Facile Synthesis of Leaf-Like CuO Nanoparticles and TheirApplication on Glucose Biosensor

Ying Li, Yinyin Wei, Guoyue Shi, Yuezhong Xian, Litong Jin*

Department of Chemistry, East China Normal University, Shanghai 200062, P. R. China*e-mail: ltjin@chem.ecnu.edu.cn

Received: May 28, 2010;&Accepted: September 23, 2010

AbstractAn efficient amperometric biosensor based on well-crystallized leaf-like CuO nanoparticles for detecting glucosehas been proposed. The leaf-like CuO nanoparticles, synthesized by a simple one-step hydrothermal method, werecharacterized by X-ray diffraction (XRD), scanning electron microscope (SEM) and transmission electron micros-copy (TEM) for the morphology study. Under the optimal condition, the electrochemical behaviour of the leaf-likeCuO nanoparticles modified electrode for detection of glucose exhibited high sensitivity of 246 mA/mM/cm2, shortresponse time (within 5 s), linear dynamic range from 1.0 to 170 mM (R2 =0.9995), and low limit of detection(LOD) (S/N=3) of 0.91 mM. The high sensitivity, good reproducibility, stability, and fast amperometric sensing to-wards oxidation of glucose, make this biosensor promising for future application.

Keywords: CuO, Nanoparticles, Glucose oxidase, Biosensors

DOI: 10.1002/elan.201000343

1 Introduction

Glucose is an essential nutrient substance for biologicalmetabolism. It�s important for human health to maintainthe concentration of blood sugar at a definite level, thusglucose detection is extremely important especially to thepatients suffering from diabetes [1, 2]. Glucose oxidase(GOD) has been widely applied to fabricate diverse am-perometric biosensors for glucose detection [3–9] due toits high sensitivity and selectivity to glucose and stable ac-tivity. “Finger pricking” is commonly used method by dia-betic patients for measuring their blood sugar level. How-ever, it is not a continuous, convenient and non-invasivemonitoring method. Usually, continuous glucose monitor-ing do not measure blood glucose directly, but rely in-stead on measurement of the glucose levels in other bio-logical fluids such as tears, mucus, sweat and saliva [10–15]. Glucose concentrations in these biological fluids arelow, thus a glucose biosensor with high sensitivity and lowdetection limit is a pressing need.

During the past few years, various nanomaterials haveattracted extensive attention for their small size, largesurface area and correspondingly special electronic,chemical and optical properties [16–21]. CuO, an impor-tant p-type metal oxide semiconductor with narrow band-gap (1.2 eV), has been investigated as an extraordinaryand attractive monoxide material due to its plentifulunique characters [22–26]. Owing to its exceptional elec-trochemical activity and the possibility of promoting elec-tron transfer at a low potential, nanostructured CuO is agood candidate for glucose sensing. Further more, the

properties of nanomaterials are highly size and morpholo-gy dependent, thus original structured nanomaterialswere much interesting for their new physicochemical be-haviours and promise for the fabrication of materials withadvanced functionalities [27,28]. It is therefore significantto synthesize novel size and shape of CuO nanoparticlesand to further improve its application on glucose detec-tion.

Nafion has been widely used as a selective modifyingmaterial owing to its good chemical stability and biocom-patibility [29,30] and network structure which providesenough room that nanoparticles can infiltrate into it[31,32] to enhance electronic conduction. In addition,Nafion is a kind of negative charge film adept in repellinginterfering species such as ascorbic acid and uric acid, soit often act as a protective film to improve anti-interfer-ence ability of modified electrode.

In this paper, we introduced a leaf-like CuO nanoparti-cles based amperometric glucose biosensor with high sen-sitivity and low detection limit. In general, the direct elec-tron transfer between the electrode and the active site ofthe enzyme is difficult, and catalyst for oxidation of H2O2

produce during the enzymatic reaction is needed, there-fore we have employed leaf-like CuO nanoparticles aselectron mediator and catalyst to construct the glucosebiosensor. The leaf-like CuO nanoparticles were first syn-thesized by a simple, convenient, surfactant-assisted, andone-step hydrothermal route, and then utilized to modifythe glassy carbon electrode (GCE) with GOD andNafion successively. The electrochemical performancewas investigated by characterizing its sensitivity, linearity

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range, detection limit, selectivity, stability and reproduci-bility of the fabricated glucose biosensor.

