Biosensors based on direct electron transfer of protein - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 548

to enable electron transfer to the electrode in the absence of artifi cial electron mediator
by mimicking the domain structure of the quinohemoprotein ethanol dehydrogenase
(QH-EDH) from Comamonas testosteroni , which is composed of a PQQ-containing
catalytic domain and a cyt c domain. They genetically fused the cyto c domain of QH-
EDH to the C-terminal of GDH-B. The constructed fusion protein showed not only
intramolecular electron transfer, between PQQ and heme of the cyt c domain, but also
electron transfer from heme to the electrode, thereby allowing the construction of a
direct electron-transfer-type glucose sensor.
The large redox enzyme xanthine oxidase (XOD, mw 286 000), being a unimolecular,
multicomponent electron-transport biomacromolecule, features one olybdenum center,
two Fe 2 S 2 centers, and one fl avin adenine dinucleotide (FAD), and has been the target
of extensive study, especially in the study of biosensors. XOD is implicated as a key oxi-
dative enzyme in many physiological processes. The enzyme can catalytically oxidize
many substrates, including purines, pteridines, heterocyclic molecules, and aldehydes
[184]. The oxidation of xanthine takes place at the molybdenum center and the electrons
distributed to other redox centers. The reoxidation of the reduced enzyme by the oxidant
substrate, either NAD or molecular oxygen, occurs through FAD [185]. Furthermore,
the electron transfers of O 2 , NAD , methylene blue, quinines, and nitrate are also associ-
ated with XOD. So, electrochemical researchers have studied the electron-transfer prop-
erty of XOD [186-188], and used this enzyme to detect xanthine, hypoxanthine, and
other biological molecules [189-191]. Nonetheless, the direct electron-transfer reaction
of XOD is still very diffi cult to achieve. Although various methods have been employed,
there is no fi nding that electrochemical signals both of FAD and molybdenum centers
can be observed simultaneously. And no response is observed unless denaturation of the
enzyme has occurred, in which case one fi nds a surface-confi ned chemically revers-
ible process for free FAD. Li and coworkers [192] used DNA as a matrix to embed
XOD, and obtained the electrochemical responds of FAD and molybdenum center of
XOD. Meanwhile, XOD kept its enzymatic activity to hypoxanthine. In DNA, because
p-conjugated nucleic acid bases are stacked, it is naturally considered to act as an effec-
tive molecular wire for electron transfer. The experimental results indicated that DNA
could simultaneously activate both the FAD and the molybdenum centers of XOD. Wang
[193] also obtained the direct electrochemistry of XOD at a gold electrode modifi ed
with SWNTs.
17.3 APPLICATION OF BIOSENSORS BASED ON DIRECT
ELECTRON TRANSFER OF PROTEIN
17.3.1 Biosensors based on direct electron transfer of proteins
One of the important purposes for the study of the direct electron transfer of pro-
tein is to construct the mediator-free protein-based biosensors. These biosensors
can determine many small molecules like H 2 O 2 , O 2 , NO, nitrite, small organic
peroxide, and so on. They also can determine glucose, alcohol, and amino acids by