Nano Zinc Oxide Applications in Biosensors - A Review

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Nanostructured Zinc Oxide Particles in Chemically
Modified Electrodes for Biosensor Applications
S. Ashok Kumar a; Shen-Ming Chen a
a
Department of Chemical Engineering and Biotechnology, National Taipei
University of Technology, Taipei, Taiwan (ROC)
Online Publication Date: 01 January 2008

To cite this Article: Kumar, S. Ashok and Chen, Shen-Ming (2008) 'Nanostructured
Zinc Oxide Particles in Chemically Modified Electrodes for Biosensor Applications',
Analytical Letters, 41:2, 141 — 158
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DOI: 10.1080/00032710701792612
Nanostructured Zinc Oxide Particles

in Chemically Modified Electrodes
for Biosensor Applications
S. Ashok Kumar and Shen-Ming Chen
Department of Chemical Engineering and Biotechnology, National

Taipei University of Technology, Taipei, Taiwan (ROC)
Abstract: Zinc oxide (ZnO) has received considerable attention because of its unique
optical, semiconducting, piezoelectric, and magnetic properties. ZnO nanostructures
exhibit interesting properties including high catalytic efficiency and strong adsorption
ability. Recently, the interest has been focused toward the application of ZnO in biosen-
sing because of its high isoelectric point (9.5), biocompatibility, and fast electron
transfer kinetics. Such features advocate the use of this exciting material as a
biomimic membrane to immobilize and modify biomolecules. This review highlights
the potential use of ZnO in modified electrodes and biosensing.
Keywords: Zinc oxide particles, biosensors, proteins, direct electrochemistry,

modified electrodes, electrocatalysis
INTRODUCTION
ZnO is an important member of the II –VI group of semiconductors, a wide

direct band gap semiconductor with a band gap of 3.37 eV. With a binding
energy of excitons in ZnO as high as 60 meV (Chen et al. 2001), it is an ideal
candidate for fabricating ultraviolet light-emitting diodes and lasers. ZnO has
Received 16 October 2007; accepted 17 October 2007

This project work supported by the National Science Council and the Ministry of
Education of the Taiwan (ROC).
Address correspondence to Shen-Ming Chen, Department of Chemical Engineering
and Biotechnology, National Taipei University of Technology, No. 1, Section 3,
Chung-Hsiao East Road, Taipei 106, Taiwan (ROC). E-mail: smchen78@ms15.
hinet.net
141
142 S. A. Kumar and S.-M. Chen
received attention in optics, optoelectronics, sensors, and actuators due to its

semiconducting, piezoelectric, and pyroelectric properties (Sun and Kwok
1999; Norton et al. 2004). ZnO is also a biocompatible material with a
high Isoelectric Point (IEP) of 9.5 which makes it suitable for absorption
of proteins with low IEPs where the protein immobilization is primarily
driven by electrostatic interaction (Zhang et al. 2004; Liu et al. 2005).
ZnO nanostructures possess several unique advantages such as high-
specific surface area, nontoxicity, chemical stability, electrochemical
activity, and high electron communication features. Such properties
indicate the potential applications of ZnO as one of the promising
materials for biosensor applications (Wang et al. 2006). ZnO films,
composed of nanosized metal oxide particles, have been investigated inten-
sively for dye-sensitized photoelectrochemical applications (Izaki and Omi
1996; Yoshida and Minoura 2000; Karuppuchamy et al. 2001; Yoshida
et al. 2004), optoelectronic devices (Schlettwein et al. 2000; Keis et al.
2002; Petrella et al. 2004), and inorganic acceptor dye-sensitized solar
cells (Rensmo et al. 1997; Van de Walle 2000; Bahadur and Srivastava
2003).
ZnO almost exhibits n-type conductivity with the electrons in its valence
band as charge carriers (Schmidt-Mende and MacManus-Driscoll 2007). The
structure of ZnO, for example, can be described as a number of alternating
planes composed of tetrahedrally coordinated O22 and Zn2þ ions, stacked
alternately along the c-axis. In ZnO, zinc is acting as a deep acceptor and
oxygen is acting as a deep donor. Tsukazaki et al. (2005) have developed a
technique for fabricating p-type ZnO films reliably and reproducibly using
the temperature modulation technique for doping very high crystallinity,
intrinsic ZnO films using N as a p-type dopant, a promising material in
light-emitting diode technology.
Using a solid-vapor phase thermal sublimation technique, nanocombs,
nanorings, nanohelixes/nanosprings, nanobows, nanobelts, nanowires, and
nanocages of ZnO have been synthesized under specific growth conditions.
These unique nanostructures unambiguously demonstrate that ZnO is
probably the richest family of nanostructures among all materials, both in
structures and properties. The nanostructures could have novel applications
in optoelectronics, sensors, transducers, and biomedical science because of
their biosafety (Vayssieres 2003). Nanohelices/nanosprings, seamless
nanorings, aligned nanopropellers, patterned growth of aligned nanowires,
mesoporous single-crystal nanowires, ultranarrow ZnO nanobelts, and poly-
hedral cages like structures of ZnO are characterized and reviewed by
Wang (2004).
Recently, biosafe-ZnO materials have emerged into sensor technology so
it is necessary to review the recent publications in this field. This review
focuses on the recent publications based on ZnO film modified electrodes
and their interaction/integration with biomolecules for chemical and sensor
applications.
Nanostructured Zinc Oxide Particles in Chemically Modified Electrodes 143
INTERACTION OF ENZYME AND PROTEIN WITH ZnO FILM
The flavins, an important class of biochemical compounds, are found in both

