β-Cyclodextrin polymer as the immobilization matrix for peroxidase and mediator in the fabrication of a sensor for hydrogen peroxide

Abstract

A novel immobilization approach based on the supramolecular function between β-cyclodextrin polymer (β-CDP) and cationic dyes, and the condensation polymerization among β-CDP, glutaric dialdehyde and horseradish peroxidase (HRP) in the fabrication of a hydrogen peroxide sensor is described. IR and UV–vis spectroscopy were employed to characterize the structure of the composite membrane modified on the surface of the electrode. AFM was used to visualize the morphology of the composite membrane. The characteristics of supramolecular inclusion compounds between β-CDP and cationic dyes were studied. The fabricated H2O2 sensor responded rapidly to H2O2 in the linear range from 1.0 to 1.1 mmol l−1 with a detection limit of 0.5 mol l−1.

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www.elsevier.nl/locate/jelechem
Journal of Electroanalytical Chemistry 480 (2000) 255–261
Short Communication
www.spm.com.cn
?-Cyclodextrin polymer as the immobilization matrix for
peroxidase and mediator in the fabrication of a sensor for
hydrogen peroxide
Min Zhu, Shubo Han, Zhuobin Yuan *
Department of Chemistry, Graduate School, Uni?ersity of Science and Technology of China, Beijing 100039, PR China
Received 7 June 1999; received in revised form 12 October 1999; accepted 22 October 1999
Abstract
A novel immobilization approach based on the supramolecular function between ?-cyclodextrin polymer (?-CDP) and cationic
dyes, and the condensation polymerization among ?-CDP, glutaric dialdehyde and horseradish peroxidase (HRP) in the
fabrication of a hydrogen peroxide sensor is described. IR and UV–vis spectroscopy were employed to characterize the structure
of the composite membrane modified on the surface of the electrode. AFM was used to visualize the morphology of the composite
membrane. The characteristics of supramolecular inclusion compounds between ?-CDP and cationic dyes were studied. The
fabricated H2O2sensor responded rapidly to H2O2in the linear range from 1.0 to 1.1 mmol l−1with a detection limit of 0.5 mol
l−1. © 2000 Elsevier Science S.A. All rights reserved.
Keywords: Supramolecular inclusion complex; Cationic dye mediator; Biosensor; Hydrogen peroxide
1. Introduction
The fabrication of H2O2sensors has attracted great
interest because the measurement of H2O2is the basis
of detecting many biologically active materials, such as
glucose, cholesterol and uric acid [1,2]. The immobiliza-
tion of an enzyme and an electron transfer mediator is
one of the critical steps in the construction of a H2O2
mediator bioelectrode, a kind of second-generation am-
perometric biosensor. The mediators have to be immo-
bilized firmly on the electrode in case the soluble
mediating species might diffuse away from the electrode
surface into the bulk solution. On the other hand,
sufficient mobility of the applied mediator is also vital
to the sensor when the mediator is employed to facili-
tate electron communication between the redox center
of the enzyme and the electrode. Some inorganic
porous materials, such as montmorillonite [3], zeolite
[4] and clay [5,6] have been proven to be promising as
the immobilization materials, due to their good me-
chanical, thermal and chemical stability, ion-exchanger
and electrocatalytic properties, hydrophobic property
and existing mesopores. Many researchers suggested
that the absorption and cationic exchange function of
the enzyme and the mediator be attributed to the
excellent electrochemical response using these porous
materials. However, in the view of supramolecular
chemical theory, some natural inorganic materials, such
as montmorillonite and zeolite are typical cage com-
pounds. The type of inorganic solid particles are suit-
able for the host, which includes the guest molecule to
form a cage compound. So the excellent immobilization
characteristics of the enzyme and the mediator using
montmorillonite and zeolite depend partly on their
special supramolecular structures, in which the cage
compound acted as a host, and a mediator as a guest.
With this in mind, the purpose of this work is to
illustrate that the supramolecular action could be a
novel immobilization approach in the fabrication of a
biosensor.
* Corresponding author. Fax: +86-10-68210501.
