Analytica Chimica Acta 653 (2009) 212–216
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Analytica Chimica Acta
journal homepage: www.elsevier.com/locate/aca
Zinc oxide–potassium ferricyanide composite thin film matrix for
biosensing applications
Shibu Saha a , Sunil K. Arya b,1 , S.P. Singh c , K. Sreenivas a , B.D. Malhotra b , Vinay Gupta a,∗
a
b
c
Department of Physics & Astrophysics, University of Delhi, Delhi 110007, India
Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, New Delhi 110012, India
Department of Engineering Science and Materials, University of Puerto Rico, Mayaguez, PR 00680, USA
a r t i c l e
i n f o
Article history:
Received 26 June 2009
Received in revised form 28 August 2009
Accepted 2 September 2009
Available online 6 September 2009
Keywords:
Biosensor
Pulsed laser deposition
Potassium ferricyanide
Zinc oxide
Composite matrix
a b s t r a c t
Thin film of zinc oxide–potassium ferricyanide (ZnO–KFCN) composite has been deposited on indium tin
oxide (ITO) coated corning glass using pulsed laser deposition (PLD). The composite thin film electrode
has been exploited for amperometric biosensing in a mediator-free electrolyte. The composite matrix
has the advantages of high iso-electric point of ZnO along with enhanced electron communication due to
the presence of a redox species in the matrix itself. Glucose oxidase (GOx) has been chosen as the model
enzyme for studying the application of the developed matrix to biosensing. The sensing response of
the bio-electrode, GOx/ZnO–KFCN/ITO/glass, towards glucose was studied using cylic voltammetry (CV)
and photometric assay. The bio-electrode exhibits good linearity from 2.78 mM to 11.11 mM glucose
concentration. The low value of Michaelis–Menten constant (1.69 mM) indicates an enhanced affinity of
the immobilized enzyme towards its substrate. A quassireversible system is obtained with the composite
matrix. The results confirm promising application of the ZnO–KFCN composite matrix for amperometric
biosensing applications in a mediator-less electrolyte that could lead to the realization of an integrated
lab-on-chip device.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The last two decades have seen a tremendous growth in the
field of biosensor research due to their potential use in the
field of health-care, biological analysis, environment-monitoring
and food industries. The critical aspects in the designing of
biosensor are the selection of the material onto which the
biorecognition elements are to be immobilized and the binding
chemistry behind the biomolecule immobilization on the chosen material. Therefore, designing of an electrode system with
a compatible microenvironment for the bio-molecule and good
electron transfer capability is desired to obtain a biosensor with
enhanced sensitivity [1–3]. With the advancement of research in
material science a variety of materials such as metal oxides, selfassembled monolayers, conducting polymers and nanocomposites
have been used as platform to enhance the electron transfer rate
and obtain a good stability of biomolecules, such as enzymes,
nucleic acids and proteins, on its surface [4–11]. Materials that
exhibit excellent properties such as stability, chemical resistivity
∗ Corresponding author. Tel.: +91 11 27667725.
E-mail address: vgupta@physics.du.ac.in (V. Gupta).
1
Presently working at BioMEMS and Microsystems Lab., Dept. Elect. Eng., University of South Florida, Tampa, Florida, USA as Post Doctoral fellow.
0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.09.002
and biocompatibility are promising candidates for bio/chemical
sensors.
Zinc oxide has been attracting much attention due to its
wide range of applications [11–15]. Recently it has emerged as
a suitable matrix for immobilization of biomolecules due to its
properties like high catalytic efficiency, strong adsorption ability,
bio-compatibility, high isoelectric point (IEP ∼9.5) and abundance
in nature [4,11,15–17]. Because of high isoelectric point ZnO provides a suitable environment for immobilization of low isoelectric
point (4.2) enzyme such as GOx (at pH 7.0) [4,15]. A large number of
biomolecules have been reported to be immobilized on ZnO matrix.
Zhang et al. reported a uric acid biosensor based on ZnO nanorods
[18]. Immobilization of glucose oxidase on ZnO nanocomb matrix
has been reported by Wang et al. [16]. Cholesterol oxidase has
been immobilized on ZnO nanostructure, for the fabrication of a
cholesterol biosensor [4,19]. Heme proteins have been immobilized on ZnO for H2 O2 sensing [20–23]. Deng et al. have studied
the direct electron transfer of SOD which is strikingly facilitated
at the nanostructured ZnO [24]. Gu et al. reported the immobilization of tyrosinase on ZnO for detection of phenolic compound
[25]. Immobilization of DNA on ZnO has also been reported [26].