plants and animals. The flavoprotein coenzyme plays an important biological
role in many oxidoreductases and reversible redox conversion in biochemical
reactions. It consists of a nucleotide adenine, a sugar ribose, and two
phosphate groups. Due to their importance, most of the flavins have been
the subject of intensive studies. In particular, isoalloxazine, riboflavin,
flavin mononucleotide, and Flavin Adenine Dinucleotide (FAD) have been
thoroughly investigated using various types of electrodes because of their
strong electrochemical signal response over a wide pH range (Underwood
and Burnett 1973; Kamal et al. 1991; Zhang et al. 1995; Wang et al. 1997;
Kubota et al. 1998; Chen and Liu 2006a, 2006b; Lin and Chen 2006a; Chen
and Song 2007; Kumar and Chen 2007a).
Codeposition of FAD/ZnO films were obtained on Glassy Carbon (GC)
and indium tin oxide coated glass electrodes by mixing 10 mM FAD, 0.1 M
zinc nitrate, and 0.1 M NaNO3 solutions (pH 6.5). By this method, FAD/
ZnO hybrid film modified electrodes can be prepared and their electrochemi-
cal properties are reported (Lin and Chen 2005; Kumar and Chen 2007b).
Surface characterizations of the FAD/ZnO modified electrode can be
performed by Scanning Electron Microscopy (SEM) as shown in Fig. 1.
The electrode is covered by a strong yellow colored film and the native
Figure 1. SEM image of FAD/ZnO hybrid film. Reused with permission from S. A.
Kumar et al., Journal of Solid State Electrochemistry; 11, 993– 1006 (2007).
flavin enzyme behavior can be characterized by cyclic voltammetry. The

FAD/ZnO modified GC electrode exhibits excellent electrocatalytic activities
toward the oxidized form of nicotinamide adenine dinucleotide, reduced form
of nicotinamide adenine dinucleotide, nitrite, hydrogen peroxide, S4O22 6 ;
2 2 2
SO22 22
5 ; S2O8 ; ClO3 ; BrO3 ; and IO3 ions (Lin and Chen 2005; Kumar and
Chen 2007b). The FAD/ZnO modified GC electrode also displays an
excellent stable electrochemical signal over a wide pH range (Lin and Chen
2005; Kumar and Chen 2007b). The zero-point charge (zpc) for ZnO is 9.0.
Based on the pHzpc value, the ZnO surface is positively charged at pH
below 9. At low pH values, electrostatic interactions between the positive
ZnO surface and the phosphate group of FAD in aqueous solutions may
lead to strong adsorption of the FAD on the ZnO surface (Fig. 2).
Adsorption of Heme Protein on FAD/ZnO Films
In the presence of hemoglobin (Hb) in phosphate buffer solution, the FAD/

