Biosensors based on direct electron transfer of protein - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 547

The metal colloidal fi lms have been used to construct the interface for direct elec-
tron transfer of redox-active proteins and retaining their bioactivity [171]. Colloidal gold
is an extensively used metal colloid, which has been used for the study of direct elec-
trochemistry of proteins [172-173]. It provides an environment similar to that of redox
proteins in native systems and gives the protein molecules more freedom in orientation,
thus reducing the insulating property of the protein shell for the direct electron transfer
and facilitating the electron transfer through the conducting tunnels of colloidal gold. Ju
[174] combined the advantageous features of colloidal gold and carbon paste technol-
ogy to achieve the direct electron transfer of GOD. GOD adsorbed on a colloidal gold-
modifi ed carbon paste electrode displayed a pair of redox peaks with a formal potential
of 1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a

surface-controlled electrode process with an electron-transfer rate constant of (38.99

(449

5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold
colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD
could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase
of the reduction peak current.

17.2.3.4 Direct electron transfer of other active enzymes
Several dehydrogenases harboring pyrroloquinoline quinone (PQQ) as their prosthetic
group have been reported, such as glucose dehydrogenases, ethanol dehydrogenases,
and methanol dehydrogenases [175-179]. They are divided into two categories, qui-
noprotein (PQQGDHs, PQQMDH, and type I PQQADH) and quinohemoprotein
(types II and III PQQADH), the latter containing an additional heme c prosthetic
group together with PQQ. These quino- and quinohemoproteins form protein com-
plexes composed of catalytic subunits, with PQQ as their redox center, and electron
acceptors, such as cyt c , which transfer electrons from reduced PQQ to the respira-
tory chain. The 3D structure of the quinohemoprotein ethanol dehydrogenase (QH-
EDH) from Comamonas testosteroni was recently determined [180]. This enzyme
has two domains separated with a peptide linker region, an eight-bladed b propeller
fold catalytic domain containing PQQ, and a cyt c domain that is located at the
C-terminal region. The heme is located on top of the catalytic site of the fi rst domain,
thereby allowing smooth electron transfer from the catalytic site via heme to the exter-
nal electron acceptor. Due to their superior electron transferability, such heme-contain-
ing, multicofactor dehydrogenases, consisting of an FAD- or PQQ-harboring catalytic
subunit and a heme-containing electron transfer subunit/domain, were recently shown
to display direct electron transfer with the electrode [181-182]. The direct electron
transfer mechanism has been investigated as the ultimate enzyme sensor format, allow-
ing the direct monitoring of the catalytic reaction by the electrode. Unfortunately, only
a limited number of enzymes, such as multicofactor enzymes, are capable of direct
electron transfer to electrode. Among the numerous dehydrogenases, the water-soluble
PQQGDH from Acinetobacter calcoaceticus (GDH-B) is one of the most industrially
attractive enzymes, as a sensor constituent for glucose sensing, because of its high cat-
alytic activity and insensitivity to oxygen. Okuda [183] attempted to engineer GDH-B