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A highly reversible and sensitive tyrosinase inhibition-based amperometric biosensor for benzoic acid monitoring
Sensors and Actuators B 134 (2008) 1016–1021
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A highly reversible and sensitive tyrosinase inhibition-based amperometric biosensor for benzoic acid monitoring Dan Shan a , Qingbo Li a , Huaiguo Xue a,∗ , Serge Cosnier b a b
College of Chemistry & Chemical Engineering, Yangzhou University, Jiangsu 225002, China Départment de Chimie Moléculaire UMR-5250, ICMG FR-2607, CNRS Université Joseph Fourier, BP-53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Received 9 May 2008 Received in revised form 1 July 2008 Accepted 8 July 2008 Available online 17 July 2008 Keywords: Inhibition Benzoic acid Tyrosinase Calcium carbonate Nanoparticles Biosensor
a b s t r a c t In this present work, a highly reversible and sensitive amperometric biosensor, based on the immobilization of tyrosinase (Tyro) by calcium carbonate nano-materials (nano-CaCO3 ), was applied for determination of food preservative, benzoic acid. The detection of benzoic acid was performed via its inhibiting action on the Tyro/nano-CaCO3 modified glassy carbon electrode. The effects of enzyme substrate type and substrate concentration on the inhibitory were investigated in detail. A potential value of −0.20 V versus SCE, and a constant catechol concentration of 6 M were selective to carry out the amperometric inhibition measurement. The inhibitor biosensor had a fast response to benzoic acid (<5 s) with a wide linear range of 5.6 × 10−7 to 9.2 × 10−5 M and a high sensitivity of 1061.4 ± 13 mA M−1 cm−2 . The inhibiting action of benzoic acid on the Tyro/nano-CaCO3 electrode was highly reversible (100%) and of the typical competitive type, with an apparent inhibition constant of 17 M. This inhibitor biosensor was successfully applied for the determination of benzoic acid in some real beverage sample, such as CocaCola, Pepsi-Cola, Sprite and Yoghurt. Results were compared to those obtained using high performance liquid chromatography, showing a good agreement. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Preservatives having antimicrobial properties are permitted food additives in various food products to preserve them from decay. Benzoic acid is widely regarded as the most active against yeasts, moulds and the least active against bacteria [1]. Thus, benzoic acid and its salt are extensively used as preservatives in foods, beverages, toothpastes, mouthwashes, dentifrices, cosmetics, and pharmaceuticals. However, the presence of the preservatives at higher than permitted safety levels can be harmful to human health [2]; benzoic acid can accumulate in the human body and postpone the growth of child. The maximum permitted concentration of benzoic acid in each type of food is controlled by legislation [3]. Therefore, the analytical determination of benzoic acid is not only important for quality assurance purposes but also for consumer interest and protection. A variety of analytical methods for determining benzoic acid have been reported to date. The most common analytical method for detecting and quantifying benzoic acid is high performance liquid chromatography (HPLC) [4], although other analytical methods such as thin layer chromatography (TLC) [5], capillary electrophore-
∗ Corresponding author. Tel.: +86 514 87971023; fax: +86 514 87975244. E-mail address: [email protected] (H. Xue). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.07.006
sis [6] and gas chromatography (GC) have also been reported. The main disadvantages of these methods are the complexity and cost of the equipments needed and the high level of operator’s skill required. Owing to their exceptional performance capabilities, which include high specificity and sensitivity, rapid response, low cost, relatively compact size and user-friendly operation, electrochemical sensors and biosensors have become an important tool for detection of chemical and biological components. Most recently, several enzyme inhibition-based sensors have appeared in scientific literature for the determination of benzoic acid [7–12]. Among them, the composite biosensors based on graphite-Teflon [10] and polyaniline–polyacrylonitrile [12] have been realized to determine benzoic acid in foodstuffs. Nevertheless, the analytical performance to benzoic acid is still not satisfactory enough. The high sensitivity of biosensor could open up new opportunities for purposeful modification of the sensitivity and selectivity of the determination of inhibitors [11]. Encourage by this opinion, in this context, we purposed to the further development of the amperometric benzoic acid assay by a high sensitive Tyro/nanoCaCO3 electrode. Tyro was immobilized in attractive matrix calcium carbonate nanoparticles via cross-linking procedure by glutaraldehyde. Due to the special excellent properties of the nano-CaCO3 such as good biocompatibility, high surface activity, and high hydrophilicity, the immobilized Tyro maintained its bioactivity and
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native structure [13]. The determination of benzoic acid was carried out through its inhibitory effect on immobilized Tyro. Optimization of benzoic acid inhibition with respect to factor such as enzyme substrate, substrate concentration, biocoating thickness and temperature was presented. The kinetic study of inhibition was also described in detail.
(In%) corresponding to the ratio of the current decrease (I0 –I) versus the original current I0 (no inhibition) in the steady state.
2. Experimental
2.5. Sample pretreatment
2.1. Materials
All of the samples studied were bought at a local supermarket. Yoghurt was first centrifuged to remove insoluble residue. The carbonated drinks, including Coca-Cola, Pepsi-Cola and Sprite, were diluted 10 times with water and degassed by ultrasonication for 5 min.
