Amperometric detection redox mediator

There are several demands that must be more or less fulfilled by the mediator before a successfull amperometric detection of NADH with CMEs can be realized. Despite having a E° lower or comparable with the optimal working potential range for amperometric detection, the mediator should exhibit fast reaction rates both with the electrode proper and NADH, and also be chemically stable at any redox state. Furthermore, the redox reaction of the mediator should involve two electrons and at least one proton making possible, at least theoretically, a fast inner sphere hydride transfer in the homogeneous reaction with NADH. 

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.

The redox chemistry of PQQ has been investigated by a number of research groups. Duine et al. [14,15] performed potentiometric titrations of PQQH2 at several pHs and measured the redox potential of PQQ/PQQH2. Eckert et al. [16,17] compared the redox properties of PQQ with those of o-phenanthroline quinones. Kano et al. [18] performed cyclic voltammetry at acidic pH. Bergethon [19] investigated the amperometric detection of PQQ as a tool for HPLC. From pulse radiolysis experiments, McWhirter and Klapper [20] derived a value of -122 mV (NHE) for Em PQQ/PQQH at pH 7, as compared to the value of - 218 mV calculated from mediator-linked potentiometric titrations [15],... 

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). 

FIG. 6 Schematic illustrating the difference between (A) GC mode and (B) feedback mode detection of immobilized enzyme activity. The example enzyme is glucose oxidase (GOx) with a ferrocenyl redox mediator (Fc) detected at a Pt tip. (A) The bulk solution contains oxidized mediator (Fc+) and the tip detects the reduced form amperometrically which is generated continually wherever active enzyme is present on the specimen. (B) The bulk solution contains reduced mediator, the tip generates ferrocenium locally and detects the enhanced flux due to feedback when there is active enzyme on the portion of the specimen beneath the tip. , Glucose o, gluconolactone. 

Monitoring of the intracellular redox activity in eukaryotic cells imposes the requirement that the utilized mediator is capable of readily crossing the plasma membrane into the intracellular environment to communicate with the enzyme(s), the activity of which is to be monitored. This strictly requires the utilization of a lipophilic mediator that can diffuse through the plasma membrane. Using chip based amperometric detection on S. cerevisiae, menadione was shown to possess the desired properties [28]. Figure 3 depicts the functional principle of the chip based detection technique to monitor CRE in eukaryotic cells, which aside from the lipophilic menadione,... 

The concentration of hydrogen peroxide can be measured directly using amperometric detection. A change in H2O2 concentration in the medium appears as a variarion in the output current. The quantified parameters are m nitude of the sensor response, response time, and current response. It is desirable to measure signals in conditions when the linear relationship exists between the current value and the analyte concentration. At that point, the reactions are considered to be in steady state when pseudoequilibrium occurs between the species close to the sensor and their consumption at the indicative electrode. One of the serious problems associated with measurement of complex analytes is the possible interference of the redox species present in the sample. Several methods have been reported which aimed at reducing level of interference. These methods include use of perm-selective coatii, use of artificial mediators, or selective electrocatalysis. The use of mediators or selective electrocatalysis helps to lower the detection potential to the level when the majority of interferii species are electroinactive. ... 

In some cases the polymer incorporates the mediator, producing a redox macromolecule, e.g., poly[(ferrocenyl)amidopropyl]pyrrole, ferrocene-modified poly(ethylene oxide), " and highly flexible ferrocene-modified silox-ane. These redox polymers have short chains and low molecular weights (< 20 kDa) they function as a special kind of diffusional mediators and do not alleviate the loss of a mediator to the bulk solution during amperometric detection. 

Pseudohomogenous amperometric displacement immunosensors have also been developed. The displacement assay is performed on a disposable screen printed carbon electrode and takes advantages of the cross-reactivity of some monoclonal antibodies. As an example, the monoclonal anti-2,4-D antibody exhibits a relative cross-reactivity toward immobilized MCPA. In the presence of the target analyte (2,4-D) a displacement effect is observed. Consequently, the peroxidase label of the remaining anti-2,4-D antibodies is detected at the carbon electrode, when adding hydrogen peroxide and potassium iodide as redox mediator, at OV versus Ag/AgCl. 

New developments in this area include uric acid sensors based on the mediation of urate oxidase by a novel redox polymer, poly(N-methyl-o-phenylenediamine) [145], and by the freely diffusing mediator 1-methoxy-5-methylphenazinium [146], continued research on the direct amperometric detection of NADH [147] and the use of redox mediators [148] for dehydrogenase enzymes, to allow practical sensors that exploit this large class of enzymes, and the use of cytochrome P450-modified glassy carbon electrodes as drug metabolism biosensors [149]. 

Redox mediators can be used in this case for cathodic detection. An amperometric sensor for choline based on electron transfer between horseradish peroxidase and a redox polymer has been described (19, 20). The use of redox mediators facilitate electron exchange but leads to system complication. 

The lateral resolution of SECM is defined by the ability to resolve two nearby objects and is different from the ability to detect an isolated small object, that is, detectability. The lateral resolution and detectability of SECM in the feedback mode were assessed theoretically, when a disk-shaped tip was positioned over the disk-shaped conductive spot embedded in an insulating substrate (Figure 1.1a) [18]. In the feedback mode, a redox mediator in solution, 0, is electrolyzed amperometrically at the tip (0 + e R) to yield steady-state diffusion-limited current. When the tip is positioned within a tip diameter from the conductive spot, tip current is enhanced... 

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