2 Experimental

2.1 Reagents and Apparatus

Glucose oxidase type VII (136,000 units/g, EC1.1.3.4.from Aspergillus niger) and Nafion were purchased fromSigma. Glucose and other reagents were of analyticalgrade, purchased from Shanghai Chemical Corp (Shang-hai, China). Doubly distilled water was used in all experi-ments.

Amperometric detections were carried out on CHI660C electrochemical workstation (Chenhua InstrumentCorporation, China) with a three-electrode system, aNafion/GOD/CuO modified electrode as working elec-trode, a Ag/AgCl as reference electrode and a platinumelectrode as counter electrode. Bruker D8 Advanced X-ray diffraction (XRD) using Cu-Ka radiation, S-4800scanning electron microscope (SEM) (Hitachi, Japan) andJEM-2100 high resolution transmission electron micro-scope (HRTEM) (JEOL Co. Ltd., Japan) were used forcharacterization of the CuO nanoparticles. Sonicationwas performed in a KQ-2100DA ultrasonic cleaner with afrequency of 40 kHz and a nominal power 100 W.

2.2 Synthesis of Leaf-Like CuO Nanoparticles

Leaf-like CuO nanoparticles were prepared as follows:0.34 g CuCl2·2H2O and 0.232 g Sodium dodecyl benzene-sulfonate (SDBS) were first dissolved in 40 mL doublydistilled water entirely. 1.6 g NaOH was then added intothe above-mentioned solution slowly under constant stir-ring at room temperature. After 10 minutes, the mixturewas transferred into 50 mL sealed Teflon autoclave,which was heated at 100 8C for 16 hours subsequently andthen cooled to room temperature naturally. The blackprecipitates were collected to centrifuge and rinse withdoubly distilled water and ethanol several times. Finally,the products were dried in vacuum at 60 8C.

2.3 Preparation of Nafion/GOD/CuO ModifiedElectrode

Firstly, 4.0 mg/mL CuO nanoparticles (in ethanol) and0.1 mg/mL GOD (in PBS, pH 7.0) solutions were pre-pared. The GCE was firstly polished carefully with 0.3and 0.05 mm alumina slurry and rinsed thoroughly with bi-distilled water. Then the electrode was ultrasonicated in1 : 1 nitric acid, acetone and doubly distilled water for5 min respectively, and dried at room temperature [33].5.0 mL CuO nanoparticles solution was dropped onto thesurface of the pretreated GCE and dried by high-puritynitrogen gas. For immobilization of GOD, cross-linkingmethod was employed and in a typical reaction process,100.0 mL GOD solution 50.0 mL (2.5 %) glutaraldehydeand 50.0 mL (0.5 %) Nafion solutions were mixed thor-

oughly. Thereafter, 2.0 mL above mixture solution was ap-plied onto the GCE/CuO electrode surface and dried inair at room temperature. And then 2.0 mL 0.5% Nafionwas further coated on the modified electrode to eliminatethe possible fouling and prevent the leaching of theenzyme. Finally, the Nafion/GOD/CuO modified elec-trode was rinsed thoroughly with doubly distilled waterand stored in PBS (pH 7.0) at 4 8C when not in use.