Tyro (EC 1.14.18.1) from mushroom (807 units mg−1 ) was purchased from Amresco (America). Nano-CaCO3 was prepared by the high-gravity reactive precipitation as reported in literature [14]. All other reagents were of analytical grade and used as received without further purification. All aqueous solutions were prepared in deionized distilled water. Phenolic solutions in 0.1 M phosphate buffer solution (PBS) were prepared daily. 2.2. Apparatus A CHI 660 electrochemical workstation was used for amperometric measurement. A three-electrode cell was used with a saturated calomel electrode (SCE) as reference electrode, a platinum foil as counter electrode. The working electrode was a glassy carbon electrode (diameter 3 mm), which was polished carefully with 0.05 m alumina particles on silk and was then rinsed with distilled water and dried in air before use. All measurements were carried out in a thermostated cell at 25 ◦ C, containing phosphate buffer solution. HPLC measurements were performed on HPLC-10A VP (Shimadzu, Japan). 2.3. Construction of biosensor and enzyme immobilization The nano-CaCO3 colloidal suspension (2 mg ml−1 ) was prepared by dispersing nano-CaCO3 in deionized water with stirring overnight. Tyro was also dissolved in deionized water with a concentration of 4 mg ml−1 . A defined amount of aqueous Tyro/nano-CaCO3 mixture (containing 30 g Tyro, 15 g nanoCaCO3 ) was spread on the surface of the glassy carbon electrode. The mixture was dried at room temperature, leading to an adherent nano-CaCO3 film in which enzymes were entrapped. The resulting electrode was placed in saturated glutaraldehyde vapor at room temperature for 15 min in order to induce the chemical crosslinking of the entrapped enzyme molecules. Before use, the enzyme electrode was rinsed under stirring for 20 min with buffer solution to remove the enzyme not firmly immobilized. After use, the biosensor was stored in phosphate buffer solution overnight at 5 ◦ C.
In% =
I0 − I × 100 I0
The detection limit is based on a signal-to-noise ratio of 3.
3. Results and discussion 3.1. Effect of the enzyme substrate on benzoic acid inhibition In the literature, several substrates for Tyro with different affinities for the active site of the enzyme have been used for amperometric inhibition biosensor designs [15]. The analytical performance for inhibitor varied considerably depending on the characteristics of the substrate. Therefore, five phenolic compounds, catechol, p-cresol, m-cresol, phenol and p-chlorophenol were used as the enzyme substrates for the evaluation of benzoic acid inhibition. The concentration of the phenolic compounds was fixed at 3 M in the present study. Fig. 1 presents their influence on the resulting calibration curves obtained at a Tyro/nano-CaCO3 (40 g/20 g) electrode for the inhibitive detection of benzoic acid. The decreased current due to the presence of benzoic acid is symbolized as I. It appears that the maximum decreased current can be obtained when catechol was used as the enzyme substrate. According to the previous report in the literature, the sensitivity for inhibition can be calculated from the slope of the linear part of the curve obtained from I versus the inhibitor concentration [15]. The sensitivity of Tyro/nano-CaCO3 for benzoic acid follows the sequence: catechol > p-cresol > m-cresol > phenol > pchlorophenol, namely, 532.9 ± 8, 335.7 ± 6, 322.9 ± 5, 317.1 ± 5 and app 94.3 ± 3 mA M−1 cm−2 . The KM value can give information on the enzyme-substrate kinetics for the enzyme electrode. They are
2.4. Benzoic acid measurement procedure To perform each measurement, the Tyro/nano-CaCO3 electrode was dipped into an electrochemical cell containing 10 ml of 0.1 M phosphate buffer solution (pH 6.0) maintained under constant magnetic stirring. The applied potential was fixed to −0.2 V versus SCE. Once the baseline was established (10 min approximately), a defined amount of phenolic compound stock solution was added to the measuring cell. A large reduction current was observed due to the addition of phenolic compound, and a plateau corresponding to the steady-state response was reached in approximately 20 s. Then, the benzoic acid stock solutions were added consecutively, each time a plateau was reached (approximately every 30 s). The addition of benzoic acid solution resulted in a current decrease. The benzoic acid effect was quantified as an inhibition percentage
Fig. 1. The effect of enzyme substrate on the inhibitory of Tyro/nano-CaCO3 bioelectrode to benzoic acid: relations of current change (the difference between the steady-state current in the absence and in the presence of benzoic acid) versus concentration of benzoic acid in 0.1 M PBS (pH 6) containing 3 M enzyme substrate at −0.2 V.
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Table 1 Influence of catechol concentration on benzoic acid inhibition at Tyro/nano-CaCO3 electrode (40 g/20 g) Catechol (M) 1 3 6 12
Inmax (%)
Reversibility (%)
70 80 78 77
70.8 68.2 66.5 27.8
Sensitivity (mA M−1 cm−2 ) 132.9 532.9 1050.2 2374.3
0.256, 0.157, 0.063, 0.043 and 0.03 mM for catechol, p-cresol, mcresol, phenol and p-chlorophenol, respectively [13]. The smaller app KM value means the higher affinity between enzyme and substrate. These data implies that there may be relative between enzyme–substrate affinity and efficiency of inhibition process. The substrate exhibiting the highest affinity to the active enzyme site leads to a less efficient system for benzoic acid determination. In contrast with other substrates, the lowest detection limit of benzoic acid (4 × 10−8 M) can be also obtained when catechol was used as the substrate. These experimental results indicate that catechol is the most appropriate substrate in the inhibition test. As a consequence, catechol was chosen as the enzyme substrate in the following procedure. For an inhibitor biosensor, substrate concentration has to be adjusted carefully in order to obtain correct results [16]. The dependence of catechol concentration on the inhibitory effect was thus investigated under the same conditions but in the presence of different substrate concentrations and the results were summarized in Table 1. The maximum inhibition percentage (Inmax ) is found independent with the catechol concentration, which arrives the similar inhibition degree (about 80%). While the sensitivity of Tyro/nano-CaCO3 for benzoic acid seems to have the inherent relation to catechol concentration, the sensitivity increases with increasing of the substrate concentration in the same amplification. The highest sensitivity is found when catechol concentration being fixed as 12 M. Reversibility is one of the important parameter for purpose of biosensor application. In this present work, reversibility is defined as I0 /I0 , where I0 and I0 correspond to the biosensor response to catechol before and after benzoic acid determination. However, with too concentrated enzyme substrate, the poor reversibility is observed. It might stem from the substrate inhibition [17]. When the enzyme substrate being fixed as 6 M, the biosensor shows more sensitive and exhibits wider linear range of benzoic acid. Therefore, catechol concentration of 6 M was chosen as the fixed concentration in the following procedure. 3.2. Effect of biomembrane thickness on the benzoic acid inhibition In order to optimize the amperometric biosensor for inhibitive determination of benzoic acid, we studied a set of five membranes with different amounts of Tyro loading. The Tyro/nano-CaCO3 ratio being fixed at 2 according to our previous work [13], the effect of film thickness on the biosensor response to benzoic acid was easily examined by varying the amount of Tyro/nano-CaCO3 mixture deposited on the electrode surface. The sensitivity to benzoic acid increased firstly with big amplitude (about three times
