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Biosensors and Bioelectronics 22 (2007) 2121–2126

A hydroquinone biosensor using modified core–shell magneticnanoparticles supported on carbon paste electrode

Yi Zhang, Guang-Ming Zeng ∗, Lin Tang, Dan-Lian Huang,Xiao-Yun Jiang, Yao-Ning Chen

College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China

Received 11 May 2006; received in revised form 12 September 2006; accepted 27 September 2006Available online 1 November 2006

bstract

A hydroquinone biosensor was developed and used to determine hydroquinone concentration in compost extracts based on the immobilizationf laccase on the surface of modified magnetic core–shell (Fe3O4–SiO2) nanoparticles. Laccase was covalently immobilized on the magneticanoparticles by glutaraldehyde, which was modified with amino groups on its surface. The obtained magnetic bio-nanoparticles were attached tohe surface of carbon paste electrode with the aid of a permanent magnet to determine hydroquinone. A good microenvironment for retaining the

ioactivity of laccase was provided by the immobilization matrix. The linear range for hydroquinone determination was 1 × 10−7 to 1.375 × 10−4 M,ith a detection limit of 1.5 × 10−8 M. The current reached 95% of the steady-state current within about 60 s. Hydroquinone concentration in compost

xtracts was determined by laccase biosensor and HPLC, the results of the two methods were approximately the same.2006 Elsevier B.V. All rights reserved.

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eywords: Fe3O4 magnetic nanoparticles; Laccase; Hydroquinone; Biosensor;

. Introduction

Phenols are important raw materials and byproducts inarge-scale chemical industry, some of which are highly toxicCanofeni et al., 1994). Many of them are resistant to biotic andbiotic degradation. As a result, their wide existence in the envi-onment brings on undesirable ecological effects (Kulys andidziunaite, 2003). As proverbial hazardous substances, phe-ols are harmful to human health and environment. At present,pectrophotometry, gas chromatography and high performanceiquid chromatography (HPLC) based on the absorbance spectraf phenols (Faure et al., 1996; Kim and Kim, 2000; Di Corcia etl., 1996) are the commonly used analytical methods. Becausef the interference from turbidity and UV–vis-light-absorbingubstances, the accuracy and detection range of spectrophotom-try are limited. In chromatography methods, the pretreatment

f the samples is cumbrous and time-consuming. In addition,he instruments are expensive and ponderous, which cannot besed in the in vivo and in situ determination.

∗ Corresponding author. Tel.: +86 731 8822754; fax: +86 731 8823701.E-mail addresses: ezhangyi123@yahoo.com.cn (Y. Zhang),

gming@hnu.cn (G.-M. Zeng).

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956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2006.09.030

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Laccase is a multi copper-containing oxidase catalysing thexidation of a variety of substrate (Yaropolov et al., 1994; Xu,999), and the enzyme can be used in the analytical chemistryBogdanovskaya et al., 2002). Laccase biosensors have beenroved to be sensitive and convenient for environmental in vivond in situ analysis (Kulys and Vidziunaite, 2003; Vianello etl., 2004). Laccase does not need H2O2 as co-substrate or anyther co-factors for its catalysis. It can directly catalyse thexidation of phenols accompanied by the reduction of oxygen.henolic substrate is subjected to a one-electron oxidation lead-

ng to an aryl radical. The active species can be converted touinone in the second stage of the oxidation (Marko-Varga etl., 1995). The key aspect is the immobilization procedure ofaccase on the sensor surface. Many efforts have been made tobtain a simple and effective immobilization method to retainood bioactivity, such as physical adsorption (Peralta-Zamorat al., 2003), sol–gel technique (Li et al., 1998), covalent cross-inking (Roy et al., 2005; Rogalski et al., 1999), immobilizationn polymer films (Timur et al., 2004) and so on. However, some

f these methods lead to sensors of poor stability and/or weak inetaining the bioactivity of enzyme and complicated in manipu-ation. At present, magnetic nanoparticles as special biomoleculemmobilizing carriers have aroused great interest in research

