Biomedical Engineering Reference
In-Depth Information
14.2.2.1.3 The Attachment of Biomolecules to the Underlying Film
Being different from the above two strategies that can be proposed as “one-step procedure,” this
immobilization method could be called as a “two-step procedure” that involves electropolymeriza-
tion of functionalized conducting polymers and then the attachment of biomolecules to the polymer
surfaces.
For the two-step procedure, immobilization permits the selection of the optimum reaction con-
ditions for each step. Since immobilization of biomolecules only takes place on the outer surface
of the polymer, the access of polymer to biomolecules can be improved, and the attachment of the
biomolecules to the functionalized polymer could be carried out in aqueous buffer solutions con-
taining additives and stabilizers that could avoid damage to the biological entity.
There are three kinds of interactions between biomolecules and the underlying polymer for
the attachment: adsorption, covalent bonding, and affi nity interaction. The main binding forces
of adsorption are static interactions between the polycationic matrix of the oxidated polymer and
the total negative enzyme charge provided that the pH of the solution is higher than the isoelectric
point (IEP) of the enzyme [234]. However, this technique suffers from desorption of enzyme from
the immobilizing material into the sample solution during measurement. This procedure has been
used for the preparation of cholesterol biosensors [235], urea sensors [236], and glucose biosen-
sors [237,238].
Covalent linking is another interaction for biomolecules to be attached to the functionalized
membrane. Apart from preventing the loss of biomolecules, this procedure improves the effi ciency
of the charge transfer process and increases the stability of the device [239]. Chemically reactive
sites of a protein may be amino groups, carboxyl groups, phenol residues of tyrosine, sulfhy-
dryl groups or the imidazole group of histidine, and in most cases amino and carboxyl groups of
enzymes can be used for stable immobilizations by covalent bonding to polymer fi lms functional-
ized by carboxyl or amino groups [240]. The covalent reaction conditions often partly denature
the protein or lead to incomplete derivatization of the polymer surface. Therefore, effort has been
focused on improving polymers for the grafting of biomolecules in terms of the performance of
their chemically activated surfaces. An alternative method called postfunctionalization has been
developed. The functional groups are attached onto the conducting polymer surface by electropoly-
merizing functioned thiophene, bithiophene, and pyrrole monomers with easy leaving groups such
as
N
-hydroxysuccinimide (NHS) and NHP esters [241]. And the replaceable NHS or NHP groups
can react easily by nucleophilic substitution with amines to form amide bonds. Thus, it is possible
to anchor biomolecules with terminal amino functions onto the surface of conducting polymers
functionalized with an activated ester. By this procedure, various biosensors have been prepared,
such as enzymes [234] and oligonucleotides [242].
The affi nity between biomolecules and fi lms is another potent interaction for the surface modi-
fi cation of membranes. Compared with chemical modifi cations, it could improve the selectivity
and orientation more effectively. One of the most important affi nity interactions is the metal ion
affi nity, which originated from the “immobilized metal (ion) affi nity chromatography” (IMAC)
[243]. The technique is based on the differences in the affi nity of proteins for metal ions bound
to a metal-chelating substance, which is immobilized on a support membrane. Cooper et al. [244]
further developed metal ion affi nity binding for the immobilization of proteins onto the conducting
polymers. In that procedure, Ni(II) was immobilized through its coordination to electrogenerated
PPy fi lms,
N
-substituted by carboxylate or imidazole groups, and then poly(histidine) and bacterial
alkaline phosphatase bearing a chain of six histidines immobilized on the PPy-localized metal ions
through specifi c interaction. Since proteins are now routinely engineered with terminal polyhisti-
dine sequences, this immobilization procedure offers the great convenience of oriented immobili-
zation of protein.
Electropolymerized PANI-poly(acrylate) and PANI-poly(vinyl sulfonate) fi lms with electro-
chemically incorporated Ni(II) have been used to anchor the histidine-tagged enzyme [245,246].
