Microchimica Acta

, Volume 174, Issue 3–4, pp 303–309 | Cite as

Biosensor based on a glassy carbon electrode modified with tyrosinase immmobilized on multiwalled carbon nanotubes

  • Jing Ren
  • Tian-Fang Kang
  • Rui Xue
  • Chao-Nan Ge
  • Shui-Yuan Cheng
Original Paper


We describe a biosensor for phenolic compounds that is based on a glassy carbon electrode modified with tyrosinase immobilized on multiwalled carbon nanotubes (MWNTs). The MWNTs possess excellent inherent electrical conductivity which enhances the electron transfer rate and results in good electrochemical catalytic activity towards the reduction of benzoquinone produced by enzymatic reaction. The biosensor was characterized by cyclic voltammetry, and the experimental conditions were optimized. The cathodíc current is linearly related to the concentration of the phenols between 0.4 μM and 10 μM, and the detection limit is 0.2 μM. The method was applied to the determination of phenol in water samples.


A tyrosinase and carbon nanotubes (MWNTs) modified glassy carbon electrode was fabricated and used for the sensitive detection of phenol. The reduction peak of benzoquinone produced by enzymatic reaction of phenol was greatly enhanced due to the presence of MWNTs(c)


Tyrosinase Multiwalled carbon nanotubes Electrochemical biosensors Phenols 


Phenolic compounds often exist in the wastewaters of many industries. Many of them are very toxic, showing adverse effects on animal and plants [1]. High levels of phenols have detrimental effects on animal health. Prolonged oral or subcutaneous exposure causes damage to the lungs, liver, kidney and genitourinary tract [2]. Therefore, the determination of phenolic compounds is of great importance. Spectrophotometry and chromatography are commonly used to determine phenols [3, 4]. However, these methods are complicated in sample pretreatment and unsuitable for in situ monitoring. Biosensor based on tyrosinase (Tyr) have been proven to be a promising method for a simple, fast and sensitive detection of phenolic compounds [5, 6, 7, 8, 9, 10].

Tyrosinase can catalyse two reactions: ortho-hydroxylation of phenols to catechol and the further oxidation of catechols to ortho-quinones, both in the presence of molecular oxygen. The phenol can be detected via the reduction of the produced quinone at low potential [11, 12, 13].
$$ {\text{phenol}} + {\text{tyrosinase}}\left( {{{\text{O}}_2}} \right) \to {\text{catechol}} $$
$$ {\text{catechol}} + {\text{tyrosinase}}\left( {{{\text{O}}_2}} \right) \to {\text{o}} - {\text{quinone}} + {{\text{H}}_2}{\text{O}} $$
$$ {\text{o}} - {\text{quinone}} + {2}{{\text{H}}^{+} } + {2}{{\text{e}}^{-} } \to {\text{catechol}} $$

Recently many tyrosinase biosensors for phenols have been presented. Various materials such as magnetic nanoparticles [14], metal nanoparticles [15, 16] and polymers [12, 17] have been used to fabricate tyrosinase biosensors. Many methods have been employed for stabilizing and immobilizing the enzyme. Polymer entrapment [18, 19], electropolymerization [20, 21], sol–gels [22, 23, 24], self-assembled monolayers [25, 26], covalent linking [2, 27] and incorporation within carbon paste [11, 16, 28] have been used to construct tyrosinase biosensors. Although these materials have their own advantages in enzyme immobilization, they also have some drawbacks. Take silica sol–gel films as a example, it can retain the catalytic activities of enzymes to a large extent. But the fabrication of silica sol–gel films is time consuming and the silica sol–gel matrix is fragile, it easily shrinks, cracks and delaminates from the electrode surface [29]. Many methods which have been reported about biosensor fabrication are time consuming and consist of many steps, which result in difficulties in obtaining sensors with comparable sensitivity. Thus, more efforts are needed to develop a simple and reliable method to fabricate biosensors.