± ± ± ±
Detection limit (M) −8
9.9 × 10 4.0 × 10−8 9.9 × 10−8 5.0 × 10−8
4 8 11 16
Linear range (M) 8.9 × 10−7 –8.0 × 10−5 1.6 × 10−6 –1.0 × 10−4 6.9 × 10−7 –1.5 × 10−4 1.3 × 10−6 –1.2 × 10−4
higher) up to 30 g of Tyro loading, and then decreased gradually (Table 2). In addition, the change of reversibility exhibited the same tendency. With thicker biocoating, benzoic acid remained in the biocoating seemed more difficult to be washed out in short time, which showing the poor reversibility. While with too thin biocoating (Tyro/nano-CaCO3 , 20 g/10 g), the immobilized enzyme might release easily from the biocoating, which induced also the poor reversibility. Moreover, too low enzyme loading, significant substrate inhibition could occur [17]. The highest sensitivity to inhibitor and reversibility were both found for a membrane containing relative low enzyme loading (30 g Tyro). The relative low enzyme loading in the sensor element could provide an inhibitor biosensor with the best possible analytical performance, which was consistent with the experiment result obtained by Evtugyn et al. [18]. Therefore, the configuration with 30 g Tyro loading was chosen for the further experiments. 3.3. Analytical properties of the Tyro/nano-CaCO3 electrode to benzoic acid Curve a in Fig. 2 demonstrates the time-dependent response of the Tyro/nano-CaCO3 -modified electrode to benzoic acid. The left part of the response curve shows the amperometric response increase as 6 M catechol being added. The right part shows the response decrease due to the successive addition of benzoic acid into the catechol solution, clearly indicating that benzoic acid inhibits the activity of Tyro immobilized in nano-CaCO3 matrix. For each injection, a quite fast decrease in the current can be observed. The response time of the biosensor to benzoic acid reaching almost 100% of its maximum response is less than 5 s. The inhibition (In%) of benzoic acid increases with increasing its concentration (inset A, Fig. 2). A typical inhibition calibration curve for the determination of benzoic acid is shown in inset B of Fig. 2. The concentration of the substrate (catechol) is fixed at 6 M. There is a linear dependence between the logarithm of the benzoic acid concentration and the inhibition (%). The linear range covers from 5.6 × 10−7 to 9.2 × 10−5 M (R = 0.9936, n = 15), which is broader than those obtained by Tyro-single walled carbon nanotube sensor [19]. The detection limit is 8.0 × 10−8 M, which is much lower that those obtained by the traditional determination method of HPLC (0.5 mg L−1 ) [20] and GC (1 mg L−1 ) [1]. I0.5 , i.e. the concentration of the inhibitor corresponding to 50% of the inhibition signal is calculated to be 12.9 M from the data of Fig. 2. This value is much lower than the value determined for Tyro in solution [21] and that obtained via screen-printed Tyro-containing electrode [22]. The lower I0.5 of inhibition for benzoic acid than the reported results may be contributed to the
Table 2 Influence of biocoating thickness on benzoic acid inhibition at Tyro/nano-CaCO3 electrode. Catechol concentration was fixed as 6 M Thickness (Tyro/nano-CaCO3 )
Inmax (%)
Reversibility (%)
Sensitivity (mA M−1 cm−2 )
20 g/10 g 30 g/15 g 40 g/20 g 60 g/30 g 80 g/40 g
73.5 89.6 78.3 83.3 83.8
68.8 100 89.6 66.2 57.4
331.4 1061.4 1050.2 994.3 982.9
± ± ± ± ±
4 13 11 9 7
Detection limit (M)
Linear range (M)
8.0 × 10−8 8.0 × 10−8 9.9 × 10−8 8.0 × 10−8 8.0 × 10−8
9.5 × 10−7 –9.2 × 10−5 5.6 × 10−7 –9.2 × 10−5 6.9 × 10−7 –1.5 × 10−4 9.5 × 10−7 –1.5 × 10−4 9.5 × 10−7 –2.1 × 10−4
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Fig. 2. The reversibility of inhibition of benzoic acid to the Tyro/nano-CaCO3 modified GCE. (a) Typical current-time recordings at the Tyro/nano-CaCO3 modified GCE upon increasing various concentrations of benzoic acid. Applied potential, −200 mV; 0.1 M PBS containing 6 M catechol. (b) Another response of the same bioelectrode to 6 M catechol after contacting with benzoic acid and rinsing with PBS simply. Inset A: relationship between the inhibition percentage and the concentration of benzoic acid obtained from the data of curve a. Inset B: linearization of the calibration curve (Inset A) obtained on a semilogarithmic scale.
good microenvironment of nano-CaCO3 bring about a considerable enhancement of sensitivity to substrates. These results again further imply that the Tyro activity is well retained at Tyro/nanoCaCO3 hybrid nanocomposite. Especially, the high sensitivity to benzoic acid makes the Tyro/nano-CaCO3 developed as an effective inhibition biosensor to detect benzoic acid at low concentration. Furthermore, the initial response for catechol, i.e. restoration of the enzyme activity, could be obtained again by a short incubation period (10 min) in a phosphate buffer solution. The data indicates the response of Tyro/nano-CaCO3 to catechol retains 100% of its original response (curve b in Fig. 2). This behavior suggests the inhibition of benzoic acid is reversible, which is quite in agreement to the previous report [10,12]. This makes it possible to use continuous measurement methodology for benzoic acid assay. The reproducibility of the biosensor fabrication was evaluated via the comparison of the sensitivity of different electrodes. Nine different enzyme electrodes were tested independently for the determination of benzoic acid, providing a R.S.D. value of 5.6%. This indicates, in particular, an efficient and reproducible immobilization process of tyrosinase in the host matrix calcium carbonate nano-materials although the procedure used was a non-automatic handmade process.
bioelectrode in the presence of different catechol concentrations (Fig. 4). The value obtained for Ki , 17.1 M, agrees with fairly well with that found using a graphite–Teflon–Tyro composite biosensor in a reversed micelle-working medium [10].