E-mail address: hanshb@es1.gsbustc.ac.cn (Z. Yuan)
0022-0728/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0022-0728(99)00442-8

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M. Zhu et al. / Journal of Electroanalytical Chemistry 480 (2000) 255–261256
?-CD is a cyclic oligomer comprised of seven ?-D-
glucopyranose units linked 1?4, as in an amylose. The
interior cavity of the doughnut-shaped molecule pro-
vides a relatively hydrophobic environment into which
various organic substrates, especially hydrophobic sub-
stances can be trapped selectively. ?-CD cross-linked
polymer was selected as the immobilization matrix in
this work due to its special structural features. Such a
porous three-dimension network provides not only a
large hydrophobic surface for enzyme and mediator
loading, but also a desirable microenvironment to
transform enzymatically-produced hydrogen peroxide
and proton more efficiently. The inclusion of ?-CDP
and heteroanthracene ring cationic dyes as the media-
tor, eg phenazine, phenoxazine and phenoxthine dyes,
such as methylene blue (MB), azure A (AA), toluidine
blue (TB), resorcinol blue (RB), neutral red (NR),
safranine T (ST) and indigo carmine (IC) were investi-
gated. The inclusion formation constants were deter-
mined. Then the cationic dyes and the enzyme were
co-immobilized on the electrode by supramolecular
function between ?-CDP and the guest molecule, as
well as the condensation polymerization among ?-CDP,
glutaric dialdehyde and HRP [7]. The fabricated H2O2
sensor exhibited excellent characteristics on the firm
immobilization and a fair electronic mobility. So a
novel immobilization approach to the enzyme and the
mediator was provided, and its attractive properties
would find various practical applications.
2. Experimental
2.1. Material
Peroxidase from horseradish (HRP) (Type IV) was
purchased from Sigma. The cross-linked polymer of
?-CD and epoxy chloropropane was synthesized ac-
cording to the literature [8] and milled to a fine resin
with a grain of 150 mesh. Cellulose triacetate (average
degree of polymerization 200–400) was obtained from
BeijingChemicalReagent
buffer solution (pH 7.0) served as the supporting elec-
trolyte. All aqueous solutions were prepared in twice
distilled water.
Co. Britton–Robinson
2.2. Apparatus
Cyclic voltammetric and amperometric signals were
obtained with a PAR polarographic analyzer, Model
174 (EG&G, Princeton Applied Research Corporation,
USA), equipped with a glassy carbon electrode (GCE)
as a working electrode, a saturated calomel electrode as
the reference electrode, and a platinum wire as the
auxiliary electrode. Spectral measurements were made
on a Shimadzu UV-240 spectrophotometer with a 10
mm pathlength fused silica cuvette (Tokyo, Japan). IR
spectra were recorded on a FT-JR 5DX spectrometer
(Nicolet). Atomic force microscope (AFM) images were
obtained with a Benyuan 930 b scanning probe micro-
scope (Benyuan Co., China).
2.3. Construction of H2O2sensor
The dye solution, buffer solution and ionic strength
conditioning agent were mixed with ?-CDP, and the
mixture was incubated at 25°C for 40 min. Then the
supramolecular inclusion compound of ?-CDP and the
mediator molecule was obtained. Firstly, 5 ?l of 40 g
l−1?-CDP-mediator inclusion compound colloid was
mixed carefully and thoroughly with 10 ?l of 5% cellu-
lose triacetate which acted as a supporting carrier in
acetone. Then 5.0 mg HRP and 5.0 mg BSA were
dissolved into this colloid and subsequently 5 ?l of 5%
glutaric aldehyde solution was mixed completely with it
as soon as possible. The composite membrane coated
GCE was prepared by spin-coating the colloid on the
GCE surface at 3000 rpm and allowed to dry under
ambient conditions for 3 h. A 5 ?l aliquot of such a
mixture was pipetted onto the surface of mica and
allowed to dry for AFM observation. The sensor was
kept in 0.1 mol l−1phosphate buffer (pH 7.0) at 4°C.
2.4. E?aluation of EC50and absorption and the amount
included (Q)
The experiments were performed on a vibrator by
treating for 40 min 0.1000 g of ?-CDP, a certain
amount of the cationic dye, and buffer solutions under
different pH values at an ionic strength of 0.1 mol l−1.
