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Synthetic Metals

A polypyrrole-biotin

102 (1999)


based biosensor: elaboration and characterization

L. M. Torres-Rodriguez, M. Billon, A. Roget and G. Bidan

dElectrochimie Moldculaire.
CllMR 5819 CEA-CNRS-Universitt
Joseph Fourier, Dipartement de recherche fondamentale
sur la Mati&e Condenske, CEA Grenoble, I7 Av. des Martyrs, 38054 Grenoble Ckdex 09 France

In this paper, we present the electrosynthesis and the electrochemical characterization of a polypyn-ole including biotin units in
order to realize a versatile avidin biosensors. The accessibility of immobilized biotin entities toward avidin conjugates has been
shown by the quarzt crystal microbalance.




and derivates, Biosensors, Biotin.

1. Introduction
The elaboration
of enzyme sensors has been a topic of
interest in recent years due to their potential
applications [ 11. Their fabrication needed the immobilization
enzymes on the electrode surface. Several techniques have been
developed such as (i) the anchoring of biotinylated enzymes to
the avidin-modified
electrode thanks to the strong affinity
between avidin and biotin [2, 31 or (ii) the insertion of enzymes
within an electronic conducting polymer (ECP) matrix by
entrapment during its electropolymerization
We report herein a novel strategy based on copolymerization
of pyrrole monomers and pyrrole covalently linked to biotin
through a spacer arm (Scheme 1).

could elaborate a large number of biosensors due to a wide

variety of commercial avidin conjugates and on the other hand it
goes the possibility
of the miniaturization
thanks to the
electrochemical addressing of the electropolymerization
as we
have previously shown for the polypyrrole DNA chip [5].

CoPoLY] I 1 (


Jj s i
I Q 1
- __- ___
Scheme 1 : The pyrrole-biotin


The synthetic strategy leads (reaction 1, scheme 2) to an

ECP film anchored on the electrode surface where biotin entities
are grafted on the polypyrrole network. Then the immobilization
on the film of biomolecules such as avidin enzyme conjugates,
can be realized. Indeed these immobilized biotin entities appear
as anchoring points since biomolecules bearing avidin units can
be easily linked on the polypyrrole
surface thanks to the
interaction (reaction 2) and this in a one step only
whitout any chemical coupling.
This approach shows several features. On the one hand, one

* Corresponding



- see front
PII: SO379-6779(98)01053-4


Scheme 2 : Synthetic strategy towards a avidin biosensor.

After the description of the electropolymerization
and the
electrochemical properties of the biotin polypyrrole, we present
in this report the results achieved by quartz crystal microbalance
(QCM) showing that the film provides biotin sites capable of
selectively binding avidin.
2. Experimental
Electrochemical syntheses of the biotinylated copolypyrrole
and their characterizations by cyclic voltamperometry were made
with a PAR 273 potentiostat from EG&G Princeton Applied
Research controlled by a computer. The working electrode was a
platinum electrode (diameter, 3 mm). All potentials are relative

: billon@drfmc.ceng.cea.fr)

matter 0 1999 Elsevier



All rights



M. Billon

et al. I Synthetic

to a saturated calomel electrode (SCE). The electrolyte was an

aqueous solution containing 3% CHsCN with 0.1 mol L-i LiC104
(Fluka Purum) which was purged by argon bubbling before each
The quartz crystal is AT-cut with a basic resonance
frequency of 9 Mhz and sandwiched between two Pt electrodes
(diameter, 5 mm). The frequencies were measured by a Hp
53424 A frequency counter from Hewlett Packard.
3. Results and Discussion
The pyrrole-biotin
1 was synthesised by coupling an amino
alkyl pyrrole and a biotm entity [6].
Concerning the electrosynthesis of the biotinylated copolypyrrole
(noticed copoly[l]), it was realized by repeated potentiel linear
scanning between -0.25 and +0.85 V from a solution containing
20 mM pyrrole and 10 mM pyrrole-biotin
monomers 1. The
cyclic voltammogram recorded during the electropolymerization
shows two responses. One is an irreversible and sharper anodic
peak due to monomer oxidation at approximatively
+0.81 V and
the other is constituted of an oxidation peak at +0.55 V and a
reduction peak at -0.10 V. Both responses increased in intensity
with subsequent scans.
ARer synthesis and thoroughy rinsing, the modified electrode
is transferred to an electrolytic solution free of monomers. As
shown Figure 1, the electrochemical response of this modified
electrode is composed of one reversible and stable response with
a shape typical of that observed with an ECP deposed on the
surface electrode.
In comparison
with the electroactivity
associated to an polypyrrole film, we observed that the response
of copolyu] film is shifted to the more positive potential values
and characterized by a higher value (340 mV) of AEp (AEp
=Epc-Epa). And more, the electroactivity
of the biotinylated
copolypyrrole occurs in a large potential range in agremment
with a mixed behaviour of poly(pyrrole)
and poly(N-substitued

102 (1999)



anchor to the surface of the biotinylated copolypyrrole via the

After the electrosynthesis on the platinum surface of the
QCM, the copoly[l] film was placed in a aqueous solution
containing the Denhardts reagent in order to avoid non-specitic
adsorption of avidin. The response of QCM (Figure 2) before
and after the injection of an avidin solution shows a fast
decreasing of frequency just after the addition
of avidin
conjuguate. From this result, we have assessed that the mass of
avidin immobilized on the the copoly[l] film equal to 48.03 ng
cooresponding to 0.7 pmol of avidin conjugates immobilized on
the copolyu] film.

Fig. 2. Variation of frequency with the time of the copoly[l] film

in aqueous solution containing the Denhardts reagent, before (a)
and after (b) the injection of an avidin solution.
The same experiment has been realized with a polypyrrole film.
In this case, the injection of avidin did not induced a revealing
variation of the frequency in comparison with this observed for
the biotinylated copolypyrrole.
4. Conclusion
This study has demonstrated
shows the possibility
biotin units linked to the polypyrrole
Although these biotin units were inserted first in the ECP, some
of their were accessible on the polypyrrole surface to avidin
conjugates. Thus the biotinylated polypyrrole can be use in order
to elaborate a versatile avidin biosensor.



E vs


Fig. 1. Cyclic voltammetry of copoly[l] film on a platinum

electrode (diameter 3 mm) in H20/LiC104 0.1 M (v = 50 mV/s).
Synthesis of the film (see text) was performed by repeated
potentiel linear scanning by passing 34 mC/cm.

In order to check the ability of recognition characteristic of

the copolyu] film towards avidin conjugates, a study by the
quartz microbalance was undertaken. Indeed the great sensitivity
of this device allows to determine the mass of avidin which can

5. References
[l] G. Bidan in Gabor Harsanyi (Ed.), Polymer films in sensor
applications, Ph. D. Gabor Harsanyi, Hungary, (1995) 206.
[2] T. Hoshi, J.-I. Anzai, T. Osa, Anal. Chem., 67 (1995) 770.
[3] M. Vreeke, P. Rocca, A. Heller, Anal. Chem., 67 (1995) 303.
[4] P. Bartlett, J. Cooper, J. Electroanal. Chem., 362 (1993) 1.
[5] T. Livache, B. Fouque, A. Roget, J. Marchand, G. Bidan, R.
Teoule, G. Mathis, Anal. Biochem., 255 (1998) 188.
[6] L. M. Torres-Rodrigez, A. Roget, M. Billon, T. Livache, G.
Bidan, submitted for publication.