ZnO film modified electrode shows high-cathodic catalytic current for
Oxygen Reduction Reaction (ORR). By using spectroelectrochemistry, the
Hb adsorption on the FAD/ZnO film can be confirmed. The Hb/FAD/
ZnO film electrode is used for electroanalysis of hydrogen peroxide, trichlor-
oacetic acid, and SO22 3 whereas the electrocatalytic oxygen reduction
reaction is conducted using the Hb/FAD/ZnO modified electrode (Lin and
Chen 2006c).
ZnO NANOCOMB BIOSENSOR FOR GLUCOSE DETECTION
Single crystal ZnO nanocombs can be synthesized in bulk quantity by vapor

phase transport. A glucose biosensor is constructed using these nanocombs
Figure 2. Interactions between FAD with ZnO film. Reused with persmission from
K. C. Lin et al. Journal of Electroanalytical Chemistry, 577, 213– 222 (2007).
as supporting materials for Glucose Oxidase (GOx) loading. The ZnO nanocomb
glucose biosensor shows high sensitivity (15.33 mA/cm2 mM) for glucose
detection and high affinity of GOx to glucose (the apparent Michaelis-Menten
constant KMapp ¼ 2.19 mM). With a detection limit of 0.02 mM, such features
imply that zinc oxide nanostructures have potential applications toward the
development of high-performance biosensors (Wang et al. 2006).
The X-ray diffraction pattern of the as-prepared product by vapor phase
transport is shown in Fig. 3(a), where all the diffraction peaks can be
indexed to the hexagonal wurtzite phase of ZnO with a lattice constant of
a ¼ 0.325 nm and c ¼ 0.520 nm, matching well the standard XRD data file
(JCPDS 79-2205). No other crystalline forms, such as Zn or other Zn
compounds, can be detected. Figures 3(b – d) shows typical SEM images of
the ZnO product in low, medium, and high magnifications, respectively.
The product consists of numerous comblike nanostructures with single mor-
phology (nanocomb) and uniform size in the area examined. The stems of
nanocombs are ribbons with a thickness of 50 nm, the length of the main
stems reaches several tens of micrometers, and the width of the stems
decreases gradually along the growth direction to form sharp tips. The
branching nanorods grow on one side of the nanoribbon with a diameter of
200 nm. The distance between two adjacent nanorods is 500 nm. Wang
Figure 3. (a) XRD pattern of ZnO nanocombs and SEM images of ZnO nanocombs
with (b) Low, (c) Medium, and (d) High magnifications, respectively. Reused wih
permission from J. X. Wang, Applied Physics Letters, 77, 233106 (2006). Copyright
2006, American Institute of Physics.
et al. (2006) reported that the ZnO nanocombs with ribbonlike stems possess a
high-specific surface area and good biocompatible properties, providing a
favorable microenvironment for high GOx loading. At a certain pH range,
ZnO nanocombs are positively charged and display electrostatic interaction
with negatively-charged GOx.
Enzymatic Glucose Biosensor Based on ZnO Nanorod Array Grown

by Hydrothermal Decomposition
Wei et al. (2006) reported a glucose biosensor based on GOx immobilized on a

ZnO nanorod array grown by hydrothermal decomposition. At pH 7.4,
negatively-charged GOx was immobilized on positively charged ZnO
nanorods through the electrostatic interaction. At þ0.8 V versus Ag/AgCl,
the ZnO nanorod based biosensor exhibits high and reproducible sensitivity
of 23.1 mA cm22 mM21 with a response time of , 5 sec. The biosensor
shows a linear range from 0.01 to 3.45 mM and a detection limit of
0.01 mM. The apparent Michaelis-Menten constant of 2.9 mM illustrates
high affinity between glucose and Gox immobilized on ZnO nanorods.
Figure 4(a) displays the amperometric response of the glucose biosensor
for successive addition of glucose with 0.01, 0.03, 0.05, 0.1, 0.5, and 1 mM/
step in 0.01 M PBS, pH 7.4 at þ 0.8 V (vs. Ag/AgCl) under stirring. The
biosensor exhibits a rapid and sensitive response to glucose. As shown in
Fig. 4, the addition of 0.01 mM glucose provokes a visible increase above
Figure 4. Amperometric responses of GOx/ZnO nanorods/Au electrodes with

successive addition of glucose to the 0.01 M pH 7.4 PBS buffer under stirring.
The applied potential was þ0.8 V (vs. Ag/AgCl reference). The inset shows the
enlarged response for low concentration (0.01, 0.03, and 0.05 mM) of glucose
additions. Reused with permission from A. Wei, Applied Physics Letters, 79,
123902 (2006). Copyright 2006, American Institute of Physics.
the background current, confirming good catalytic behavior of Gox/ZnO

nanorods. The sensor achieves 95% of steady-state current in less than 5
sec, indicating a fast electron exchange between Gox and ZnO nanorods
(Wei et al. 2006).
TAILORING ZINC OXIDE NANOWIRES FOR HIGH-

