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AN AMPEROMETRICBIOSENSOR FOR HYDROGENPEROXIDASE BASED ON THECO-IMMOBILIZATION OFCATALASE AND METHYLENEBLUE IN AN AL2O3 SOL-GELMODIFIED ELECTRODEDandan Chen a , Baohong Liu a , Zhengjiu Liu a &Jilie Kong ba Department of Chemistry , Fudan University ,Shanghai, 200433, People's Republic of Chinab Department of Chemistry , Fudan University ,Shanghai, 200433, People's Republic of ChinaPublished online: 02 Feb 2007.

To cite this article: Dandan Chen , Baohong Liu , Zhengjiu Liu & Jilie Kong (2001)AN AMPEROMETRIC BIOSENSOR FOR HYDROGEN PEROXIDASE BASED ON THE CO-IMMOBILIZATION OF CATALASE AND METHYLENE BLUE IN AN AL2O3 SOL-GEL MODIFIEDELECTRODE, Analytical Letters, 34:5, 687-699, DOI: 10.1081/AL-100103212

To link to this article: http://dx.doi.org/10.1081/AL-100103212

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ANALYTICAL LETTERS, 34(5), 687–699 (2001)

CHEMICAL AND BIO-SENSORS

AN AMPEROMETRIC BIOSENSOR FOR

HYDROGEN PEROXIDASE BASED ON THE

CO-IMMOBILIZATION OF CATALASE AND

METHYLENE BLUE IN AN AL2O3 SOL-GEL

MODIFIED ELECTRODE

Dandan Chen, Baohong Liu, Zhengjiu Liu, and Jilie Kong*

Department of Chemistry, Fudan University, Shanghai,People’s Republic of China 200433

ABSTRACT

A novel biosensor for the amperometric detection of hydro-gen peroxide was developed based on the co-immobilizationof catalase and methylene blue on an Al2O3 sol-gel fabricatedglassy carbon electrode. The membrane structure of thesol-gel-immobilized catalase and methylene blue was studiedwith scanning electron microscopy. Cyclic voltammetric andamperometric measurements demonstrated that methyleneblue co-immobilized with catalase in this way displayedgood stability and efficiently shuttled electron between theimmobilized enzyme and the electrode. Electrocatalytic reduc-tion of H2O2 at the electrode was evaluated with respect tosolution pH, operating potential and selectivity. The biosen-sor was stable at least for 3 weeks.

687

Copyright & 2001 by Marcel Dekker, Inc. www.dekker.com

*Corresponding author.

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Key Words: Al2O3 sol-gel; Catalase peroxidase; Methylene

blue; H2O2 detection.

INTRODUCTION

The quantitative determination of hydrogen peroxide is of importance

in many areas. The available systems to detect H2O2 include spectrophoto-

metry(1), chemiluminesceme(2,3,4) and electrochemical monitoring(5,6).

Among the procedures mentioned above, the biosensor method based on

enzyme immobilization offers several advantages for H2O2 detection(4,7–

13). Many researchers employed horseradish peroxidase (HRP) as the

enzyme because it catalyzes four kinds of reaction, i.e., peroxidation, oxida-

tion, dismutation and hydroxylation(4,7–13). Peroxidases provide electro-

chemical H2O2 sensor with a detection limit as low as 10-8�10-7M(14). The

mainly employed mediators are methylene green(12,13), methylene

blue(12,13), (2-aminoethyl) ferrocene(14), {Ru(NH3)5py}2+(14), o–phenyl-

enediamine(15), ferrocene(16), and tetrathiafulvalene(17). Horseradish

peroxidase involves compound [POD-I] containing Fe(IV) and a por-

phyrin-radical cation and compound [POD-II] with Fe(IV) in the following

consecutive reactions:

H2O2 þ POD ! H2Oþ POD-I

POD-IþAH ! POD-IIþA

POD-IIþAH ! PODþA�

Where AH represents the hydrogen donor and A� is the free radical formedduring the reaction(18-20). Although most researches on H2O2 biosensor

focused on horseradish peroxidase, other peroxidases have the same effect

on the H2O2 sensor. Catalase is a peroxidase that also contains redox Fe

couple in its reaction center. Its electron transfer in-between the mediator

and H2O2 follows the similar pathway.