3.5. Interferences In order to demonstrate the selectivity of the biosensor, the potential interference from other substances which can be found in drinks on the determination of benzoic acid was checked. The effect of the interferents on the detection signal was checked using 20 M catechol, 20 M benzoic acid and 100 M interferents. Under the experimental conditions, the influence of lactic acid, sorbic acid, citric acid, saccharin sodium, caffeine to the inhibition response of benzoic acid were acceptable, namely 1.3–4.7%, only ascorbic acid influenced on the benzoic acid response producing an increase in the signal of 10.6%.
3.4. Kinetic study and mechanism of the inhibition It is well known that the preparation procedure of a sensor affects the substrate as well as inhibitor kinetics of an enzyme. Thus, in this present work, the kinetic study of the inhibitory for benzoic acid to the Tyro/nano-CaCO3 electrode was also carried out. The inhibition mechanism can be studied by examining the relationship between the response of the Tyro/nano-CaCO3 electrode to the inhibitor and to the substrate concentration. As it can be deduced from Fig. 3, approximately the same value for the maximum current (Imax = 151.5 M), but different apparent app Michaelis–Menten constant (KM ), are obtained in the absence and presence of different benzoic acid concentrations. Moreover, app the KM value increases with increment in benzoic acid concentration (Inset II, Fig. 3). It is concluded that benzoic acid exerted a competitive inhibition; located at Tyro cresolase active site [23]. The apparent inhibition constant, Ki , was calculated from the Dixon plots of the benzoic acid inhibition at Tyro/nano-CaCO3
Fig. 3. Lineweaver–Burk plots for the catechol without (a) and with 20 M (b), app 40 M (c), 60 M (d) and 100 M benzoic acid (e). Inset II shows the plot of KM against concentration of benzoic acid.
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Acknowledgements The authors are grateful to the financial supports of National Natural Science Foundation of China (Grant NO. 20773108 and 20505014), the Key Project of Chinese Ministry of Education (NO. 207041), and the project sponsored for D. Shan by the scientific research foundation for the returned overseas Chinese scholars, state education ministry. References
Fig. 4. Dixon plots of the benzoic acid inhibition at Tyro/nano-CaCO3 bioelectrode in the presence 20 M (a), 30 M (b), 40 M (c) and 80 M catechol (d).
Table 3 Determination of benzoic acid in some drinks by Tyro/nano-CaCO3 electrode and comparison with HPLC method Sample
HPLC method (mg/kg)
Coca-Cola Pepsi-Cola Sprite Yoghurt
127 129 121 11
± ± ± ±
3.4 0.1 1.3 0.1
Biosensor (mg/kg) 130 131 113 12
± ± ± ±
0.4 0.5 0.8 0.2
Relative error (%) +2.3 +1.5 −7.1 +8.3
3.6. Analysis of real samples with the benzoic acid inhibition system The feasibility of practical use of the Tyro/nano-CaCO3 modified electrode was assessed by the determination of benzoic acid concentration in real beverage sample. Four kinds of samples were assayed in order to demonstrate the practical usage of the biosensor. The contents of benzoic acid can then be calculated from the calibration curve and were listed in Table 3. Results were compared with these obtained by HPLC official method [24]. The relative errors are acceptable. The calculated results show that the results determined by Tyro/nano-CaCO3 -modified electrode were in satisfactory agreement with those given by HPLC method, indicating that it is feasible to apply the developed Tyro/nano-CaCO3 -modified electrode to determine benzoic acid in real beverage sample. 4. Conclusion In this work, we have described the development of a benzoic acid biosensor based on the entrapment of Tyro into the nanoCaCO3 membrane. The kinetic interpretation of the amperometric response to catechol for the Tyro/nano-CaCO3 electrode, recorded in the absence and in the presence of benzoic acid, allowed identification of an inhibition process of a competitive type. It is envisaged that the developed biosensor will be conversed to a commercially available and marketable device suitable for food application, due to its advantages such as low cost, ease of fabrication, fast response time, high sensitivity and excellent reversibility.
