Biomedical Engineering Reference
In-Depth Information
usually implies higher operation costs than for direct label-free detection, as the
costs of these compounds have to be added. Another point to consider is the fact
that real-time monitoring of the analyte binding to the surface is not possible
[
45
,
82
-
84
]. Finally, when it comes to deciding whether to use labels or not,
the transduction principle also has to be taken into account, as some of the
transduction principles require the use of labels. Examples are transduction
principles based on fluorescence, which would require the use of fluorophores.
On the other hand, a label-free gravimetric transduction principle, for instance,
can be used without a label, but a compound labeled with a nanoparticle as a
mass label may help to increase the signal respone, if required [
51
].
In a sandwich assay (Fig.
1
b), the analyte-related signal response is obtained
when a second biorecognition element binds to the analyte after the analyte has
bound to the biosensor surface. This requires analyte molecules which are large
enough for two independent biorecognition elements to bind. Whether the second
biorecognition element has to be labeled or not depends on the transduction
principle. It is also possible to use a third biorecognition element binding to the
second one [
51
] (''indirect sandwich''). The use of more than three biorecognition
elements is not common [
81
]. In a competitive assay (Fig.
1
c), the analyte and a
known concentration of the labeled analyte (derivative) compete for the surface-
bound analyte-specific binding sites provided by the biorecognition element. This
test format including the corresponding transduction principles requires labels. In
the binding inhibition assay (Fig.
1
d), a preincubation step, in which the analyte
and a known concentration of the unbound biorecognition element equilibrate,
precedes the actual measurement. Again, whether the biorecognition element has
to be labeled or not depends on the underlying transduction principle. After
equilibrium has been attained, the binding sites of the remaining free biorecog-
nition elements are detected with a biosensor providing an immobilized analyte
(derivative), allowing one to derive the original analyte concentration in the
sample [
82
]. This method is particularly suitable for label-free detection of small
molecules, as the signal response is achieved by binding of the corresponding
biorecognition element. The latter is usually a protein of higher mass and size and
hence should create a higher signal response [
86
].
2.3.2 Test Formats Based on Molecular Switches
Today, 85% of all biosensors sold are glucose sensors [
8
]. The reason for this
success story might be that this is one of the very few biosensor types in which the
interaction between the analyte and the biorecognition element creates a reagent
which can easily be detected: in this process the chemical transformation of glu-
cose by glucose oxidase results in the formation of hydrogen peroxide, which can
be monitored amperometrically [
2
,
28
,
87
]. In contrast, other biosensor formats,
such as those mentioned earlier, are mainly based on physical effects near the
transducer surface, i.e., no reagent is released, and there are few significant con-
formal changes supporting the signal response. From a technical point of view, the