Although these studies suggest the potential of ZnO in the field of
biosensor applications, but the poor electrochemical behavior of
ZnO is still a barrier to develop efficient electrochemical biosensor. The enzymatic electrochemical biosensor relies on shuttling
S. Saha et al. / Analytica Chimica Acta 653 (2009) 212–216
of the electrons, generated in the biochemical reaction, from the
redox center of the enzyme to the electrode. Enzymes, because of
their insulated-shell redox centers, are unable to show direct electrochemistry. The presence of a redox species (mediator), in the
system, provides the path for electron transfer from the redox centers of the enzyme to the electrode. Potassium ferricyanide (KFCN)
is one of the most commonly used mediators and its excellent redox
properties have been exploited in number of biosensor studies and
environment monitoring [27–29]. However the leaching of mediators from the integrated electrode, because of their low molecular
weight, remains a long standing problem [30]. Efforts are continuing towards the realization of an efficient mediator-less biosensor,
either by changing the matrix material that has redox properties or
by using novel design structure [6]. Designing a composite material
of ZnO with such a mediator may provide a better material system
with inherent semiconductor as well electrochemical properties for
biosensor applications. To the best of our knowledge no attempt
has been made till date towards the realization of such a composite
material system.
In this paper, we are reporting the development of a composite matrix of ZnO–KFCN by pulsed laser deposition (PLD) technique.
GOx has been chosen as the model enzyme to study the application
of the matrix towards biosensing. The sensing response characteristic of the prepared bio-electrode confirms that the composite
ZnO–KFCN matrix exhibits a linear sensing response at a relatively lower potential over a wide range of concentration of glucose
(2.78–11.11 mM).
2. Experimental
2.1. Materials
GOx (200 U mg−1 ), horseradish peroxidase (HRP, 200 U mg−1 )
was purchased from Sigma–Aldrich. Sodium phosphate monobasic
anhydrous and sodium phosphate dibasic dihydrate were obtained
from Sisco chemical, India. ZnO powder (99.9% purity) and Pottasium ferricyanide was acquired from Merck & Co. Inc., and was
used for making targets for PLD. All chemicals were used without
further purification. Deionized water was used in the preparation
of aqueous solutions.
213
2.4. Preparation of ZnO/K3 [Fe(CN)6 ] film and immobilization of
GOx
Composite thin films of ZnO–K3 [Fe(CN)6 ] (ZnO–KFCN) were
deposited by PLD, at room temperature, onto indium tin oxide
(ITO) coated corning glass plates. PLD target pellet (1 in. diameter) was prepared using 4 g of ZnO powder having 3% of potassium
ferricyanide. The composite films of ZnO–KFCN were prepared by
ablating the ceramic oxide target with fourth harmonic of NdYAG laser ( = 266 nm) at a fluence of 1.2 J cm−2 in 50 mT oxygen
pressure. The composite films were annealed at 200 ◦ C. Thin films
of pure ZnO was also prepared under similar processing condition on ITO coated glass plates for comparison with ZnO–KFCN
matrix. GOx was immobilized onto the ZnO–KFCN composite film
by physical adsorption technique. For immobilization of GOx, 30 ␮L
of the freshly prepared GOx solution was dropped on the surface
of the composite thin film and kept overnight at 4 ◦ C followed
by extensive washing with buffer to remove any unbound GOx.
The prepared bio-electrode (GOx/ZnO–KFCN/ITO/glass) was dried
under dry nitrogen flow and kept at 4 ◦ C when not in use.
3. Results and discussions
3.1. Physical properties
The ZnO–KFCN composite thin films (∼80 nm) deposited by PLD
under optimized processing condition were found to be smooth,
transparent and strongly adherent to the surface of ITO coated
glass. The films were highly c-axis oriented in hexagonal wurtzite
structure having a bandgap of 3.47 eV. The fact that the preferred
orientation of the film is preserved on inclusion of KFCN into the
ZnO matrix suggests that the KFCN does not disturb the crystal
structure of ZnO.