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Tsang et al., 2000; Khin and Si-Shen, 2005). In this paper,e3O4 magnetic nanoparticles were synthesized and silylanized

o form core–shell (Fe3O4–SiO2) structure. After modificationith amino groups, the nanoparticles were cross-linked to lac-

ase by glutaraldehyde. Then with the help of magnetic force,he bio-nanoparticles were immobilized on the surface of elec-rode. The biosensor was successfully used for the detectionf hydroquinone. Compared with other laccase amperometriciosensors and HPLC, the sensor prepared by immobilizing lac-ase on the modified core–shell magnetic nanoparticles showeddvantages of high sensitivity, wide determining range and lowost in manipulation.

. Materials and methods

.1. Reagents and apparatus

Laccase (EC 1.10.3.2, 23.3 U/mg) was from Fluka.etraethoxysilane (TEOS), 3-aminopropyltriethoxysilaneAPTES), polyethylene glycol (PEG) and all other chemicalsere of analytical grade and used as received. The supporting

lectrolyte was phosphate buffer solution (PBS) prepared with7 mM KH2PO4 and 67 mM Na2HPO4 and tartrate bufferolution prepared with 0.1 M tartaric acid and 0.1 M sodiumartrate. All solutions were prepared with doubly distilled water.

Cyclic voltammetric measurement and amperometric mea-urement were carried out on CHI660B electrochemistry systemChenhua Instrument, Shanghai, China). The three-electrodeystem used in this work consists of a carbon paste electrodediameter of 8 mm) as working electrode of interest, a saturatedalomel electrode (SCE) as reference electrode and a Pt foiluxiliary electrode. All the work was done at room temperature25 ◦C) unless otherwise mentioned.

Transmission electron micrographs (TEM) of magneticanoparticles were obtained with a Hitach-800 transmissionlectron microscope (Hitachi, Japan). Agilent 1100 high per-ormance liquid chromatograph was used to determine the con-entration of hydroquinone in compost extract. Model PHSJ-3Faboratory pH meter (Leici Instrument, Shanghai, China) wassed to test pH value. A Sigma 4K15 laboratory centrifuge, aacuum freezing dryer and a mechanical vibrator were used inhe assay. In amperometric measurement, a magnetic stirrer wassed to stir the solution.

.2. Preparation of magnetic bio-nanoparticles

The synthesis of Fe3O4 magnetic nanoparticles was achievedn a typical procedure. Before mixing, 0.4 M hydrochloric acidnd 0.7 M ammonia solution were bubbled by nitrogen for0 min. 8.5 g FeCl3·6H2O and 3 g FeCl2·4H2O were dissolvedn 38 ml of hydrochloric acid, then added quickly to 375 mlmmonia solution under vigorous stirring (non-magnetic) atoom temperature. After half-an-hour stirring, the precipitate

as isolated by magnetic force. The precipitate was washed

hree times with water then diluted with 150 ml doubly distilledater. Next, the silica was coated on the magnetic Fe3O4 core

Kuang et al., 2002). Fe3O4 suspension (20 ml) was added to

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ectronics 22 (2007) 2121–2126

00 ml of 2-propanol and sonicated for 20 min. Under continu-us stirring, 5.36 g PEG, 20 ml water, 10 ml ammonia solution28 wt.%) and 1.2 ml TEOS were respectively added into theuspension, and continuously reacted for 24 h under stirring atoom temperature. PEG is added to avoid the agglomerationf colloidal silica, steer the silica polymerization throughilica–PEG interactions, and improve the biocompatibility anduspensibility of Fe3O4 nanoparticles (Sun et al., 2004). Afterhe reaction was completed, the products were collected throughentrifugation at 4000 rpm for 5 min, sonicated with ethanol andater two times, and then lyophilized to obtain the core–shellagnetic nanoparticles. To modify the surface of core–shellagnetic nanoparticles with amino groups, 0.5 g core–shellagnetic nanoparticles were suspended in 10 ml methanol, and

