2.6 Immobilization of Enzymes and Mediators
45
2.6.1 Bifunctional Reagents
For immobilization of enzymes and mediators, bifunctional reagents are usually
employed. Amino and carboxy groups on the surface of enzymes and some mediators
are feasible positions for covalent linkages. In addition, the surface of electrodes can
also be functionalized with various groups by several approaches. In this part, we
introduce some of common bifunctional reagents utilized for enzymes and mediators
immobilization.
Glutaraldehyde (1,5-pentanedial, GA) is one of the most widely used bifunctional
reagents with the capacity to polymerize and immobilize enzymes and mediators.
GA may react with different moieties of enzymes, mainly involving primary amino
groups, but it may eventually react with other groups such as thiols, phenols, and
imidazoles [37]. The exact structure of GA is not fully clarified. Some of proposed
structures in solution and their reactions schemes are given in Panel A in Fig. 2.6
[37]. With the aid of GA, HRP and thionine were co-immobilized onto an l-Cys
self-assembled monolayer (SAM) gold electrode (AuE) for fabrication of an H 2 O 2
biosensor [38]. Here, amino groups (i.e. l-Lys residues) on HRP, two primary amino
groups on thionine, and the amino tails of l-Cys SAM are crosslinked by GA and
finally formed covalent leakages (Fig. 2.6B). In a similar manner, a stable matrix
at glassy carbon electrode (GCE) constructed by cross-linking HRP, thionine, and
bovine serum albumin (BSA) with GA [39].
On the other hand, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) mediates the formation of covalent linkages between
carboxy and amino groups to form amide bonds through activated ester intermediates.
(Fig. 2.6C) [40] One of ferrocene derivatives, common electrochemical mediators,
with a carboxy tail thus can form an amide bond with amino groups on the surface of
enzymes [41]. A glucose-oxidizing bioanode was fabricated by co-immobilization
with 1,1
-ferrocenedicarboxylic acid between GOD onto an aminated glassy carbon
electrode with EDC/NHS [42]. In this case, 1,1
-ferrocenedicarboxylic acid with two
carboxy groups act as a bridge for covalent-bonding of GOD to an aminated electrode
by forming amide bonds with them.
Poly(ethylene glycol) diglycidyl ether (PEGDGE), which has two epoxy groups
that can react with amino, hydroxy, and carboxy groups, is another common
bifunctional reagents for enzymes and mediators immobilization (Fig. 2.6D) [43].
GOD was covalently linked to a ferrocenecarboxaldehyde functionalized linear
poly(ethylenimine) (PEI) with PEGDGE for construction of a amperometric glucose
biosensors [44].
45
2.6.1 Bifunctional Reagents
For immobilization of enzymes and mediators, bifunctional reagents are usually
employed. Amino and carboxy groups on the surface of enzymes and some mediators
are feasible positions for covalent linkages. In addition, the surface of electrodes can
also be functionalized with various groups by several approaches. In this part, we
introduce some of common bifunctional reagents utilized for enzymes and mediators
immobilization.
Glutaraldehyde (1,5-pentanedial, GA) is one of the most widely used bifunctional
reagents with the capacity to polymerize and immobilize enzymes and mediators.
GA may react with different moieties of enzymes, mainly involving primary amino
groups, but it may eventually react with other groups such as thiols, phenols, and
imidazoles [37]. The exact structure of GA is not fully clarified. Some of proposed
structures in solution and their reactions schemes are given in Panel A in Fig. 2.6
[37]. With the aid of GA, HRP and thionine were co-immobilized onto an l-Cys
self-assembled monolayer (SAM) gold electrode (AuE) for fabrication of an H 2 O 2
biosensor [38]. Here, amino groups (i.e. l-Lys residues) on HRP, two primary amino
groups on thionine, and the amino tails of l-Cys SAM are crosslinked by GA and
finally formed covalent leakages (Fig. 2.6B). In a similar manner, a stable matrix
at glassy carbon electrode (GCE) constructed by cross-linking HRP, thionine, and
bovine serum albumin (BSA) with GA [39].
On the other hand, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) mediates the formation of covalent linkages between
carboxy and amino groups to form amide bonds through activated ester intermediates.
(Fig. 2.6C) [40] One of ferrocene derivatives, common electrochemical mediators,
with a carboxy tail thus can form an amide bond with amino groups on the surface of
enzymes [41]. A glucose-oxidizing bioanode was fabricated by co-immobilization
with 1,1
-ferrocenedicarboxylic acid between GOD onto an aminated glassy carbon
electrode with EDC/NHS [42]. In this case, 1,1
-ferrocenedicarboxylic acid with two
carboxy groups act as a bridge for covalent-bonding of GOD to an aminated electrode
by forming amide bonds with them.
Poly(ethylene glycol) diglycidyl ether (PEGDGE), which has two epoxy groups
that can react with amino, hydroxy, and carboxy groups, is another common
bifunctional reagents for enzymes and mediators immobilization (Fig. 2.6D) [43].
GOD was covalently linked to a ferrocenecarboxaldehyde functionalized linear
poly(ethylenimine) (PEI) with PEGDGE for construction of a amperometric glucose
biosensors [44].