PERFORMANCE AMPEROMETRIC GLUCOSE SENSOR
ZnO nanowires can be tailored physically and chemically to immobilize GOx

for the construction of a glucose sensor with high performance. High-specific
surface area and isoelectric point of ZnO facilitate enzyme immobilization
with high loading and the mediating effect of the redox reaction of ZnO
nanowires (Zang et al. 2007). Electrochemical methods and Field Emission
Scanning Electron Microscopy (FESEM) can be used to systematically
study the sensor structure and electrode catalytic reactions. Through
enzyme kinetics, they demonstrated that the apparent Michaelis constants
could be tuned for making nanostructured ZnO on biosensors with different
specifications. Linear Response Sensitivity (LRS) is correlated with the
enzyme loadings, ranging from 0.8 to 11.3 mA cm22 mM21. Interestingly,
the KM value significantly decreases over a wide range with increased GOx
loading. As the enzyme loading increases, a bigger fraction of the GOx is
located near to the surface of the electrode. The consequent increase in
enzyme-substrate binding effects a decrease in KM (McMohan et al. 2005,
2006). In clinical diagnosis, some enzyme sensors need to offer high sensi-
tivity in a relatively narrow range, whereas other applications require a
broad linear response range. As KM and LRS can be correlated to the
enzyme loadings over a wide range, the ZnO nanowire glucose sensor could
be easily tailored for different specifications. The GOx/ZnO sensor exhibits
high current responses at glucose levels below 1 mM. The ZnO nanowire
based glucose sensor demonstrated superior performance, which could be
used in clinical diagnosis. The sensor retains 95% of its original current
response after being kept at 48C for 1 month, indicating very satisfactory
stability (Zang et al. 2007).
DIRECT ELECTROCHEMISTRY WITH THE HELP OF NANO-

ZnO
Direct electron transfer reactions of microperoxidase (MP) can be achieved

with the help of semiconductive ZnO nanoparticles on a pyrolytic graphite
electrode. Notice that MP is not ready to take redox reactions at any
electrode surface. With ZnO NPs, the protein can exhibit direct fast electron
transfer reactivity. As is shown in Fig. 5 (the solid curve), a pair of
well-defined, quasi-reversible redox peaks are obtained using a MP/ZnO NP
modified electrode for 0.1 M PBS, pH 7. In contrast, only a small pair of
Figure 5. Cyclic voltammograms for 0.1 M PBS with pH 7.0 obtained at the PG elec-
trode modified with ZnO NPs alone (The dashed curve), MP alone (The dotted curve),
MP/ZnO NPs (The solid curve), or MP/Agarose (The short dashed Curve) with VMP/
VZnO or VMP/Vagarose proportion of 1/1. Scan rate: 200 mV sec21. Reused with
permission from X. Zhu, Biosensors and Bioelectronics, 22, 1600– 1064 (2007).
redox waves is observed at the MP alone modified electrode. There is no peak on