After equilibrating the guest concentration in the liquid
phase, the equilibrium concentration of guest ([G]eq)
was determined at its maximum absorption wavelength
by observing the supernatant liquid. EC50, i.e. the dye
concentration that gives 50% maximal saturation of
?-CDP, as well as absorption and the amount included
(Q) was calculated from the these experimental results.
2.5. The measurement of H2O2
All experiments were conducted in a cell containing
10 ml B-R buffer solution as the supporting electrolyte
at 30.0?0.2°C. All experimental solutions were deoxy-
genated thoroughly by bubbling N2through the solu-
tion for at least 6 min prior to use. In the potentiostatic
experiment, successive additions of stock H2O2solution
in the buffer were made and current–time data were
recorded after a constant residual current had been
established under stirring. The sensor response was
measured by the difference between the total and the
residual current.

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M. Zhu et al. / Journal of Electroanalytical Chemistry 480 (2000) 255–261 257
3. Results and discussion
3.1. The surface characteristics of ?-CDP-cationic
dye-HRP modified electrode
The IR absorption bands of ?-CDP and its absorp-
tion and inclusion compounds with MB, glutaric alde-
hyde and HRP are presented in Table 1. After the
formation of an inclusion compound with MB, the
absorption band of the hydroxyl group in ?-CDP split
into peaks at 3422 and 3179 cm−1. The strength of the
absorption band decreased significantly. ?-CDP-MB
had absorption bands at 1368 and 1346 cm−1; a shift
to lower wavenumbers in comparison with those of
?-CDP, which was attributed to the formation of a
hydrogen bond between a ?CH2? group and other
relative groups. ?-CDP-MB also had absorption bands
at smaller wavenumbers at 1643 and 1137 cm−1, and
the strength of the absorption band decreased greatly.
These facts indicated that intermolecular interaction
existed between them. The original association of hy-
drogen bonds in ?-CDP molecules was destroyed and a
new hydrogen bond appeared between ?-CDP and the
guest, MB. In the IR spectrum of ?-CD-MB, the
appearance of some absorption bands (1592, 1176
cm−1) was attributed to the characteristic absorption of
MB. However, the fact that the absorption band of MB
at 2834?2917 cm−1could not be observed, indicated
that the stretching vibration of ?CH2? was being hin-
dered by the formation of the inclusion compound. It
was seen from the IR spectrum of the ?-CDP-glutaric
aldehyde that a new absorption band at 1703 cm−1was
attributed to the formation of a carboxyl group in the
complex, probably due to the condensation between the
hydroxyl group in ?-CDP and the aldehyde group in
glutaric aldehyde. ?-CDP-glutaric aldehyde possessed
an absorption band at 1600 cm−1or so, indicating that
a part of the carbonyl group in glutaric aldehyde did
not participate in the reaction during condensation, and
eventually became a group hanging on the chain of the
polymer. These groups can be used in the immobiliza-
tion of HRP. So it is possible to immobilize HRP on
the ?-CDP resin through glutaric aldehyde. This has
been illustrated by IR spectra of ?-CDP-MB-glutaric
aldehyde-HRP with the mixture exhibiting the weaker
absorption at 1600 cm−1or so. As a result, ?-CDP
resin would incorporate together with MB, glutaric
aldehyde and the enzyme by supramolecular and cova-
lent interaction. It is an excellent immobilization matrix
for a mediator and an enzyme.
AFM is becoming increasingly useful in studies of
electrode surface characteristics. AFM images, obtained
in the present study as shown in Fig. 1, show the
morphology of the composite film modified on the
electrode surface. A porous structure was observed in
the cellulose triacetate membrane (Fig. 1(1)), with a
pore diameter of 0.60 (A) to 1.2 ?m (B). When ?-CDP
was mixed with cellulose triacetate acetone solution
before membrane formation, most of the holes in the
membrane were covered (Fig. 1(2)), and the pore di-
ameter was reduced to 0.34 ?m (A). However, there still
existed about 10% pores in the composite membrane of
?-CDP and cellulose triacetate. The molar mass of the
inclusion compounds increased greatly when a dye,
such as MB, was included by ?-CDP. Moreover, the
aggregation of inclusion compounds was attributed to a
larger granularity of inclusion compounds than that of
?-CDP itself. So a rough and irregular topography of
the composite membrane of ?-CDP-MB-glutaric alde-
hyde-HRP was observed (Fig. 1(3)).