3.2. Electrochemical impedance studies
Fig. 1 shows the electrochemical impedance spectra of
ZnO/ITO/glass and ZnO–KFCN/ITO/glass electrodes. The plot
between the real and imaginary component of impedance for the
ZnO electrode shows a semicircle, and is similar to that observed
2.2. Measurement and apparatus
The electrochemical impedance studies (EIS) and the cyclic
voltametry (CV) studies were carried out on an potentiostat/galvanostat using a three-electrode cell configuration with
Ag/AgCl electrode as a reference electrode and platinum foil as
a counter electrode. The CVs were performed in 10 mL of phosphate buffer saline (PBS) solution (50 mM, pH 7.0, 0.9% NaCl).
However, the electrochemical impedance spectroscopic (EIS) studies were made in PBS containing 5 mM Fe(CN)6 3−/4− . The apparent
enzyme activity, shelf life and selectivity studies were carried out
by photometric assay using PerkinElmer (lambda 35) UV–vis spectrophotometer.
2.3. Preparation of solutions
Phosphate buffer saline (PBS) 50 mM, pH 7.0 (0.9% NaCl) solution
was prepared by adjusting the proportion of monobasic sodium
phosphate solution and dibasic sodium phosphate solution and
then adding 0.9% NaCl to the solution. GOx (1 mg mL−1 ) solution and HRP solution (1 mg mL−1 ) were freshly prepared in PBS
buffer of pH 7.0. Different concentrations of glucose solution and
solution of o-dianisidine (1%) were freshly prepared in deionized
water.
Fig. 1. Impedance spectra of the ZnO–KFCN/ITO/glass composite electrode and
ZnO/ITO/glass electrode (inset shows the equivalent circuit of the impedance
spectra and the impedance spectra of ZnO–KFCN/ITO/glass electrode and
GOx/ZnO–KFCN/ITO/glass bio-electrode).
214
S. Saha et al. / Analytica Chimica Acta 653 (2009) 212–216
Table 1
Fitting parameters of the impedance spectroscopy graph.
Matrix
Rs ()
Rp (k)
ZnO
ZnO–KFCN
200
220
915.16
70.37
Cp E
C (F)
3.34 × 10−6
4.31 × 10−5
P
0.89
0.95
by other workers for different electrodes. The diameter of the semicircle reduced significantly after incorporating KFCN into the ZnO
matrix (Fig. 1). The equivalent circuit model of the system is shown
in the inset of Fig. 1. The equivalent circuit consists of a series solution resistance (Rs ) and parallel combination of a resistance (Rp )
and a capacitance (Cp ). The Rp signifies the charge transfer resistance, which is inversely proportional to the charge transfer rate at
the electrode. The capacitor (Cp ) is the contribution from electrode,
its interface with electrolyte and other factors like microscopic
roughness, inhomogeneity in deposited films and adsorbed species
[31,32].
The fitting of the experimental impedance data with the equivalent electrical circuit has been made and the fitting parameters
are shown in Table 1. The value of Rs was found to be same within
experimental error for both the ZnO/ITO/glass and ZnO–KFCN/glass
electrode (Table 1), and is expected as the solution and the positions
of the electrode are kept same. It is interesting to note from Table 1
that the value of resistance Rp decreases by one order in case of the
ZnO–KFCN/ITO/glass composite matrix indicating enhancement in
the charge transfer rate and may be attributed to the presence
of redox ions in the composite matrix. The corresponding value
of Cp of the composite matrix is seen to increase in comparison
to ZnO/ITO/glass electrode and may be attributed to increase in
inhomogeniety in the prepared composite matrix. However, the
capacitive path does not play a major role in the CV study as dc
potential is applied for the CV measurements.
The inset of Fig. 1 shows the impedance spectra of the
ZnO–KFCN/ITO/glass electrode and the GOx/ZnO–KFCN/ITO/glass
enzyme electrode. The charge transfer resistance (Rp ) of the electrode increases to 83.37 k on immobilization of the enzyme. The
increase in the Rp is due to the non-conducting nature of the
macro-molecular enzyme. This is reflected in the cyclic voltammetry measurement where the oxidation current of the electrode
decreases (10.95–1.06 ␮A) on immobilization of the enzyme (inset
(ii) of Fig. 2). A similar behavior of impedance spectra is obtained
for pure ZnO electrode (supplement Fig. 1).