Entrapment of enzyme on carbon paste(21), polypyrrole(22), glassy

carbon electrode(10,11,12,13), include silk fibroin directly from silk

larvae(23), regenerated silk fibrobin(11,13), zeolite(10), eletropolymeriza-

tion(12) has been reported before. Since the low temperature sol-gel

process provides a new, available avenue to encapsulate protein by

Braun(24), a number of researchers have reported their work on the

sol-gel derived biosensors(4,24–33) due to the numerous advantages,

including tenability of physical properties, mechanical rigidity, chemical

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inertness, high photochemical and thermal stability and negligible swellingboth in aqueous and organic solutions.

In this paper, catalase instead of horseradish peroxidase as the enzymewas immobilized by sol-gel on a glass carbon electrode. The interaction

between the media and matrix was investigated. The ideal performance of

the biosensor based on sol-gel entrapment showed that the positivelycharged alumina sol-gel was an attractive material to immobilize catalase

peroxidase thereon for H2O2 detection.Our lab has reported the work of Al2O3 sol-gel derived biosensors

previously(32,33). The results showed the feasibility of methylene blue’s

mediating electron transfer between catalase peroxidase in sol-gel matrixand a glassy carbon electrode. The effect of the ratio for Al2O3 sol-gel to

water and the effect of various experimental parameters, such as pH, tem-

perature, applied potential, selection ability and lifetime were explored foroptimum analytical performance.

EXPERIMENTAL

Reagents

Catalase peroxidase, noted activity of 15,000u/mg of solid, was pur-chased from Sigma (C-40, Lot 46H7085, EC 1.11.1.6) and methylene blue

(MB) was obtained from Chroma. Aluminium iso-propoxide, L-lactate,

uric, ascorbic acid, galactose, L-cysteine, L-glutamic acid were fromShanghai Chemical Company. H2O2 (30%9w/v) solution was purchased

from Shanghai Chemical Reagent Company. The phosphate buffers were

0.2M NaH2PO4/KOH. All chemicals were of analytical grade. All the sol-utions were prepared with distilled water.

Apparatus and Measurements

All measurements were done using a conventional three-electrode

system consisting of a platinum wire counter electrode, a saturated calomel

reference electrode (SCE), and the biosensor working electrode.Amperometric measurements were carried out on CHI 660A (CH

Instrument Electrochemical Workstation, USA). A magnetic stirrer and

bar with a constant-temperature control provide the convective transport.Scanning electron microscopy (SEM) is XL30, D6716, Philip.

AMPEROMETRIC BIOSENSOR FOR HYDROGEN PEROXIDASE 689

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Preparation of an Alumina Sol-gel Solution

Al(i-PrO3) (2g chemical grade) was added to a certain volume (44.0ml)of distilled water. After stirring the mixture for 45�60 min, a certain quan-tity (1.20ml) of 1M HCl was added (the molar ratio of the three materialsis 1: 100: 0.05). The mixture was heated to 908C and the vessel was openfor several hours to let evaporate the i-PrOH, which was produced fromhydrolysis. Then the mixture was refluxed for over 16 hours under 908C. Astable and homogeneous Boemite sol (r-AlOOH) has been obtained(32,33).

Construction of the H2O2 Sensor

A 4mm glassy carbon electrode (GCE) was used as the base electrodefor the sol-gel-modified H2O2 biosensor. The GCE was polished withdiamond paper, followed by 0.3, 0.1, 0.05mm alumina particles, rinsedthoroughly with deionied water between each polishing step, thensuccessively washed with 1: 1 nitric acid, acetone and doubly distilledwater in an ultrasonic bath, and dried in air before use.

The surface of the electrode was coated with sol-gel-MB by pipteting10 ml of well mixed stock standard sol-gel solution (ratio of Al:H2O was1:100) and MB. The concentration of the MB was 1mM. After dryingunder ambient temperature for 20min, a sol-gel-MB film was formed. Theblue color showed on the GCE surface proved the immobilization of MB.Then catalase peroxidase (10mg/ml) was mixed completely with sol-gel andwas deposited on the GCE and drying under 48C in air for over 24h. Thesensor was kept in air at refrigerator between measurements.

Measurement Procedure

Cyclic voltammetric and amperometric measurements were performedusing CHI660A coupled with a P-III computer. The three-electrode systemwas used for detection. All the experimental solutions were thoroughlydeoxygenated by bubbling nitrogen through the solution all over theexperiment.

