Biomedical Engineering Reference
In-Depth Information
2.2.3 Covalent Immobilization
The transducer surfaces typically provided are metals (e.g., gold), oxidic layers
(e.g., metal oxide or quartz/glass), and polymers. First, functional groups have to
be introduced. In the case of gold layers, suitably substituted thiols forming self-
assembled monolayers (SAMs) are used as adhesion linking layers for further
coupling. A chain length of at least ten carbon atoms is recommended to obtain
well-defined and stable SAMs of high density. If oligonucleotides or aptamers
provided with thiol substituents are available, they can be coupled directly to
the gold surfaces, which will lead to complete biorecognition layers. On oxidic
layers, SAMs can be formed by treatment with silanes. Frequently used silanes
are 3-aminopropyl triethoxysilane and 3-glycidyloxypropyl trimethoxysilane,
providing amino groups and epoxy groups, respectively, for further coupling
procedures [ 74 ]. If the transducers expose polymer surfaces, they can be activated
by oxidative treatment via wet etching or plasma treatment and processed directly
[ 74 ] or silanized similarly to the oxidic layers [ 77 ]. In the next step, biomolecules
or hydrogels serving as intermediate layers can be coupled to the transducer
surfaces via the functional groups of the SAM.
As mentioned earlier, minimizing nonspecific binding on biosensor surfaces is
crucial for biosensor measurements in complex media. In this context, it was
shown that sensing layers consisting of antibodies coupled on gold surfaces by
means of 16-mercaptohexadecanoic acid can be used for analyte detection in
serum samples, because nonspecific binding was effectively reduced [ 78 ]. If gold
surfaces are not at hand, it is often advantageous to couple the biorecognition
elements via an additional hydrogel layer, as these layers are known to effectively
prevent nonspecific binding on sensor surfaces. Furthermore, they enable mild
reaction conditions, so the biomolecules will retain their physiological structure
and hence their functionality in the subsequent modification step [ 74 ]. The most
commonly used hydrogels featuring these properties are functionalized dextrans,
particularly carboxymethyl dextran, and poly(ethylene glycol)s [ 39 , 74 ].
If these hydrogels provide carboxyl groups, antibodies, enzymes, and other
protein-based biorecognition elements can easily be immobilized covalently via
their amino groups by means of carbodiimide. This coupling procedure is regarded
as flexible, simple, and robust and provides high coupling yields, which is why it is
the most frequently employed immobilization method [ 74 ]. If the transducer
(hydrogel) surface provides amino groups itself, this method can still be applied,
because the amino groups can easily be converted to carboxyl groups, e.g., by
means of dicarboxylic acid anhydrides [ 74 , 79 ]. One drawback of the carbodiimide
coupling procedure is the fact that a protein's amino groups are distributed all over
the molecule; hence, the protein will be coupled in a random orientation to the
surface, resulting in a potential blocking of the protein's binding sites. A way to
overcome this problem is the affinity binding of the biorecognition elements via
corresponding capture molecules. Examples are the coupling of biotinylated
species of biorecognition elements via streptavidin and the coupling of antibodies
via protein A or protein G. However, these capture molecules might increase the
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