onicated for 3 min. Then 0.5 ml APTES was added, and theixture was agitated for 12 h at room temperature. Then the

recipitates were respectively washed with methanol and watern an ultrasonic bath. To conjugate laccase to the magneticanoparticles, the magnetic nanoparticles were added to 5 ml.5% glutaraldehyde under gentle agitation at room temperatureor 3 h. Then the mixture was centrifugated and the precipitatesere washed with PBS several times and suspended in 5 mlBS. 0.01 g laccase was added into the mixture, and it wasgitated gently at 4 ◦C for 12 h. Excess laccase was removed byashing with PBS, then vacuum filtered, and the magnetic bio-anoparticles were obtained and stored at 4 ◦C in the refrigerator.

.3. Preparation of the enzyme electrode

A solid carbon paste electrode (CPE) was prepared accord-ng to the procedure reported elsewhere (Zhou et al., 2003).

kryptol was put into the polytetrafluoroethylene (PTFE) tubeaperture of 8 mm) at first, and a caky magnet (diameter of 8 mmnd thickness of 1.5 mm) was embedded with a depth of 8 mmrom the surface of electrode. The paraffin (400 mg) was meltedt 60 ◦C and mixed with graphite powder (500 mg) thoroughlyo get homogeneous paste. The paste was stuffed into the PTFEube to immobilize the magnet. The tube was left to harden for

day. The CPE was polished thoroughly with no. 6 diamondaper, sonicated in 1:1 (v/v) nitric acid, acetone and water suc-essively, and rinsed with water before use.

The magnetic bio-nanoparticles were suspended in 1 ml PBSpH 5.5), then 60 �l was dripped onto the surface of CPE, driedor 1 h, the surface was washed with PBS to remove the excessiveio-nanoparticles. The magnetic bio-nanoparticles were firmlyttached to the electrode surface with the help of magnetic force.hen the fabrication of the enzyme electrode was accomplished.hen not in use, the CPE was stored in a moist state at 4 ◦C.

.4. Measurement in compost extracts

The determination of hydroquinone in compost extracts wasespectively carried out by HPLC and enzyme biosensor. The

omponents of compost were 218 g soil, 2600 g straw, 988 gcraps and 52 g bran, and the water ratio was 51%. The soil wasollected from 100 cm underground on the unfrequented hill-ide of Yuelu Mountain (Changsha, China), from which large

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Y. Zhang et al. / Biosensors and

rganic scraps were removed. Then aerobic compost was man-ged 40 days under the condition of 30 ◦C temperature and.033 m3 h−1 ventilation. Compost sample (10 g) was placedn a flask and 200 ml water was added in. The suspension wasgitated on a mechanical vibrator at 200 rpm for 2 h. The super-atant was centrifuged at 10,000 rpm for 5 min, and then filteredo get the filtrate as the compost extract. All the work was donet room temperature unless otherwise mentioned (Zeng et al.,004).

The pH value of extract was adjusted to 5.5 with the phosphateuffer solution. The biosensor worked at an applied potential of0.232 V versus SCE, and the same samples was analyzed byPLC. The eluent consisted of an isocratic mixture of water,

cetonitrile and acetic acid (88:10:2) at flow rate of 0.7 ml min−1.he concentration of hydroquinone was detected by ultravioletpectrophotometer at 280 nm (Chapuis-Lardy et al., 2002).

.5. Regeneration of biosensor

To regenerate the biosensor when the bioactivity of immo-ilized laccase declined obviously, we turned the nut at the endf the carbon paste electrode to extrude a little carbon pastend polished the surface with no. 6 diamond paper. The CPE

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Fig. 1. TEM images of: (a) magnetic Fe3O4 core, (b) magnetic core

ectronics 22 (2007) 2121–2126 2123

as cleaned by sonication in water for 2 min. The addition ofagnetic bio-nanoparticles onto electrode surface was the same

s described in Section 2.3.