3 Results and Discussion

3.1 Characterization of the Leaf-Like CuO Nanoparticles

The typical XRD pattern of the leaf-like CuO nanoparti-cles is shown in Figure 1A, which reveals that all diffrac-tion peaks can be assigned to the monoclinic phase ofCuO (JCPDS 05-0661). No characteristic peaks of impuri-ties are observed in XRD pattern, indicating the highlypure CuO can be readily obtained by this hydrothermalreaction approach. The morphology and structure of theleaf-like CuO nanoparticles are characterized by SEM.As shown in Figure 1B and Figure 1C, the final CuOproducts in good dispersion are highly uniform leaf-likearchitectures, with a length of about 700–800 nm andwidth of 40–60 nm. Figure 1D and Figure 1E shows TEMand HRTEM images for the as-synthesized CuO, whichfurther confirm the results of SEM. The HRTEM analysisreveals that the clear spacing between two neighbouringfringe is ~0.25 nm, corresponding to the distance of the(002) plane, confirms that the prepared CuO nanoparti-cles were well crystallized with a monoclinic structureand grew along the preferential direction of [002]. It�sworth mentioning that the CuO leaf-like architectures arevery stable and still maintain the morphology after ultra-sonication for 30 min.

3.2 Optimal Operating Conditions

An optimization study involving the parameters of bestexperimental condition was performed. The effect of ap-plied potential on the response currents of the fabricatedbiosensor was shown in Figure 2A. The biosensor wasused to detect 30 mM glucose over the potential rangfrom 0.2 to 0.8 V. In the range of 0.2–0.6 V the responsecurrents of the biosensor increased accordingly with in-creasing potential. When applied potential increased from0.6 to 0.8 V, the response currents increased slowly. How-ever, the higher potential would result in larger interfer-ences of other coexisting electroactive impurities andnoise of the response. Thus, a potential of 0.60 V was se-lected for subsequent studies.

Figure 2B shows the influence of pH on the glucosesensing of the biosensor in a solution of 30 mM glucose.Maximum response current was obtained at pH 7.0. Thisvalue is consistent with the optimum pH 6–7.4 for mostsoluble enzymes. Thus, 0.6 V and pH 7.0 were selected forsubsequent experiments as the optimal experimental con-dition.

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3.3 Electrochemical Performance of Sensing Glucose

The schematic of glucose detection by electrochemicalmethod using CuO nanoparticles modified glassy carbonelectrode was exhibited in Figure 3A. Glucose would beoxidised by GOD to produce gluconic acid and H2O2.Redox of the electroactive H2O2 was catalysed by CuOnanoparticles, which would generate redox currents. Thusthe amperometric detection of H2O2 is performed to de-termine the concentration of glucose.

Figure 3B compares the electrocatalytic towards glu-cose at different modified electrode. In PBS (pH 7.0,

0.1 M) containing 2 mM glucose, no distinguished peakhas been observed in CV curve when Nafion/GOD/GCEelectrode was employed (green line). In contrast, theCuO nanoparticles-modified electrode exhibited a distinctpeak of current at the potential about 0.6 V (black line),indicating good electrocatalytic of the CuO nanoparticles.No distinguished peak of the Nafion/GOD/CuO/GCEelectrode has been observed in CV curve when glucosewas absent, and the curve exhibited a sharp peak of cur-rent at the potential about 0.6 V in the presence of 2 mM

Fig. 1. A) Typical X-ray powder diffraction pattern; B) low and C) high-magnification FESEM images; D) low and (E) high-resolu-tion TEM images of the as-prepared CuO nanoparticles.

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glucose, confirming the electrochemical response of theNafion/GOD/CuO modified electrode to glucose.

Figure 3C depicts the amperometric response of thisbiosensor on successive addition of glucose to a continu-ously stirring PBS. The calibration curve for the biosensorunder the optimized experimental conditions is shown inFigure 3D. The biosensor displayed a linear response toglucose in the concentration range from 1.0 to 170 mM(R2 =0.9995) with a sensitivity of 246 mA/mM/cm2, whichwas higher than values found in the literature previously[34,35]. The linear equation was I (mA)=0.7769+0.0174 C (mM), the average relative standard deviation(RSD) is 1.7 % (n=10), and the detection limit of thebiosensor was estimated to be 0.91 mM at a signal/noiseratio of 3 with a short current response time (within 5 s),much lower than the previous method [36,37]. The appar-ent Michaelis–Menten constant KM

app can be calculatedaccording to the Lineweaver–Burk equation [38]: 1/i=KM

app/imax(1/C)+ (1/imax), where i is the current, imax is themaximum current measured under saturated substrateconditions, and C is the glucose concentration. The KM

app

was calculated to be 0.29 mM, less than the previously re-ported literature [39]. The high GOD affinity to glucosemay be attributed to the biocompatible nature, high spe-cific surface area, chemical stability, high conductivity ofthe leaf-like CuO nanoparticles which provide high elec-tron communication features that enhance the direct elec-tron transfer between the active sites of enzyme and theelectrodes [40].