the voltammogram in this potential range with the ZnO NPs modified electrode
without the enzyme. Therefore, these two peaks arise from the redox reactions of
MP immobilized with ZnO NPs at the electrode surface. The response time is
1.5 sec with the anodic and cathodic peaks potentials located at 2265.9 and
0
2342.3 mV, respectively (vs. SCE). The formal potential (E0 ) is calculated to
be 2304.1 mV with a peak separation (DE) of 76.4 mV, indicating a fast
quasi-reversible one-electron heterogeneous electron transfer process. The
authors have also conducted a comparative study with agarose because this
material has been known to facilitate proteins in exhibiting electron transfer reac-
tivity. As shown in Fig. 5, the peaks are more negative and the peak separation is
larger in this case with E00 being –371.1 mV, and DE being 150 mV. Thus, nano-
ZnO can greatly promote electron transfer reactivity of MP. More interestingly,
ZnO NPs cannot only make MP exhibit electrochemical reactivity and catalytic
activity, but also enhance the catalytic ability of the enzyme. After 4 h UV
irradiation, the linear range for hydrogen peroxide detection has been enlarged
to be 1 1027 to 8 1024 M (Y ¼ 5.85 þ 0.041X, R ¼ 0.99). The detection
limit is lowered to 3 1028 M with improved sensitivity of 0.041 mA M21.
RSD is calculated to be between 0.3 and 3.1%. Such improvement can be
attributed to the photovoltaic effect from ZnO (Zhu et al. 2007). Therefore,
the photovoltaic effect might be a promising way to promote the development
of photo-controlled biotechnology. Zhang et al. (2005) observed a direct electro-

chemical response of hemoglobin and cytochrome c in the nano-ZnO film.
IMMOBILIZATION OF URICASE ON ZnO NANORODS FOR A

REAGENTLESS URIC ACID BIOSENSOR
A reagentless Uric Acid (UA) biosensor based on uricase immobilized on ZnO

nanorods was developed. Direct electrochemistry and thermal stability of
immobilized uricase were also studied. The ZnO nanorod derived electrode
retains the enzyme bioactivity and enhances the electron transfer between
the enzyme and the electrode. This sensor shows a high thermal stability up
to 858C and an electrocatalytic activity to the oxidation of uric acid without
the presence of an electron mediator. The electrocatalytic response shows a
linear dependence on the uric acid concentration ranging from 5.0 1026
to 1.0 1023 M with a detection limit of 2 mM at 3s. The apparent Kapp M
value for the uric acid sensor is 0.238 mM (Zhang et al. 2004). The positively
charged ZnO nanorod matrix not only provides a friendly microenvironment
for the negatively-charged uricase to retain its activity but also effectively
promotes the direct electron transfer between the uricase and the electrode.
The experiments indicated that both conductive and biomimetic properties
of ZnO nanorods played important roles in the electrochemical behavior of
the adsorbed enzyme (Rodriguez et al. 2000; Tian et al. 2002). The three-
dimensional assembly with ZnO nanorods achieves the direct electron
transfer of uricase and shows excellent thermal stability and anti-interference
behavior. Observed attractive features might be attributed to the unique con-
ducting ZnO nanorod matrix, which provides a favorable microenvironment
for high enzyme loading. Such successful procedures in enzyme immobiliz-
ation may lead to a novel method for biosensor construction.
LOW TEMPERATURE SELECTIVE NO2 SENSORS BY

NANOSTRUCTURED FIBERS OF ZnO
There is interest in the gas sensing property of Zn nanopowders, synthesized

by an aerosol route with two different morphologies: fiber-mats and cauli-
flower structure. Both the sensors show great sensitivity for sub-ppm con-
centrations of NO2 at low temperature. The fiber powders relative
response toward 0.4 ppm of NO2 is 50, while a relative response of the cau-
liflower structure is 8 at 1008C. Both sensors are also selective, since no
resistance variation can be observed for CO and ethanol. However, resist-
ance variations are noted with changes in relative humidity. Sensor
behavior was studied at different temperatures, from 20– 1508C. The
mechanism of NO2 interaction with the zinc oxide surface should follow
the conventional metal oxide semiconductor sensor theory (Baratto et al.
2004). The adsorption of NO2 on the zinc oxide surface creates acceptor
surface states, thus increasing the band bending at the grain surface and
result in an increase of the resistance.
DYE/ZnO FILM MODIFIED ELECTRODES FOR DETECTION

