Biomedical Engineering Reference
In-Depth Information
enzyme or adhesion-promoting peptide in an artificial
organ is designed to remain attached to or entrapped
within the support over the duration of use. Either
physical or chemical immobilization can lead to ''perma-
nent'' or long-term retention on or within a solid support,
the former being due to the large size of the biomolecule.
If the polymer support is biodegradable, then the chem-
ically immobilized biomolecule may be released as the
matrix erodes or degrades away. The immobilized bio-
molecule may also be susceptible to enzymatic degrada-
tion
in vivo,
and this remains an interesting aspect that has
received relatively little attention.
A large and diverse group of methods have been de-
veloped for covalent binding of biomolecules to soluble
or solid polymeric supports (
Weetall, 1975; Carr and
Bowers, 1980; Dean
et al.
, 1985; Shoemaker
et al.
, 1987;
Yang
et al.
, 1990; Park and Hoffman, 1990; Gombotz and
Hoffman, 1986; Schense and Hubbell, 1999; Lutolf
et al.
, 2003a
). Many of these methods are schematically
illustrated in
Fig 3.2.16-1
. The same biomolecule may be
immobilized by many different methods; specific ex-
amples of the most common chemical reactions utilized
are shown in
Fig. 3.2.16-2
.
For covalent binding to an inert solid polymer surface,
the surface must first be chemically modified to provide
reactive groups (e.g., -OH, -NH
2
, -COOH, -SH, or
-CH
ΒΌ
CH
2
) for the subsequent immobilization step. If
the polymer support does not contain such groups, then
it is necessary to modify it in order to permit covalent
immobilization of biomolecules to the surface. A wide
number of solid surface modification techniques have
been used, including ionizing radiation graft copoly-
merization, plasma gas discharge, photochemical graft-
ing, chemical modification (e.g., ozone grafting), and
chemical derivatization (
Hoffman
et al.,
1972,1986;
Hoffman, 1987, 1988; Gombotz and Hoffman, 1986,
1987
). (See also Section 3.2.14.)
A chemically immobilized biomolecule may also be
attached via a spacer group, sometimes called an ''arm'' or
a ''tether'' (
Cuatrecasas and Anfinsen, 1971; Hoffman
et al.
, 1972; Hoffman, 1987
). One of the most popular
tethers is PEG that has been derivatized with different
reactive end groups (
Kim and Feijen, 1985
), and some
companies offer a variety of chemistries of hetero-
bifunctional linkers having activated coupling end groups
such as NHS, maleimide, pyridyl disulfide, and vinyl
sulfone. Such spacer groups can provide greater steric
freedom and thus greater specific activity for the immo-
bilized biomolecule, especially in the case of smaller
biomolecules. The spacer arm may also be either hydro-
lytically or enzymatically degradable, and therefore will
release
polymeric adlayer. Such physisorbed or chemisorbed
polymers can be bound to the surface via electrostatic
interactions (
VandeVondele
et al.
, 2003
), hydrophobic
interactions (
Neff
et al.
, 1999
), or specific chemical in-
teractions, such as that between gold and sulfur atoms
(
Harder
et al.
, 1998; Bearinger
et al.
, 2003
). Metal or
ceramic surfaces may also be derivatized with functional
groups using silane chemistry, such as with functionalized
triethoxysilanes (
Massia and Hubbell, 1991; Puleo,
1997
). Plasma gas discharge has been used to deposit
polymeric amino groups for conjugation of hyaluronic
acid to a metal surface (
Verheye
et al.
, 2000
).
As noted earlier, hydrophobic interactions have been
used to functionalize surfaces, utilizing ligands attached
to hydrophobic sequences (e.g.,
Ista
et al.,
1999; Nath
and Chilkoti, 2003
). Surfaces with hydrophobic gradi-
ents have also been prepared for this purpose (
Detrait
et al.
, 1999
). An interesting surface active product was
developed several years ago that was designed to convert
a hydrophobic surface to a cell adhesion surface by hy-
drophobic adsorption; it had an RGD cell adhesion
peptide coupled at one end to a hydrophobic peptide
sequence.
Sometimes more than one biomolecule may be
immobilized to the same support. For example, a soluble
polymer designed to ''target'' a drug molecule may have
separately conjugated to it a targeting moiety such as an
antibody, along with the drug molecule, which may be
attached to the polymer backbone via a biodegradable
spacer group (
Ringsdorf, 1975; Kopecek, 1977; Gold-
berg, 1983
). In another example, the wells in an immu-
nodiagnostic microtiter plate usually will be coated first
with an antibody and then with albumin or casein, each
physically adsorbed to it, the latter acting to reduce
nonspecific adsorption during the assay. In the case of
affinity chromatography supports, the affinity ligand may
be covalently coupled to the solid packing, and in some
cases a ''blocking'' protein such as albumin or casein is
then
added
to
block
nonspecific
adsorption
to
the
support.
It is evident that there are many different ways in
which the same biomolecule can be immobilized to
a polymeric support. Heparin and albumin are two
common biomolecules that have been immobilized by
a number of widely differing methods. These are illus-
trated schematically in
Figs. 3.2.16-3
and
3.2.16-4
.
Some of the major features of the different immo-
bilization techniques are compared and contrasted in
Table 3.2.16-7
. The important molecular criteria for
successful immobilization of a biomolecule are that
a large fraction of the available biomolecules should be
immobilized, and a large fraction of those immobilized
biomolecules should retain an acceptable level of bio-
activity over an economically and/or clinically appro-
priate time period.
the
immobilized
biomolecule
as
it
degrades
(
Kopecek, 1977; Hern and Hubbell, 1998
).
Inert surfaces, whether polymeric, metal, or ceramic,
can also be functionalized through modification of an
