Thin Solid Films 519 (2010) 1141–1144

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ZnO based third generation biosensor

Vinay GuptaDepartment of Physics & Astrophysics, University of Delhi, Delhi 110007, India

E-mail address: vgupta@physics.du.ac.in.

0040-6090/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.tsf.2010.08.058

a b s t r a c t

a r t i c l e i n f o

Available online 20 August 2010

Keywords:Composite matrixZnOThird generation biosensor

Zinc oxide (ZnO) has emerged as a leading material in the field of biosensors. However, the absence of redoxcouple is proving to be the major hindrance towards the development of a ZnO based third generationbiosensor. Development of a composite matrix can be a possible way towards the realization of ZnO basedmediator-less biosensor. In the present work pulsed laser deposition, chemical method and ion implantationhas been explored for realization of ZnO based matrix which can be exploited for biosensor applications inmediator-free environment.

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1. Introduction

Biosensor has become a well-investigated field due to itsextensive application in the field of health-care, biological analysis,environment-monitoring and food industries [1–10]. The primarystage of bio-sensing is the recognition of an analyte by the receptor.The sensing receptor has to be integrated with a suitable biosensormaterial in a bio-compatible environment so that it does not lose itsbio-activity. The solid support that traps the sensing molecule(receptor) is known as matrix. In spite of being studied for quite along time there is no clear consensus on the choice of material forthe matrix for biosensor. A wide range of materials, ranging fromconducting polymers to carbon nanotubes (CNTs), nanoparticles toself-assembled monolayer (SAM), modified metals to hybrid nano-composite surfaces, has been reported as a matrix for bio-sensingapplications [1,2]. In the recent past interest has been generated toutilize the metal-oxide matrices for bio-sensing application and anumber of matrices based on TiO2, ZnO, SnO2, ZrO2 and CeO2 havebeen exploited [3–7]. Among these the study on ZnO, a wide bandgap semiconductor, gained momentum for biosensor applicationsbecause of its high isoelectric point (IEP ~9.5), biocompatibility andabundance in nature [4,8]. ZnO matrix, which is positively chargedat pH 7.0, provides a suitable environment for immobilization of thenegatively charged enzyme like glucose oxidase (GOx) havingrelatively low isoelectric point (4.2) [8]. Apart from a suitableplatform for enzyme immobilization, electron transfer betweenenzymes and electrodes via redox path is a very crucial factor in thedevelopment of an electrochemical biosensor [1,10].

However, due to absence of redox couple in ZnO, a mediator isessentially required in the electrolyte to act as a redox linker betweenthe enzyme and the ZnO matrix in amperometric sensing [4,11,12].This has emerged as a major hindrance towards the development of a

third generation biosensor based on ZnO matrix. Moreover, therealization of an integrated and miniaturized ZnO based electrochem-ical biosensor is difficult due to the presence of mediator in theelectrolyte solution. Few biosensors were developed where bio-molecules were immobilized along with the mediators, on the surfaceof the matrix. Li et.al. reported a cholesterol biosensor based on co-immobilization of enzymes with potassium ferricyanide [13]. Meth-ylene blue has been co-immobilized with enzyme on glassy carbonelectrode for the detection of mercury compounds [14]. A hydrogenperoxidase biosensor, based on co-immobilization of catalase andmethylene blue on an Al2O3 sol-gel fabricated glassy carbon electrode,has also been reported [15]. However, due to the low molecularweight and good solubility in water the mediators have a tendency toleach out from the matrix surface and thereby destabilizing the bio-electrode [16]. Therefore, it has become necessary to tailor the ZnOmatrix so that it can be exploited for realization of a third generationbiosensor.

2. ZnO-mediator composite matrix

It is expected that the redox property in ZnO electrode would beintroduced by incorporation of the mediators or suitable dopants intothe matrix itself. Various techniques including ablation of a compositetarget by pulsed laser deposition (PLD), spin coating of a compositesuspension and ion implantation have been used successfully for thefabrication of ZnO based matrix having redox couple, and arediscussed separately in the following sections.

3. PLD grown ZnO based matrix

Potassium ferricyanide (KF) embedded ZnO matrix was grown byPLD using a composite ceramic target (1 in diameter) of ZnO having3% of potassium ferricyanide by molar weight. The pure ZnO film andcomposite films of ZnO–KF were prepared on ITO coated glasssubstrate by ablating the respective target with fourth harmonic of

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Fig. 2. Variation of anodic peak current with square root of potential scan rate (Insetshows the CV spectra of ZnO–KF/ITO/glass electrode in PBS buffer at 50 mV/s scan rate).

