Biosensors and Bioelectronics 22 (2006) 544–549
Using protein templates to direct the formation of
thin-ﬁlm polymer surfaces
John Rick, Tse-Chuan Chou
Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan, ROC
Received 17 February 2006; received in revised form 28 April 2006; accepted 30 June 2006
Available online 17 August 2006
Protein imprinted electrodes formed by the cyclic voltammetric deposition of conductive polymers, on screen-printed platinum supports, in the
presence of target proteins have been fabricated. An initial layer of polypyrrole was used as a supporting polymer layer, upon which were formed
two layers of polyaminophenylboronic acid. The ﬁrst of these layers was non-imprinted and formed a barrier between the polypyrrole and the outer
layer, which was deposited in the presence of a protein template (lysozyme or cytochrome c). After protein extraction, re-binding of the template
proteins to their respective imprinted electrodes showed a distinct two-phase binding proﬁle; whereas, binding to control polymers, made in the
same way but without the addition of protein templates, showed progressive binding typical of non-speciﬁc recognition.
Reductions in the observed current transmission due to bonding to the polymer surface of non-conductive protein have been used as a measure of
re-binding. It was found that when challenged with 1 part per million protein in solution, the current reductions for the lysozyme and cytochrome
c imprinted electrodes were 30.3 and 66.2%, respectively, compared to 4.5 and 29.9% for their respective control electrodes. All measurements
carried out at −0.1 V with Ag/AgCl reference.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting; Protein; Lysozyme; Cyclic voltammetry; Polymerisation; Aminophenylboronic acid; Polypyrrole
The malformation of proteins is apparent in a wide variety
of pathological conditions (Selkoe, 2003; Thomas et al., 1995);
hence, new approaches to their detection and characterisation
are a matter of interest. However, proteins as a class of molecule
tend to be large, easily deformed and labile; for these reasons, the
construction of artiﬁcial recognition materials for their detection
Molecular imprinting is a rapidly maturing technology in
which a target molecule, termed a template, directs the arrange-
ment of functional monomers around itself to form an initial pre-
polymerisation complex. Subsequent polymerisation of these
monomers, within a scaffold formed from cross-linkers, fol-
lowed by extraction of the template, gives rise to a relatively rigid
polymeric structure containing cavities; complimentary, both in
shape and functionality, to the original template. This description
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and model of imprinted polymer formation is perfectly adequate
for small highly functionalised targets. However, proteins are
invariably large and consequently their extraction from three-
dimensional bulk monolithic structures is not easily achieved,
due largely to diffusion constraints, thus reports citing the suc-
cessful imprinting of proteins in bulk macroporous polymers are
rare (Huang et al., 2005). For this reason, recognition structures
for large, bulky macromolecules are commonly constructed as
either porous cross-linked gels, often formed from polyacry-
lamide; or, as thin-ﬁlms, in which the surface architecture of the
ﬁlm is modiﬁed, such that its shape and functionality are com-
plimentary to a given template (Chou et al., 2005; Hawkins et
al., 2005; Shi and Ratner, 2000; Shi et al., 1999; Hjert
en et al.,
1997; Liao et al., 1996).
Electro-polymerisation is a simple and convenient technique
that allows thin polymeric ﬁlms to be formed on electrode
surfaces, although some workers who have used polymers in
protocols for protein sensing have concluded that the binding
events are irreversible and as such data cannot be analysed
from the normal perspective of concentration verses binding
at a state of equilibrium. Recently however, the use of poly-
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