OF REDUCED FORM OF NICOTINAMIDE ADENINE
DINUCLEOTIDE
Meldloa’s dye/ZnO hybrid films have been electrochemically deposited onto

glassy carbon, gold, and Indium Tin Oxide coated glass (ITO) electrodes at
room temperature (25 + 28C) from a solution containing 0.1 M Zn(NO3)2,
0.1 M KNO3 and 1 1024 M Meldloa’s dye (MB). The surface morphology
and deposition kinetics of MB/ZnO hybrid films were studied by SEM,
Atomic Force Microscopy (AFM), and Electrochemical Quartz Crystal Micro-
balance (EQCM) techniques, respectively. SEM and AFM images of MB/
ZnO hybrid films reveal that the surfaces are well-crystallized, porous, and
micro structured (Fig. 6). MB is immobilized and strongly fixed in a transpar-
ent inorganic matrix. The MB/ZnO hybrid film modified GC electrode (MB/
Figure 6. (a)AFM image of (A) ZnO film and (B) MB/ZnO hybrid film, (b) SEM
image of (A) ZnO film and (B) MB/ZnO hybrid film. Reused with permission from
S. A. Kumar, Analytica Chemica Acta, 592, 36 – 44 (2007)
ZnO/GC) shows one reversible redox couple centered at a formal potential of

20.12 V at pH 6.9. The surface coverage (G) of the MB immobilized on ZnO/
GC was about 9.86 10212 mol cm22 with an electron transfer rate constant
(ks) of 38.9 sec21. The MB/ZnO/GC electrode acts as a sensor and displays
an excellent specific electrocatalytic response to the oxidation of reduced form
of Nicotinamide Adenine Dinucleotide (NADH). The linear response ranging
from 50 to 300 mM NADH at pH 6.9 is observed with a detection limit of
10 mM (S/N ¼ 3). The electrode is stable for 1 month during repeated
analysis without any noticeable decrease in the response signal (Kumar and
Chen 2007c).
Thin Toluidine Blue (TBO) and ZnO Hybrid Films Modified

Electrode for Amperometric Determination of NADH
Thin Toluidine Blue (TBO) and ZnO hybrid films have been grown on GC
electrodes and indium tin oxide coated (SnO2) glass electrodes, respectively,
by using Cyclic Voltammetry (CV). SEM images reveal a spherical and
bead-like shape of highly oriented TBO/ZnO hybrid films. Energy Disper-
sive Spectrometry (EDS) results confirm that the films consist mainly of
Zn and O. The TBO/ZnO hybrid film modified electrode is electrochemi-
cally active and dye molecules do not leak out easily from the ZnO
matrix. Hence, the hybrid films might have potential applications as sensor
for determination of NADH at 0.0 V. There is a linear correlation between
the electrocatalytic current and NADH concentration, ranging from 25 to
100 mM in phosphate buffer solution (pH 6.6). Dopamine, ascorbic acid,
Figure 7. Cyclic voltammograms of TBO/ZnO/GCE modified electrodes in pH 6.6

PBS: NADH ¼ (a) 0.0, (b) 5 mM and (a0 ) ZnO/GC electrode with 5 mM NADH. Scan
rate 10 mV sec21.
and uric acid do not interfere with the amperometric detection of NADH at
0.0 V. The TBO/ZnO hybrid film modified electrode is highly stable and its
response to NADH remains persistent.
Figure 7 shows the cyclic voltammograms of a TBO/ZnO/GCE in
phosphate buffer in the absence (curve a) and in the presence (curve b) of
5 mM NADH. The oxidation of NADH is accomplished by a noticeable
increase in the anodic current at 0.0 V, corresponding to the catalytic
oxidation of NADH. Simultaneously, a decrease in the cathodic peak
current in the presence of NADH, compared with that in the absence of
NADH can be observed. No electrochemical oxidation current was obtained
at ZnO modified (curve a0 ) or an unmodified GCE in 0.1 M phosphate
buffer solution in the presence of NADH. These results indicate that NADH
could be electrocatalytically oxidized at the TBO/ZnO hybrid film modified
GC electrode (Kumar and Chen 2007d).
CHOLESTEROL BIOSENSOR BASED ON SPUTTERED ZINC

