Food Biochemistry and Food Processing (2 edition)

BLBS102-c45 BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come

45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins 867

Table 45.5.The Ideal Characteristics and Properties of

an Electron Mediator

1. Exhibits reversible kinetics.


2. Reacts readily with the reduced form of an enzyme.


3. Has a low oxidation potential and is mediator activity is
   pH independent.


4. Is stable in both redox forms.


5. Easily retained at the surface of an electrode.


6. Unreactive towards oxygen.


7. Chemically unreactive with the immobilised biological
   material.

Table 45.5 illustrates the characteristics that are favourable in

choosing a specific mediator (Cassidy et al. 1998).

Mediators are usually low molecular redox couples that shut-

tle the electrons from the enzyme’s active site to the surface

of the transducer. Equation 2 illustrates the reaction of the me-

diator and the subsequent generation of an electron resulting

electrochemical cycling.

H 2 O 2 +2H++2e−→2H 2 O(1)

Mediatorred→Mediatorox+e− (2)

Equations 3, 4, 5 and 6 illustrate a peroxidase-mediated reac-

tion on a biosensor surface. Equation 3 shows the resting state

peroxidase (in its reduced form) reacting with hydrogen perox-

ide and two hydrogen ions to form an intermediary oxidised-

peroxidase compound and two water molecules. Equation 4

shows the reaction of the intermediary oxidised-peroxidase com-

pound with the reduced form of a mediator to produce the resting

reduced-state peroxidase and an oxidised mediator. Equation 5

shows the oxidised mediator reacting with two electrons, thus,

reverting to the reduced form of the mediator. It is these electrons

that the transducer detects and converts into an electrical signal.

The final Equation 6 shows the overall reaction, whereby one

hydrogen peroxide molecule in the presence of two electrons and

two hydrogen ions is converted to two water molecules (Ryan

et al. 2006).

H 2 O 2 +2H++POred→POox+2H 2 O(3)

POox+Mediatorred→POred+Mediatorox (4)

Mediatorox+2e−→Mediatorred (5)

H 2 O 2 +2e−+2H+→2H 2 O(6)

It is often possible to use a multi-enzyme electrochemical

biosensor system to detect the presence and determine the con-

centration of a particular compound in a matrix of interest. This

method employs two or more enzymes that are in proximity with

each other and in contact with a mediator or directly in contact

with the transducer. As illustrated in Figure 45.8, enzyme 1 (glu-

cose oxidase (Gox)) generates hydrogen peroxide (H 2 O 2 )asa

by-product through the catalysis of glucose by Gox. The H 2 O 2

generated is catalysed by a peroxidase enzyme and the redox

cycling of the mediator. The cycling of the peroxidase enzyme

in the presence of H 2 O 2 generates electrons that are detected

by the transducer. This in turn enables the concentration of the

target analyte to be determined, thereby providing quantitative

determination.

There are several well-characterised electrochemical biosen-

sor devices that have been applied in the fruit and vegetable

industry for the detection of a number of structurally di-

verse compounds, including pesticides, herbicides, insecticides,

organophosphates, organochlorines and carbamates. With ref-

erence to the detection of pesticide residues, there are many

available options that can be implemented by the end user. More

specifically, enzymes that are directly affected by the presence

of a pesticide can be incorporated into a biosensor-based plat-

form permitting detection (Amine et al. 2006). As an example,

acetylcholinesterase (AChE) presents in muscles, red blood cells

Substrate

Gox Enzyme

H 2 O 2

Product

e-

R

R'

Transducer

Electrical signal

Figure 45.8.A representation of a sensor with two enzymes operating together. The first enzyme catalyses the substrate of interest and, as a

by-product, H 2 O 2 is generated. This is then catalysed by a peroxidase enzyme. The redox activity of the peroxidase enzyme is detected by

the transducer. Gox is glucose oxidase; H 2 O 2 is hydrogen peroxide; R and R′represent the oxidised and reduced forms, respectively, of an

electron mediator and e−is an electron.