Biomedical Engineering Reference
In-Depth Information
Chemical methods of microbe immobilization include covalent binding and cross-linking.
Covalent binding methods rely on the formation of a stable covalent bond between functional
groups of the cell wall components of the microorganism and the transducer. To successfully
achieve this goal, whole cells are exposed to harsh chemical reactions that can damage the
microbial cell membrane and decrease the biological viability of the cells. Determining how
to overcome this drawback remains a practical challenge. Cross-linking, on the other hand,
involves bridging between functional groups on the outer cell membrane by multifunctional
reagents (e.g., glutaraldehyde) to form a network. Because of the speed and simplicity, the
method has found wide acceptance for the immobilization of microorganisms. The cells may
be cross-linked directly onto the transducer surface or on a removable support membrane that
can then be placed on the transducer. While cross-linking has advantages over covalent bind-
ing, the cell viability can be affected by the cross-linking agents. Therefore, cross-linking is suit-
able in constructing microbial biosensors where cell viability is not important and only the
intracellular enzymes are involved in the detection.
Physical methods of microbe immobilization include adsorption and entrapment. Because
these methods do not involve covalent bond formation with microbes and provide relatively
small perturbation of microorganism native structure and function, these methods are pre-
ferred when viable cells are required. Physical adsorption is the simplest method for microbe
immobilization. Typically, a microbial suspension is incubated with the electrode or an immo-
bilization matrix, such as glass bead. The microbes are immobilized due to adsorptive interac-
tions (i.e., ionic or polar bonding) and hydrophobic interaction. However, immobilization
using adsorption alone generally leads to poor long-term stability because of desorption of
microbes. The immobilization of microorganisms by entrapment can be achieved, for example,
by the retention of the cells in close proximity of the transducer surface using a dialysis mem-
brane. However, a major disadvantage of entrapment immobilization is the additional diffu-
sion resistance offered by the entrapment material, which will result in a lower sensitivity
and detection limit.
A number of microbial sensors have been developed mainly for online control of bio-
chemical processes in various environmental, agricultural, food, and pharmaceutical appli-
cations. Microbial biosensors typically involve the assimilation of organic compounds by
the microorganisms, followed by a change in respiration activity (metabolism) or the pro-
duction of specific electrochemically active metabolites, such as H
2
,CO
2
,orNH
3
, that are
secreted by the microorganism.
Examples of microbial biosensors include ammonia (NH
3
) and nitrogen dioxide (NO
2
)
sensors that utilize nitrifying bacteria as the biological sensing component. An ammonia
biosensor can be constructed based on nitrifying bacteria, such as
Nitrosomonas sp.,
that
use ammonia as a source of energy and oxidize ammonia as follows:
Nitrosomonas sp
:
NO
2
H
þ
NH
3
þ
1
:
5O
2
!
þ
H
2
O
þ
This oxidation process proceeds at a high rate, and the amount of oxygen consumed by the
immobilized bacteria can be measured directly by a polarographic oxygen electrode placed
behind the bacteria.
Nitric oxide (NO) and NO
2
are the two principal pollution gases of nitrogen in the
atmosphere. The principle of a NO
2
biosensor is shown in Figure 10.35. When a sample of
