The Route to Electrically Contacted Enzymes in Biosensors

The electronic coupling between redox enzymes and electrodes for the construction of enzyme electrodes has been based on the electroactivity of the enzyme cosubstrate or product (first generation) or through the use of redox mediators (second generation) [17] most typically illustrated by numerous biosensors using glucose oxidase (Figure 4.2). The drawbacks with first-generation biosensors put the focus on the use of mediators, small redox-active molecules that could diffuse in and react with the active site of the enzyme and diffuse out and react with the electrode surface, thus shuttling the electrons between the enzyme and the electrode. Diffusional electron mediators, such as ferrocene derivatives, ferricyanide, conducting organic salts (particularly tetrathiafulvalene-tetracyanoquinodimethane, TTF/TCNQ, phenothiazine

Fig. 4.2 Generations of electrically contacting enzymes to electrodes.

and phenoxazine compounds, or quinone compounds have thus been widely used to electrically contact glucose oxidase (GOD) [18]. The use of mediators made it possible to decrease the applied potential for hydrogen peroxide-producing oxidases, thus decreasing the influence from bias signals caused by electrochemically easily oxidizable interfering compounds present in real samples. The use of mediators also opened up the possibility for the use of various dehydrogenases, peroxidases, and even whole cells. However, the redox mediators are general redox catalysts facilitating not only the electron transfer between electrode and enzyme but also various interfering reactions. Further progress in the development of second-generation biosensors was achieved with the use of flexible polymers onto which mediating functionalities were covalently bound [19, 20].

Chemical conversion of a single-center redox enzyme, for example, by introducing a mediator molecule (ferrocene [21], pyrrolo quinoline quinone (PQQ)) [5] located half way between the prosthetic group and the molecule surface produced electroactive hybrids. The procedure was demonstrated to result in almost oxygen-independent electrodes, probably due to the fact that an artificially introduced redox center is shielded by the protein as well as the rapid electron exchange with the electrode. Obviously, the rate of electron transfer via the 'wired' route is high compared to the rate of reaction of the enzyme with endogenous oxygen. Most mediator-based sensors involving oxidases do not meet this requirement and exhibit parasitic effects of oxygen on their response. The approach requires, however, a high-skilled manipulation for unfolding-modifying-refolding of the enzyme.

Efficient direct electron transfer - the basic principle of third generation enzyme electrodes - has been reported for several electron transport proteins but only for a restricted number of redox enzymes [17, 22]. Proteins and enzymes transferring electrons to other proteins typically function in ordered structures such as mitochondria, and the redox-active centers are generally accessible to the outer surface of the protein and therefore able to communicate with electrodes (Table 4.3).

It has been demonstrated that promoters, such as aminoglycosides, are effective for accelerating the electron transfer of cytochrome c peroxidase, flavohemoproteins, and methylamine dehydrogenase at graphite electrodes and modified gold electrodes. In the absence of promoters, peroxidase, laccase, cytochrome c peroxidase, methylamine dehydrogenase, and ferredoxin-NADP oxidoreductase produce catalytic currents by the bioelectrocatalytic oxidation or by reduction of their substrates at carbon electrodes. Flavohemoproteins and quinohemoproteins from bacterial cytoplasmic membranes immobilized by adsorption produce catalytic currents in the presence of the substrates based on direct electron transfer [23]. These enzymes contain a subunit of a c-type cytochrome and another subunit of flavine adenine dinucleotide (FAD), or PQQ. The heme group accepts electrons from the FAD or PQQ by intramolecular electron transfer and donates them to the respiratory chain in vivo. Approximate orientation of the enzymes in the electrodes allows bioelectrocatalytic oxidation of the substrates based on the direct electron transfer between the electrode and the heme group of the adsorbed enzyme.

Tab. 4.3 Redox enzymes for which direct mediator-free reactions with electrodes have been shown

Enzyme

Prosthetic group

Substrates

Ascorbate oxidase Laccases

Theophylline oxidase Superoxide dismutase

Cu Cu Cu

Cu-Zn

O2 O2

Cytochrome c

Ascorbate oxidase Laccases

Theophylline oxidase Superoxide dismutase

Cu Cu Cu

Cu-Zn

O2 O2

Cytochrome c

Fe Mn

Diaphorase

FMN

NADH

Pentachlorophenol hydroxylase

FAD

O2, pentachlor phenol

Putidaredoxin reductase

FAD

Putidaredoxin

Methylamine dehydrogenase

TTQ

Methylamine

Phospholipidhydroperoxide

Selenocysteine

Glutathione, H2O2

Glutathione peroxidase

Catalase

Heme

H2O2/O2

Cytochrome P450

Heme b

O2, aminopyrine, benzphetamine

Methane monooxygenase

Binuclear heme

Acetonitrile, methane

Peroxidases

Heme

H2O2

Chloroperoxidase

Heme

H2O2

Cytochrome c peroxidase

Heme b

H2O2

Fungal peroxidase

Heme

H2O2

Horseradish peroxidase

Heme

rho2

Lignin peroxidase

Heme

H2O2

Manganese peroxidase

Heme

H2O2

Peanut peroxidase

Heme

H2O2

Soybean peroxidase

Heme

H2O2

Sweet potato peroxidase

Heme

H2O2

Tobacco peroxidase

Heme

H2O2

Pentachlorophenol hydroxylase

FAD

O2, PCP

Putidaredoxin reductase

FAD

Putidaredoxin

Multi Center Enzymes

Amine oxidase

Cu, topa quinone

Amines

Cytochrome c oxidase

cua, CuB, heme a3,

O2, cytochrome c

Cellobiose dehydrogenase

FAD, heme c

Cellobiose, lactose

p-Cresolmethylhydrolase

FAD, heme c

p-Cresol

l-lactate dehydrogenase

FMN, heme b2

Lactate

(flavocytochrome b2)

Flavocytochrome cs22

FAD, heme

Sulfide

Flavocytochrome c3

FAD, heme c

Fumarate

Fumarate reductase

FAD, Fe-S FAD, heme c

Fumarate/succinate

d-gluconate dehydrogenase

FAD, heme c, Fe-S

d-gluconate

Alcohol dehydrogenase

PQQ, heme c

Ethanol

Aldose dehydrogenase

PQQ, heme

Aldose

d-fructose dehydrogenase

PQQ, heme c

d-fructose

Amine oxidase

Cu, topa quinone

Amines

Cytochrome c oxidase

cua, CuB, heme a3,

O2, cyt c

Nitrite reductase

Cu, multi heme

no2-

DMSO-reductase

Mo-pterin, Fe-S

DMSO

Tab. 4.3 (Continued)

Enzyme

Prosthetic group

Substrates

Sulfite oxidase

Mo-pterin heme b5

SO32-

Sulfite dehydrogenase

Mo-pterin, heme c

SO32-

Hydrogenase

Fe-S

H2, H+, NAD

Ni, (Se), Fe-S

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