Fig. 2. CV spectra of ZnO–KFCN/ITO/glass electrode at different scan rate (inset:
(i) graph between the square root of potential scan rate and the peak current;
(ii) CV spectra of ZnO–KFCN/ITO/glass electrode and GOx/ZnO–KFCN/ITO/glass bioelectrode at 0.05 V s−1 scan rate).
peak in a mediator-free electrolyte. The oxidation peak was found
to shift slowly towards higher potential and the reduction peak
towards lower potential, with increase in scan-rate from 0.01 V s−1
to 0.1 V s−1 (Fig. 2). The redox peak currents are found to be proportional to the square root of scan rate (inset (i) of Fig. 2), indicating
a quasi reversible system and diffusion assisted electron-transfer
process [35]. Further it may be noted that the Ep (=Epa − Epc ) of the
system is 200 mV, which clearly indicates the quassi-reversibility
of the system.
The enzymatic activity of the prepared GOx/ZnO–KFCN/ITO/
glass bio-electrode was investigated by CV in a mediator-free PBS
solution (Fig. 3). The enzyme immobilization is confirmed by a relative decrease in current in the CV of GOx/ZnO–KFCN/ITO/glass
bio-electrode as compared to that of ZnO–KFCN/ITO/glass electrode
(inset (ii) of Fig. 2). In the present study, no peak corresponding
to the oxidation of H2 O2 is seen in the working potential window of the CVs of the prepared composite bio-electrode as the
electron transfer channel is provided by the K3 [Fe(CN)6 ] present
in the matrix and it acts as an efficient electron acceptor, over
bio-oxygen, resulting in a peak at a potential of −0.2 V. The oxidation current in CV of GOx/ZnO–KFCN/ITO/glass bio-electrode
increases linearly with an increase in the glucose concentration
3.3. Cyclic voltammetric studies
The electrochemical response of the prepared ZnO–KFCN/ITO/
glass electrode was investigated by cyclic voltammetry. A welldefined redox peak was observed at −0.2 V (at 50 mV s−1 scan-rate)
in a mediator-free PBS buffer (Fig. 2). Oxidation peaks in the similar potential range were observed for heme-proteins [24,33] which
indicates the fact that the Fe-ions, obtained from KFCN, may be
playing the role in the generation of the redox peak. Also the possibility of formation of Zn–Fe complex cannot be completely ruled out
[34]. However, the present composite, owing to its low oxidation
potential, reduces the interference from other oxidisable species.
The studies on the interactions between ZnO and KFCN, and the
mechanisms responsible for such behavior are in progress. Biosensor based on the pure ZnO electrode has already been reported
where the electrochemical response of the pure ZnO electrode has
been studied [4]. It is important to note that the pure ZnO electrode does not show any redox peak in PBS due to the absence of a
redox couple. For sensing of bio-molecules a redox species (KFCN) is
added in the buffer solution. However, the presence of KFCN in the
composite matrix (ZnO–KFCN) facilitates the generation of redox
Fig. 3. CV response of GOx/ZnO–KFCN/ITO/glass bio-electrode with varying glucose
concentration (inset shows the sensing response curve at a fixed bias of −0.2 V with
varying glucose concentration).
S. Saha et al. / Analytica Chimica Acta 653 (2009) 212–216
215
Table 2
Comparison of the sensing response parameters of GOx/ZnO–KFCN/ITO/glass bio-electrode with other ZnO matrices for glucose sensing.
S.No.
Material
Deposition technique
1
2
3
4
5
6
Native GOx in solution
ZnO
Hydrothermal decomposition
ZnO
Vapor phase transport
ZnO
Physical coating
ZnO:Co
Nanocluster beam deposition
ZnO–KFCN
Pulsed laser deposition
Structure of electrode
Linearity
Km
References
GOx/ZnO/Au
Naffion/GOx/ZnO/Au
Naffion/GOx/ZnO
GOx/ZnO:Co/Au/Ti/PET
GOx/ZnO–KFCN/ITO/glass
0.01–3.45
0.02–4.5 mM
–
0–4 mM
0–11.1 mM
27 mM
2.9 mM
2.19 mM
1.0–13.4 mM
21 mM
1.69 mM
[36]
[15]
[16]
[37]
[36]
Present work
from 2.78 mM to 11.11 mM (inset of Fig. 3). The continuous rise
in the oxidation current with increasing glucose concentration is
attributed to the release of more number of electrons in the catalytic
oxidation of glucose by glucose oxidase. The response of bioelectrode is found to be relatively fast ∼10 s and is due to the high
electron communication feature of the ZnO and the presence of
KFCN in the matrix. The sensitivity of the composite bio-electrode
is estimated to be 0.078 ␮A mM−1 cm−2 with a detection limit of
0.23 mM.
