Electrocatalysis for Fuel Cells at Enzyme-Modified Electrodes - Fuel Cell Catalysis: A Surface Science Approach - page 594

solution at near-neutral pH and ambient temperature. Examples include a direct
covalent bond between cysteine thiols and metals such as gold and silver (see, e.g.,
Hasan et al. [2006]) and direct amide bonding between activated surface carboxylates
or hydroxides on an electrode and lysine amines on the surface of an enzyme (see, e.g.,
Cardosi [1994]) or between amine functionalities introduced onto an electrode surface
and carboxylic acid side chains (of glutamic or aspartic acid) on the surface of an
enzyme [Alonso-Lomillo et al., 2007]. In addition to covalent coupling approaches,
metal coordination chemistry can sometimes be exploited; for example, a protein
with a genetically introduced His 6 tag will coordinate Ni 2 þ introduced onto an elec-
trode surface by chelation to nitrilotriacetic acid (NTA) [Mayer et al., 2005].
Reconstitution of the ligand sphere at a metal site within a protein by a
coordinating head group of a linker provides another strategy for protein attachment.
For example, Canters and co-workers generated a genetic mutant of azurin, a copper
electron transfer protein, that lacked a histidine ligand to the metal and were able to
attach an imidazole-capped linker to this site [de Jongh et al., 2006]. Specific examples
of coupling strategies used in enzyme fuel cell electrocatalysis are discussed in
Sections 17.2 and 17.3.
Covalent attachment of enzymes to surfaces is often intuitively perceived as being
more reliable than direct adsorption, but multisite physical interactions can in fact
yield a comparably strong and stable union, as demonstrated by several biological
examples. The biotin/streptavidin interaction requires a force of about 0.3 nN to be
severed [Lee et al., 2007], and protein/protein interactions typically require 0.1 nN
to break, but values over 1 nN have also been reported [Weisel et al., 2003]. These
forces are comparable to those required to rupture weaker chemical bonds such as
the gold - thiolate bond (1 nN for an alkanethiol, and even only 0.3 nN for a 1,3-
alkanedithiol [Langry et al., 2005]) and the poly(His) - Ni(NTA) bond (0.24 nN,
[L´vy and Maaloum, 2005]).
Immobilized substrates, cofactors, or their analogs have been used to orientate,
anchor, and electronically couple enzymes to conducting surfaces in an approach
that has been developed extensively by Katz, Willner, and co-workers. Examples
include attachment of apo-glucose oxidase (lacking the flavin adenine dinucleotide
(FAD) cofactor) to a gold surface modified with FAD attached via a pyrroloquin-
oline quinone (PQQ) linker (reviewed in Katz et al. [2003]) and attachment of apo-glu-
cose dehydrogenase (lacking a PQQ cofactor) to gold nanoparticles via a PQQ-capped
linker [Zayats et al., 2005]. These are described in further detail in Section 17.3.1.
More recently, a substrate analog linker approach was applied to fungal laccase. A
2-aminoanthracene modifier on the electrode surface was found to stabilize laccase
films, probably by interacting with the hydrophobic surface pocket where organic
substrates are oxidized by the enzyme in its native environment (see Section 17.2.1
for further details) [Blanford et al., 2007].
In some cases, small biological redox partner proteins such as heme-containing
cytochromes, ferredoxins comprising an iron - sulfur cluster, or azurin with a mono-
nuclear Cu site have been used as natural mediators to facilitate fast electron exchange
with enzymes. A specific surface site on the redox protein often complements a region
on the enzyme surface, and enables selective docking with a short electron tunneling