Biomedical Engineering Reference
In-Depth Information
layer, i.e., biorecognition element including any intermediate layers above the
transducer, must be taken into account. For instance, the thickness may be required
not to be too great, particularly if diffusion through the layer is hindered. Other-
wise, with increasing distance from the transducer surface, analyte binding to the
biorecognition element might lead to a reduced effect on the near-surface signal
transduction and hence to reduced signal changes [
74
].
Ideally, sensing layers should be stable, robust, and possess a certain lifetime.
Surface-bound binding sites must be accessible and able to bind the analyte to be
detected. On the other hand, nonspecific binding on the transducer surface has to
be inhibited to prevent false-positive signals. This applies in particular for non-
analyte molecules in complex sample matrices, such as serum, as used in diag-
nostic applications. However, this also applies, for example, for secondary
detection antibodies, which may be used for signal amplification (for details, see
Sect. 2.3
), i.e., for any nonanalyte molecule involved in the measurement which
may interfere with the biosensor signal response. Strategies to avoid nonspecific
binding include a high surface density of the surface-bound biorecognition ele-
ment, if permitted by its type and availability, and additional intermediate layers
with nonstick properties as provided, e.g., by hydrogels. Furthermore, the use of
blocking solutions which contain surfactants, proteins (such as bovine serum
albumin), or even analyte-free serum (if available) is recommended. These solu-
tions are supposed to occupy the nonspecific binding sites; however, the binding
efficiency might be reduced and the blocking proteins themselves might interact
with the sample components. Another strategy is to dilute the serum sample to
minimize nonspecific binding. However, this also dilutes the concentration of the
analyte to be detected and therefore might not be suitable if the analyte concen-
trations which are expected are too low [
76
].
In the following sections typical immobilization strategies for biorecognition
elements—including nonstick intermediate layers—will be outlined.
2.2.2 Noncovalent Immobilization
Direct adsorption of the biorecognition elements on the surfaces would be the
easiest way; however, owing to unavoidable denaturation of the biomolecules and
insufficient shielding against nonspecific binding of sample matrix components to
the substrate, this method is generally not recommended. Electrostatic binding of,
for instance, oligonucleotides might not be stable enough for use in biosensors, as
in this case potential shifts in the solution's pH value or ion strength may lead to
detachment of biorecognition elements from the surface. Therefore, immobiliza-
tion protocols based on covalent coupling procedures are typically preferred [
74
].
To ensure the binding ability of enzymes, however, it has proven to be useful to
encapsulate them, e.g., via hydrogel, sol-gel, or lipid bilayers, instead of coupling
them covalently to the surface [
28
,
31
].
