Sensors and Actuators B 101 (2004) 155–160

Development of an amperometric biosensor based on glucoseoxidase immobilized through silica sol–gel film onto

Prussian Blue modified electrodeTong Lia,∗, Zihua Yaoa, Liang Dinga,b

a College of Chemistry and Environmental Science, Hebei University, No. 1, Hezou road, Baoding 071002, Chinab Hebei Medical College for Continuing Education, Baoding 071000, China

Received 17 October 2003; received in revised form 15 February 2004; accepted 26 February 2004

Available online 17 April 2004

Abstract

A new approach is described that provides the immobilization of glucose oxidase (GOD) on a Prussian Blue (PB)-modified glassycarbon electrode with a silica sol–gel outer layer. A ‘sandwich’ configuration has been employed in the glucose biosensor. The sensorexhibits high sensitivity and selectivity, which due to both electroreduction of PB-modified electrode for H2O2 at lower potential andsol–gel microenviroment. The sol–gel film outside the enzyme layer contributes to both intensified stability and permselectivity. Theresulting biosensor is highly sensitive to glucose with a linear calibration plot in the concentration range of 0–4.75 mM glucose, slope1.182�A mM−1 l and the detection limit 0.02 mM glucose (S/N = 3). It is performed with excellent reproducibility and long-storagestability. It is attempted being used for clinical assay of diabetic patients blood glucose.© 2004 Elsevier B.V. All rights reserved.

Keywords:Amperometric biosensor; Glucose; Silica sol–gel immobilization; Prussian Blue

1. Introduction

Because of its excellent characteristics, Prussian Blue(PB)-modified electrode has been extensively studied andused, such as electrocatalytic activity, high stability, and easypreparation[1–6]. Since the electrochemically depositedPrussian Blue film exhibits high activity and selectivity forH2O2 electroreduction, i.e., property of biocatalyst, it is alsoknown as an “artificial peroxidase”[7]. Hydrogen peroxideis present in many biological reactions as the main productof several oxidase. Therefore, its determination is of greatimportance for monitoring these bio-processes. Generally,the detection mode involved in oxidase-based biosensors isoften based on the electrochemical detection of hydrogenperoxide, which is produced during the enzyme-catalyzedoxidation of substrates by dissolved oxygen. The direct de-tection of hydrogen peroxide is usually done at platinum orplatinised electrodes through its oxidation at anodic poten-tial (>+0.6 V, Ag/AgCl)[8,9]. Many substances, such as uric

∗ Corresponding author. Tel.:+86-312-5079385;fax: +86-312-5079385.E-mail address:litong1018@sohu.com (T. Li).

acid, acetaminophen, and ascorbic acid, normally presentin biologic samples. These substances can also be electro-chemically oxidized at such a potential, which may cause aninterfering response in the quantitation of substrate concen-tration. PB-modified electrode catalyzes the electrochemicalreduction and decreases the working potential for H2O2analysis, the latter in turn reduces the interfering influence ofmost oxidizable species presented in the sample. Due to itshigh sensitive and selective detection for H2O2, PB-modifiedelectrode has been used in constructing biosensors[6,10–12]. Moreover, Prussian Blue is a relatively cheap andstable electrocatalyst comparing with an enzyme (peroxi-dase). As a result, it is an attractive material for possible massproduction of the base electrode material for biosensors.

On the other hand, one of the key steps in designingand fabrication of biosensors is a simple and reliable pro-cedure of immobilizing and stabilizing reactive enzymes onthe electrode. In previous work, various methods had beenused, including crosslinking with glutaraldehyde, and en-trapment in polymerized films or hydrogel to immobilizethe enzyme for the fabrication of biosensors based on Prus-sian Blue[11–15]. It is well known that crosslinking withglutaraldehyde is an efficient means of enzyme immobiliza-

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.snb.2004.02.047

156 T. Li et al. / Sensors and Actuators B 101 (2004) 155–160

tion. However, glutaraldehyde is detrimental to enzyme, sothe sensitivity of these biosensors is relatively low. As forpolymerized films being used in entrapment of enzymes,such as Nafion and hydrogel, low penetration of polymers,and the swelling property of the hydrogel are still majorproblems.