3.4 Selectivity, Stability and Reproducibility

The selectivity of this biosensor was evaluated. As shownin Figure 3E, in an air-saturated and stirred 0.1 M pH 7.0PBS containing 0.1 mM glucose, the responses arisingfrom 0.1 mM uric acid (UA), 0.1 mM l-cysteine (l-cys),

0.1 mM glutamate (GA) and 0.1 mM ascorbic acid (AA)were negligible.

The stability of the biosensor was examined by measur-ing consecutive amperometric responses nine times. TheRSD was 3.28%, indicating that the sensor was stable.The biosensor also exhibited a good long-term stability.After each experiment, the sensor was washed with PBSand stored in PBS (pH 7.0) at 4 8C. The sensor retainedabout 90% of its original bioactivity after three months.

Reproducibility tests were carried out. Five Nafion/GOD/CuO/GCE electrodes were investigated to comparetheir amperometric current responses. The RSD was2.97%, confirming that the preparation method washighly reproducible.

3.5 Application to Real Samples

The applicability of this biosensor was explored by de-tecting glucose concentration in human serum samplesutilizing the standard addition method. The samples fromone donor were diluted 100 fold by PBS (pH 7.0) toensure the glucose concentrations fall in the workingrange of the calibration curve. The results were given inTable 1. The recoveries were in the range of 93–101 %,

Fig. 2. The influences of applied potential A) and pH value B) on the response currents of the Nafion/GOD/CuO/GCE biosensor atglucose concentration of 30 mM.

Table 1. The detection results of real samples.

Sample Glucose added (mM) by thefabricated biosensor [a](mM)

Glucosedetermined

RSD(%)

Human serumdiluted 100fold

0 45.7 0.710 55.0 1.520 66.1 0.930 74.7 1.140 86.2 1.9

[a] Average value from triplicate.

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which demonstrated that the glucose biosensor offered anexcellent method for the determination of glucose in di-luted human serum and could be used clinically.

4 Conclusions

In this paper, we have synthesized the leaf-like CuOnanoparticles by a fast, reliable, and one-step hydrother-mal method. The as-prepared CuO nanoparticles utilized

Fig. 3. A) Schematic of glucose detection by electrochemical method using CuO nanoparticles modified electrode; B) CV sweepcurve for Nafion/GOD/CuO/GCE electrode in absence (red line) and presence of 2.0 mM glucose (black line) and for Nafion/GOD/GCE electrode in presence of 2.0 mM glucose (green line); C) Current response of the biosensor up on various concentrations of glu-cose in PBS at 0.6 V (vs. Ag/AgCl); D) Calibration curve of the biosensor in pH 7.0, 0.1 M PBS at 0.6 V(vs. Ag/AgCl); E) Current–time profile of the biosensor with addition of 0.1 mM glucose, 0.1 mM UA, 0.1 mM l-cys, 0.1 mM GA and AA.

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to enhance the electron conducting and catalyse the oxi-dation of H2O2 produce were employed to construct glu-cose biosensor. The proposed glucose biosensor exhibitedexcellent oxidation of glucose and advantages over someearlier reported glucose biosensors, especially the low de-tection limit and high sensitivity to glucose concentrationchange. These features make the as-synthesized leaf-likeCuO nanoparticles an attractive and potential materialfor the fabrication of efficient amperometric biosensors.

Acknowledgements

This work was supported by the Science and TechnologyCommission of Shanghai Municipality (no. 06dz05824),the National Natural Science Foundation of China (no.20475017), and Ph.D Program Scholarship Fund of (no.2010031).

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