. Results and discussions

.1. Characterization of core–shell magnetic nanoparticles

To confirm the structure of nanoparticles, TEM was per-ormed to identify Fe3O4 magnetic nanoparticles, Fe3O4–SiO2agnetic nanoparticles and the bio-nanoparticles, respectively.

n Fig. 1(a), the Fe3O4 powder consisted of particles of 4–6 nm,hich were spherical with irregular polyhedrons and wellispersed. The TEM images of Fe3O4 nanoparticles with SiO2hells were shown in Fig. 1(b). The core–shell (Fe3O4–SiO2)anoparticle diameter was 14–18 nm. The thickness of the SiO2hell was 5–6 nm, and the enlarged particles were still keptpheric. The coating thickness of shell could be adjusted whenome experimental parameters varied. However, to ensure the

articles could be caught up with the help of magnetic force,he shells should not be too thick. The proper shell thicknessas favourable to keep the high magnetic-field intensity and

tability; meanwhile it was convenient for modification on the

–shell (Fe3O4–SiO2) nanoparticles and (c) bio-nanoparticles.

2 Bioelectronics 22 (2007) 2121–2126

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d(best signal-to-background current was detected in the pH rangeof 5.0–6.0. The result was consistent with detection by cyclicvoltammetry. And it was observed that the current response intartrate buffer was not as steady as in PBS, so the optimum cur-

124 Y. Zhang et al. / Biosensors and

urface of the core–shell nanoparticles (Liu et al., 2005). Lac-ase was cross-linked on the nanoparticles by glutaraldehyde,nd the size of particles (Fe3O4–SiO2-laccase) was slightlyncreased to 22–25 nm, which could be seen in Fig. 1(c). Theio-nanoparticles were so small that they could offer a greatpecific surface area. This could extend the contact area ofeaction, available for detection.

.2. Characterization of the immobilization of enzyme

Laccase was covalently immobilized on the magneticanoparticles by glutaraldehyde, which was modified withmino groups on its surface by APTES. The immobilizationf enzyme could enhance stability of laccase and extend the lifepan of electrode. In contrast with other methods of enzymemmobilization, covalent cross-linking kept 90% bioactivity ofree laccase and had few effects on its kinetic constant. Immo-ilized enzyme behaved with a favorable stability in a longpplication procedure (Leonowicz et al., 1988). Taking advan-age of the paramagnetism, the magnetic bio-nanoparticles werettached to the surface of carbon paste electrode, which wasonvenient not only for isolation of the analyte, but also forperation and recovery of the biosensor. Due to the presencef external magnetic field, superparamagnetic bio-nanoparticlelusters were attracted to the CPE surface to form an evenayer on the surface for robust electrochemical determination.n addition, different from the other methods to form a mono-ayer laccase, a multilayer laccase could be obtained. The filmf the bio-nanoparticles on the surface of electrode was about.04 mm thick. Because the diameter of single bio-nanoparticleas around 25 nm that had been proved from the TEM photo-raph, it could be estimated that the thin film was multilayer. Sohe CPE was more sensitive, and the lineation detection rangeas augmented (Jarosz-Wilkołazka et al., 2005; Vianello et al.,004).

.3. Catalytic reaction mechanism of biosensor

Laccase catalysed the oxidation of a great variety of hydrox-benzene dyestuff, chlorophenol, sulfophenol, aromatic amine,tc. The redox reaction of hydroquinone catalysed by laccase isescribed as follows (Yaropolov et al., 1994):

ydroquinone + O2laccase−→ P-benzoquinone + 2H2O (1)

The redox process of laccase on the surface of the CPE washown in Fig. 2. Laccase is a multicopper phenol oxidase. Itan oxidize hydroquinine and utilize dioxygen as an oxidant,educing it to water. In the reaction, hydroquinone as electrononor for the oxidized form of the enzyme, was mainly converted

nto quinone and/or free radical product, and then was reducedn the surface of the electrode at potentials below 0 V (versusCE) (Roy et al., 2005; Haghighi et al., 2003), which efficientlyhuttled electrons between laccase redox center and CPE surfacen a dynamical equilibrium, leading to the detectable responseurrent (Tang et al., 2005).