3.2. Characteristics of ?-CDP-cationic dye mediator
supramolecular inclusion compounds
3.2.1. Absorption spectra of inclusion compounds of
?-CDP and cationic dyes
The incorporation of the dye and the polymer resin
was identified by resin phase absorption spectra with
?-CDP resin as the background, since no apparent
absorption band of ?-CDP blank resin was observed in
the range of 350–750 nm. The absorption band of MB
showed a red shift of 10 and 5 nm at 605 and 610 nm,
respectively, when it was included and absorbed on the
resin from the aqueous solution. Similarly, the absorp-
tion bands of AA, TB, RB, NR, ST and IC exhibited
shifts to 620, 607, 620, 502, 504 and 613 nm from 648,
629, 611, 544, 521 and 604 nm, respectively, when
incorporated in the ?-CDP resin. The absorption
strength of the solid phase increased greatly compared
with the dye solution, probably due to the enrichment
Table 1
IR absorption bands of ?-CDP and its absorption and inclusion
compound
IR absorption band/cm−1
Samples
3382?3190(?-OH, s), 2917?2834(?-C?H, s),
1643(?-CH?O, s), 1396?1368(?CH2, m),
1137(?aC?O?C, s), 1039(?aR-C?OH, s)
3422, 3179(?-OH, s), 1624(?CH?O, s),
1592(?C?N?CH), 1381, 1346(?CH2, m),
1176(?(CH2)3N, m), 1098(?aC?O?C, s),
1014(?aR-C?OH, s)
3402, 3201(?-OH, s), 2881(?-C?H, s),
1703(?C?O, vs), 1640 (?C?O, 1598(?CH?O,
s), 1375, 1336(?CH2, m), 1078(?aC?O?C, s),
1029(?aR-C?OH, s)
3376(?-OH, s), 2898(?-C?H, s), 1601
(?CH?O, s), 1383,1359 (?CH2, m), 997(?aR-
C?OH, s)
?-CDP-MB-glutaric 3422, 3181 (?-OH, s), 2908(?-C?H, s),
aldehyde-HRP1699(?C?O, vs), 1601(?C?O, vs),
1592(?C?N?C, m), 1372, 1331(?CH2, m),
1232(?(CH2)3N, m), 1147, 1008(?aR-C?OH)
?-CDP
?-CDP-MB
?-CDP-glutaric
aldehyde
?-CDP-HRP

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M. Zhu et al. / Journal of Electroanalytical Chemistry 480 (2000) 255–261258
Fig. 1. A series of AFM images of cellulose triacetate membrane (1) and composite membranes of ?-CDP (2), or ?-CDP-MB-glutaric
aldehyde-HRP (3) and cellulose triacetate membrane.
of ?-CDP resin suggesting the formation of inclusion
compounds, consistent with the results from the IR
spectrum.
3.2.2. Effect of pH on EC50and adsorption and
included amount (Q)
As noted earlier, ?-CD exhibited a peculiar structure
with the interior hydrophobic, and the exterior hy-
drophilic. The stability of the inclusion compound of
?-CDP was associated with the hydrophobicity of the
guest molecule, so it depended greatly on the pH of the
solution. EC50values were measured in the solution of
different pH, with the results shown in Table 2.
The absorption of the guest per gram of ?-CDP is
given by the following equation:
Q=([G]0−[G]eq)V/mA
where [G]eq, [G]0are equilibrium and initial concentra-
tion of a guest, cationic dye compound, respectively, V
is the volume of the dye solution in liters, and mAis the
mass of ?-CDP (host) in grams. The effect of pH on the
adsorption and inclusion amount of azo molecule, Q
was studied, with the results shown in Table 2.