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Biographies Dan Shan is an Associated Professor in College of Chemistry and Chemical Engineering, Yangzhou University, PR China. She received her MS in physical chemistry from Yangzhou University, China, in 2001 and her PhD in electrochemistry from Joseph Fourier University of Grenoble (France) in 2004. Her current fields of interest include biosensor, interfacial electrochemistry and electroconductive polymer. Qingbo Li received his bachelor’s degree in 2000 from College of Xuzhou Normal University, China. He is currently studying for degree of MS at Yangzhou University. Huai-Guo Xue is presently employed as a Professor in School of Chemistry & Chemical Engineering, Yangzhou University, PR China. He obtained PhD from Institute
1021
of Polymer Science and Engineering, Zhejiang University, PR China, in 2002. His research interests biosensor, polymer chemistry and electrochemistry. Serge Cosnier obtained in 1982 his doctoral degree in chemistry from the Paul Sabatier University of Toulouse (France). In 1983, he joined the Joseph Fourier University of Grenoble (France), where he is currently in charge of bioelectrochemistry team. Since 1983, he has been working in CNRS and is presently Directeur de Recherche. In 2001, he was elected President of the French Group of Bioelectrochemistry and is the Director of the CNRS unit GDR 2619 “Microbiosensors”. His current researches are in the area of electrodes modified by inorganic materials and electrogenerated conducting polymers whose applications are focused on biomimetic electrochemistry, biosensors and biochips.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A highly reversible and sensitive tyrosinase inhibition-based amperometric biosensor for benzoic acid monitoring Dan Shan a , Qingbo Li a , Huaiguo Xue a,∗ , Serge Cosnier b a b
College of Chemistry & Chemical Engineering, Yangzhou University, Jiangsu 225002, China Départment de Chimie Moléculaire UMR-5250, ICMG FR-2607, CNRS Université Joseph Fourier, BP-53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Received 9 May 2008 Received in revised form 1 July 2008 Accepted 8 July 2008 Available online 17 July 2008 Keywords: Inhibition Benzoic acid Tyrosinase Calcium carbonate Nanoparticles Biosensor
a b s t r a c t In this present work, a highly reversible and sensitive amperometric biosensor, based on the immobilization of tyrosinase (Tyro) by calcium carbonate nano-materials (nano-CaCO3 ), was applied for determination of food preservative, benzoic acid. The detection of benzoic acid was performed via its inhibiting action on the Tyro/nano-CaCO3 modified glassy carbon electrode. The effects of enzyme substrate type and substrate concentration on the inhibitory were investigated in detail. A potential value of −0.20 V versus SCE, and a constant catechol concentration of 6 M were selective to carry out the amperometric inhibition measurement. The inhibitor biosensor had a fast response to benzoic acid (<5 s) with a wide linear range of 5.6 × 10−7 to 9.2 × 10−5 M and a high sensitivity of 1061.4 ± 13 mA M−1 cm−2 . The inhibiting action of benzoic acid on the Tyro/nano-CaCO3 electrode was highly reversible (100%) and of the typical competitive type, with an apparent inhibition constant of 17 M. This inhibitor biosensor was successfully applied for the determination of benzoic acid in some real beverage sample, such as CocaCola, Pepsi-Cola, Sprite and Yoghurt. Results were compared to those obtained using high performance liquid chromatography, showing a good agreement. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Preservatives having antimicrobial properties are permitted food additives in various food products to preserve them from decay. Benzoic acid is widely regarded as the most active against yeasts, moulds and the least active against bacteria [1]. Thus, benzoic acid and its salt are extensively used as preservatives in foods, beverages, toothpastes, mouthwashes, dentifrices, cosmetics, and pharmaceuticals. However, the presence of the preservatives at higher than permitted safety levels can be harmful to human health [2]; benzoic acid can accumulate in the human body and postpone the growth of child. The maximum permitted concentration of benzoic acid in each type of food is controlled by legislation [3]. Therefore, the analytical determination of benzoic acid is not only important for quality assurance purposes but also for consumer interest and protection. A variety of analytical methods for determining benzoic acid have been reported to date. The most common analytical method for detecting and quantifying benzoic acid is high performance liquid chromatography (HPLC) [4], although other analytical methods such as thin layer chromatography (TLC) [5], capillary electrophore-
∗ Corresponding author. Tel.: +86 514 87971023; fax: +86 514 87975244. E-mail address: [email protected] (H. Xue). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.07.006
sis [6] and gas chromatography (GC) have also been reported. The main disadvantages of these methods are the complexity and cost of the equipments needed and the high level of operator’s skill required. Owing to their exceptional performance capabilities, which include high specificity and sensitivity, rapid response, low cost, relatively compact size and user-friendly operation, electrochemical sensors and biosensors have become an important tool for detection of chemical and biological components. Most recently, several enzyme inhibition-based sensors have appeared in scientific literature for the determination of benzoic acid [7–12]. Among them, the composite biosensors based on graphite-Teflon [10] and polyaniline–polyacrylonitrile [12] have been realized to determine benzoic acid in foodstuffs. Nevertheless, the analytical performance to benzoic acid is still not satisfactory enough. The high sensitivity of biosensor could open up new opportunities for purposeful modification of the sensitivity and selectivity of the determination of inhibitors [11]. Encourage by this opinion, in this context, we purposed to the further development of the amperometric benzoic acid assay by a high sensitive Tyro/nanoCaCO3 electrode. Tyro was immobilized in attractive matrix calcium carbonate nanoparticles via cross-linking procedure by glutaraldehyde. Due to the special excellent properties of the nano-CaCO3 such as good biocompatibility, high surface activity, and high hydrophilicity, the immobilized Tyro maintained its bioactivity and
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native structure [13]. The determination of benzoic acid was carried out through its inhibitory effect on immobilized Tyro. Optimization of benzoic acid inhibition with respect to factor such as enzyme substrate, substrate concentration, biocoating thickness and temperature was presented. The kinetic study of inhibition was also described in detail.
(In%) corresponding to the ratio of the current decrease (I0 –I) versus the original current I0 (no inhibition) in the steady state.
2. Experimental
2.5. Sample pretreatment
2.1. Materials
All of the samples studied were bought at a local supermarket. Yoghurt was first centrifuged to remove insoluble residue. The carbonated drinks, including Coca-Cola, Pepsi-Cola and Sprite, were diluted 10 times with water and degassed by ultrasonication for 5 min.