Braun et al.[16] reported the first attempt to encapsu-late proteins inside silica glasses in 1990. Since then thesenew kinds of inorganic materials are particularly attractiveto the development of electrochemical biosensor becausethey can be prepared under ambient conditions and exhibittunable porosity, high thermal stability, chemical inertness,and negligible swelling in aqueous and non-aqueous solu-tion [17]. The enzymes and proteins can be immobilizedwithin sol–gel matrices whilst maintaining their nativeproperties and reactivities, which makes a potential tool forthe development of biosensors[18–23]. Moreover, suffi-cient amount of trapped interstitial water in gels plays animportant role in the retention of the tertiary structure andactive reactivity of encapsulated biomolecules. The poresize in sol–gel can be controlled into an appropriate sizefor the diffusion of the analyte to the redox active sites, atthe same time preventing enzyme leakage. This property issuitable of development of new biosensors. As we know,it has not reported any attempt has been made to fabricatethe biosensor applying the enzyme immobilized by sol–geltechnique in association with a Prussian Blue modifiedelectrode.

Now there is a chance to develop a biosensor combiningthe merits of the sol–gel technique and Prussian Blue mod-ified electrode. Due to highly-sensitive electrocatalysis ofPB-modified electrode for H2O2 and the improved activityof enzyme, appropriate penetration provided by the sol–gelimmobilization process, a novel, highly sensitive, and stablebiosensor can be developed. In this paper, we described thefabrication of an amperometric biosensor based on glucoseoxidase (GOD) immobilized through silica sol–gel film ontoPrussian Blue modified electrode in a ‘sandwich’ configu-ration. The enzyme electrode was found to improve the ac-tivity of enzyme, to reduce the interference, and to enhancethe sensitivity and stability. This paper also reports the op-timization, the characterization and the application of theprepared sensors in clinical assay of diabetic patients bloodglucose.

2. Experimental

2.1. Reagents

Glucose oxidase (EC 1.1.3.4. 234,900 U g−1, puri-fied from Aspergillus niger) was purchased from Sigma.�-d(+)-glucose was obtained from Beijing chemical fac-tory. Tetramethylortho-silicate (TMOS 98% purity) waspurchased from Wuhan University Silicone New MaterialCo., Ltd. All of the other chemical reagents used were ana-

lytical grade without further purification. All solutions wereprepared with doubly distilled water.

Stock glucose solution of 0.5 M was allowed to mutaro-tate at room temperature for 24 h before use. The phos-phate buffer contains 0.05 M KH2PO4, 0.05 M K2HPO4, and0.1 M KCl.

2.2. Apparatus

All measurements were performed using a conventionalthree-electrode system consisting of a platinum wire counterelectrode, an Ag/AgCl (saturated KCl) reference electrodeand a glassy carbon electrode (3 mm diameter). Electro-chemical experiment and amperometric measurements werecarried out on a microcomputer-based electrochemical an-alyzer (Tianjin Lanlike Chemical and Electron High Tech-nology Co., Ltd). A magnetic stirrer and bar provide theconvective transport. All potentials were reported versus theAg/AgCl (saturated KCl) electrode.

2.3. Preparation of a typical stock standard silica sol–gelsolution

A homogeneous stock standard sol–gel solution was pre-pared within 5 min by vigorously mixing 1 ml of methonal,100�l of TMOS, 20�l of 3.85% methanolic cetyltrimethy-lammonium bromide (CTAB) solution, 20�l of 5×10−3 MNaOH and 300�l H2O in a small test tube at room temper-ature. CTAB serves as a surface-active agent to protect theouter sol–gel film from fracture. The stock standard sol–gelsolution was freshly prepared prior to the fabrication of thebiosensor.

2.4. Preparation of the enzyme electrode

The glass carbon electrode (GCE) was used as the baseelectrode for the sol–gel modified glucose biosensor. A GCEwas polished first with 0.05�m alumna powder and rinsedthoroughly with deionized water after polishing, then suc-cessively washed with 1:1 nitric acid, acetone, and doubledistilled water in an ultrasonic bath, and dried in air beforeuse.

Preparation of the PB-modified electrode was accom-plished according to the literature[24]: the actual depositionof the PB was accomplished in containing 2.5 × 10−3 MFeCl3, 2.5 × 10−3 M K3[Fe(CN)6], 0.1 M HCl, and 0.1 MKCl solution by applying a constant potential to the glassycarbon electrode of 0.4 V for 20 s. The electrode was thencarefully washed with water and transferred into a 0.1 MHCl and 0.1 M KCl solution and electrochemically cycledfor 25 times between 0.35 and−0.05 V at 0.05 V s−1. Afterwashing with water it was dried at 100◦C for 1 h.