In the constant potential experiments, successive additions of stockH2O2 solution to the phosphate buffer were made and the current-time data were recorded as a function of time following the addition ofH2O2.

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RESULTS AND DISCUSSION

Interaction Between Al2O3 Sol-gel and Catalase

Al2O3 sol-gel had a structure of matrix and when it was mixed with

catalase, the enzyme was enclosed by the matrix whatever the size of

enzyme. This was one of the advantages that interested scientists. The inter-

action between sol-gel and enzyme was investigated by SEM. A typical SEM

picture of the sol-gel membrane showed a three-dimensional porous open-

cell network (Fig. 1a). This porous network provided a significantly

AMPEROMETRIC BIOSENSOR FOR HYDROGEN PEROXIDASE 691

(b)

Figure 1. Scanning electron microscopic photographs for the catalase peroxidase(a) and Al2O3 sol-gel fabricated catalase peroxidase (b).

(a)

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increased effective electrode surface for high enzyme loading. When enzymewas immobilized in the Al2O3 sol-gel matrix, many bright particles, whichwas probably the embedded enzyme, appeared in Fig. 1b.

Amperometric Response of H2O2 Sensor

No electrocatalytical catalytic reduction current was recorded on ancatalase –free Al2O3 sol-gel derived electrode when H2O2 was added to thephosphate buffer solution. Fig. 2 showed the effect of the scan rate on thevoltammograms . As the scan rate increased, both the cathodic and anodiccurrent increased and the peak current vs. square root of scan rate (ip /v

1/2)was constant, which represented the redox behavior contributed from MB.

Fig. 3 displayed a typical cyclic voltametric response for the integratedH2O2 sensor. In the absence of the H2O2, the peroxidase yielded no responseand only typical oxidation and reduction peaks for MB fabricated in the sol-gel derived electrode were observed (Fig. 3a). With H2O2 added to the cell, adramatic change occurred in cyclic voltammogram, an increase in cathodiccurrent and a concomitant decreased in anodic current was recorded asshown in Fig. 3b. Differences between the voltammograms suggested that

692 CHEN ET AL.

1.0 0.5 0.0 -0.5 -1.0

-10

-5

0

5

10

15

20

Cur

rent

/uA

Protencial(V vs SCE)

Figure 2. A cyclic voltammograms of the H2O2 sensor at various scan rates (frominner curve to outer curve): 50, 100, 200, 300, 400, 500 mv/s in 0.2 M phosphate

buffer.

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MB effectively shuttled electrons between peroxidase in sol-gel matrix andthe glassy carbon electrode. The mechanism of the sensor was summarizedas in Scheme.1 where the peroxidase (POD) reduced hydrogen peroxide towater and then oxidized peroxidase converted the MBH to MB+, MB+ wasreduced at the sensor, resulting in cathodic current.

The potential dependence of the sensor response was investigated,showing that the steady-state current increased with a decrease of the poten-tial from 70.05 to 70.2V, which maybe attribute to an increase drivingforce for fast reduction of POD. Fig. 4 shows the calibration (Fig. 4a) andthe dynamic response (Fig. 4b) of the sensor at applied potential of 70.2V

AMPEROMETRIC BIOSENSOR FOR HYDROGEN PEROXIDASE 693

1.0 0.5 0.0 -0.5 -1.0

-2

0

2

4

6

8

b

a

Cur

rent

(uA

)

Potential(V vs SCE)

Figure 3. Cyclic votalmmograms of the H2O2 sensor at the scan rate of 50 mv/s in

0.2 M phosphate buffer (pH 0.7) in the absence of H2O2 (a) and in the presence of 0.2mM H2O2 (b).

MBH

MBH+

2e 2e

PODred

PODox

H2O2

H2O

Electrode

Scheme 1. Response mechanism for the H2O2 Sensor.

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with successive injection of H2O2. The response time required to reach 95%of maximum response was less than 30s. The sensor response to H2O2 waslinear in the range of 0.01 mM to 0.10 mM.

Effect of pH and Other Parameters on the H2O2 Sensor

The pH dependence of the sensor was illustrated in Fig.5, indicatingthat the optimum pH was 7.0, which exhibited that the pH profile wascontrolled by the enzymatic activity. This proved that the sol-gel matrixhad no effect on the enzyme bioactivity.

