Biomedical Engineering Reference
In-Depth Information
1. Direct detection (Fig.
1
a)
2. Sandwich assay (Fig.
1
b)
3. Competitive assay (Fig.
1
c)
4. Binding inhibition assay (Fig.
1
d).
By definition, biosensors provide immobilized biorecognition elements; hence,
biosensor test formats are per se heterogeneous. Therefore, they can generally be
derived from test formats used for traditional immunoassays, such as RIA and
enzyme linked immunoassay (ELISA), which include the use of labels [
81
,
82
].
However, instead of a ''passive'' substrate, such as a microplate, a biosensor
provides an ''active'' transducer on which the biorecognition element is immo-
bilized. Hence, biosensors permit an additional test format, i.e., the direct and
label-free detection of the analyte (Fig.
1
a). In this case, provided a label-free
transduction principle is used, the signal response changes as soon as the analyte
binds to the biorecognition element immobilized on the transducer surface. It is a
one-step process without any other reagents which even allows real-time moni-
toring of binding to the biosensor surface, if required. The simplicity, speed, and
the inexpensiveness of this approach are the major advantages of this procedure
and are among the driving forces for the development of label-free transduction
principles. However, the performance in the detection of low concentrations or of
small molecules (or both) may be limited. Furthermore, when one is working with
complex sample matrices, such as serum, the simplicity of the direct approach
might turn out to be a considerable disadvantage, because nonanalyte components
from the sample matrix may also bind to the transducer surface, leading to false-
positive results. This has to be strictly avoided [
45
,
83
,
84
]. To prevent nonspecific
binding, specific procedures for the preparation of biosensor recognition layers are
required which have to ensure that only the analyte to be detected will bind on the
biosensor surface. Details are described in
Sect. 2.2
.
Widely used labels for biosensors involve enzymes, nanoparticles, and fluo-
rescent or electrochemiluminescent probes [
82
,
83
]. Details about corresponding
transduction principles are described in
Sect. 2.1
. If labeled test formats are used,
undesired effects resulting from nonspecific binding of sample matrix compo-
nents are supposed to be reduced. This is because the final biosensor signal
response is usually determined by the labeled compound, whose binding
behavior is assumed to be largely independent of the matrix components.
However, nonspecific adsorption of labeled compounds will also lead to false-
positive results and therefore has to be avoided as well. An important advantage
of labeled test formats (including the use of transduction principles requiring
labels) is the higher potential for the detection of lower analyte concentrations
due to the amplification afforded by the respective label or label-induced reac-
tion, such as a fluorescent signal or an enzyme-catalyzed redox reaction [
83
].
However, a significant disadvantage of labels is that they can interfere with the
analyte binding, leading to distorted results. In the case of TIRF or other fluo-
rescence-based transduction principles, autofluorescence of biological samples
can be a problem as well [
85
]. Furthermore, the need for labeled compounds