It was concluded from Table 2 that, when RB or IC
were the guest of ?-CDP, EC50increased with pH,
probably due to increasing ionization of the guest
molecule as a result of the formation of the ion R-SO3
Conversely, when AA, TB or ST was the guest of
?-CDP, EC50values decreased with the increase of pH,
because the positive charge the tertiary amine possessed
in acidic or neutral conditions was neutralized at high
−.
pH and the electroneutral product formed. For the
same reason, when MB or NR was the guest, EC50
values first decreased with the increase of pH in acidic
or neutral solution, but finally increased slowly in a
strong basic condition, probably resulting from the
increasing ionization with excessive OH−.
3.3. Electrochemical characteristic of the biosensor
From the cyclic voltammograms of 5.85×10−4mol
l−1MB, 7.80×10−5mol l−1AA, 1.11×10−4mol l−1
TB, 2.25×10−4mol l−1RB, 1.04×10−4mol l−1NR,
1.00×10−4mol l−1ST and 2.57×10−5mol l−1IC in
0.1 mol l−1phosphate buffer at GCE and that of the
cationic dye incorporated in ?-CDP-modified mem-
brane, two rather different quasi-reversible waves were
observed for the free and membrane-incorporated dye
with scan potential between 0 and −1.00 V. The
?-CDP inclusion compound modified electrode dis-
played more negative peak potentials but larger ?Ep
values resulting from the interaction of the dyes with
?-CDP resin. However, almost all of ?Epvalues were
less than 100 mV, except for that of RB less than 120
mV under the optimal conditions, which indicated that
there was relatively rapid charge transfer through the
modified membrane as well as the charge transfer from
the membrane to the electrode. In addition, NR and ST
displayed two pairs of quasi-reversible waves, which
were attributed to two consecutive reactions. The linear
relationship of peak current to the square root of scan
rates indicated that the electrode reactions initiated by
dyes in the working buffers were controlled mainly by

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M. Zhu et al. / Journal of Electroanalytical Chemistry 480 (2000) 255–261
259
Table 2
The effects of pH on EC50and Q of cationic azo dyes
NRe
MBa
STf
ICg
AAb
TBc
RBd
pH
105EC50/
mol l−1
Q/?mol
b−1
Q/?mol
b−1
105EC50/
mol l−1
Q/?mol
b−1
Q/?mol
b−1
105EC50/
mol l−1
105EC50/
mol l−1
105EC50/
mol l−1
Q/?mol
b−1
105EC50/
mol l−1
Q/?mol
b−1
Q/?mol
b−1
105EC50/
mol l−1
1.27
1.26
1.28
1.30
1.28
1.25
1.24
1.23
1.20
1.15
9.39
9.34
9.19
8.82
8.58
8.34
7.35
8.35
8.78
8.83
19.1
19.2
18.9
18.3
18.7
19.4
19.5
19.7
20.3
21.4
6.15
7.49
7.54
7.98
8.17
8.34
8.61
8.93
10.7
11.0
10.5
9.82
7.72
6.84
6.46
6.12
5.58
4.94
1.45
0.739
11.2
9.55
9.35
8.85
8.70
8.60
8.40
8.70
8.80
8.95
7.69
10.9
11.3
12.3
12.6
12.8
13.2
12.6
12.4
12.1
7.94
7.82
7.66
7.39
6.65
6.38
6.23
6.14
5.95
5.90
4.05
4.29
4.62
5.16
6.63
7.18
7.47
7.65
8.04
8.13
4.75
4.89
4.96
5.03
5.06
5.08
5.14
5.26
5.27
5.39
4.80
4.52
4.37
4.24
4.19
4.13
4.02
3.77
3.75
3.51
7.42
7.52
7.82
8.56
9.04
9.52
11.5
9.14
8.63
8.54
7.03
6.25
6.16
6.14
6.03
5.85
5.79
5.72
5.59
5.35
5.94
7.49
7.68
7.72
7.93
8.29
8.42
8.56
8.81
9.29
2.56
3.78
4.56
5.75
6.80
7.40
8.69
9.37
10.38
11.20
a[MB]0=1.31×10−4mol l−1.
b[AA]0=1.00×10−4mol l−1.
c[TB]0=2.22×10−4mol l−1.
d[RB]0=1.14×10−4mol l−1.
e[NR]0=1.50×10−4mol l−1.
f[ST]0=9.97×10−5mol l−1.
g[IC]0=7.15×10−5mol l−1.