Nanomaterials possess extreme small size, a high specific surface area, a high surface-to-volume ratio and unique physicochemical characteristics. The use of nanomaterials superstructures for the creation of electrochemical devices is an extremely promising prospect. They hold important applications as catalysts. Carbon nanotubes (CNTs), as a new form of carbon, are of great interest for many applications in, for example, batteries and chemical sensors [30, 31, 32, 33]. Furthermore, high accessible surface area, low resistance and high stability suggest that CNTs are suitable for the potential catalyst supports [34, 35, 36]. After acid treatment, CNTs possess more active sites. CNTs can promote the electron transfer of the active site of biological molecules, and improve relative activity of the enzyme.

Recently, there has been growing interest in using CNTs to fabricate biosensors. Korkut et al. reported an amperometric biosensor based on horseradish peroxidase and CNTs/polypyrrole nanobiocomposite film on a gold surface for determination of phenol derivatives [37]. Yin et al. fabricated an amperometric biosensor based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine (MWNTs-CoPc) silk fibroin film to determine bisphenol A [38].

We describe sensitive biosensors for phenols here which based on tyrosinase immobilized onto MWNTs modified glassy carbon electrodes (Tyr/MWNTs/GCE). The novel characteristics of the biosensors are simple fabrication, excellent stability and high sensitivity for the determination of phonlic compounds. The characterization and performance of this tyrosinase biosensor was studied. Additionally, the biosensor has been successfully used for the detection of phenol in real samples.



Tyrosinase (EC 232-653-4, 3933 unit·mg−1 from mushroom) was purchased from Aldrich (http://www.sigmaaldrich.com, USA). Multiwalled carbon nanotubes (95%) were purchased from Shenzhen Nanotech Port Co. Ltd. (http://www.seasunnano.com, China). Pretreatment of MWNTs was performed by refluxing in HCl + HNO3 solution (3:1) at 110 °C for 8 h, followed by filtrating and washing with doubly distilled water till pH 7.0, dried at 110 °C for 2 h. After then, 1.0 mg MWNTs was dispersed in 10 mL DMF with the aid of ultrasonic agitation. 0.1 mg ·mL−1 treated MWNTs suspension was therefore obtained [34]. Phosphate buffer solution of pH 7.5, 0.1 M was prepared using K2HPO4 and KH2PO4 in redistilled water. All of the chemicals were of analytical reagent grade and all the solutions were prepared with redistilled deionized water.


All electrochemical measurements were performed with CHI842B electrochemical analyzer (Shanghai Chenhua Co., China) with a conventional three-electrode cell. A glassy carbon electrode (GCE) with diameter of 3 mm was used as working electrode, saturated calomel electrode (SCE) as reference electrode, and a platinum wire was used as auxiliary electrode.

Preparation of the electrodes and amperometric measurements

The bare GCE was polished successively with 0.5 μm Al2O3 slurry to a mirror, and ultrasonically cleaned in ethanol and water for 5 min, respectively, then washed with redistilled deionized water and dried in air before use. A 2.0 μL aliquot of the treated MWNTs solution was cast on the GCE surface, dried in air. The chemically modified electrode was denoted as MWNTs/GCE.

To dissolve 1.2 mg of tyrosinase (3,933 units·mg−1) and 3.6 mg of bovine serum albinum (BSA) in 0.2 mL phosphate buffer solution, 10 μL of this solution was mixed with 2 μL of 2.5% glutaraldehyde. 4 μL of this mixture was then deposited upon the MWNTs/GCE surface and allowed to cross-link to dryness at room temperature. This enzyme activity was calculated and it approximately equal to 80 units on each electrode surface. The electrode was denoted as Tyr/MWNTs/GCE. For comparison, Tyr/GCE was fabricated with the same method. When not in use, the biosensor was stored at 4 °C in a refrigerator.

Amperometric experiments were carried out in an electrochemical cell containing 10 mL of 0.1 M pH 7.5 phosphate buffer solution. All measurements were performed at room temperature.