1142 V. Gupta / Thin Solid Films 519 (2010) 1141–1144

Nd-YAG laser (λ=266 nm). The details of the processing conditionsare reported elsewhere [17]. GOx, as a test bio-enzyme wasimmobilized onto the surface of ZnO based matrix by physicaladsorption technique. The prepared bio-electrodes (GOx/ZnO–KF/ITO/glass, GOx/ZnO/ITO/glass) after washing was dried under nitro-gen flow and stored at 4 °C.

X-ray diffraction (XRD) study of the composite matrix (Fig. 1)shows growth of c-axis oriented ZnO thin film. No other phases wereseen in the XRD.

The charge transfer resistance (RCT)measured from the impedancespectra for the ZnO–KF/ITO/glass and ZnO/ITO/glass electrodes,prepared by PLD technique, were about 66 kΩ and 895 kΩ respec-tively. The decrease of charge transfer resistance by an order for ZnO–KF electrode in comparison to pure ZnO matrix is due to the presenceof redox ions into the matrix.

The electrochemical response of the ZnO–KF/ITO/glass electrodewas investigated by cyclic voltammetry (CV). A well-defined redoxpeak was seen at−0.2 V in PBS buffer (inset of Fig. 2). The redox peakcurrents are proportional to the square root of the scan rate in therange of 0.01 V/s to 0.10 V/s (Fig. 2), indicating a quasi reversiblesystem and diffusion assisted electron-transfer process in the ZnO-based composite electrode.

The CV response of GOx/ZnO–KF/ITO/glass bio-electrode fordifferent concentrations of glucose (50 to 300 mg/dl) in buffersolution was recorded. A relative decrease in the oxidation currentwas observed with immobilization of GOx, on the surface of ZnO–KF/ITO/glass, which is a protein having a macro-molecular structure ofnon-conducting nature. A continuous increase in the oxidationcurrent in GOx/ZnO–KF/ITO/glass bio-electrode with increasingglucose concentration was observed and is attributed to the releaseof more number of electrons in the catalytic oxidation of glucose byglucose oxidase. Fig. 3 shows the variation of oxidation currentmeasured at a fixed potential (−0.2 V) for GOx/ZnO–KF/ITO bio-electrode as a function of glucose concentration. The observed linearresponse with glucose concentration indicates that the bio-electrodebased on composite matrix (ZnO–KF) can be efficiently used to detectglucose over the range of 50 to 300 mg/dl without using any mediatorin the buffer solution.

The Michaelis–Menten kinetic parameter (Kmapp) of enzymatic

reaction estimated for the GOx immobilized on the ZnO–KF compositeelectrode was 1.69 mM, and its relatively low value shows that theenzyme immobilized on the matrix has a high affinity towards thesensing analyte.

Photometric assay (Fig. 3) has also been performed to identify theapparent enzyme activity of GOx/ZnO–KF/ITO/glass bio-electrode andwas about 1.75×10−2 Ucm−2. The shelf life studies carried out on thebio-electrode based on ZnO–KF composite matrix at regular interval

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Fig. 1. XRD of ZnO–KF matrix.

for 10 weeks indicate that more than 80% of activity is retained evenafter two and a half months.

4. Spin coating of composite suspension

To explore the incorporation of another mediator, methyleneblue (MB) into the ZnO matrix, a wet chemical route was employed.ZnO nanoparticles (~5 nm diameter) were prepared as reported byYadav et al. [18] and subsequently capped with methylene blue(TEM image shown in inset Fig. 4). The ZnO–MB thin films wereprepared by spin coating on ITO coated glass slide using the nano-composite ZnO–MB sol. The immobilization of GOx was carried outphysically on the surface of ZnO–MB/ITO/glass as explained earlier.

The charge transfer resistance (RCT) at the ZnO/ITO/glass electrodeand ZnO–MB/ITO/glass electrode was about 3 kΩ and 11 kΩ,respectively. The decrease in the value of RCT of ZnO–MB compositematrix in comparison to pure ZnO matrix suggests an increase incharge transfer from the active sites to the electrode.

Cyclic voltammograms of the ZnO–MB electrode were recorded atdifferent scan rates (0.01 V/s to 0.08 V/s) and shows a linear variationof peak current suggesting improved electrocatalytic behavior (Fig. 4).For the ZnO–MB/ITO/glass electrode a well-defined redox wave peakwas obtained in mediator-less PBS buffer. The oxidation peak was at−0.24 V and attributed to the oxidation of MB in the nano-compositehybrid ZnO matrix (inset of Fig. 4).

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Fig. 3. Variation of current with glucose concentration for ZnO–KF electrode. Thephotometric assay of GOx/ZnO–KF/ITO/glass bio-electrode is also included (Inset showsCV of GOx/ZnO–KF/ITO/glass with varying glucose concentration).

