Biology Reference
In-Depth Information
dry chemistry techniques, avoiding tedious, expensive, and time-
consuming immobilization procedures. The rigid conducting bio-
composite acts not only as a transducer but also as a reservoir for
avidin. After use, the electrode surface can be renewed by a simple
polishing procedure for further uses, highlighting a clear advantage
of this new material with respect to surface-modified approaches
such as classical biosensors and other common biological assays.
DNA probe can be easily immobilized on the surface of the avidin-
modified transducer through the avidin-biotin reaction, since both
nucleic acids as well as short oligonucleotides can be readily linked
to biotin without serious effects on their biological, chemical, or
physical properties (Fig. 3.3B). The knowledge about the avidin-
biotininteractionhasadvancedsignificantlyandoffersanextremely
versatiletool.Moreover,thisinteractionpresentsavarietyofspecific
advantages over other single-point immobilization techniques. In
particular, the extremely specific and high a
nity reaction between
biotin and the glycoprotein avidin (association constant Ka
=
10
15
)
leads to strong associations similar to the formation of a cova-
lent bonding. This interaction is highly resistant to a wide range of
chemicals (detergents, protein denaturants), pH range variations,
and high temperatures [84]. In addition, much progress has been
done in the modification of biomolecules with biotin. Moreover, the
strept(avidin) could be considered as a universal a
nity biomole-
culebecauseitisabletolinknotonlybiotinylatedDNAorODNsbut
also enzymesorantibodies[23].
Biotinylated DNA can be firmly single-point attached in Av-
GEB (Fig. 3.3B). In this case, a capture format was used in which
the immobilization of the biotinylated probe together with the
hybridization was performed in a one-step procedure [74]. Briefly,
the three-step experimental procedure consists of (i) one-step
immobilization/hybridizationprocedureinwhichthebiotin-labeled
capture probe is immobilized onto the electrode surface through
a biotin-avidin interaction, while the hybridization with the target
and with a second complementary probe—in this case labeled with
digoxigenin—is occurring at the same time; (ii) enzymatic labeling
using as enzyme label the antibody anti-DIG-HRP; and (iii) ampero-
metric determination based on the enzyme activity by adding H
2
O
2
and using hydroquinoneas a mediator.