Results and discussion

Electrocatalysis of MWNTs modified electrodes to quinone

The biosensor based on MWNTs with immobilized tyrosinase was first characterized using cyclic voltammetry (CV) to test its response to phenol. The immobilized tyrosinase in the presence of molecular oxygen can catalyse the oxidation of phenol to form benzoquinone. Cathodic potential scan was used to measure the reduction current of benzoquinone. The experimental results indicated that the electrochemical reduction peak of the quinone was sensitive. The reduction peak current increased with the amount of phenol added. Figure 1 shows cyclic voltammetric responses of Tyr/MWNTs/GCE in absence of phenol(a) and in presence of phenol (c). Moreover, cyclic voltammetric responses of phenol at Tyr/GCE (b) is also showed for comparison. No reduction peak for quinone was observed when the phenol is absence. After phenol solution was added into Phosphate buffer solution, benzoquinone, the enzymatic reaction product of phenol, at both Tyr/MWNTs/GCE and Tyr/GCE appeared as a reduction peak. But the reduction peak of benzoquinone at Tyr/MWNTs/GCE was much higher than that at Tyr/GCE, the peak potentials shifted positively from −0.08 V at Tyr/GCE to −0.05 V at Tyr/MWNTs/GCE, which could be attributed to that MWNTs possess excellent catalytic activity towards the electroreduction of benzoquinone. Tyr/MWNTs/GCE was, therefore, chosen as the working electrode in the following research.
Fig. 1

Cyclic voltammograms of a Tyr/MWNTs/GCE in absence of phenol in 0.1 M phosphate buffer solution (pH 7.5), b Tyr/GCE and c Tyr/MWNTs/GCE in 0.1 M Phosphate buffer solution (pH 7.5) containing 0.1 mM phenol at a scan rate of 50 mVs−1

The effect of the amounts of immobilizing tyrosinase and MWNTs on the response current

The effect of enzyme loading on the biosensor from 20 U to 120 U was investigated. As shown in Fig. 2, the current increased with the increase of the amount of tyrosinase immobilized on the electrode up to 80 U. The phenomena are similar to those previously reported [24]. When the amount of tyrosinase was more than 80 U, the current decreased with the increase of the enzyme amount. The facts can be attributed that the increase of the thickness of enzymatic film will lead to an increase of interface electron transfer resistance and make the electron transfer more difficult [39]. Therefore, 80 U tyrosinase immobilized on the modified electrode was chosen for all subsequent experiments.
Fig. 2

Effect of enzyme amount on the current response of the biosensor to 0.1 mM phenol in 0.1 M phosphate buffer solution (pH 7.5). Other conditions are defined in Fig. 1

The effect of MWNTs amount on the on reduction peak current of phenol in 0.1 M phosphate buffer solution (pH 7.5) was shown in Fig. 3. The amount of tyrosinase immobilizing on each electrode modified with MWNTs was 80 U. It can be seen that the current increased as the MWNTs amount from 0 μg to 0.2 μg, following an obvious decrease when the MWNTs amount was increased from 0.2 μg to 0.4 μg. The maximum response was obtained when the biosensor was prepared with 0.2 μg MWNTs. The reason may be that with the amount of MWNTs increasing, the enrichment active sites on electrode surface increase, leading to the current increases. However, when the amount is more than 0.2 μg, the peak current decreased, the reason may be that the thicker film of MWNTs on the electrode surface increases the diffusion distance of quinone, hinder the mass transport and charge-transfer rate at electrode. So 0.2 μg MWNTs was used to modified each electrode.
Fig. 3

Effect of multiwalled carbon nanotubes amount on the current response of the biosensor to 0.1 mM phenol at the biosensor. Other conditions are defined in Fig. 1