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ig. 2. Schedule of the catalytic reaction mechanism of laccase on electrodeurface. QH2 = hydroquinone, Qox = quinone and/or free radical product.

.4. Optimization of experimental variables

Fig. 3 displays the cyclic voltammograms of the CPE in PBS67 mM, pH 4.6–6.5). It could be observed that a low back-round current in the absence of hydroquinone. On the additionf 0.4 M hydroquinone to the buffer solution, a reduction peakppeared according to the reduction of quinone species producedy laccase-catalysed hydroquinone oxidation on the surface oflectrode (Freire et al., 2001). The peak current varies with pHalues of PBS, and the response peak potentials are different,oo. The peak potentials shift to negative values at higher pHalues from 4.6 to 6.5. It can be seen that the reduction currentt pH 5.5 was the largest.

In the presence of 10 �M hydroquinone, the pH depen-ence of the enzyme electrode was investigated in tartrate buffer0.1 M) with pH 4.0–4.6 and in PBS (67 mM) with pH 5.0–7.0y chronoamperometry with relative applied potential. The high-

ig. 3. Cyclic voltammograms of the electrode at a scan rate of 100 mV s−1

n blank PBS (67 mM, pH 5.5) and in PBS (67 mM, pH 4.6–6.5) with 0.4 mMydroquinone.

Y. Zhang et al. / Biosensors and Bioel

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ydroquinone concentration by 10 �M in each step in PBS (67 mM, pH 5.5) atn applied potential of −0.232 V vs. SCE.

ent response was obtained in the pH range 5.0–6.0. In order tochieve the maximum sensitivity, pH 5.5 and the correspond-ng applied potential −0.232 V (versus SCE) were chosen inubsequent experiments.

.5. Electrode response characteristics

Fig. 4 shows a typical current–time plot of the biosensornder the optimized experimental conditions after the additionf successive aliquots of hydroquinone to the PBS under stir-ing. With the increase of the concentration of substrate, the

iosensor responded rapidly and 95% steady-state current waseached within 60 s. Fig. 5 shows the calibration of responseurrent and the concentration of hydroquinone. The responseurrent increases linearly with the hydroquinone concentration

ig. 5. Calibration plot of current response vs. hydroquinone concentration inBS (67 mM, pH5.5) at an applied potential of −0.232 V vs. SCE. The verticalars designate the standard deviation for the mean of three replicate tests. (Inset)he plot of current response vs. hydroquinone between 15 nM to 1 �M.

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n the range of 1 × 10−7 to 1.375 × 10−4 M. The correspondingegression equation is

= (0.2118 ± 0.0045)x + (0.9834 ± 0.2496) (2)

here y is the current change (�A), x the hydroquinoneoncentration (�M), and the coefficient is 0.9933. Accordingo the generally accepted definition, the lower detection limits 1.5 × 10−8 M, which resulted in a current signal that equalshe mean value of background signals plus three times standardeviation of background signals. As compared with other laccaseiosenseors in the references, the linear range of detection of thenzyme biosensor in this work was enlarged, and the detectionimit was lowered. For example, Vianello et al. (2004) devel-ped an amperometric laccase biosensor with the linear rangeor hydroquinone of 1 × 10−7 to 2 × 10−5 M and the detectionimit of 1 × 10−7 M. And the linear range for hydroquinoney another laccase biosensor was 1 × 10−6 to 1 × 10−5 M andhe detection limit was 5.8 × 10−7 M (Jarosz-Wilkołazka et al.,005).