The construction of enzyme electrode was accomplishedas follows. Firstly, to coat the surface of the PB-modifiedelectrode with 2�l of GOD solution; After drying for20 min, to depose 6�l of the stock standard sol–gel solution

T. Li et al. / Sensors and Actuators B 101 (2004) 155–160 157

Electrode

Sol-gel protective layerEnzyme layer Prussian Blue layer

Fig. 1. Sandwich configuration in the biosensor.

on the enzyme surface then to dry it. Fig. 1 illustrates the‘sandwich’ configuration employed in the biosensor. Theenzyme electrode was then left under damp conditions at4 ◦C for 24 h before used.

2.5. Amperometric measurement

Amperometric experiments were carried out in an electro-chemical cell holding 10 ml of 0.05 mol l−1 phosphate buffer(pH 6.5). A magnetic stirred and a stirring bar provided theconvective transport in the amperometric experiment. Theenzyme electrode was maintained at a constant temperaturefor 10 min and a holding potential of −0.05 V was applied tothe working electrode. The background current was allowedto decay to a steady state before aliquots of glucose standardsolution were added to the cell. Current–time curves for theamperometric experiments were all recorded at 25±0.5 ◦C.When not being used, the electrode was stored in the refrig-erator at 4 ◦C.

For investigating the performance of the enzyme elec-trode and applying it to the determination of blood glucose,the calibration curve was obtained by its amperometric re-sponses which added aliquots of glucose standard solutioninto phosphate buffer containing 0.05 M KH2PO4, 0.05 MK2HPO4, and 0.1 M KCl.

3. Results and discussion

3.1. Electrodeposition of the PB film

Prussian Blue is deposited on the electrode from aque-ous solutions containing iron (Fe3+) and hexacyanoferrate([Fe(CN)6

3−]) ions. The process takes place on the anodeat a constant potential of 0.4 V versus Ag/AgCl (saturatedKCl) electrode. The reaction is as follow:

4Fe3+ + 3[Fe(CN)63−] + 3e → (FeIII)4[FeII(CN)6

4−]3 ↓

The cyclic voltammogram of the PB-modified electrode reg-istered between 350 and −50 mV in a 0.1 M KCl and 0.1 MHCl solution reveals well-defined cathodic and anodic peakscorresponding to the reduction and the oxidation of PB, re-spectively [24]:

Fig. 2. Cyclic voltammograms of the glucose sensor at the scan rate of0.05 V s−1 in phosphate buffer (pH 6.5) in (a) the absence of H2O2 andin (b) the presence of 5 mmol l−1.

(FeIII)4[FeII(CN)64−]3 + 4K+ + 4e

↔ K4(FeII)4[FeII(CN)64−]3

3.2. Cyclic voltammetry

Fig. 2 showed the cyclic voltammograms obtained withthe enzyme electrode in an unstirred 0.05 M phosphatebuffer (pH 6.5) without H2O2 and with 5 mmol l−1 H2O2.In the absence of H2O2, the enzyme electrode gives noresponse and only the electrochemical behavior of PB wasobserved (Fig. 2a). The redox behavior of PB at the enzymeelectrode showed a reversible electrochemical response. Onaddition of 5 mmol l−1 H2O2, the voltammogram changed,with an increase in the reduction current and a decrease inthe oxidation current (Fig. 2b). Electrrocatalytic reductionof hydrogen peroxide produced by enzymatic oxidation ofglucose was preferable for construction of glucose biosen-sor, because a low applied potential was helpful for prevent-ing interference resulting from easily oxidizable speciessuch as ascorbic acid, uric acid, and acetylaminophenol.Therefore, we used the current response basing on elec-trocatalytic reduction of hydrogen peroxide as analyticalsignals for determination of glucose, because the hydrogenperoxide was produced by enzymatic oxidation of glucose.

3.3. Optimization of experimental conditions

Various experimental parameters affecting the ampero-metric determination of glucose are the amount of enzymeloading, the applied potential, pH of the solution, andtemperature.

Enzyme loading on the electrode has a large effect on theresponse. We compared the responses of the enzyme elec-trode loading different contents of GOD (10, 20, 30, and60 �g). The study showed that current response increasesobviously with increasing of the amount of enzyme load-ing while linear range diminished. Since the oxidase-based

158 T. Li et al. / Sensors and Actuators B 101 (2004) 155–160

100 50 0 -50 -100 -1500.0

0.5

1.0

1.5

E/mV (vs.Ag/AgCl)

I/µ

A

Fig. 3. Effect of applied potential on the enzyme electrode response.

sensor requires oxygen as the co-substrate to carry out ox-idation, the oxygen tension may effect on sensor response.While increasing of the glucose concentration, lack ofoxygen makes oxygen the rate limiting substrate. There-fore, when the enzyme reaction reaches the maximum rate,there is a saturated glucose concentration. This is the rea-son that the linearity is limited [25]. With increasing of theamount of enzyme loading the reaction rate accelerated onthe enzyme electrode. The saturated substrate concentrationwhich reached the reaction maximum rate decreased due tothe lack of oxygen. The linear range diminished. Consider-ing simultaneously both the sensitivity and the linear range,20 �g of enzyme loading was selected.