The effect of pH and temperature of the solution on the response current

The effect of pH value on the current response of the biosensor to 0.1 mM phenol in 0.1 M phosphate buffer solution was investigated at the pH range from 5.0 to 9.0 and the results were shown in Fig. 4. It can be seen that the reduction peak current gradually increased with an increasing pH value from 5.0 to 8.0. However, the reduction peak current decreased when the pH further increased to 9.0. At low pH range, the increase in the current response with an increasing pH could be attributed to the activity increase of tyrosinase. The optimum biosensor response was obtained at pH 8.0, which was similar to the optimum pH range of 5.0–8.0 reported for free tyrosinase [40]. This indicated that the immobilization procedure has not altered the activity of tyrosinase. Therefore, 0.1 M pH 7.5 phosphate buffer was chosen as the supporting electrolyte solution in this work.
Fig. 4

Effect of pH on the current response of the biosensor to 0.1 mM phenolin 0.1 M phosphate buffer solution at the biosensor. Other conditions are defined in Fig. 1

Since the catalytic activity of enzymes can be affected by temperature [41], the relationship between temperature and response current of the biosensor is examined in 0.1 M phosphate buffer solution (pH 7.5) containing 0.1 mM phenol. The results are displayed in Fig. 5. With an increasing temperature from 5 to 25 °C the response increased, afterwards the response decreased as the temperature further increased. The maximum response was obtained at 25 °C. Therefore, further measurements were performed at room temperature (25 °C).
Fig. 5

Effect of temperature on the current response of the biosensor to 0.1 mM phenol at the biosensor. Other conditions are defined in Fig. 1

Linear range and detection limit

Amperometry was used to estimate the detection limit of phenol. Figure 6 showed a typical amperometric current–time response of the biosensor under the optimum experimental conditions after successive addition of phenol to phosphate buffer solution under stirring every 150 s. With the increase of phenol concentration, the biosensor could achieve the steady state current within less than 20 s, which indicated a fast electron transfer process. The current linearly increases with the concentration of phenol from 4.0 × 10−7 to 1.0 × 10−5 M and the regression equation can be expressed as i(μA) = 0.2261c(μM)−0.0758, with a correlation coefficient of r = 0.9995. The detection limit was estimated to be 2.0 × 10−7 M (S/N = 3).
Fig. 6

Current–time response curve of the biosensor upon successive additions of phenol with different concentrations. Applied potential: −0.05 V

In order to know the response of the biosensor for other phenolic compounds, the experiments on other three phenolic compounds (hydroquinone, catechol and bisphenol A) were also performed by the Tyr/MWNTs/GCE under the same conditions. The experimental results showed that the biosensor we prepared has a good response to them. The linear range and detection limit of the compounds are shown in Table 1.
Table 1

Linear range and detection limit


Linear range (M)

Detection limit (M)

Correlation coefficient


4.0 × 10−7 to 1.0 × 10−5

2.0 × 10−7



2.0 × 10−7 to 6.0 × 10−5

2.0 × 10−7



2.0 × 10−7 to 1.0 × 10−5

2.0 × 10−7


Bisphenol A

2.0 × 10−6 to 1.0 × 10−4

5.0 × 10−7


Reproducibility, stability and interference

The repeatability of the current response of one enzyme electrode to 50 μM phenol was examined. The relative standard deviation (RSD) was 3.4% for eight successive assays. The electrode-to-electrode reproducibility was determined from the response to 50 μM phenol at five different enzyme electrodes, an acceptable reproducibility was obtained with a variation coefficient of 4.0%.

The enzyme electrode was stored in a dry state at 4 °C in a refrigerator when not in use. The enzyme electrode retained 72% of its original response after 1 month testing.

In order to evaluate the selectivity of the biosensor, the influence of some possible interfering substances was examined in phosphate buffer solution (pH 7.5) containing 20 μM phenol. The results suggested that 100-fold concentration of K+, Ca2+, Mg2+, Zn2+, Pb2+, Cu2+, Cl, Br, I, SO42−, PO43−, CO32−, NO3, 100-fold concentration of glucose, 50-fold concentration of L-cysteine and uric acid, 20-fold concentration of aminoacetic acid, 5-fold concentration of benzoic acid did not interfere with the determination of phenol.

