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Nanostructured Zinc Oxide Particles in ChemicallyModified Electrodes for Biosensor ApplicationsS. Ashok Kumar a; Shen-Ming Chen aa Department of Chemical Engineering and Biotechnology, National TaipeiUniversity of Technology, Taipei, Taiwan (ROC)

Online Publication Date: 01 January 2008

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

To link to this article: DOI: 10.1080/00032710701792612URL: http://dx.doi.org/10.1080/00032710701792612

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Nanostructured Zinc Oxide Particlesin 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

Analytical Letters, 41: 141–158, 2008

Copyright # Taylor & Francis Group, LLC

ISSN 0003-2719 print/1532-236X online

DOI: 10.1080/00032710701792612

141

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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, andnanocages 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.

S. A. Kumar and S.-M. Chen142

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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).

Nanostructured Zinc Oxide Particles in Chemically Modified Electrodes 143

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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, S4O622;

SO522; S2O8

22; ClO32; BrO3

2; and IO32 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 SO322 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).

S. A. Kumar and S.-M. Chen144

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as supportingmaterials for GlucoseOxidase (GOx) loading. The ZnOnanocomb

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.

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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 ZnONanorod 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 atþ 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.

S. A. Kumar and S.-M. Chen146

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the background current, confirming good catalytic behavior of Gox/ZnOnanorods. 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 ELECTROCHEMISTRYWITH 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

Nanostructured Zinc Oxide Particles in Chemically Modified Electrodes 147

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redox waves is observed at theMP 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

2342.3 mV, respectively (vs. SCE). The formal potential (E00) 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 casewithE00 being –371.1 mV, andDE 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 5.85þ 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

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).

S. A. Kumar and S.-M. Chen148

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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 KappM

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.

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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)

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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.

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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)).

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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

Figure 8. (A) AFMmicrographs of (a) ZnO thin film (1 � 1 mm) and (b) ChOx/ZnOthin film (3�3 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.

Nanostructured Zinc Oxide Particles in Chemically Modified Electrodes 153

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Table 1. Data’s obtained for various analytes (biomoleeules) studied using ZnO nanoparticles coated electrodes

Serial

nos.

Electrode

substrate

Nature of

substrate

Dye, enzyme or

proteins used for

modification

Analyte

studied

pH used

for

analysis

Detection

limit (mM)

Linear range

(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

dinucleotide,

hemoglobin

NADH,

NADþ, H2O2

6.8 NA NA (Lin and Chen 2005,

2006c; Kumar and

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

electrode

Nano-ZnO Microperoxidase H2O2 7.0 0.3 1–700 (Zhu et al. 2007)

7 Gold electrode ZnO Nanorod

array

GOx Glucose 7.4 0.01 mM 0.01–3.45 mM (Wei et al. 2006)

8 Au electrode Sputtered ZnO

nanoporous

Cholesterol

oxidase

Cholesterol 7–7.5 20 0.65–10.34 mM (Singh et al. 2007)

9 Au electrode ZnO:Co

nanoclusters

GOx Glucose 7.4 20 0–4 mM (Zhao et al. 2007)

10 Au electrode ZnO nanocomb GOx Glucose 7.4 20 0.02–4.5 mM (Wang et al. 2006)

Abbreviations: NA-Not available.

S.A.KumarandS.-M

.Chen

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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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