The influence of temperature on the sensor had been examinedbetween 15–508C. The immobilized enzyme lost about 50% of its activityat 508C. The experiment showed that the current response increased withtemperature, reaching a maximum value at 458C. A higher temperaturecaused a decrease in response current due to the partial denaturation ofthe enzyme, so all the experiments were tested under the room temperaturedue to the activity of the enzyme.

Other parameters, such as the peroxidase concentration, MB concen-tration and the ratio of Al2O3 to water were also tested (Table 1). To get the

694 CHEN ET AL.

0.00 0.02 0.04 0.06 0.08 0.10

3.9

4.0

4.1

4.2

4.3

4.4

4.5

4.6

4.7

a

Cur

rent

(uA

)

Concentration of H2O2 (mM)

-20 0 20 40 60 80 100 120 140

3.9

4.0

4.1

4.2

4.3

4.4

4.5

4.6

4.7

b

C u

r r e

n t

( u A

)

T i m e ( )

Figure 4. a. The calibration plot for the H2O2 sensor to successive addition of 0.2mMH2O2 steps in 0.2 M phosphate buffer solution at 208C. b. Dynamic response forthe H2O2 sensor.

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most efficient results of the H2O2 sensor, the optimum value was selected asin Table 1.

Analysis and Stability of the H2O2 Sensor

The H2O2 sensor showed high selectivity to several substances, whichmay cause a possible interference at a concentration of 0.2 mM. The resultsshowed that the presence of folic acid, ascorbic acid, tyrosine, lysine,

AMPEROMETRIC BIOSENSOR FOR HYDROGEN PEROXIDASE 695

5.5 6.0 6.5 7.0 7.5 8.0

5

6

7

8

9

10

11

Cur

ren

(uA

)

pH

Figure 5. Effect of pH on the H2O2 sensor. The steady-current is measured in the

presence of 0.2 mM H2O2 in phosphate buffer (pH 7.0) at 208C.

Table 1. Optimization of Peroxidase Immobilization Condition

Experimental Variable Testing Range Selected Value

Peroxidase concentration(mg/ml) 8–12 10MB concentration(M) 1� 10-3� 5� 10-7 1� 10-6

Ratio of Al2O3 to water 1:50� 1:500 1:100Ratio of Al2O3 to enzyme 1:0.1� 1:5 1:1Ratio of Al2O3 to MB 1:0.1� 1:5 1:2

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guanine, glutamic acid and glucose did not cause any observable inter-ference. The lower operating potential by using MB as electron transfermediator minimized interference from other electroactive species.

In addition, experiments showed good reproducibility for the H2O2

biosensor. Over 20 measurements, the sensor exhibited no apparent changein the response to H2O2. In a series of 6 biosensors prepared under the sameconditions, a relative standard deviation of 15% was obtained for the sameH2O2 solution.

The precision of the biosensor was obtained by the determination ofthe recoveries of H2O2 in the range from 0.005mM to 0.097mM by thestandard calibration method. The result was presented in Table 2, the recov-eries were in the range of 96.4–105.0%.

The operational stability of the electrode was evaluated by comparingthe response to 0.02M H2O2 samples. It was found that within 30measurements, the sensitivity of the sensor had decreased less than 10%.The sensor was stored in air at 48C, the decrease in the response current wasabout 10% after one month.

CONCLUSION

In this paper, we concluded that Al2O3 sol-gel immobilization wasquite efficient in retaining the enzyme activity. Additionally, the experimentsshowed that the stability of sensor was also improved by sol-gel immobiliza-tion. While the experiment is illustrated in connection with peroxidase, itcould be extended to other enzyme. It is hoped that this Al2O3 sol-gelderived electrode would find various practical applications.

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Table 2. Recovery for the Hydrogen Peroxide Biosensor

H2O2 Concentration(mM)

Added Found Recovery(%)

0.005 0.0051 102.0

0.012 0.0126 105.00.025 0.0241 96.40.058 0.0574 98.9

0.076 0.0762 100.20.097 0.0981 101.3

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ACKNOWLEDGMENTS

This work was supported by the National Science Foundation ofChina (Projects 39970195 and 29905001) and the ElectroanalyticalChemistry Open Laboratory of Changchun Institute of AppliedChemistry, Chinese Academy of Sciences.

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Received: September 11, 2000Accepted: October 7, 2000

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