The effect of applied potential on the enzyme electroderesponse showed that the sensitivity of the enzyme electrodeincreased slightly with diminishing the applied potentialfrom +100 to −150 mV (see Fig. 3). Taking into accountsensitive response, effective avoiding interference and op-erational stability, −50 mV was selected as the appliedpotential in subsequent experiments.

The pH dependence of the enzyme electrode over the pHrange 5.0–7.5 in 0.05 M phosphate buffer in the presenceof 0.5 mM glucose was studied. The optimum response wasachieved in the pH range 6.5–7.5, which is close to the opti-mum pH 7.0 observed for free GOD. Therefore, we selectedpH 6.5 for this study so as to assure higher sensitivity andthe stability of PB.

The effect of temperature on the enzyme electrode re-sponse was studied in the range 15–55 ◦C in a phosphatebuffer (pH 6.5)(see Fig. 4.). The experiment showed thatthe response current increased with temperature, reaching amaximum value at about 40 ◦C. The further increase of tem-perature gave rise to a decrease of the response because ofthe partial denaturation of the enzyme. We observed that thebiosensors exhibited relatively high sensitivity, long-termstability, and operated conveniently at 25 ◦C, so this tem-perature was used throughout the experimental work.

10 20 30 40 50 600.0

0.5

1.0

1.5

Temperature / ˚C

I/µA

Fig. 4. Effect of temperature on the enzyme electrode.

3.4. Electrode response characteristics

Fig. 5 showed a typical current–time response curve ob-tained by using the enzyme electrode under the optimizedexperimental condition, i.e., a holding potential of −0.05 Vin phosphate buffer (pH 6.5) at 25 ◦C. The biosensor ex-hibited a fast and sensitive response to changes in the glu-cose concentration which allowed convenient quantificationof glucose. Resulting calibration curve for glucose over theconcentration range of 0–6.25 mM was presented in the in-set in Fig. 5. The linear calibration range of the biosensorwas from 0 to 4.75 mM with a slope of 1.182 �A mM−1 land a correlation coefficient of 0.9993. The sensitivity of thebiosensor was high comparing with other glucose biosen-sors based on Prussian Blue [11,15,24]. Higher sensitiv-ity of the sensor can be attributed to the biosensor basedon the enzyme immobilized by the sol–gel on PrussianBlue modified electrode in a ‘sandwich’ construction (seeFig. 1). There are two main points: on the one hand, the bio-

200 400 600 800 1000 12000

1

2

3

4

5

6

7

0 2 4 60

2

4

6

I/¦ÌA

[Glucose]/mmol/L

t/s

I/µA

I/µA

Fig. 5. Typical steady-state response of the enzyme electrode to successiveaddition of glucose in steps of 0.25 mmol l−1. The inset illustrates resultingcalibration curve. Conditions: −0.05 V (vs. Ag/AgCl), 25 ◦C, pH 6.5 PBS.

T. Li et al. / Sensors and Actuators B 101 (2004) 155–160 159

500n

A20s

dcb

a

curr

ent

time

Fig. 6. Current-time recording at the enzyme electrode for an additionof (a) 1.0 mmol l−1 glucose, followed by additions of (b) 0.2 mmol l−1

ascorbic acid, (c) 0.5 mmol l−1 acetaminophen, and (d) 0.6 mmol l−1 uricacid, respectively. Other conditions are same as in Fig. 5.

compatible microenviroment provided by the silica sol–gelis helpful to maintain the activity of the enzyme and thisleads to high sensitivity. On the other hand, the enzymeexposes directly to PB film in the ‘sandwich’ construc-tion, and this makes higher sensitive electrocatalysis of PBfilm for H2O2. At a signal-to-noise ratio of 3, the detectionlimit of the biosensor was found to be 0.02 mmol l−1. Theapparent Michaelis–Menten constant (Kapp

m ) of the biosen-sor was found to be 6.7 mM, according to basing on theelectrochemical version of the Lineweave–Burk equation[26].