Tyro (EC 1.14.18.1) from mushroom (807 units mg−1 ) was purchased from Amresco (America). Nano-CaCO3 was prepared by the high-gravity reactive precipitation as reported in literature [14]. All other reagents were of analytical grade and used as received without further purification. All aqueous solutions were prepared in deionized distilled water. Phenolic solutions in 0.1 M phosphate buffer solution (PBS) were prepared daily. 2.2. Apparatus A CHI 660 electrochemical workstation was used for amperometric measurement. A three-electrode cell was used with a saturated calomel electrode (SCE) as reference electrode, a platinum foil as counter electrode. The working electrode was a glassy carbon electrode (diameter 3 mm), which was polished carefully with 0.05 m alumina particles on silk and was then rinsed with distilled water and dried in air before use. All measurements were carried out in a thermostated cell at 25 ◦ C, containing phosphate buffer solution. HPLC measurements were performed on HPLC-10A VP (Shimadzu, Japan). 2.3. Construction of biosensor and enzyme immobilization The nano-CaCO3 colloidal suspension (2 mg ml−1 ) was prepared by dispersing nano-CaCO3 in deionized water with stirring overnight. Tyro was also dissolved in deionized water with a concentration of 4 mg ml−1 . A defined amount of aqueous Tyro/nano-CaCO3 mixture (containing 30 g Tyro, 15 g nanoCaCO3 ) was spread on the surface of the glassy carbon electrode. The mixture was dried at room temperature, leading to an adherent nano-CaCO3 film in which enzymes were entrapped. The resulting electrode was placed in saturated glutaraldehyde vapor at room temperature for 15 min in order to induce the chemical crosslinking of the entrapped enzyme molecules. Before use, the enzyme electrode was rinsed under stirring for 20 min with buffer solution to remove the enzyme not firmly immobilized. After use, the biosensor was stored in phosphate buffer solution overnight at 5 ◦ C.
In% =
I0 − I × 100 I0
The detection limit is based on a signal-to-noise ratio of 3.
3. Results and discussion 3.1. Effect of the enzyme substrate on benzoic acid inhibition In the literature, several substrates for Tyro with different affinities for the active site of the enzyme have been used for amperometric inhibition biosensor designs [15]. The analytical performance for inhibitor varied considerably depending on the characteristics of the substrate. Therefore, five phenolic compounds, catechol, p-cresol, m-cresol, phenol and p-chlorophenol were used as the enzyme substrates for the evaluation of benzoic acid inhibition. The concentration of the phenolic compounds was fixed at 3 M in the present study. Fig. 1 presents their influence on the resulting calibration curves obtained at a Tyro/nano-CaCO3 (40 g/20 g) electrode for the inhibitive detection of benzoic acid. The decreased current due to the presence of benzoic acid is symbolized as I. It appears that the maximum decreased current can be obtained when catechol was used as the enzyme substrate. According to the previous report in the literature, the sensitivity for inhibition can be calculated from the slope of the linear part of the curve obtained from I versus the inhibitor concentration [15]. The sensitivity of Tyro/nano-CaCO3 for benzoic acid follows the sequence: catechol > p-cresol > m-cresol > phenol > pchlorophenol, namely, 532.9 ± 8, 335.7 ± 6, 322.9 ± 5, 317.1 ± 5 and app 94.3 ± 3 mA M−1 cm−2 . The KM value can give information on the enzyme-substrate kinetics for the enzyme electrode. They are
2.4. Benzoic acid measurement procedure To perform each measurement, the Tyro/nano-CaCO3 electrode was dipped into an electrochemical cell containing 10 ml of 0.1 M phosphate buffer solution (pH 6.0) maintained under constant magnetic stirring. The applied potential was fixed to −0.2 V versus SCE. Once the baseline was established (10 min approximately), a defined amount of phenolic compound stock solution was added to the measuring cell. A large reduction current was observed due to the addition of phenolic compound, and a plateau corresponding to the steady-state response was reached in approximately 20 s. Then, the benzoic acid stock solutions were added consecutively, each time a plateau was reached (approximately every 30 s). The addition of benzoic acid solution resulted in a current decrease. The benzoic acid effect was quantified as an inhibition percentage
Fig. 1. The effect of enzyme substrate on the inhibitory of Tyro/nano-CaCO3 bioelectrode to benzoic acid: relations of current change (the difference between the steady-state current in the absence and in the presence of benzoic acid) versus concentration of benzoic acid in 0.1 M PBS (pH 6) containing 3 M enzyme substrate at −0.2 V.
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Table 1 Influence of catechol concentration on benzoic acid inhibition at Tyro/nano-CaCO3 electrode (40 g/20 g) Catechol (M) 1 3 6 12
Inmax (%)
Reversibility (%)
70 80 78 77
70.8 68.2 66.5 27.8
Sensitivity (mA M−1 cm−2 ) 132.9 532.9 1050.2 2374.3
0.256, 0.157, 0.063, 0.043 and 0.03 mM for catechol, p-cresol, mcresol, phenol and p-chlorophenol, respectively [13]. The smaller app KM value means the higher affinity between enzyme and substrate. These data implies that there may be relative between enzyme–substrate affinity and efficiency of inhibition process. The substrate exhibiting the highest affinity to the active enzyme site leads to a less efficient system for benzoic acid determination. In contrast with other substrates, the lowest detection limit of benzoic acid (4 × 10−8 M) can be also obtained when catechol was used as the substrate. These experimental results indicate that catechol is the most appropriate substrate in the inhibition test. As a consequence, catechol was chosen as the enzyme substrate in the following procedure. For an inhibitor biosensor, substrate concentration has to be adjusted carefully in order to obtain correct results [16]. The dependence of catechol concentration on the inhibitory effect was thus investigated under the same conditions but in the presence of different substrate concentrations and the results were summarized in Table 1. The maximum inhibition percentage (Inmax ) is found independent with the catechol concentration, which arrives the similar inhibition degree (about 80%). While the sensitivity of Tyro/nano-CaCO3 for benzoic acid seems to have the inherent relation to catechol concentration, the sensitivity increases with increasing of the substrate concentration in the same amplification. The highest sensitivity is found when catechol concentration being fixed as 12 M. Reversibility is one of the important parameter for purpose of biosensor application. In this present work, reversibility is defined as I0 /I0 , where I0 and I0 correspond to the biosensor response to catechol before and after benzoic acid determination. However, with too concentrated enzyme substrate, the poor reversibility is observed. It might stem from the substrate inhibition [17]. When the enzyme substrate being fixed as 6 M, the biosensor shows more sensitive and exhibits wider linear range of benzoic acid. Therefore, catechol concentration of 6 M was chosen as the fixed concentration in the following procedure. 3.2. Effect of biomembrane thickness on the benzoic acid inhibition In order to optimize the amperometric biosensor for inhibitive determination of benzoic acid, we studied a set of five membranes with different amounts of Tyro loading. The Tyro/nano-CaCO3 ratio being fixed at 2 according to our previous work [13], the effect of film thickness on the biosensor response to benzoic acid was easily examined by varying the amount of Tyro/nano-CaCO3 mixture deposited on the electrode surface. The sensitivity to benzoic acid increased firstly with big amplitude (about three times