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Fig. 4. Variation of anodic peak current with potential scan rate (Inset shows CV of ZnO–MB/ITO/glass in PBS buffer at 50 mV/s scan rate and TEM of ZnO–MB particles).

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Fig. 6. Variation of current with glucose concentration for ZnO–MB electrode.Photometric assay of GOx/ZnO–MB/ITO/glass bio-electrode is also included.

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Fig. 5 shows the cyclic voltammograms of the GOx/ZnO–MB/ITO/glass bio-electrode in PBS solution at different glucose concentra-tions. The oxidation current increases continuously with an increasein the glucose concentration over the range 0 to 350 mg/dl. Thevariation of currents measured at a fixed potential of −0.24 V as afunction of glucose concentration is shown in Fig. 6. The observedlinear response up to 300 mg/dl indicates that the bio-electrodebased on ZnO–MB composite matrix can also be efficiently used forglucose sensing. The sensitivity estimated from the linearity curve(Fig. 6) is 0.2 μAmM−1 cm−2. The value of Km

app is 2.65 mM for theGOx immobilized on the nano-composite ZnO–MB matrix. Theobserved value of Km

app of GOx/ZnO–MB/ITO/glass bio-electrode isslightly higher as observed in the case of GOx/ZnO–KF/ITO/glassbio-electrodes for glucose. However, these values are still lower ascompared to the corresponding value for free GOx (27 mM) andshow the advantage of the incorporation of KF or MB in the ZnOmatrix, which is due to the excellent high electron communicationfeature of ZnO and the direct electron transfer through the redoxcouple of the mediator incorporated into the matrix.

Photometric assay for the GOx/ZnO–MB/ITO/glass bio-elec-trode (Fig. 6) gives the apparent enzyme activity of about1.65×10−2 Ucm−2.

The shelf life studies carried out at regular interval with 200 mg/dlglucose concentration indicate that GOx/ZnO–MB/ITO/glass bio-electrode also retains 80% of activity even after two and a half months.

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Fig. 5. CV of GOx/ZnO–MB/ITO/glass with varying glucose concentration.

5. Fe-implanted ZnO matrix

Ion implantation is a powerful method to embed suitable ions ina controlled manner into a thin film matrix. Preliminary studieswere carried out in modifying a ZnO thin film by implantation withFe ion at 15 keV and up to a fluence of 1015 cm−2, for therealization of third generation bio-electrode. Fig. 7 shows the CVresponse obtained on both ZnO/ITO/glass (without implantation)and ZnO–Fe/ITO/glass (Fe implanted) electrodes in mediator-lessPBS buffer. It may be noted that no redox peak was observed in themeasured potential window in the case of ZnO/ITO/glass electrodeconfirming the essential requirement of a mediator in the PBS buffersolution to obtain the redox property. However, the appearance of aredox peak for ZnO–Fe/ITO/glass electrode (Fig. 7) indicates theimportance of implantation of Fe in ZnO thin film to introduceredox property in the matrix. Preliminary CV studies on electrodesbased on implanted ZnO matrix are encouraging having shownpromising results in a mediator-less PBS solution.

6. Summary

ZnO thin film matrix has been modified successfully using varioustechniques including PLD, wet chemical route and ion implantation.Suitable redox species has been incorporated into ZnO. The ZnO–MBcomposite matrix has been prepared by wet chemical route which hasthe advantage of low working potential. The low working potential

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Fig. 7. CV of Fe–ZnO/ITO/glass and ZnO/ITO/glass electrodes.

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makes the sensor stable against interference from other biologicalspecies. Another composite matrix of ZnO–KF was prepared by PLDtechnique. The bio-electrode prepared using ZnO–KFmatrix exhibiteda Km

app value of 1.69 mM which was much lower than thecorresponding value obtained for ZnO–MB bio-electrode (2.65 mM).Therefore the activity of immobilized bio-molecule is expected to beenhanced on the ZnO–KF sensing matrix as compared to thechemically grown ZnO–MB composite matrix. The peak potential ofthe ZnO–KF composite film indicated that the redox couple was beingprovided due to presence of Fe in the matrix. Therefore, Fe wasimplanted into the pure ZnO thin film matrix and further bio-sensingstudies on the modified matrix are in progress. The results show thatZnO on incorporation of redox species does not lose its bio-compatibleand electron-transfer properties and provides a suitable platform forthe immobilized bio-molecules. This will lead towards application ofZnO based thin film matrix in third generation biosensors andrealization of integrated lab-on-chip devices.

Acknowledgement

The financial support of DST and UGC, Govt. of India is gratefullyacknowledged. Author is thankful to Dr. B. D. Malhotra (NPL, India)and Dr. S. P. Singh (UPRM, USA) for fruitful discussions.

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