The biosensor attained 95% of the steady-state responsewithin 12 s for a concentration of 0.5 mM glucose. This rapidresponse was due to the close contact between the PB andbiocatalytic sensing sites of GOD, which allowed the steadystate to be established rapidly due to reducing H2O2, andindicated that the catalytic properties of the enzyme was nothindered by the sol–gel matrix.

The fabrication reproducibility, investigated at 0.5mmol l−1 glucose, was the R.S.D. of 7.2% for five differentbiosensors. For eight replicate measurement at 0.5 mmol l−1

glucose using a typical biosensor, the R.S.D. was 2.4%.The number of interfering species depends on the work-

ing potential and the nature of sample. The species selectedfor interference studies included those likely to appear inbiological, food sample. Addition of sucrose, fructose, lac-tose, maltose individually to 0.5 mM glucose did not alterthe biosensor response. Addition of 0.2 mM ascorbic acid,0.6 mM uric acid, 0.5 mM acetaminophen to 1.0 mM glucosedid not produce any observable interference in the enzymeelectrode response (see Fig. 6).

The storage stability of the biosensor was investigated.The steady-state response current of 0.5 mmol l−1 glucosewas determined every 2 days. This sensor was stored in at4 ◦C when in not use. The result showed the response sensi-tivity was reduced to 90% of its initial value after 45 days.This was because that the PB deposed film had excellentlong-term stability and the sol–gel immobilization was quiteeffective for retaining the activity of GOD. The sol–gelfilm acted as a protective layer preventing from swelling,

Table 1Glucose contents in human serum sample

Simple no. Cglucose (mM) R.S.D. (%)a

Biosensors Spectrophotometric

1 12.07 11.77 2.562 8.95 9.07 1.613 5.69 5.57 2.054 6.78 6.20 2.835 25.19 23.50 1.75

a The R.S.D. of the five measurements by the enzyme electrode.

Table 2Recovery of glucose in human serum

Cglucose (mM) Recovery (%)

Added Founda

5.0 4.91 98.205.0 5.49 109.85.0 4.81 96.207.5 8.03 107.17.5 7.16 95.507.5 7.44 99.20

a The average value of the three measurements.

that gives additional advantage on the immobilizationtechnique.

3.5. Application of the enzyme electrode

Human serum samples were assayed in order to demon-strate the practical usage of the glucose oxidase electrode.Fresh serum samples, taken from human diabetics were firstanalyzed based on spectrophotometric method for the de-tection of glucose. The samples were then reassayed withthe glucose oxidase electrode. Serum sample (200 �l) wasadded into 10 ml of phosphate buffer with pH 6.5, and theresponse was obtained at −0.05 V, 25 ◦C. The contents ofglucose in blood can then be calculated from the calibra-tion curve (see Table 1). The results showed a good agree-ment with those measured by GOD–POD spectrophotomet-ric method (reagent kit for glucose provided by BaodingGreat Wall Clinical Reagents CO. Ltd.).

The accuracy of the enzyme electrode was evaluated bydetermining the recoveries of glucose in human serum sam-ple by a standard addition method. The results presented inTable 2 indicated the method had exhibited reasonable se-lectivity and produced satisfactory results with an averagerecovery of 101.0% and the R.S.D. of 5.9%.

4. Conclusions

A new technique for fabricating glucose biosensors hasbeen developed which features an effective combination ofPB-modified electrode and silica sol–gel immobilization. A‘sandwiched’ configuration was used to construct a stableenzyme electrode. The new technique was reliable, simple,

160 T. Li et al. / Sensors and Actuators B 101 (2004) 155–160

and cost-effective. The silica sol–gel was found to be a bet-ter alternative than expensive Nafion in the case of biosensorbased on Prussian Blue. Using this technique, a biosensorhas been fabricated and exhibited high sensitivity, fast re-sponse and excellent stability. Moreover, the enzyme elec-trode showed good performance for clinical assay of bloodglucose of diabetic patients. However, in terms of linearrange, it is not very satisfying. Further work on this aspectis in progress.

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Biographies

Tong Li received her BSc from Nankai University, Tianjin, in 1984. Thenshe taught in Hebei Medical University. She is a doctoral candidate atpresent in Hebei University. Her current interests are in the area of elec-trochemistry and biosensors.

Zihua Yao is currently a professor of chemistry in College of Chem-istry and Environmental Science, Hebei University, Baoding, PR China.He graduated from Department of Physics, Beijing Normal University,Beijing, in 1965. His research interests are bioinformatics and surfacemicroanalysis.

Liang Ding is currently an associate professor of chemistry in HebeiMedical College for Continuing Education. She received MSc and PhDdegree in chemistry from Hebei University. Her research interests arebioinformatics.