± ± ± ±
Detection limit (M) −8
9.9 × 10 4.0 × 10−8 9.9 × 10−8 5.0 × 10−8
4 8 11 16
Linear range (M) 8.9 × 10−7 –8.0 × 10−5 1.6 × 10−6 –1.0 × 10−4 6.9 × 10−7 –1.5 × 10−4 1.3 × 10−6 –1.2 × 10−4
higher) up to 30 g of Tyro loading, and then decreased gradually (Table 2). In addition, the change of reversibility exhibited the same tendency. With thicker biocoating, benzoic acid remained in the biocoating seemed more difficult to be washed out in short time, which showing the poor reversibility. While with too thin biocoating (Tyro/nano-CaCO3 , 20 g/10 g), the immobilized enzyme might release easily from the biocoating, which induced also the poor reversibility. Moreover, too low enzyme loading, significant substrate inhibition could occur [17]. The highest sensitivity to inhibitor and reversibility were both found for a membrane containing relative low enzyme loading (30 g Tyro). The relative low enzyme loading in the sensor element could provide an inhibitor biosensor with the best possible analytical performance, which was consistent with the experiment result obtained by Evtugyn et al. [18]. Therefore, the configuration with 30 g Tyro loading was chosen for the further experiments. 3.3. Analytical properties of the Tyro/nano-CaCO3 electrode to benzoic acid Curve a in Fig. 2 demonstrates the time-dependent response of the Tyro/nano-CaCO3 -modified electrode to benzoic acid. The left part of the response curve shows the amperometric response increase as 6 M catechol being added. The right part shows the response decrease due to the successive addition of benzoic acid into the catechol solution, clearly indicating that benzoic acid inhibits the activity of Tyro immobilized in nano-CaCO3 matrix. For each injection, a quite fast decrease in the current can be observed. The response time of the biosensor to benzoic acid reaching almost 100% of its maximum response is less than 5 s. The inhibition (In%) of benzoic acid increases with increasing its concentration (inset A, Fig. 2). A typical inhibition calibration curve for the determination of benzoic acid is shown in inset B of Fig. 2. The concentration of the substrate (catechol) is fixed at 6 M. There is a linear dependence between the logarithm of the benzoic acid concentration and the inhibition (%). The linear range covers from 5.6 × 10−7 to 9.2 × 10−5 M (R = 0.9936, n = 15), which is broader than those obtained by Tyro-single walled carbon nanotube sensor [19]. The detection limit is 8.0 × 10−8 M, which is much lower that those obtained by the traditional determination method of HPLC (0.5 mg L−1 ) [20] and GC (1 mg L−1 ) [1]. I0.5 , i.e. the concentration of the inhibitor corresponding to 50% of the inhibition signal is calculated to be 12.9 M from the data of Fig. 2. This value is much lower than the value determined for Tyro in solution [21] and that obtained via screen-printed Tyro-containing electrode [22]. The lower I0.5 of inhibition for benzoic acid than the reported results may be contributed to the
Table 2 Influence of biocoating thickness on benzoic acid inhibition at Tyro/nano-CaCO3 electrode. Catechol concentration was fixed as 6 M Thickness (Tyro/nano-CaCO3 )
Inmax (%)
Reversibility (%)
Sensitivity (mA M−1 cm−2 )
20 g/10 g 30 g/15 g 40 g/20 g 60 g/30 g 80 g/40 g
73.5 89.6 78.3 83.3 83.8
68.8 100 89.6 66.2 57.4
331.4 1061.4 1050.2 994.3 982.9
± ± ± ± ±
4 13 11 9 7
Detection limit (M)
Linear range (M)
8.0 × 10−8 8.0 × 10−8 9.9 × 10−8 8.0 × 10−8 8.0 × 10−8
9.5 × 10−7 –9.2 × 10−5 5.6 × 10−7 –9.2 × 10−5 6.9 × 10−7 –1.5 × 10−4 9.5 × 10−7 –1.5 × 10−4 9.5 × 10−7 –2.1 × 10−4
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Fig. 2. The reversibility of inhibition of benzoic acid to the Tyro/nano-CaCO3 modified GCE. (a) Typical current-time recordings at the Tyro/nano-CaCO3 modified GCE upon increasing various concentrations of benzoic acid. Applied potential, −200 mV; 0.1 M PBS containing 6 M catechol. (b) Another response of the same bioelectrode to 6 M catechol after contacting with benzoic acid and rinsing with PBS simply. Inset A: relationship between the inhibition percentage and the concentration of benzoic acid obtained from the data of curve a. Inset B: linearization of the calibration curve (Inset A) obtained on a semilogarithmic scale.
good microenvironment of nano-CaCO3 bring about a considerable enhancement of sensitivity to substrates. These results again further imply that the Tyro activity is well retained at Tyro/nanoCaCO3 hybrid nanocomposite. Especially, the high sensitivity to benzoic acid makes the Tyro/nano-CaCO3 developed as an effective inhibition biosensor to detect benzoic acid at low concentration. Furthermore, the initial response for catechol, i.e. restoration of the enzyme activity, could be obtained again by a short incubation period (10 min) in a phosphate buffer solution. The data indicates the response of Tyro/nano-CaCO3 to catechol retains 100% of its original response (curve b in Fig. 2). This behavior suggests the inhibition of benzoic acid is reversible, which is quite in agreement to the previous report [10,12]. This makes it possible to use continuous measurement methodology for benzoic acid assay. The reproducibility of the biosensor fabrication was evaluated via the comparison of the sensitivity of different electrodes. Nine different enzyme electrodes were tested independently for the determination of benzoic acid, providing a R.S.D. value of 5.6%. This indicates, in particular, an efficient and reproducible immobilization process of tyrosinase in the host matrix calcium carbonate nano-materials although the procedure used was a non-automatic handmade process.
bioelectrode in the presence of different catechol concentrations (Fig. 4). The value obtained for Ki , 17.1 M, agrees with fairly well with that found using a graphite–Teflon–Tyro composite biosensor in a reversed micelle-working medium [10].