OXIDE NANOPOROUS THIN FILM
Cholesterol Oxidase (ChOx) has been immobilized onto ZnO nanoporous thin
films grown on a gold surface. A preferred c-axis oriented ZnO thin film with
porous surface morphology has been fabricated by rf sputtering under high
pressure. Optical studies and cyclic voltammetric measurements show that
the ChOx/ZnO/Au bioelectrode is sensitive to the detection of cholesterol
in 25 –400 mg/dl range. The relatively low value of the Michaelis-Menten
constant of 2.1 mM indicates enhanced enzyme affinity of ChOx to choles-
terol. The observed results show the promising application of the nanoporous
ZnO thin film for biosensing application without any functionalization (Singh
et al. 2007).
The AFM image of as-grown ZnO thin film (Fig. 8A(a)) reveals the
formation of rough microstructures having uniformly distributed nanopores.
The formation of rough and porous microstructure is attributed to the proces-
sing of ZnO thin films under high sputtering pressure. The intensive in situ
bombardment of energetic species under high pressure is likely to embed a
large oxygen concentration at the grain boundaries or interstitial sites,
thereby changing the microstructure of the deposited film. The average
roughness of the film surface is 4 nm. The presence of uniformly distributed
globular structures over the ZnO porous surface can be clearly seen
(Fig. 8A(b)) in the AFM image and is attributed to the functionalization of
the oxide layer by cholesterol oxidase. Figures 8B(a) and 8B(b) show the
FTIR spectra of ZnO/Au and ChOx/ZnO/Au layered structures. The
presence of a sharp and intense band at 578 cm21 confirms the formation of
ZnO thin film on gold surface (Fig. 8B(a)). The appearance of additional
absorption bands at 1561, 1637, and 3330 cm21 can be attributed to the
amide bond of ChOx (Fig. 8B(b)).
Figure 8. (A) AFM micrographs of (a) ZnO thin film (1 1 mm) and (b) ChOx/ZnO
thin film (33 mm). (B) FTIR spectra of (a) ZnO/Au and (b) ChOx/ZnO/Au films.
Reused with permission from S. P. Singh, Applied Physics Letters, 91, 063901
(2007). Copyright 2007, Ameican Institute of Physics.
AMPEROMETRIC BIOSENSOR BASED ON ZnO:Co

NANOCLUSTERS FOR GLUCOSE
ZnO:Co nanoclusters have been synthesized by nanocluster-beam deposition

with a averaged particle size of 5 nm and porous structure, which were for the
first time adopted to construct a novel amperometric glucose biosensor.
Glucose oxidase was immobilized into the ZnO:Co nanocluster assembled
thin film through the Nafion-assisted cross-linking technique. Due to the
high-specific active sites and high electrocatalytic activity of the ZnO:Co
nanoclusters, the constructed glucose biosensor shows a high sensitivity of
13.3 mA/mA cm2. The low detection limit of 20 mM (S/N ¼ 3) and the
apparent Michaelis –Menten constant of 21 mM, indicate the high activity
of the enzyme on ZnO:Co nanoclusters to glucose. Consequently, the
ZnO:Co nanocluster-assembled thin films with nanoporous structures and
nanocrystallites have potential applications as a platform for enzyme immo-
bilization (Zhao et al. 2007). Several biomolecules have been studied with
154
Table 1. Data’s obtained for various analytes (biomoleeules) studied using ZnO nanoparticles coated electrodes
Dye, enzyme or pH used

Serial Electrode Nature of proteins used for Analyte for Detection Linear range
nos. substrate substrate modification studied analysis limit (mM) (mM) Reference
1 GC electrode ZnO film Meldola’ dye NADH 6.9 10 50 – 300 (Kumar and Chen 2007c)
2 GC electrode ZnO film Toluidine blue NADH 6.6 2.4 25 – 400 (Kumar and Chen 2007d)
3 GC electrode ZnO film Favin adenine NADH, 6.8 NA NA (Lin and Chen 2005,
dinucleotide, NADþ, H2O2 2006c; Kumar and
hemoglobin Chen 2007b)
4 GC electrode ZnO nanorods Uricase Uric acid 6.9 2.0 5 – 1000 (Zhang et al. 2004)
5 Gold electrode ZnO nanowires GOx Glucose 7.4 0.2 1 – 0.76 mM (Zang et al. 2007)
6 Pyrolytic graphite Nano-ZnO Microperoxidase H2O2 7.0 0.3 1 – 700 (Zhu et al. 2007)
electrode
7 Gold electrode ZnO Nanorod GOx Glucose 7.4 0.01 mM 0.01 – 3.45 mM (Wei et al. 2006)
S. A. Kumar and S.-M. Chen

array
8 Au electrode Sputtered ZnO Cholesterol Cholesterol 7 – 7.5 20 0.65 – 10.34 mM (Singh et al. 2007)
nanoporous oxidase
9 Au electrode ZnO:Co GOx Glucose 7.4 20 0 – 4 mM (Zhao et al. 2007)
nanoclusters
10 Au electrode ZnO nanocomb GOx Glucose 7.4 20 0.02 – 4.5 mM (Wang et al. 2006)
Abbreviations: NA-Not available.

