Biomedical Engineering Reference
In-Depth Information
in preventing non-specifi c binding. To solve this problem, various blocking agents have
been employed to saturate the free binding sites on the surface and thus reduce non-
specifi c binding. For example, the use of high salt conditions [48], high detergent con-
centrations [49], cocoating of surfaces with anionic proteins [50], milk proteins [51] or
denatured proteins [52] can effectively passivate the surface while maintaining specifi c
binding. However, despite rigorous blocking, it seems that the level of non-specifi c
binding still depends on the intrinsic protein-binding capability of a surface, refl ect-
ing the fact that it is impossible to completely block a surface. Another drawback in
using an adsorption method is that some proteins may not bind depending on the nature
of the protein. A weakly bound protein may be washed away during the washing step
especially if stringent washes such as those with high salt or high detergent concen-
trations are used. All the above indicate that adsorption can only provide low protein
loading and therefore is unsuitable for high throughput applications.
Compared with the adsorption method, affi nity binding can offer a strong and
highly selective attachment through the use of specifi c biological interactions, for
instance between biotin and avidin or between protein A and IgG. Note that both the
surface and the protein to be attached should be derivatized with the components of
the interaction. Moreover, if the derivatized surface is resistant to non-specifi c pro-
tein adsorption, a lower background can be achieved while keeping specifi c binding.
It has been reported that a polyethylene glycol (PEG) coated surface which is func-
tionalized with biotin and streptavidin can adsorb spotted antibodies containing biotin
easily but non-specifi c background proteins can be repelled by PEG. A main drawback
of attachment by affi nity binding is that proteins to be attached have to be modifi ed
before spotting, adding steps to protein preparation and sometimes changing the pro-
tein structure.
Covalent binding is the most promising method for microarray technology for the
reason that it provides the strongest attachment of proteins to surfaces [53]. For exam-
ple, PDMS surface with (3-aminopropyl)-triethoxysilane (APTES) treatment offered
effi cient protein immobilization and high sensitivity through succinic acid anhydride
(SAA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or
glutaraldehyde (GA) for covalent binding [54]. In such a mood, proteins are attached
to the surface through the reaction between the amino or carboxyl groups on proteins
and immobilized functional groups on the substrate such as aldehydes or succinim-
ides. Methods to form aldehyde groups on surfaces include oxidation of the carbohy-
drate groups of adsorbed polyacrylamide or agarose and cross-linking glutaraldehyde
to immobilize polyacrylamide or aminosilanes. Similarly, succinimide groups were
formed using a cross-linker attached to aminosilanes or a bovine serum albumin (BSA)
coating. However, the requirement of accessible amino groups on the spotted proteins
may, in some cases, limit this approach. Besides, other covalent attachment techniques
that have been popular in immunosensors and chromatography can also be used for
protein microarrays. IgG molecules can be covalently bound by oxidizing the carbo-
hydrate group on the Fc region, creating an aldehyde and reacting the aldehyde with
hydrazide-activated surface. Attention should be paid in this treatment process because
oxidation of the antibody may occasionally damage the antigen-binding site.
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