3.5. Interferences In order to demonstrate the selectivity of the biosensor, the potential interference from other substances which can be found in drinks on the determination of benzoic acid was checked. The effect of the interferents on the detection signal was checked using 20 M catechol, 20 M benzoic acid and 100 M interferents. Under the experimental conditions, the influence of lactic acid, sorbic acid, citric acid, saccharin sodium, caffeine to the inhibition response of benzoic acid were acceptable, namely 1.3–4.7%, only ascorbic acid influenced on the benzoic acid response producing an increase in the signal of 10.6%.
3.4. Kinetic study and mechanism of the inhibition It is well known that the preparation procedure of a sensor affects the substrate as well as inhibitor kinetics of an enzyme. Thus, in this present work, the kinetic study of the inhibitory for benzoic acid to the Tyro/nano-CaCO3 electrode was also carried out. The inhibition mechanism can be studied by examining the relationship between the response of the Tyro/nano-CaCO3 electrode to the inhibitor and to the substrate concentration. As it can be deduced from Fig. 3, approximately the same value for the maximum current (Imax = 151.5 M), but different apparent app Michaelis–Menten constant (KM ), are obtained in the absence and presence of different benzoic acid concentrations. Moreover, app the KM value increases with increment in benzoic acid concentration (Inset II, Fig. 3). It is concluded that benzoic acid exerted a competitive inhibition; located at Tyro cresolase active site [23]. The apparent inhibition constant, Ki , was calculated from the Dixon plots of the benzoic acid inhibition at Tyro/nano-CaCO3
Fig. 3. Lineweaver–Burk plots for the catechol without (a) and with 20 M (b), app 40 M (c), 60 M (d) and 100 M benzoic acid (e). Inset II shows the plot of KM against concentration of benzoic acid.
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Acknowledgements The authors are grateful to the financial supports of National Natural Science Foundation of China (Grant NO. 20773108 and 20505014), the Key Project of Chinese Ministry of Education (NO. 207041), and the project sponsored for D. Shan by the scientific research foundation for the returned overseas Chinese scholars, state education ministry. References
Fig. 4. Dixon plots of the benzoic acid inhibition at Tyro/nano-CaCO3 bioelectrode in the presence 20 M (a), 30 M (b), 40 M (c) and 80 M catechol (d).
Table 3 Determination of benzoic acid in some drinks by Tyro/nano-CaCO3 electrode and comparison with HPLC method Sample
HPLC method (mg/kg)
Coca-Cola Pepsi-Cola Sprite Yoghurt
127 129 121 11
± ± ± ±
3.4 0.1 1.3 0.1
Biosensor (mg/kg) 130 131 113 12
± ± ± ±
0.4 0.5 0.8 0.2
Relative error (%) +2.3 +1.5 −7.1 +8.3
3.6. Analysis of real samples with the benzoic acid inhibition system The feasibility of practical use of the Tyro/nano-CaCO3 modified electrode was assessed by the determination of benzoic acid concentration in real beverage sample. Four kinds of samples were assayed in order to demonstrate the practical usage of the biosensor. The contents of benzoic acid can then be calculated from the calibration curve and were listed in Table 3. Results were compared with these obtained by HPLC official method [24]. The relative errors are acceptable. The calculated results show that the results determined by Tyro/nano-CaCO3 -modified electrode were in satisfactory agreement with those given by HPLC method, indicating that it is feasible to apply the developed Tyro/nano-CaCO3 -modified electrode to determine benzoic acid in real beverage sample. 4. Conclusion In this work, we have described the development of a benzoic acid biosensor based on the entrapment of Tyro into the nanoCaCO3 membrane. The kinetic interpretation of the amperometric response to catechol for the Tyro/nano-CaCO3 electrode, recorded in the absence and in the presence of benzoic acid, allowed identification of an inhibition process of a competitive type. It is envisaged that the developed biosensor will be conversed to a commercially available and marketable device suitable for food application, due to its advantages such as low cost, ease of fabrication, fast response time, high sensitivity and excellent reversibility.
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Biographies Dan Shan is an Associated Professor in College of Chemistry and Chemical Engineering, Yangzhou University, PR China. She received her MS in physical chemistry from Yangzhou University, China, in 2001 and her PhD in electrochemistry from Joseph Fourier University of Grenoble (France) in 2004. Her current fields of interest include biosensor, interfacial electrochemistry and electroconductive polymer. Qingbo Li received his bachelor’s degree in 2000 from College of Xuzhou Normal University, China. He is currently studying for degree of MS at Yangzhou University. Huai-Guo Xue is presently employed as a Professor in School of Chemistry & Chemical Engineering, Yangzhou University, PR China. He obtained PhD from Institute
1021
of Polymer Science and Engineering, Zhejiang University, PR China, in 2002. His research interests biosensor, polymer chemistry and electrochemistry. Serge Cosnier obtained in 1982 his doctoral degree in chemistry from the Paul Sabatier University of Toulouse (France). In 1983, he joined the Joseph Fourier University of Grenoble (France), where he is currently in charge of bioelectrochemistry team. Since 1983, he has been working in CNRS and is presently Directeur de Recherche. In 2001, he was elected President of the French Group of Bioelectrochemistry and is the Director of the CNRS unit GDR 2619 “Microbiosensors”. His current researches are in the area of electrodes modified by inorganic materials and electrogenerated conducting polymers whose applications are focused on biomimetic electrochemistry, biosensors and biochips.