Analytical applications

To study the real feasibility of the biosensor, we checked the biosensor in determination of phenol in real water samples (obtained from Beijing south moat). No special sample pretreatment was required, after the water samples were filtered with a 0.45 μm filter to eliminate particulate matters, the determination can be proceeded. The results were shown in Table 2. In addition, standard addition method was adopted to estimate the accuracy of the method, and the results were also presented in Table 2. It can be seen that the results were satisfactory.
Table 2

Recovery of tyrosinase biosensor (n = 6)


Found (M)

Phenol added (M)

Found (M)

Recovery (%)


3.98 × 10−7

4.00 × 10−7

4.14 × 10−7



3.70 × 10−7

4.00 × 10−7

4.22 × 10−7



3.55 × 10−7

4.00 × 10−7

3.77 × 10−7


Comparison of the biosensor for determination of phenol with others

Table 3 summarises some characteristics of recent phenol biosensors compared to our biosensor. As it can be observed, the analytical range and detection limit values of the biosensors fabricated by us are better than many methods reported in recent publications. The immobilization matrix is easy to prepare and cheaper than other materials like polyacrylamicrogels microgels [12], sol-gel silicate/Nafion composite [29] and PDDA [42]. The biosensor exhibited better long-term stability than that reported in references [24, 37, 43]. Moreover, the method can be applied to the determination of phenol in real water samples.
Table 3

Comparison of the biosensor fabricated by this paper for determination of phenol with others (GCE, glassy carbon electrode; CPE, carbon paste electrode; MWNTs, multiwalled carbon nanotubes; PDDA, poly(dimethyldiallylammonium) chloride; Tyr, tyrosinase)




Immobilization technique


Analytical range(M)




70% after 30 days

Covalently immobilized to MgFe2O4-SiO2


1.0 × 10−6–2.5 × 10−4



Polyacrylamicrogels microgels

100% after 20 days



5.0 × 10−6–2.2 × 10−5


Carbon electrode

Alumina sol–gel

Loss 100% after 3 weeks

Mix Tyr and sol–gel solution


5.0 × 10−7–3.0 × 10−5



Sol–gel silicate/Nafion composite

74% after 2 weeks

MixTyr and sol–gel silicate/Nafion


5.0 × 10−6–1.0 × 10−4




Layer by layer self-assembled


2.0 × 10−6–1.0 × 10−4



MWNTs, Nafion

Mix Tyr, MWNTs and Nation


1.0 × 10−6–1.9 × 10−5




80% after 60 days

Mix Tyr and palygorskite

5.0 × 10−8–1.0 × 10−4




72% after 30 days

Cross-linking with glutaraldehyde


4.0 × 10−7–1.0 × 10−5

This work


We describe an amperometric biosensor here which was fabricated based on tyrosinase immobilized onto MWNTs film for the determination of phenolic compounds. The MWNTs film enhanced the direct electron transfer rate of benzoquinone produced by enzymatic reaction at the electrode. Compared with those biosensors reported in other literatures, the biosensors reported in this work possess main merits are simple fabrication method, good performances in terms of response rate, high sensitivity and operational stability.



This work was supported by the National Natural Science Foundation of China (No. 20247002), Beijing Natural Science Foundation (No.8102009), the Beijing Municipal Education Commission Scientific Technological Project Foundation (No. KZ201110005006) and Beijing Municipal institution of higher learning academic innovating group projects (No. PHR 201007105).


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jing Ren
    • 1
  • Tian-Fang Kang
    • 1
  • Rui Xue
    • 1
  • Chao-Nan Ge
    • 1
  • Shui-Yuan Cheng
    • 1
  1. 1.College of Environmental and Energy EngineeringBeijing University of TechnologyBeijingChina

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