been tested. 

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