app
3.4. Estimation of Michaelis–Menten kinetic parameters (Km )
app
The Michaelis–Menten kinetic parameter (Km ) of enzymatic
reaction has been estimated using Hanes plot [6] (i.e. graph
between [substrate concentration] and [substrate concentration/current]). The graph is a straight line represented by the
app
equation y = 0.574x + 17.463. The value of Km is estimated to be
1.69 mM for the GOx immobilized on the composite bio-electrode
and is comparatively lower than the value reported for pure ZnO
app
matrix (Table 2). The low value of Km reflects enhanced activity of GOx enzyme towards its substrate. The increased interaction
between the enzyme’s active sites and the substrate is due to the
favorable conformational changes in the enzyme structure on being
immobilized on the matrix, which are facilitated by the suitable
biocompatible microenvironment provided by the composite thin
film.
3.5. Photometric enzyme assay
To carry out the photometric enzyme assay of immobilized
GOx, the bio-electrode was dipped in 3 mL PBS solution containing 20 ␮L horseradish peroxidase (HRP), 20 ␮L o-dianisidine dye
and 100 ␮L of biosubstrate (glucose). The difference between the
initial and final absorbance value at 500 nm after 3 min incubation of biosubstrate is recorded (supplement Fig. 2) as a function
of glucose concentration. The enzyme activity increases with
increase in glucose concentration. The apparent enzyme activity,
i.e. the amount of enzyme bound on the surface of the compos−2
ite matrix, has been calculated using the equation aenz
app (U cm ) =
AV/εts, where A is the difference in absorbance before and after
incubation, V is the total volume (3.17 cm3 ), ε is the millimolar extinction coefficient (7.5 for o-dianisidine at 500 nm), t is
the reaction time (min) and s is the surface area (cm2 ) of the
electrode [4]. The apparent enzyme activity was estimated to be
1.75 × 10−2 U cm−2 .
The shelf life study of the prepared GOx/ZnO–KFCN/ITO/glass
bio-electrode has been carried out at regular interval for 10 weeks.
Keeping a fixed substrate concentration, the value of absorbance
obtained at different time intervals is compared with that recorded
in the initial stage (data not shown). The study indicates that the
composite bio-electrode retains more than 80% of activity even
after 10 weeks. The selectivity of the bio-electrode has been studied
by adding 8.33 mM solution of different interferants like cholesterol, urea and ascorbic acid to 8.33 mM glucose solution. The
presence of interferants was found to have a negligible effect on
the performance of the composite bio-electrode towards glucose
(inset of supplement Fig. 2).
4. Conclusions
ZnO–K3 [Fe(CN)6 ] composite thin films are a promising matrix
for biosensing application. The matrix has been successfully
utilized for the immobilization of the model enzyme, glucose
oxidase, and efficiently exploited for glucose biosensing. The
presence of the redox species in the matrix enhances the charge
transfer rate and provides a quassireversible system. The lower
value of Michaelis–Menten constant (1.69 mM) proves that the
composite matrix provides a platform in which the enzyme not
only retains its bioactivity but maintains a confirmation that
enhances the enzymes’ activity. Moreover, the fabricated composite bio-electrode (GOx/ZnO–KFCN/ITO/glass) shows a good
linear response in a mediator-free electrolyte. The shelf life of
the composite bio-electrode is more than 10 weeks. The present
study suggests that ZnO–KFCN composite thin film proves to be
a promising matrix for biosensing applications and may lead to
development of MEMS and ISFET based miniaturized sensors.
Acknowledgements
The financial support of DST, UGC and CSIR is gratefully acknowledged. Authors thank Dr. Vikram Kumar for providing facilities.
SS acknowledges UGC for fellowship and University of Delhi for
teaching assistantship. SKA is thankful to University of Alberta,
Edmonton, Canada for postdoctoral fellowship.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.aca.2009.09.002.
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