Each of the calibration is done three times with the standardeviations of current response not more than 4%.

.6. Reproducibility and stability

The repeatability of the current response of enzyme elec-rode to 10 �M hydroquinone was examined. The relativetandard deviation (R.S.D.) was 3.13% for five replicatessays.

The CPE was stored in a moist state at 4 ◦C in a refrigeratorhen not in use. The stability of the biosensor was investi-ated by measuring the enzyme electrode response with 10 �Mydroquinone. The current of the biosensor kept an invariablealue during about 15 days with a neglectable decrease. Afterbout 40 days, the enzyme electrode retained 70% of its orig-nal response. The phenomenon indicated the good stability ofhe biosensor. This observation indicates that the stability ofhis biosensor may be sufficient for practical applications. Theioactivity was remained owing to the structure of the multilayernd the biocompatibility in the microenvironment of core–shellatrix.

.7. Interference

To evaluate the selectivity of the biosensor, the influence ofome possible interfering substances, such as phenol, vanillin,uaiacol, 3,5-dinitrosalicylic acid and N,N-dimethylaniline werexamined under the same condition for the determination ofydroquinone. Measurements were carried out by adding 10 �Marious substrate in PBS (67 mM, pH 5.5) at an applied potentialf −0.232 V (versus SCE). Compared with the response currenthange of biosensor to hydroquinone, the relative response cur-

ent changes were 1.87% for phenol, 0.31% for vanillin, 2.14%or guaiacol, 0.02% for 3,5-dinitrosalicylic acid and 1.79% for,N-dimethylaniline. It can be inferred that these substances in

ower concentrations will not cause interference. So the enzymeiosensor performed with good selectivity.

2126 Y. Zhang et al. / Biosensors and Bioel

Table 1Hydroquinone concentration in compost extracts determined by laccase biosen-sor and HPLC

Samples Hydroquinone concentration (�M) Relative signaldeviation (%)

Laccase biosensora HPLCb

1 6.93 6.70 3.432 12.33 12.53 1.603

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a An average of three replicate measurement.b An average of two replicate measurement.

.8. Application in compost extracts

Hydroquinone concentration of three compost extract sam-les were determined by laccase biosensor and HPLC, the resultsf the two methods were approximately the same, shown inable 1.

From the results, it is easy to find that the two methods dis-layed a good correlation. Consequently, the biosensor offeredsimple, fast and sensitive method for hydroquinone detectionith favorable accuracy and specificity.

. Conclusion

A hydroquinone biosensor had been developed and used toetect compost extracts on the basis of the immobilization ofaccase on the surface of modified magnetic core–shell nanopar-icles. In fabrication of the laccase biosensor, the enzyme wasovalently immobilized onto the magnetic nanoparticles andhen attracted to CPE surface by dint of magnetic force. Hydro-uinone efficiently shuttles electrons between laccase redox cen-er and CPE surface. The optimized experimental conditions forhe operation of the enzyme biosensor had been studied. Its supe-ior sensitivity, stability, reusability, selectivity were obtainedith obvious advantages for hydroquinone determination. In theetermination of hydroquinone in the real sample of compostxtracts, the detection results of the biosensor and the paral-el HPLC method were in close propinquity, while the methodf biosensor was more simple, convenient, rapid and sensitive.urther more, it could avoid the interference from turbidity andV–vis-light-absorbing substances in the detection process in

he complex compost system. All of these clearly illustrate thathis laccase biosensor offered a possible and economical methodor “on-the-spot” monitoring of hydroquinone in compostingystem.

cknowledgements

The study was financially supported by the National63 High Technologies Research Foundation of China (no.

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004AA649370), the National Basic Research Program (973rogram) (no. 2005CB724203), and the Natural Foundation foristinguished Young Scholars (nos. 50425927, 50225926).

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