ZnO nanoparticles modified electrodes (Table 1), implying the potential appli-
cation of ZnO nanoparticles in biosensing.
CONCLUSIONS
The unique properties of ZnO and the ease of ZnO nanostructure fabrication
make this material extremely interesting for biosensor applications. In recent
years, the number of applications of ZnO nanostructures has risen dramati-
cally and this trend will continue. The examples shown in this review give
only a brief indication of the possible applications of biosensors. The next
challenge of modified electrodes is their applicability to process “real-
world” sample analysis. Mainly researchers concentrate to prepare ease,
user-friendly, less interference, long shelf-life, cheaper, and commercially
available electrodes. In this aspect, ZnO nanostructures have a primary role
to play in near future. The versatility of properties and formation of nanostruc-
tures makes it likely that new ideas will also come up in the future, leading to
new chemical and biochemical device applications.
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Nano Zinc Oxide Applications in Biosensors - A Review

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Nanostructured Zinc Oxide Particles

S. Ashok Kumar and Shen-Ming Chen

Department of Chemical Engineering and Biotechnology, National

Keywords: Zinc oxide particles, biosensors, proteins, direct electrochemistry,

ZnO is an important member of the II –VI group of semiconductors, a wide

Received 16 October 2007; accepted 17 October 2007

received attention in optics, optoelectronics, sensors, and actuators due to its

INTERACTION OF ENZYME AND PROTEIN WITH ZnO FILM

The flavins, an important class of biochemical compounds, are found in both

flavin enzyme behavior can be characterized by cyclic voltammetry. The

Adsorption of Heme Protein on FAD/ZnO Films

In the presence of hemoglobin (Hb) in phosphate buffer solution, the FAD/

ZnO NANOCOMB BIOSENSOR FOR GLUCOSE DETECTION

Single crystal ZnO nanocombs can be synthesized in bulk quantity by vapor

Enzymatic Glucose Biosensor Based on ZnO Nanorod Array Grown

Wei et al. (2006) reported a glucose biosensor based on GOx immobilized on a

Figure 4. Amperometric responses of GOx/ZnO nanorods/Au electrodes with

the background current, confirming good catalytic behavior of Gox/ZnO

TAILORING ZINC OXIDE NANOWIRES FOR HIGH-

ZnO nanowires can be tailored physically and chemically to immobilize GOx

DIRECT ELECTROCHEMISTRY WITH THE HELP OF NANO-

Direct electron transfer reactions of microperoxidase (MP) can be achieved

redox waves is observed at the MP alone modified electrode. There is no peak on

of photo-controlled biotechnology. Zhang et al. (2005) observed a direct electro-

IMMOBILIZATION OF URICASE ON ZnO NANORODS FOR A

A reagentless Uric Acid (UA) biosensor based on uricase immobilized on ZnO

LOW TEMPERATURE SELECTIVE NO2 SENSORS BY

There is interest in the gas sensing property of Zn nanopowders, synthesized

DYE/ZnO FILM MODIFIED ELECTRODES FOR DETECTION

Meldloa’s dye/ZnO hybrid films have been electrochemically deposited onto

ZnO/GC) shows one reversible redox couple centered at a formal potential of

Thin Toluidine Blue (TBO) and ZnO Hybrid Films Modified

Figure 7. Cyclic voltammograms of TBO/ZnO/GCE modified electrodes in pH 6.6

CHOLESTEROL BIOSENSOR BASED ON SPUTTERED ZINC

AMPEROMETRIC BIOSENSOR BASED ON ZnO:Co

ZnO:Co nanoclusters have been synthesized by nanocluster-beam deposition

Dye, enzyme or pH used

S. A. Kumar and S.-M. Chen

Abbreviations: NA-Not available.

Bahadur, L. and Srivastava, P. 2003. Efficient photon-to-electron conversion with

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